Ocular delivery of active agents via microalgae extracellular vesicles

Abstract

Provided are compositions containing microalgae extracellular vesicles (MEVs) formulated for administration to the eye for treatment of diseases, disorders, and conditions of the eye, particularly for diseases, disorders, and conditions that involve tissues and cells in the back of the eye, which generally cannot be treated with eyedrops. Upon administration to the eye, the MEVs deliver therapeutics to tissues that include the choroid, retinal pigment epithelium cells, and photoreceptor cells. The compositions contain extracellular vesicles from microalgae (MEVs) that are loaded with bioactive cargo for treating or diagnosing or monitoring a disease, disorder, or condition of the eye or involving the eye. Methods of treatment of diseases, disorders, and conditions of the eye are provided.

Description

[0001] OCULAR DELIVERY OF ACTIVE AGENTS VIA MICROALGAE EXTRACELLULAR VESICLES

[0002] RELATED APPLICATIONS

[0003] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 556,325, filed February 21, 2024, entitled “OCULAR DELIVERY OF ACTIVE AGENTS VIA MICROALGAE EXTRACELLULAR VESICLES” to inventors Lila Drittanti and Manuel Vega, and to Applicant AGS Therapeutics SAS.

[0004] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 562,941, filed March 08, 2024, entitled “OCULAR DELIVERY OF ACTIVE AGENTS VIA MICROALGAE EXTRACELLULAR VESICLES,” to inventors Lila Drittanti and Manuel Vega, and to applicant AGS Therapeutics SAS.

[0005] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 663,030, filed June 21, 2024, entitled “MICROALGAE EXTRACELLULAR VESICLE BASED GENE THERAPY VECTORS (MEV-GTVS), THEIR PREPARATION, AND USES THEREOF” to inventors Lila Drittanti and Manuel Vega, and to Applicant AGS Therapeutics SAS.

[0006] Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.

[0007] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0008] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on February 18, 2025, is 542,492 bytes in size, and is titled 5509SEQPCl.xml.

[0009] FIELD

[0010] The field is ophthalmology. Provided are compositions containing microalgae extracellular vesicles (MEVs) formulated for administration to the eye for treatment of diseases, disorders, and conditions of the eye, particularly for diseases, disorders, and conditions that involve tissues and cells in the back of the eye, which generally cannot be treated with eyedrops. Upon administration to the eye, the MEVs deliver therapeutics to tissues that include the choroid, retinal pigment epithelium cells, and photoreceptor cells. The compositions contain extracellular vesicles from microalgae (MEVs) that are loaded with bioactive cargo for treating or diagnosing or monitoring a disease, disorder, or condition of the eye or involving the eye. Methods of treatment of diseases, disorders, and conditions of the eye are provided.

[0011] BACKGROUND

[0012] Extracellular vesicles (EVs) are natural particles produced by most cells. EVs include exosomes (generally about 30-150 nm in size), which are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane, and include microvesicles (about 50-1000 nm), which are produced by the outward budding of membrane vesicles from the cell surface. Exosomes and microvesicles have similar properties, and, in general, are referred to as EVs. Mammalian EVs facilitate intercellular communication via cell-cell transfer of proteins and nucleic acids, such as microRNAs (miRNAs). By virtue of this, EVs derived from mammals and higher plants have been used as carriers for short interfering RNA (siRNA) delivery, microRNA (miRNA).

[0013] SUMMARY

[0014] Provided are cargo-loaded extracellular vesicles (EVs) from microalgae (referred to as MEVs) for use for administration to subjects in vivo. The MEVs are formulated for administration to the eye by any route. In contrast to other delivery compositions and / or delivery vehicles, the MEVs can be administered as drops for treatment of any region of the eye, and particularly can be used for treating diseases, disorders, and conditions of the back of the eye. It is shown herein that upon drop instillation onto or into the eye, the MEVs deliver cargo to tissues and cells of the back of the eye that include the choroid, retinal pigmentosa epithelium (RPE) cells, and photoreceptor cells (see, e.g., Figures 10 and 15A-15D). Other therapeutics and delivery vehicles for treatment of diseases, disorders, and conditions involving such tissues require injection, such as intravitreal injection. It is also shown herein that the MEVs administered as drops are more effective in targeting the choroid, RPE, and photoreceptor cells that other vehicles, such as AAV, following intravitreal injection.

[0015] Topical ophthalmic instillation is an appealing strategy to deliver drugs to the back of the eye because it avoids injection thereby avoiding the risks thereof and difficulties in compliance and use. The drugs are used to treat retinal diseases such as neovascular age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, and glaucomatous optic neuropathy. Topical instillation is non-invasive and allows for self-administration. Heretofore topical instillation has not been feasible because it is not possible to achieve therapeutic drug concentrations in the retina because of protective mechanisms, flows, and barriers of and in the eye (see, del Amo (2022) Frontiers in Drug Delivery, DOI 10.3389 / fddev.2022.954771). Less than 4% of the instilled drug dose enters the anterior chamber, and much less reaches the posterior segment. The MEVs also can be formulated or administered via injections, including intravitreal and subretinal injection or any other route known to those of skill in the art.

[0016] The MEVs provide numerous advantages compared to other delivery vehicles. They deliver different types of therapeutics, including nucleic acids, polypeptides, complexes, and small molecule drugs, to the cells that are targets for the treatment of retinal diseases, including neo-vascularization diseases. These cells include, but are not limited to, retinal pigmented epithelial (RPE) cells, neuronal cells, and endothelial cells. For example, the MEVs deliver (and express) different kinds of modalities relevant to the treatment of retinal diseases, such as proteins, peptides, mRNA, siRNA, and expression plasmids. The MEVs deliver (and express) cargos designed to interfere with different (alternative) mechanisms involving retinal diseases, such as, for example trapping of VEGF or VEGFR, inhibition of CD44, inhibition of the HIF- la pathway, inhibition of complement pathways, such as inhibitors of complement protein 3 (C3) and / or complement protein 5 (C5), which are validated targets for inhibition of complement, including for treatment of complement-mediated diseases, disorders, and conditions.

[0017] Provided herein are compositions containing MEVs, the compositions are formulated topical or local instillation or injection into the eye, and the MEVs contain cargo for treating, diagnosing, and / or monitoring diseases, disorders, and conditions diseases, disorders, and conditions of the eye, including diseases, disorders, and conditions of the back of the eye, including, but are not limited to, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, glaucomatous optic neuropathy, edema, (DME), retinal vein occlusion (RVO), and optical neuropathy glaucoma disease, and other diseases, disorders, and conditions that involve and / or require delivery to the back of the eye, including one or more of the retina, choroid, retinal pigmentosa epithelium (RPE) and photoreceptor cells. Diseases, disorders, and conditions of the back of the eye, include, for example, retinal diseases, such as retinal detachment where fluid leaks through a tear in the retina, causing it to detach from the eye's other tissues; diabetic retinopathy, generally a complication of diabetes that can lead to blindness; various forms of macular degeneration, including age- related disease, which cause vision loss; retinitis pigmentosa, a genetic disease that causes vision loss; retinoblastoma, which is a cancer of the retina; uveitis, which is an inflammation inside the eye, usually caused by the immune system fighting an infection; and glaucoma.

[0018] The MEVs are loaded with cargo that includes bioactive molecules, including biomolecules and small molecules, including diagnostic and / or therapeutic molecules for treatment or diagnosis of diseases, disorders, and conditions of the front (anterior) of the eye, including, but are limited to: corneal dystrophy, which is a genetic disorder that causes cloudy vision due to protein or fluid buildup on the cornea; keratitis, which is an inflammation of the cornea that can cause redness, swelling, blurry vision, and sensitivity to light; dry eye, which is a condition where the eyes don't produce enough tears; cataracts; keratoconus, which is a condition where the cornea weakens and becomes cone-shaped, causing blurry vision; photokeratitis, which is an inflammation of the cornea caused by exposure to light, such as from the sun or welding; viral infections, such as herpes simplex and herpes zoster (shingles) infections; pterygium; and Fuchs' endothelial dystrophy.

[0019] The MEVs, thus, are loaded with therapeutics, cargo, including proteins, peptides, nucleic acids, and RNAs and plasmids, that are therapeutics for treating these diseases. Such therapeutics include any described herein and any known to the those of skill in the art.

[0020] For purposes herein, the MEVs can be formulated as eyedrops or for injection, including formulations in phosphate buffered saline and other known ophthalmic formulations. The MEVs contain cargo for treatment of diseases, disorders, and conditions of the eye. In particular, the treatments are for diseases, disorders, and conditions of the eye that heretofore have required injection for delivery of therapeutics. The MEVs contain, as cargo, therapeutics that are for treatment of diseases, disorders, and conditions of the front of the eye. These include, but are not limited to, macular degeneration, glaucoma, and diseases, disorders, and conditions involving the retina. Therapeutics, include small molecules, nucleic acids, aptamers, and gene therapy treatments and vectors. Any known therapeutics can be introduced into the MEVs, which are provided in compositions formulated for administration to the eye, particularly as eye drops for instillation into the eye. The treatments generally are those that heretofore were administered by injection. The MEVs advantageously traffic to the back of the eye.

[0021] The MEVs herein are extracellular vesicles from microalgae and are referred to as MEVs. Microalgae are unicellular green algae, and include those that belong to the order Chlorellales, in particular, the Chlorellaceae family, and in particular those that belong to the Chlorella genus, such as Chlorella vulgaris. Microalgae extracellular vesicles (MEVs) can be manufactured on a large scale. The MEVs provided herein are formulated for administration to the eye. It is shown herein that MEVs can be administered as eye drop and deliver the MEVs and their cargo to tissues in the eye, including the retina. Hence, the MEVs can be administered as eye drops to treat diseases, disorders, and conditions of the eye that generally are treated by injection, including intravitreal injection and subretinal injection. Known therapeutics, including nucleic acid, polypeptides, proteins, small molecules for treatment of ophthalmological diseases, disorders, and conditions can be packaged in the MEVs and formulated as eyedrops.

[0022] The MEVs can be endogenously loaded (endo-loaded) by producing them in genetically-modified microalgae that encode or express proteins, polypeptides, small peptides, various RNA molecules, and / or other biomolecules that the microalgae can be genetically programmed to express and thereby package in MEVs.

[0023] The MEVs can be exogenously loaded with the bioactive molecule cargo following production of the MEVs. The MEVs can be exogenously loaded following isolation or partial purification / isolation of the MEVs from microalgae by contacting the MEVs with the cargo to produce the compositions in which substantially all the MEVs, generally on the average, have substantially the same exogenously-loaded heterologous cargo. Upon administration, the biodistribution pattern does not depend upon the how the MEVs are loaded. Biodistribution of the MEVs is different from mammalian EVs and other EVs and / or nanoparticles. The MEVs herein are formulated for administration to the eye, generally as a liquid, such as a suspension or emulsion, or other formulation that can be administered. In general, the MEVs are provided in ophthalmologic compositions for administration to the eye. For most of the applications, the MEVs can be administered as eye drops, and do not require injection. The skilled person, however, can administer the MEVs by any methods deemed suitable for a particular application, including injection.

[0024] Provided is a microalgae extracellular vesicle (MEV) that contains cargo that comprises a therapeutic for treatment of a disease, disorder, or condition of the eye. In general, the cargo is heterologous to the MEV or microalgae in which the MEV is produced, and is for treatment of a disease, disorder, or condition of the eye. For example, the disease, disorder, or condition is a disease, disorder, or condition of the back of the eye or posterior eye, such as, but not limited to, a disease, disorder, or condition that involves the choroid-retina region, retinal pigment epithelium (RPE) cells, and / or photoreceptor cells. When MEVs are administered topically they traffic to the choroid-retina region, retinal pigment epithelium (RPE) cells, and / or photoreceptor cells. The MEVs can be administered for treatments of other regions of the eye, and can be injected intravitreally and subretinally or by another route. An advantage of the MEVs is that they can be topically administered to deliver cargo used to treat a disease, disorder, or condition of the back of the eye or the posterior region of the eye. The MEVs deliver a therapeutically sufficient or therapeutically effective amount of the cargo.

[0025] Diseases, disorders, and conditions include, but are not limited to, one or more of an age-related macular degeneration (AMD), a retinal degenerative disease, disorder, or condition, or a disease, disorder, or condition involving choroidal neovascularization, an inheritable disease, disorder, or condition involving the retina, and an ocular hypertension. For example, the disease, disorder, or condition is selected from one or more of an age-related macular degeneration (AMD), diabetic retinopathy, retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion (RVO), and optical neuropathy glaucoma disease, such as, for example, wet AMD, geographic atrophy, diabetic retinopathy, macular edema, retinitis pigmentosa, glaucoma, and / or ocular hypertension. Diseases, disorders, and conditions include a disease of the retina that leads to blindness, including inheritable diseases that can be treated by gene therapy or a gene editor.

[0026] The cargo comprises therapeutics for treatment of any of these diseases, disorders, and conditions. The cargo can comprise a nucleic acid molecule, a polypeptide, a protein, an aptamer, or an antisense oligonucleotide, or nucleic acid that comprises or encodes a therapeutic for treatment of a disease, disorder, or condition of the eye.

[0027] Cargo in the MEV can include any of a)-t) and combinations thereof: a) the cargo comprises DNA or RNA that encodes a therapeutic product or double-stranded RNA; or b) the cargo comprises mRNA or modified mRNA; or c) the cargo comprises a gene editing system; or d) the cargo comprises a plasmid; or e) the cargo comprises a viral vector; or f) the cargo comprises a therapeutic or diagnostic protein or polypeptide; or g) the cargo comprises a protein complex; or h) the cargo comprises a vaccine; or i) the cargo comprises or encodes a protein that is an antibody or antigenbinding fragment thereof; or j) the cargo is a product that stimulates the immune system of a subject treated with the composition; or k) the cargo is a cosmeceutical or a cosmetic or cosmetically active product; or l) the cargo comprises a small molecule bioactive molecule; or m) the cargo is a chemotherapeutic agent; or n) the cargo is an immunotherapeutic agent; or o) the cargo comprises a diagnostic marker or detectable product; or p) the cargo comprises a prodrug; or q) the cargo comprises an antibody, or an scFv or another antigen-binding fragment of an antibody, or a bi- specific antibody, or r) the cargo comprises a peptide or a polypeptide; or s) the cargo comprises a siRNA, or a miRNA, or another active small RNA, or t) the cargo comprises a IncRNA. The MEV wherein the cargo comprises a diagnostic reporter marker that comprises a luciferase or nucleic acid encoding a luciferase, a fluorescent protein or nucleic acid encoding a fluorescent protein, or a luciferase operon; and the reporter detects a marker indicative of a disease, disorder, or condition of the eye, such as, for example, a cell surface marker or an enzyme.

[0028] Exemplary of therapeutic cargo is a therapeutic that inhibits a VEGFR or inhibits VEGF. Cargo includes a protein or polypeptide or peptide therapeutic or nucleic acid encoding a therapeutic protein or therapeutic RNA. The cargo can comprise a detectable product or a reporter gene, such as, for example, a fluorescent protein or nucleic acid encoding the fluorescent protein. Exemplary of the fluorescent protein or encoding nucleic acid is enhanced green fluorescent protein (EGFP) of SEQ ID NO:63 or a variant thereof that is fluorescent and has at least 95% sequence identity to SEQ ID NO:63; mRNA encoding EGFP of SEQ ID NO:64 or a fluorescent variant thereof having at least 95% sequence identity to the protein encoded by SEQ ID NO:64, a luciferase or encoding nucleic acid of SEQ ID NO:65 or an enzymatically active variant thereof having at least 95% sequence identity to SEQ ID NO:65. Other therapeutic cargo comprises siRNA targeting HIF-la (SEQ ID NO:66 and 67), siRNA targeting VEGF (SEQ ID NOs:68 and 69), FKBPL (FK506 Binding Protein-Like) peptide (SEQ ID NO:72), Aflibercept (a.k.a. VEGF Trap-Eye) protein (SEQ ID NO:73) or its encoding mRNA ( SEQ ID NO:74 or 75), Norrin protein (SEQ ID NO:76), siRNA targeting SRPK1 (SEQ ID NOs:77, 78, and 79), pBCB-23-0041- CDS-EGFP protein (SEQ ID NO:80), pBCB-23-0070-CDS-Aflibercept-l(SEQ ID NO: 81), pBCB-23-0065-CDS-Aflibercept-2 (SEQ ID NO:82), or variants thereof having at least 95% sequence identity thereto or to the encoded protein. Other therapeutics include (and their disease targets) include any known in the art, including any listed in the following table:

[0029] Other such drugs are known to those of skill in the art and can be delivered in the MEVs.

[0030] Other cargo includes, but is not limited to cargo selected from among a prostaglandin (latanoprost, travoprost, bimatoprost), a beta-blocker (timolol, carteolol, levobunolol, betaxolol, nebivolol), a parasympathomimetic (pilocarpine), alpha2- agonists (apraclonidine, brimonidine), a carbonic anhydrase inhibitor (CAI) (brinzolamide, dorzolamide), and an anti-oxidant selected from among nordihydroguaiaretic acid, meso-nordihydroguaiaretic (masoprocol). Other cargo includes, for example, Voretigene neparvovec-rzyl, EA-2353, OCU400, AGTC-501, 4D-125, CNTO 2476, SAR421869, CEP290, GUCY2D, AAV2-repl, SAR422459, Emixustat HC1, STG-001, ATSN-201, rAAV2tYF-CB-hRSl, CNGB3.

[0031] Of interest is cargo that comprises a VEGF inhibitor, such as, for example, one or more of Bevacizumab (Avastin®), nibizumab, aflibercept (Eyela®), ranibizumab, Avacincaptad pegol, and Pegcetacoplan. Exemplary thereof are nucleic acid or nucleic acid encoding a polypeptide or a polypeptide of any of SEQ IDS NOs: 73, 74, and 83-95 or comprising a plasmid of any of SEQ ID NOs:80-82.

[0032] Provided is an MEV or MEVs that comprise cargo for treating a disease, disorder, or condition that involves one or more of choroidal cells, RPE cells, and photoreceptor cells, wherein the MEV is formulated as eyedrops for topical administration to the eye. For example, cargo can comprise siRNA or other inhibitory RNA targeting HIF-la (SEQ ID NOs:66 and 67), siRNA targeting VEGF (SEQ ID NOs:68 and 69), FKBPL (FK506 Binding Protein-Like) peptide (SEQ ID NO:72), Aflibercept (a.k.a. VEGF Trap-Eye) protein (SEQ ID NO:73) or its coding mRNA (SEQ ID NO:74 or 75), Norrin protein (SEQ ID NO:76), siRNA targeting SRPK1 (SEQ ID NOs:77, 78 and 79), 1.99.25 antibody, and / or the F4L5.13 antibody, or plasmid encoding aflibercept (SEQ ID NO: 81 or 82), or plasmid encoding eGFP (SEQ ID NO:82).

[0033] The cargo can be the cargo is endogenously (endo-loaded) by genetically modified microalgae. Such cargo generally comprises nucleic acid or a polypeptide; the microalgae is genetically modified to encode such product or a plasmid or vector encoding the product is introduced into the microalgae for expression and packaging o insertion or embedding in the MEV.

[0034] Cargo also can be exogenously loaded, following production of the MEV. The MEV generally is isolated or partially purified for exogenous loaded. Such products can include nucleic acids, polypeptides, proteins, peptides, aptamers, small molecules, biological molecules, vectors, plasmids, and other product of interest.

[0035] The MEV is produced in a microalgae, such as a microalgae that is a species of the family Chlorellaceae. Such microalgae include members of the genus Chlorella or Parachlorella. Exemplary thereof are: Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis, a species of Parachlorella, such as Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hussii.

[0036] Provided are compositions that contain any of the MEVs. In general, the compositions are formulated for administration to the eye, such as eyedrops. Of interest are formulations for topical administration, such as eyedrops. The compositions can be formulated for administration to the eye drop instillation on the eye surface, or intraocular injection, intravitreal injection, supra-choroidal, subretinal injection, or intraocular injection. Compositions for administration to the eye are generally formulated at a suitable pH and can contain phosphate buffered saline (PBS). They can contain surfactants and can be formulated as a suspension or as an emulsion, including a microemulsion or a nanoemulsion.

[0037] Provided are MEVs and compositions for use for treating a disease, disorder, or condition of the eye, such as a condition of the back of the eye, including any known to those of skill in the art, including any listed or described herein above and below. Provided are methods of treatment of a disease, disorder, or condition of the eye, comprising administering an MEV or composition provided herein, where the MEVs comprise a therapeutic or detectable or diagnostic product for treating, detecting, diagnosing, and / or monitoring a disease, disorder, or condition of eye. The MEV or composition in accord with the methods and uses herein can be administered topically to the eye. The disease, disorder, or condition is a disease, disorder, or condition includes any known to those of skill in the art, and / or described herein. Included are diseases, disorders, and conditions of the back of the eye, such as, but are not limited to, a disease, disorder, or condition of the choroid-retina region, RPE cells, and / or photoreceptors, includes rods and cones. The MEVs and compositions can be administered topically. It is shown herein that, upon topical administration the MEVs traffic to the ciliary body and then to the choroid.

[0038] Diseases, disorders, and conditions that can be treated by administering an MEV or composition provided herein or using an MEV or composition provided herein are, for example, a disease, disorder, or condition of the eye or involving the eye is selected from among Adie’s pupil, adult strabismus, age-related macular degeneration (AMD), albinism, amblyopia, anisocoria, aphakia, arcus senilis, astigmatism, bacterial keratitis, Bell’s palsy, black eye, blepharitis, blocked tear duct, branch retinal vein occlusion (BRVO), carotid artery disease, cancer of the eye, cataracts, cellulitis, central retinal vein occlusion (CRVO), central serous chorioretinopathy, chalazia and stye, Charles Bonnet syndrome, choroidal neovascular membranes, chronic angle-closure glaucoma, coloboma, color blindness, conjunctivitis (pink eye), contact lens-related eye infections, convergence insufficiency, corneal abrasion, corneal dystrophies, corneal erosion, corneal laceration, corneal ulcer (keratitis), crossed eyes (strabismus), cytomegalovirus retinitis, dementia and the eye, detached retina, diabetes, drusen, dry eye, ectropion, endophthalmitis, entropion, excessive blinking in children, eye allergies, eye lymphoma, eyelid spasm and twitching, farsightedness, Fuchs’ dystrophy, fungal keratitis, giant cell arteritis, giant papillary conjunctivitis, glaucoma, headache and eye problems, hemangioma, herpes keratitis, herpes zoster (shingles), heterochromia, histoplasmosis, HIV / AIDS and the eye, hyperopia, hyphemia, idiopathic intracranial hypertension, iridocorneal endothelial syndrome, ischemic optic neuropathy, juvenile idiopathic arthritis uveitis, juvenile macular dystrophy, keratoconus, lattice degeneration, Leber congenital amaurosis, low vision, macular edema, macular hole, macular pucker, macular telangiectasia, Marfan syndrome, microvascular cranial nerve palsy, migraine, milia, myasthenia gravis, myopia (nearsightedness), neuropathic corneal pain, nevus, nystagmus, ocular hypertension, ocular melanoma, ocular rosacea, onchocerciasis (African River Blindness), optic neuritis, orbital fracture, photokeratitis, pigment dispersion syndrome, pinguecula and pterygium, posterior vitreous detachment, presbyopia, pseudoexfoliation syndrome, ptosis, retinal artery occlusion, retinal detachment, retinal vein occlusion, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, scleritis, Sjogren’s syndrome, sleep crust, Stargardt disease, Stickler syndrome, subconjunctival hemorrhage, thyroid eye disease (Grave’s disease), torn retina, toxoplasmosis, trachoma, trichiasis, trichotillomania, Usher syndrome, uveitis, vitamin A deficiency, vitreomacular traction, and xanthelasma. The methods, uses, MEVs, compositions can be used to treat, detect, diagnose, monitor treatment of AMD or a diabetic retinopathy or a disease, disorder, or condition involving the photoreceptors, including a disease, disorder, or condition is treated with an inhibitor of a VEGF receptor or an inhibitor of VEGF. The disease, disorder, or condition is one that can be treated by a therapeutic delivered to a cell or tissue in the eye that can accessed by delivery of the MEV or therapeutic to the ciliary body and to the choroid, including by topical administration the MEV or composition.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0041] Figure 1 shows the purification of MEVs by Size Exclusion Chromatography (SEC).

[0042] Figure 2 shows exemplary images of MEVs obtained using Transmission Electron Microscopy (TEM; the images show that the MEVs are round shaped vesicles sized -50-250 nm in diameter, enveloped in a single lipid bilayer membrane, their lumen had slightly higher electron density, and the thickness of the membrane is estimated to be -5-10 nm, which matches the thickness of a plasma membrane.

[0043] Figures 3A and 3B show images obtained with MEVs labelled with lipophilic dyes by confocal microcopy. Figure 3A shows, by confocal microscopy, MEVs labeled with PKH26 dye; PKH26 fluoresces in the yellow-orange region of the spectrum by confocal microscopy. Figure 3B shows confocal microscopy of MEV labelled with DiD (ThermoFisher Scientific), a fluorochrome in the red spectrum with peak excitation (650 nm) and emission (670 nm).

[0044] Figures 4A-4C show the uptake of PKH26-labelled MEVs into cells after 16 hours of incubation with visualization using Zeiss LSM8 confocal microscope using 63X objective. Blue: Hoechst Nucleus) / Red: PKH26 (MEVs).

[0045] Figure 4A: cells uptake of MEVs (2D image); Figure 4B: individual cell image (2D); and Figure 4C: individual cell image (3D).

[0046] Figure 5 shows the distribution of MEVs labelled with lipophilic dyes by cytometry; the top panels shows the DiR dye incorporated into membranes of the MEV, and lower panels show the percentage of labelled-MEVs.

[0047] Figures 6A and 6B show the high uptake of MEVs in different type of cells Figure 6A: epithelial cells; Figure 6B: monocytes) as percentage of fluorescence cells after the treatment with DiR-labelled-MEVs by cytometry. Figure 7 shows the components and order thereof from LB to RB in the plant transformation vectors of Table 3.

[0048] Figure 8 shows a profile of GFP fluorescence signal intensity in choroidretina at different timepoints after a single dose of MEVs loaded with EGFP-encoding mRNA, administered by drop instillation. EGFP is a basic (constitutively fluorescent) green fluorescent protein originally derived from Aequorea Victoria.

[0049] Figure 9 shows the kinetics of the expression of EGFP protein (fluorescence measured) in choroid-retina region provide by the delivery of mRNA loaded in MEVs.

[0050] Figure 10 shows an immunohistochemistry analysis of choroid-retina section, including Retinal Pigmented Epithelium (RPE), after a single dose of MEVs loaded with EGFP-mRNA, administered by drop instillation; black areas - cell nuclei strained with DAPI, grey zones - EGFP stained with anti-EGFP mouse antibodies.

[0051] Figures 11A and 11B depict the anatomical parts of the eye. Figure 11A is a schematic cross-section of the human eye and a magnification of retina including the choroid, Bruch’s membrane, retinal pigment epithelium (RPE), photoreceptor rod and cone cells, horizontal cells, bipolar cells, amacrine cells, Muller cells and ganglion cells (adopted from: Murphy et al. (2020) Acte Biomaterialia 104:1-16, modified). Figure 11B provides a depiction with additional anatomical parts of the eye identified.

[0052] Figures 12A and 12B provide a schematic representation of retinal damage in choroidal neovascularization (CNV) (adopted from: allaboutvision.com / condi- tions / choroidal-neovascularization-cnv); Figure 12A shows a healthy eye, and Figure 12B shows an eye with retinal damage.

[0053] Figure 13 shows routes of administration for delivery of therapeutics to the eyes via (1) Topical, (2) Subconjunctival, (3) Suprachoroidal, (4) Subretinal, (5) Intravitreal administration.

[0054] Figure 14 shows topical administration of MEVs by drop instillation on the surface of the eye globe; the black arrows indicate the pathway followed by the MEVs from the site of administration to the site of measurement delivery.

[0055] Figures 15A-D show a comparison between distribution of AAVs following intravitreal injection and topical instillation of drops of MEVs. Figures 15A and 15B show that, upon administration of eye drops containing MEVs, the encoded product is translated and delivered to the back of the eye to tissues including the choroid, RPE, and other layers of tissues in the back of the eye. Figures 15C and 15D show the tissue distribution of encoded GFP following intravitreal injection. The encoded protein following injection of the AAV vector is delivered to different tissues or follow a different gradient of trafficking from drop instillation of MEVs.

[0056] Figures 16A and 16B show anti-anti-angiogenic activity of MEVs loaded with mRNA encoded Aflibercept (SEQ ID NO:73); the MEVs enter cells and the mRNA is translated. Figure 16A shows the anti-angiogenic activity following the exposure of HUVEC cells to MEVs loaded with a mRNA encoding Aflibercept (SEQ ID NO:73), and presents images from the tube formation assay: 1- HUVEC cells pre-treated with VEGF 20ng / mL; 2- HUVEC cells non-pre-treated with VEGF; 3- HUVEC cells pretreated with VEGF and with non-loaded MEVs; 4- HUVEC cells pre-treated with VEGF and with MEVs loaded with mRNA encoding Aflibercept. Figure 16B shows the images of nodes and networks obtained in the tube formation assay; is shows the results of analysis using Angiogenesis plugin ImageJ: 5-6 from cells pre-treated with VEGF; 7-8 from cells non-pre-treated with VEGF; 9-10 from cells pre-treated with VEGF and with non-loaded MEVs; and 11-12 from cells pre-treated with VEGF and with MEVs loaded with mRNA encoding Aflibercept.

[0057] Figure 17 (with panels labeled A1-E2): Images obtained 4 hours after the scratch showing the effect on wound healing (anti-angiogenic activity) of MEVs loaded with mRNA encoding aflibercept: Al-A2= original scratch sample; Bl-B2= cells +VEGFA 150 pg / mL; Cl-C2= cells +VEGFA 150 pg / mL + non-loaded MEVs; Dl- D2= Cells +VEGFA 150 pg / mL + MEVs loaded with mRNA coding for aflibercept SEQ ID 74; El-E2= Cells +VEGFA 150 pg / mL + MEVs loaded with mRNA coding for aflibercept SEQ ID 75, where Al, Bl, Cl, DI, and El are images from overlay of wound-healing boundary 4X magnification A2, B2, C2, D2, and E2 are images of the mask of the overlay.

[0058] Figure 18 provides a bar graph showing the quantified wound healing percentages following treatment with MEVs loaded with mRNA encoding aflibercept (payload 1, mRNA Aflibercept (SEQ ID NO:74), payload 2, mRNA Aflibercept (SEQ ID NO:75)). Figure 19 depicts the interaction of the AMD drug EBIN with its target EB3. inhibits the End Binding-3 (EB3) to prevent neovascular leakage and choroidal neovascularization (Lee et al. (2023) Cell Reports Medicine 4(10)). PAN-90806 (PanOptica) is an orally-bioavailable small molecule that binds and inhibits VEGFR2 and increases the interval between standard intravitreal injections (Khachigian et al. (2023) J Transl Med 21(133)).

[0059] Figure 20 shows the images of nodes and networks obtained from the tube formation assay and anti- angiogenic effect of MEVs loaded particularly with aflibercept protein, or mRNAs coding for aflibercept protein. The figure illustrates anti-angiogenic activity following the exposure of HUVEC cells to MEVs loaded either with the aflibercept protein or with mRNAs coding for aflibercept. It presents images from the tube formation assay: A- Cells (Negative control (w / o VEGFa)); B- Cells (Positive control (Ing / mL VEGFa)); C- Cells (Positive control (2ng / mL VEGFa)); D- Cells treated with aflibercept protein 0.5 ng / mL + VEGFa Ing / mL; E- Cells treated with MEVs loaded with mRNA (coding for aflibercept) +VEGFa Ing / mL; F- Cells treated with MEVs loaded with aflibercept protein + VEGFa Ing / mL.

[0060] Figure 21 illustrates the anti-angiogenic activity following the exposure of HUVEC cells to MEVs loaded with a plasmids coding for aflibercept or MEVs loaded with a peptide: the FKBP-L (FK506 Binding Protein-Like) peptide (SEQ ID NO:72)). The figure illustrates anti-angiogenic activity following the exposure of HUVEC cells to MEVs loaded with a plasmids coding for aflibercept or MEVs loaded with a peptide (FKBP-L). It presents images from the tube formation assay: A- Cells (Negative control (w / o VEGFa)); B- Cells (Positive control (Ing / mL VEGFa)); C- Cells (Positive control (2ng / mL VEGFa)); D- Cells treated with MEVs loaded with pITR (coding for aflibercept)+ VEGFa Ing / mL; E- Cells treated with MEVs loaded with pITR’ (coding for aflibercept) +VEGFa Ing / mL; F- Cells treated with MEVs loaded with FKBP-L peptide + VEGFa Ing / mL.

[0061] Figure 22 are the quantified results of the wound healing assay showing the efficacy of treatment with MEVs loaded with different payloads for the prevention of neo-angiogenesis in HUVEC primary cells. The graph quantifies the results obtained four hours after the scratch and the respective treatment. The bars show the percent of wound healing (anti- angiogenic or cell migration inhibition) with respect to the treatment : no treatment, MEVs without VEGFa (negative control), MEVs loaded with VEGFa Ing / mL (positive control), MEVs loaded with VEGFa 2ng / ML (positive control), Aflibercept protein (0.5ng / mL) alone, MEVs loaded with Aflibercept protein, and MEVs loaded with Aflibercept mRNA.

[0062] Figure 23 are the quantified results of the wound healing assay showing the efficacy of treatment with MEVs loaded with different payloads for the prevention of neo-angiogenesis in HUVEC primary cells. The graph quantifies the results obtained four hours after the scratch and the respective treatment. The bars show the percent of wound healing (anti- angiogenic or cell migration inhibition) with respect to the treatment : no treatment, MEVs without VEGFa, MEVs loaded with VEGFa Ing / mL, MEVs loaded with VEGFa 2ng / ML, MEVs loaded with the peptide FKBP-L (FK506 Binding Protein-Like), and MEVs loaded with Aflibercept pITRs.

[0063] Figure 24 is a diagram that shows the experimental design for the Matrigel® tube formation assay with HUVEC cells.

[0064] Figure 25 shows the images of nodes and networks obtained from the tube formation assay. The figure indicates that anti-angiogenic activity following the exposure of HUVEC cells to MEVs loaded with a mRNA for aflibercept is observed not only in cells treated (condition 3) but also in fresh cells incubated with conditioned medium 3 (condition 6). It presents images from the tube formation assay: 1- HUVEC cells negative control (untreated cells); 2- HUVEC cells treated with VEGF Ing / mL; 3- HUVEC cells pre-treated with VEGF Ing / mL and with MEVs loaded with mRNA coding for Aflibercept; 4- HUVEC cells+ conditioned medium 1 ; 5- HUVEC cells+ conditioned medium 2; 6- HUVEC cells+ conditioned medium 3.

[0065] Figures 26A-C, Figure 26A shows the presence and distribution of the MEVs in the neuronal body as well as along the axons. The alignments of MEVs in the axons is fully compatible the axonal transport of MEVs along the olfactory tract, observed in vivo upon intranasal administration of the MEVs. This figure shows penetration of MEVs (labelled with PKH26) into NSCs cells: 1- shows NSCs cells, negative control (Alexa 546); 2- NSCs cells treated with MEVs labelled with PKH26 (Alexa 546); 3- NSCs cells treated with MEVs labelled with PKH26 (Alexa 546); 4- NSCs cells, negative control (merge DAPI / Alexa 546); 5- NSCs cells treated with MEVs labelled with PKH26 (merge DAPI / Alexa 546); 6- NSCs cells treated with MEVs labelled with PKH26 (merge DAPI / Alexa 546).

[0066] Figure 26B shows the delivery and the presence of Ovalbumin protein (OVA), (Ovalbumin, Alexa Fluor™ 488 Conjugate (034781, ThermoFisher) into NSCs cells: 1- NSCs cells treated with MEVs loaded with OVA (Alexa 488); 2- NSCs cells treated with MEVs loaded with OVA (merge DAPI / Alexa 488); 3- NSCs cells, negative control (Alexa 488); 4- NSCs cells, negative control (merge DAPI / Alexa 488). The presence of GFP protein is observed in the body of the neurons, only (not in the axons).

[0067] Figure 26C shows the penetration, delivery and the expression of GFP protein from the mRNA loaded MEVs in NSCs cells: 1- NSCs cells treated with MEVs loaded with mRNA coding for GFP (Alexa 488); 2- NSCs cells treated with MEVs loaded with mRNA coding for GFP (merge DAPI / Alexa 488); 3- NSCs cells, negative control (Alexa 488); 4- NSCs cells, negative control (merge). These results demonstrate the ability of MEVs to deliver their active payload into neurons and the endosomal scape of the payload (mRNA) to be translated to protein.

[0068] Figures 27A-27B show MEVs penetration and delivery of payload. Figure 27A shows the penetration of MEVs (labelled with PKH26) into HUVEC cells: 1- T=Oh, HUVEC cells treated with MEVs labelled with PKH26 (merge DAPI / Actin (ALEXA 488) / MEVs labelled PKH26 (ALEXA 546)); 2- T=48h, HUVEC cells treated with MEVs labelled with PKH26 (merge DAPI / Actin (ALEXA 488) / MEVs labelled PKH26 (ALEXA 546)); 3- T=48h, HUVEC cells treated with MEVs lo labelled with PKH26 (merge DAPI / Actin (ALEXA 488) / MEVs labelled PKH26 (ALEXA 546)). Figure 27B shows the results of penetration and presence of GFP protein translated from the payload of MEVs (mRNA coding for GFP) in HUVEC cells: 1- T=Oh, HUVEC cells treated with MEVs loaded with mRNA coding for GFP protein (merge DAPI / Actin (ALEXA 546) / GFP protein (Alexa 488)); 2- T=24h, HUVEC cells treated with MEVs loaded with mRNA coding for GFP protein (merge DAPI / Actin (ALEXA 546) / GFP protein (Alexa 488)). A time response has shown that penetration is maximal at 48 hours of treatment (data not shown). These results demonstrate the capacity of MEVs to deliver their payload to endothelial cells and the ability of the payload (mRNA) to be translated into protein.

[0069] Figure 28 shows the penetration in ARPE-19 cells. A time response shows that penetration is maximal at 48 hours of treatment: 1- T=0h, ARPE-19 cells treated with MEVs labelled with PKH26 (merge DAPI / Actin (ALEXA 488) / MEVs labelled PKH26 (ALEXA 546)); 2- T=24h, ARPE-19 cells treated with labelled with PKH26 (merge DAPI / Actin (ALEXA 488) / MEVs labelled PKH26 (ALEXA 546)). GFP expression from its coding mRNA loaded in MEVs into ARPE-19 is maximal at 72h (data non shown). The results demonstrate the ability of MEVs to deliver their payload to retinal pigmented epithelial (RPE) cells and the endosomal scape of the payload (mRNA) to be translated to protein.

[0070] Figure 29 shows the long-lasting expression of GFP protein in ARPE-19 cells treated with MEVs loaded with a plasmid carrying AAV’s ITRs and coding for GFP protein at one week after the treatment: 1- T=0h, ARPE-19 cells treated with MEVs loaded with plasmid carrying AAV’s ITRs and coding for GFP (merge); 2- T=72h, ARPE-19 cells treated with MEVs loaded with plasmid carrying AAV’s ITRs and coding for GFP (merge); 3- T= 96h, ARPE-19 cells treated with MEVs loaded with plasmid carrying AAV’s ITRs and coding for GFP (merge); 4- T=144h, ARPE-19 cells treated with MEVs loaded with plasmid carrying AAV’s ITRs and coding for GFP (merge). The Figure shows the increasing expression of GFP protein from 72h to 144h, and the expression for at least 144h (longest time studied).

[0071] DETAILED DESCRIPTION

[0072] Outline

[0073] A. Definitions

[0074] B. Overview

[0075] 1. Microalgae

[0076] 2. Overview of The Eye, Diseases, Disorders, And Conditions, And Treatments a. The Eye and MEVs b. Structure and function of ocular components c. Anatomical Parts of the Eye i. Cornea ii. Conjunctiva iii. Sclera iv. Iris v. Lens vi. Retina vii. Microstructure of the retinal tissue viii. Choroid ix. Vitreous Humor x. Ciliary Body xi. Rod Cells xii. Cone cells xiii. Drainage angle xiv. Eyelid xv. Fovea xvi. Lacrimal Gland xvii. Macula xviii. Meibomian Glands xix. Optic Nerve xx. Pupil xxi. Tear duct d. Diseases, Disorders and Conditions of The Eye e. Diseases, disorders, and conditions affecting the back of the eye (posterior segment of the eye) f. Heritable Diseases, disorders, and conditions of the Eye i. Retinitis Pigmentosa ii. Usher syndrome iii. Leber congenital amaurosis (LCS) iv. Choroideremia v. Stargardt disease vi. X-linked Retinoschisis vii. Achromatopsia g. Exemplary Treatments for Heritable Diseases, Disorders, and Conditions of the Eye h. Inflammatory Diseases, Disorders, and Conditions of the Front and Outer Eye i. Bacterial, Fungal, and Viral Keratitis and Contact Lens- Related Eye Infections ii. Bell’s Palsy iii. Blepharitis iv. Conjunctivitis v. Corneal Abrasion, Laceration, and Erosion vi. Corneal Dystrophies vii. Dry Eye viii. Keratoconus ix. Neuropathic Corneal Pain x. Optical Rosacea xi. Onchocerciasis xii. Scleritis xiii. Thyroid Eye Disease (Grave’s Disease) xiv. Uveitis and Juvenile Idiopathic Arthritis (JIA) Uveitis i. Treatments of Inflammatory Diseases, Disorders, and Conditions of the Front and Outer Eye j. Glaucoma and Pressure-Related Conditions of the Inner Eye and back of the eye i. Glaucoma ii. Iridocorneal Endothelial Syndrome iii. Ocular Hypertension k. Treatment of Glaucoma and Pressure-Related Conditions of the Inner Eye l. Refractive Conditions of the Eye or Conditions of the Lens of the Eye i. Astigmatism ii. Cataracts iii. Hyperopia (Farsightedness) iv. Myopia (Nearsightedness) v. Presbyopia m. Treatments for Refractive Conditions of the Eye n. Treatments for Diseases, Disorders, and Conditions of the Posterior Eye i. Age-related Macular Degeneration (AMD): pathological mechanisms, treatment options a) Dry AMD Treatments b) Wet AMD Treatments ii. Diabetic Retinopathy iii. Retinal vein occlusions and treatment iv. Endophthalmitis v. Uveitis vi. Cystoid macular edema (CME) vii. Epiretinal Membrane viii. Macular Hole ix. Retinitis Pigmentosa x. Cytomegalovirus Retinitis xi. Retinopathy of Prematurity xii. Lattice Degeneration xiii. Macular Telangiectasia xiv. Optic Neuritis xv. Vitreomacular Traction

[0077] C. ADMINISTRATION OF THERAPEUTICS TO THE EYE

[0078] 1. Intravitreal injection for administration of the back-of-the-eye therapeutics 2. Other routes of administration that have been used for treating diseases, disorders, and conditions of the posterior (back-of-the-eye) a. Subre tinal injection b. Suprachoroidal injection c. Periocular Administration d. Systemic Administration e. Intravenous Infusion f. Intraocular Implants: Intraocular Implants g. Topical Administration

[0079] 3. Gene therapy and other treatments of the back-of-the-eye conditions and their limitations a. Mesenchymal stem cell-derived extracellular vesicles

[0080] (MSC-EVs) b. Injected EVs for treatment of the back of the eye c. Other Delivery Systems i) Lipid nanoparticles (LNPs) ii) Viral Vectors

[0081] D. OCULAR BARRIERS LIMITING THE APPLICABILITY OF TOPICAL EYE TREATMENTS (DROP INSTILLATIONS)

[0082] 1. Corneal barrier

[0083] 2. Conjunctival and scleral barriers

[0084] 3. Tear film and clearance

[0085] 4. Blood-Retinal Barrier

[0086] 5. Enzymatic degradation

[0087] 6. Nasolacrimal drainage system

[0088] E. EXTRACELLULAR VESICLES

[0089] 1. Types of Extracellular Vesicles (EVs) a. Exosomes b. Microvesicles c. Apoptotic Bodies

[0090] 2. Uptake of EVs

[0091] 3. General Methods for Isolating EVs a. Ultracentrifugation b. Size-Based Techniques c. Immunoaffinity Capture-Based Techniques d. Exosome Precipitation e. Microfluidic Based Isolation Techniques

[0092] 4. Microalgae and Microalgae-Derived Extracellular Vesicles (MEVs)

[0093] 5. Green algae - Chlorella species a. Life Cycle b. Genomic Analyses of Chlorella Species c. Commercial and Biotechnological Uses of Chlorella d. Chlorella MEVs

[0094] F. EXOGENOUSLY LOADED MICROALGAE EXTRACELLULAR VESICLES (MEVs), CARGO, AND TARGETS

[0095] 1. Isolation of MEVs

[0096] 2. MEV Loading and Cargos

[0097] 3. Reporter genes, reporter proteins, and / or modulators thereof can be delivered in the MEVs.

[0098] 4. Generation of Payload-Loaded MEVs

[0099] 5. Exemplary Cargo and Exemplary Uses of the Exogenously Loaded MEVs a. Cargo

[0100] 1) RNA Cargo

[0101] 2) Antibody Cargo b. Diseases and Methods of Treatment

[0102] G. MEV TRAFFICKING FOLLOWING TOPICAL ADMINISTRATION TO THE EYE

[0103] H. OPHTHALMIC FORMULATIONS

[0104] 1. Pharmaceutical formulations for topical (drop instillation) administration to the eye

[0105] 2. Exemplary Formulations for topical administration to the eye a. Preservatives b. Viscosity-Enhancing Agents c. Tonicity Agents d. pH Adjusters e. Buffers f. Surfactants g. Chelating Agents h. Stabilizers i. Lubricants j. Tears Components k. Drug Carriers l. Other excipients

[0106] I. CLINICAL APPLICATIONS OF MEVs AS DELIVERY VEHICLES TO THE BACK OF THE EYE

[0107] 1. Eye conditions / diseases for MEV-based treatments a. Age-related Macular Degeneration (AMD) b. Geographic Atrophy (GA). c. Choroidal neovascularization d. Diabetic retinopathy e. Macular edema f. Retinal degenerative diseases. g. Retinal gene therapy

[0108] 2. Dosage of MEV-based formulations

[0109] J. EXAMPLES

[0110] A. Definitions

[0111] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event, that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0112] As used herein, formulated for administration to the eye refers compositions that can be administered to the directly, generally, as eye drops. Numerous suitable or compatible ophthalmic formulations are known to those of skill in the art.

[0113] As used herein, cargo refers to exogenous molecules, such as bioactive molecules, including biomolecules, and small molecules, that are loaded into the microalgae extracellular vesicles (MEVs) provided herein after the MEVs have been isolated. This includes cargo that is heterologous to the MEVs.

[0114] As used herein, in general, heterologous with respect to cargo in an MEV refers to cargo in the MEVs that does not naturally-occur in the MEVs but is loaded exogenously, as discussed above. It also refers to cargo in MEVs that have been loaded endogenously in the MEVs by genetically-modified microalgae. MEVs with heterologous cargo comprise cargo that does not occur naturally in the MEVs. Cargo that is heterologous to the microalgae and / or the MEVs is cargo that in nature does not occur in the microalgae so that it is packaged in the MEVS, and / or that does not occur in the MEVs absent modification of the microalgae or by exogenous loading into isolated MEVs. As used herein, a bioactive molecule or bioactive agent refers to any molecule or agent that can have a biological activity, such as therapeutic activity, or as a detectable marker, or that can act in vivo on a subject. Bioactive agents and molecules include biomolecules, such as DNA, RNA, proteins, other biopolymers, and small molecules, such as small molecule drugs and pharmaceuticals, immunogens, and any molecules that would be delivered to a subject, such as a human or other animal or a plant or a microorganism (bacteria or other), in connection with a therapy, a diagnostic application, or other such uses, such as a cosmetic. The bioactive agent or molecule can function as or have an activity as, for example, a therapeutic, an immunogen, a diagnostic, a detectable marker, or a cosmetic. The bioactive molecules for use herein are any that can be loaded into a microalgae extracellular vesicle (MEV). Bioactive molecules include therapeutics and also diagnostics, such as reporter molecules and detectable molecules, whereby a disease, disorder, or condition can be monitored or diagnosed.

[0115] As used herein, a biomolecule refers to any biologically active biopolymer or molecule that occurs, or can occur, in a living organism or virus or that is a modified form of such biopolymer or molecule. Biomolecules, thus, include modified naturally- occurring biomolecules, such as, for example proteins that include a modified primary sequence, such as by deletions, insertions, and / or replacements of amino acids to alter the primary sequence, and or by modification, such as post-translational modifications of the protein.

[0116] As used herein, recitation of therapeutic refers to any molecule that has any effect in vivo to ameliorate the symptoms of a disease, disorder, or condition or affects the progress of a disease, disorder, or condition, or prevents or reduces the risk of a disease, disorder, or condition or side effects of a treatment. Therapeutics includes, but are not limited to, nucleic acids encoding mRNA or protein, where the mRNA or protein is the therapeutic product, DNA that is the therapeutic product, aptamers, proteins, small molecules, and any such product as understood by those of skill in the art.

[0117] As used herein, when it is stated that MEVs have the same or substantially the same loaded cargo or amount thereof, it is understood that this refers to an average among the population of MEVs in a composition. It is understood, that when MEVs are loaded exogenously the ratio of cargo / MEV can be selected so that each MEV has, on average, a pre-determined amount of cargo. As a simple example, to load an average of one molecule of cargo / MEV, the skilled person could calculate the amount of cargo to load into a composition of MEVs, and understands that in the composition of MEVs, some would have more than one molecule of cargo / MEV, and others would have none. On average, the MEVs would have one molecule of cargo / MEV. The skilled person understands, that, in general, the amount of cargo / MEV will be more than the one molecule / MEV, and that the amount of cargo depends upon a variety of parameters, including the cargo, the target tissues and / or cells, the disease, disorder, or condition treated, and the subject treated. Generally, more than one molecule of cargo per MEV, on the average, such as at least 10 or about 10 molecules / MEV are loaded. Substantially more cargo, 100, 500, 1000, 104molecules / MEV and more, also can be loaded. The amount loaded depends upon the target, disease, disorder, or condition, the subject, the cargo, and the capacity of the MEV. It is within the skill in the art to select the amount.

[0118] As used herein, a subject is any organism, generally an animal or plant, into which or on which the composition containing the 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, and zoo animals.

[0119] As used herein, disease or disorder or condition refers to a pathological or undesirable or undesired condition in an organism resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and those characterized by identifiable symptoms.

[0120] As used herein, treating a subject with a disease, disorder, or condition means that the subject’s symptoms or manifestations of the disease or conditions are partially or totally alleviated, or remain static following treatment.

[0121] As used herein, a patient refers to a human subject.

[0122] As used herein, a subject includes an animal; an animal includes any animal, such as, but not limited to, primates including humans, gorillas, and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, and sheep; and pigs and other animals. Non-human animals exclude humans as the contemplated animal. As used herein, treatment refers to any effects that ameliorate symptoms of a disease or disorder. Treatment encompasses prophylaxis, therapy and / or cure. Treatment also encompasses any pharmaceutical use of any MEV or composition provided herein. Treatment refers to any effects that ameliorate or prevent or otherwise reduce or eliminate any symptom or manifestation of a disease or disorder. Treatment also encompasses any pharmaceutical use of any MEV or composition provided herein.

[0123] As used herein, prophylaxis refers to prevention of a potential disease and / or a prevention of worsening of symptoms or progression of a disease. Prevention or prophylaxis, and grammatically equivalent forms thereof, refer to methods in which the risk or probability of developing a disease or condition is reduced or eliminated and products that reduce or eliminate the risk or probability of developing a disease or condition.

[0124] As used herein, a disease, disorder, or condition of the eye or involving the eye is a disease, disorder, or condition in which the etiology of a disease, disorder, or condition involves the eye such that delivery of a therapeutic to the eye can effect treatment, which includes amelioration of or relief from symptoms, and / or treating cause or a manifestation of the disease, disorder, or condition, or delivery of a diagnostic molecule, such as a labeled molecule, a reporter, or an enzyme, that can be used to aid in or to effect diagnosis, or that can be used to monitor the progress of or effectiveness of treatment.

[0125] As used herein, a modification with reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule refers to and includes deletions, insertions, and replacements of amino acids or nucleotides, respectively. These include modifications of the primary sequence of a polypeptide or protein. Methods of modifying a polypeptide and nucleic acid molecule are routine to those of skill in the art, such as by using recombinant DNA methodologies. Modifications, when referring to polypeptide or protein, not to a sequence, refer to post-translational or post-purification changes, such as conjugation or linkage of moieties that alter properties of polypeptide or protein, such as half-life extending moieties, glycosylation, purification tags, detectable reporters, and other such moieties. As used herein, a modification of a genome or a plasmid or gene includes deletions, replacements, insertions, and translocations of nucleic acid. These include any changes to the native or naturally-occurring nucleic acid sequence.

[0126] As used herein, RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules to inhibit translation and thereby expression of a targeted gene.

[0127] As used herein, RNA molecules that act via RNAi are referred to as inhibitory by virtue of their silencing of expression of a targeted gene. Silencing expression means that expression of the targeted gene is reduced or suppressed or inhibited.

[0128] As used herein, gene silencing via RNAi is said to inhibit, suppress, disrupt, or silence expression of a targeted gene. A targeted gene contains sequences of nucleotides that correspond to the sequences in the inhibitory RNA, whereby the inhibitory RNA silences expression of mRNA. Small interfering RNAs (siRNAs) are small pieces of double-stranded (ds) RNA, usually about 21 nucleotides long, with 3' overhangs (2 nucleotides) at each end that can be used to interfere with the translation of proteins by binding to and promoting the degradation of messenger RNA (mRNA) at specific sequences. In doing so, siRNAs prevent the production of specific proteins based on the nucleotide sequences of their corresponding mRNAs. The process is called RNA interference (RNAi), and also is referred to as siRNA silencing or siRNA knockdown. A short-hairpin RNA or small-hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.

[0129] As used herein, non-coding RNAs are RNAs that do not encode a protein. Classes of non-coding RNA, include, but are not limited to, small interfering RNAs (siRNAs) and microRNAs (miRNAs). As used herein, inhibiting, suppressing, disrupting, or silencing a targeted gene refers to processes that alter expression, such as translation, of the targeted gene, whereby activity or expression of the product encoded by the targeted gene is reduced. Reduction includes a complete knock-out or a partial knockout, whereby, with reference to the MEVs provided herein and administration herein, treatment is effected. As used herein, a tumor microenvironment (TME) is the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). Conditions that exist include, but are not limited to, increased vascularization, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure and altered metabolites or metabolism, such as higher levels of adenosine, indicative of a tumor.

[0130] As used herein, recitation that a nucleic acid or encoded RNA targets a gene means that it inhibits or suppresses or silences expression of the gene by any mechanism. Generally, such nucleic acid includes at least a portion complementary to the targeted gene, where the portion is sufficient to form a hybrid with the complementary portion.

[0131] As used herein, deletion, when referring to a nucleic acid or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence, such as a target polynucleotide or polypeptide or a native or wild-type sequence.

[0132] As used herein, insertion, when referring to a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids, within a target, native, wild-type or other related sequence. Thus, a nucleic acid molecule that contains one or more insertions compared to a wild-type sequence, contains one or more additional nucleotides within the linear length of the sequence.

[0133] As used herein, additions to nucleic acid and amino acid sequences describe addition of nucleotides or amino acids onto either terminus compared to another sequence.

[0134] As used herein, substitution or replacement refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule. Thus, one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule. Amino acid replacements compared to a particular polypeptide can be expressed in terms of the number of the amino acid residues along the length of the polypeptide sequence.

[0135] As used herein, at a position corresponding to, or a recitation that nucleotides or amino acid positions correspond to nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part i, Griffin, A.M., and Griffin, H. G., 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).

[0136] As used herein, alignment of a sequence refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence. Related or variant polypeptides or nucleic acid molecules can be aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods, such as using manual alignments and by using the numerous alignment programs available e.g., BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides or nucleic acids, one skilled in the art can identify analogous portions or positions, using conserved and identical amino acid residues as guides. Further, one skilled in the art also can employ conserved amino acid or nucleotide residues as guides to find corresponding amino acid or nucleotide residues between and among human and non-human sequences. Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. In other instances, corresponding regions can be identified. One skilled in the art also can employ conserved amino acid residues as guides to find corresponding amino acid residues between and among human and non-human sequences.

[0137] As used herein, a property of a polypeptide, such as an antibody, refers to any property exhibited by a polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermal tolerance, and tolerance to pH conditions. Changes in properties can alter an activity of the polypeptide. For example, a change in the binding specificity of the antibody polypeptide can alter the ability to bind an antigen, and / or various binding activities, such as affinity or avidity, or in vivo activities of the polypeptide.

[0138] As used herein, an activity or a functional activity of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization, or enzymatic activity, for example, kinase activity or proteolytic activity. For an antibody (including antibody fragments), activities include, but are not limited to, the ability to specifically bind a particular antigen, affinity of antigen-binding (e.g., high or low affinity), avidity of antigen-binding (e.g., high or low avidity), on-rate, off-rate, effector functions, such as the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activities, such as the ability to prevent infection or invasion of a pathogen, or to promote clearance, or to penetrate a particular 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 on- or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cellbased assays, flow cytometry and binding assays (e.g., panning assays).

[0139] As used herein, bind, bound, and grammatical variations thereof refer to the participation of a molecule in any interaction with another molecule or among molecules, resulting in a stable association in which the molecules are in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds including drugs.

[0140] As used herein, antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly produced, including any fragment thereof containing at least a portion of the variable heavy chain and light region of the immunoglobulin molecule that is sufficient to form an antigen binding site and, when assembled, to specifically bind an antigen. Hence, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). For example, an antibody refers to an antibody that contains two heavy chains (which can be denoted H and H’) and two light chains (which can be denoted L and L’), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site (e.g., heavy chains include, but are not limited to, VH chains, VH-CH1 chains and VH-CH1-CH2- CH3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen binding site (e.g., light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H’) pairs with one light chain (L and L’, respectively). Typically, antibodies minimally include all or at least a portion of the variable heavy (VH) chain and / or the variable light (VL) chain. The antibody also can include all or a portion of the constant region.

[0141] For purposes herein, the term antibody includes full-length antibodies and portions thereof including antibody fragments, such as anti-tumor antibody 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 Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibody also includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or sub-subclass (e.g., IgG2a and IgG2b).

[0142] As used herein, nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Also included in the term nucleic acid are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a backbone bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term also includes, as equivalents, derivatives, variants, and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double- stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxy thymidine. For RNA, the uracil base is uridine.

[0143] As used herein, an isolated nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. An isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding RNAi or a therapeutic protein.

[0144] As used herein, operably linked with reference to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0145] As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0146] As used herein, the residues of naturally occurring a-amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.

[0147] As used herein, polypeptide refers to two or more amino acids covalently joined. The terms polypeptide and protein are used interchangeably herein.

[0148] As used herein, a peptide refers to a polypeptide that is from 2 to about or 40 amino acids in length.

[0149] As used herein, reference to proteins, unless otherwise specified, includes all forms of peptides, polypeptides, small peptides, and proteins.

[0150] As used herein, an amino acid is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids contained in the antibodies provided include the twenty naturally-occurring amino acids (see Table below), non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the a-carbon has a side chain). As used herein, the amino acids, which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations (see Table below). The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single-letter designations used routinely in the art.

[0151] As used herein, amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the L isomeric form. Residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821 - 1.822, abbreviations for amino acid residues are shown in the following Table:

[0152] Table of Correspondence

[0153] All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl- terminus. The phrase amino acid residue is defined to include the amino acids listed in the above Table of Correspondence, modified, non-natural and unusual amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an aminoterminal group such as NH2 or to a carboxyl-terminal group such as COOH.

[0154] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in the art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson el al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0155] Such substitutions can be made in accordance with the exemplary substitutions set forth in the following Table:

[0156] Exemplary conservative amino acid substitutions

[0157] Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0158] As used herein, naturally occurring amino acids refer to the 20 L-amino acids that occur in polypeptides.

[0159] As used herein, the term non-natural amino acid refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non- naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art, and include, but are not limited to, 2- Aminoadipic acid (Aad), 3-Aminoadipic acid (bAad), P-alanine / p-Amino-propionic acid (Bala), 2- Aminobutyric acid (Abu), 4- Aminobutyric acid / piperidinic acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2- Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr), N-Ethylglycine (EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl), allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N- Methylglycine, sarcosine (MeGly), N-Methylisoleucine (Melle), 6-N-Methyllysine (MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn).

[0160] As used herein, a DNA construct is a single or double stranded, linear, or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.

[0161] As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified 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 sequence of amino acids of the specified polypeptide.

[0162] As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5’ to the 3’ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated nt) or base pairs (abbreviated bp). The term nucleotides is used for single- and double- stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double- stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus, all nucleotides within a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.

[0163] As used herein, production by recombinant methods refers means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0164] As used herein, heterologous nucleic acid is nucleic acid that encodes products (z.e., RNA and / or proteins) that are not normally produced in vivo by the cell in which it is expressed, or nucleic acid that is in a locus in which or at which it does not normally occur, or that mediates or encodes mediators that alter expression of endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acid, such as DNA, also is referred to as foreign nucleic acid. Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed, is herein encompassed by heterologous nucleic acid; heterologous nucleic acid includes exogenously added nucleic acid that is also expressed endogenously. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically or is introduced into a genomic locus in which it does not occur naturally, or its expression is under the control of regulatory sequences or a sequence that differs from the natural regulatory sequence or sequences.

[0165] Examples of heterologous nucleic acid herein include, but are not limited to, a DNA molecule, an RNA molecule, a plasmid, and an antisense oligonucleotide. In the MEV, the heterologous nucleic acid can be encoded on a plasmid. Heterologous nucleic acid, such as DNA, includes nucleic acid that can, in some manner, mediate expression of DNA that encodes a therapeutic product, or it can encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. As used herein, cell therapy involves the delivery of MEVs to a subject to treat a disease or condition. The MEVs are exogenously loaded with cargo, so that they deliver or express products when introduced to a subject. The MEVs also can be endogenously loaded with cargo (see, e.g., copending U.S. provisional application Serial No. 63 / 349,006, filed on June 03, 2022, which details preparation of endogenously-loaded MEVs and producer cell lines thereof), and used as described herein. The trafficking of MEVs generally is independent of manner in which they are loaded with cargo. The microalgae can be modified to alter properties of the resulting MEVs. Endogenously-loaded MEVs can be used in the methods and compositions described herein.

[0166] As used herein, genetic therapy involves the transfer of heterologous nucleic acid, such as DNA, into certain cells, such as target cells, of a mammal, particularly a human, with a disorder or condition for which such therapy is sought. The nucleic acid, such as DNA, is introduced into the selected target cells in a manner such that the heterologous nucleic acid, such as DNA, is expressed and a therapeutic product(s) encoded thereby is produced. Genetic therapy also can be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid can encode a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor thereof, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous nucleic acid, such as DNA, encoding the therapeutic product, can be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy can also involve delivery of an inhibitor or repressor or other modulator of gene expression.

[0167] As used herein, expression refers to the process by which polypeptides are produced by transcription and translation of polynucleotides. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.

[0168] As used herein, a host cell is a cell that is used to receive, maintain, reproduce and / or amplify a vector. A host cell also can be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.

[0169] As used herein, a vector is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide or RNA. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well- known to those of skill in the art. A vector also includes virus vectors or viral vectors. Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0170] As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well-known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0171] As used herein, primary sequence refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.

[0172] As used herein, sequence identity refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. The alignment can be local or global. Matches, mismatches, and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequence x 100.

[0173] For purposes herein, sequence identity is determined by aligning the test polypeptide or nucleic acid molecule with the reference molecule and counting the number of differences, including gaps and insertions. The number of differences is divided by the length of reference molecule, generally the molecule whose sequence is claimed, and percentage determined. For example, if the polypeptide that is claimed is 100 amino acids in length, and variants include those with 90% sequence identity, then the variants can have 10 amino acid differences, including gaps and insertions.

[0174] As used herein, a global alignment is an alignment that aligns two sequences from beginning to end, aligning each letter in each sequence only once. An alignment is produced, regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on global alignment means that in an alignment of the full sequence of two compared sequences each of 100 nucleotides in length, 50% of the residues are the same. It is understood that global alignment also can be used in determining sequence identity even when the length of the aligned sequences is not the same. The differences in the terminal ends of the sequences will be taken into account in determining sequence identity, unless the no penalty for end gaps is selected. Generally, a global alignment is used on sequences that share significant similarity over most of their length. Exemplary algorithms for performing global alignment include the Needleman- Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplary programs for performing global alignment are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ), and the program available at deepc2.p si hastate . edu / aat / align / align .html .

[0175] As used herein, a local alignment is an alignment that aligns two sequences, but only aligns those portions of the sequences that share similarity or identity. Hence, a local alignment determines if sub-segments of one sequence are present in another sequence. If there is no similarity, no alignment will be returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For example, 50% sequence identity based on local alignment means that in an alignment of the full sequence of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length has 50% of the residues that are the same in the region of similarity or identity.

[0176] For purposes herein, sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, or other similar variations reciting a percent identity, can be determined using known computer algorithms based on local or global alignment (see e.g., wikipedia.org / wiki / Sequence_alignment_software, providing links to dozens of known and publicly available alignment databases and programs). Generally, for purposes herein sequence identity is determined using computer algorithms based on global alignment, such as the Needleman- Wunsch Global Sequence Alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)); and program from Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, the full-length sequence of each of the compared polypeptides or nucleotides is aligned across the full-length of each sequence in a global alignment. Local alignment also can be used when the sequences being compared are substantially the same length.

[0177] Therefore, as used herein, the term identity represents a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one nonlimiting example, at least 90% identical to refers to percent identities from 90 to 100% relative to the reference polypeptide or polynucleotide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide or polynucleotide length of 100 amino acids or nucleotides are compared, no more than 10% (i.e.. 10 out of 100) of amino acids or nucleotides in the test polypeptide or polynucleotide differ from those of the reference polypeptide. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10 / 100 amino acid difference (approximately 90% identity). Differences also can be due to deletions, insertions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. Depending on the length of the compared sequences, at the level of homologies or identities above about 85-90%, the result can be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

[0178] In general, determination of sequence identity, such as between two polypeptides, is effected by aligning the polypeptides to maximize the number of matching polypeptides and counting the number of differences in between the two sequences, one sequence is the reference sequence, and the other is the sequence whose identity is determined. If the claim recites a sequence that is 100 amino acids in length and the claim recites that the claimed polypeptide has 95% sequence identity thereto, there can be 5 amino acid differences between the two sequences. The differences include insertions or deletions (missing amino acids).

[0179] As used herein, a pharmaceutically effective agent includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0180] As used herein, a therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves, or ameliorates, the symptoms of a disease or condition or that cures a disease or condition.

[0181] As used herein, a therapeutically effective amount or a therapeutically effective dose refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting, or partially arresting a symptom of a disease or disorder.

[0182] As used herein, therapeutic efficacy refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.

[0183] As used herein, a prophylactically effective amount or a prophylactic ally effective dose refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0184] As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting, or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.

[0185] As used herein, an anti-cancer agent refers to any agent that is destructive or toxic to malignant cells and tissues. For example, anti-cancer agents include agents that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. Exemplary anti-cancer agents are chemotherapeutic agents.

[0186] As used herein therapeutic activity refers to the in vivo activity of a therapeutic polypeptide. Generally, the therapeutic activity is the activity that is associated with treatment of a disease or condition.

[0187] 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.

[0188] As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0189] As used herein, combination therapy refers to administration of two or more different therapeutics. The different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.

[0190] 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 a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property.

[0191] As used herein, a unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.

[0192] As used herein, a single dosage formulation refers to a formulation for direct administration.

[0193] As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and that can be directly administered to provide several single doses of the therapeutic agent. The doses can be administered over the course of minutes, hours, weeks, days, or months. Multi-dose formulations can allow dose adjustment, dose-pooling and / or dose-splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0194] As used herein, an emulsion is a system composed of two immiscible liquid phases, intimately mixed and dispersed, one into the other. An emulsion refers to a colloidal dispersion of two immiscible liquids, for example, an oil and water (or other aqueous liquid, e.g., a polar solvent), one of which is part of a continuous phase and the other of which is part of a dispersed phase Emulsions typically are stabilized by one or more surfactants and / or co- surfactants and / or emulsion stabilizers. Surfactants form an interfacial film between the oil and water phase of the emulsion, providing stability. Microemulsion and nanoemulsion are two different types of emulsions. An emulsion is a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible. Microemulsions are thermodynamically stable, whereas nanoemulsions are thermodynamically unstable.

[0195] As used herein, a microemulsion is a two-phase system composed of a lipid phase and an aqueous phase, finely interspersed. Microemulsions are liquid mixtures, transparent, isotropic and stable, of a lipid phase and an aqueous phase, held together by a surfactant, generally in conjunction with a co-surfactant. Microemulsions are clear, thermodynamically stable, isotropic liquid mixtures of oil, water and surfactant, and optionally a co- surfactant. Microemulsions form spontaneously upon mixing of the aqueous phase and lipid phase. A microemulsion is, therefore, a thermodynamically stable system, with particles dispersed in the continuous phase. The droplet size of the dispersed phase in a microemulsion is less than 100 nm, generally in the range between 5 nm and 50 nm, inclusive. In contrast, a nanoemulsion requires input of energy to form the emulsion; a nanoemulsion is thermodynamically unstable, but is kinetically stable. Eyedrop formulations provided herein include nanoemulsions and microemulsions.

[0196] As used herein, “surfactant” refers to synthetic and naturally occurring amphiphilic molecules that have hydrophobic portion(s) and hydrophilic portion(s). Surfactants contain a hydrophilic domain and hydrophobic domain, i.e., amphiphilic molecules. Given their nature, surfactants facilitate the formation of oil-in-water emulsions where the micelles, in order to exist, need to interact with both the water and the oil phases. Due to their amphiphilic (amphipathic) nature, surfactants and cosurfactants can reduce the surface tension between two immiscible liquids, for example, the oil and water phases in an emulsion, such as a microemulsion, stabilizing the emulsion. Surfactants can be characterized based on their relative hydrophobicity and / or hydrophilicity. For example, relatively lipophilic surfactants are more soluble in fats, oils, and waxes, typically having Hydrophobic-Lipophilic Balance (HLB) values less than 10 or about 10, while relatively hydrophilic surfactants are more soluble in aqueous compositions, for example, water, and typically have HLB values greater than 10 or about 10. Relatively amphiphilic surfactants are soluble in oil and water-based liquids and typically have HLB values close to 10 or about 10. Surfactants for use in the compositions herein are biocompatible.

[0197] As used herein, a co- surfactant is a surfactant that acts in addition to another surfactant to further reduce the surface tension of a liquid. For example, recitation that microemulsions contain surfactants refers to the surfactants and the co- surfactants that are included. Co- surfactants are hydrophilic in nature, and reduce the surface tension of water. They generally are used as wetting agents, for example, to increase the spreading abilities of water-based fluid by reducing the surface tension of water. Cosurfactants also are used, and often needed, to increase the solubility of the primary surfactant.

[0198] As used herein, “particle size” and “average particle size” refer synonymously to the average diameter of particles in a provided liquid, for example, the droplet diameter or micelle diameter in an emulsion

[0199] As used herein, “oil phase” or “lipid phase” refers to the portion (or phase) of a composition hat contains one or more lipophilic ingredients and / or amphiphilic ingredients, such as an oil, and is, in general, the lipid-soluble phase. In an oil-in- water (o / w) microemulsion, the lipid phase typically is the dispersed phase while water is the dispersion phase.

[0200] As used herein, oil phase ingredient(s) refers to the components of the provided compositions that are included in the oil Typical oil phase ingredients include non-polar compounds, e.g., non-polar active ingredients; at least one surfactant; oils, such as non-polar solvents; preservatives; and microemulsion stabilizers. Other lipophilic and / or amphiphilic ingredients can be included in the oil phase.

[0201] As used herein, “water phase” or “aqueous phase” refers to the portion (phase) of a composition, such as those provided herein, that contains one or more hydrophilic ingredients and / or amphiphilic ingredients (water phase ingredients) and is, in general, the water-soluble phase. Typically, in the microemulsion compositions provided herein, the water phase is the continuous phase. “Water phase” also is used to refer to the liquid containing the water phase ingredients that is generated while preparing microemulsions.

[0202] As used herein, water phase ingredient(s) refers to the components of the compositions that are included in the water phase in the provided methods for making the compositions. Typical water phase ingredients can include, but are not limited to, polar solvents, typically polar protic solvents, such as water and alcohols, typically alcohols having more than one hydroxy group such as dihydroxy and trihydroxy alcohols, such as glycerol and propylene glycol; at least one surfactant; preservatives; and emulsion stabilizers. Other hydrophilic and / or amphiphilic ingredients can be included in the water phase.

[0203] As used herein, thermodynamic stability of the microemulsions refers to the stability of the dispersion such that the phases do not separate. The microemulsions provided herein exhibit high thermodynamic stability as shown by their stability at elevated temperatures.

[0204] As used herein, room temperature and ambient temperature are used to describe a temperature that is common in one or more enclosed spaces in which human beings typically are or reside. Room temperature can vary, but generally refers to temperatures between 19 °C or about 19 °C and 25 °C or about 25 °C. When a composition is stored at room temperature, it should be understood it is generally kept at a temperature within this range or about within this range.

[0205] As used herein, an article of manufacture is a product that is made and sold. As used throughout this application, the term is intended to encompass any of the compositions provided herein contained in articles of packaging. As used herein, a fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0206] As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof) or biologically-active portion thereof (e.g., an isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

[0207] As used herein, a cellular extract or lysate refers to a preparation or fraction which is made from a lysed or disrupted cell.

[0208] As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.

[0209] As used herein, psilocin is the active form of psilocybin. Psilocin is produced by oxidation of psilocybin in the liver. For purposes herein, in the context of MEV- mediated delivery, psilocybin and psilocin should have the same meaning: a mention to ‘psilocybin’ shall mean to ‘psilocin’ and vice versa.

[0210] As used herein, a tropism of an MEV refers to cells, tissues, and / or organs in where the MEVs, upon administration, accumulate. As used herein, natural tropism with reference to the MEVS provided herein, refers to the means: MEV are not modified to provide a specific tropism or targeting property.

[0211] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide, comprising an immunoglobulin domain includes polypeptides with one or a plurality of immunoglobulin domains.

[0212] As used herein, the term “or” is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0213] As used herein, ranges and amounts can be expressed as about a particular value or range. “About” also includes the exact amount. Hence about 5 amino acids means about 5 amino acids and also 5 amino acids. Generally, although depending upon context and the understanding of one of skill in the particular art, “about” includes a range of ±10%.

[0214] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0215] As used herein, the abbreviations for any protective groups, amino acids, and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726- 1732).

[0216] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0217] B. Overview

[0218] Provided are compositions comprising MEVs formulated for topical administration, such as eyedrops, for administration to the eye. It is shown herein that upon topical application, or local application, the MEVs traffic to the back of the eye and deliver product to choroidal cells, RPE (retinal pigment epithelium), epithelial cells, and to photoreceptor cells (rods and cones), but not other parts of the eye, particularly not in neurons or the optic nerve. This is in contrast to other delivery vehicles, such as AAV vectors, that are administered by intravitreal injection for treatment of diseases, disorders, and conditions of the back of the eye. The other vehicles are not trafficked to choroidal cells, or the RPE, or retina. The AAV vectors, following administration, are found in the internal parts of the eye MEVs reach through the choroid and layers adjacent to the choroid and the retina that contact the optic nerve and neurons. AAV vectors and other such delivery vehicles must be administered by injection, and, by virtue of their pathways are not suitable for treatment of diseases, disorders, and conditions involving, for example, the choroid, RPE, photoreceptor cells. See Example 13 below, which provides exemplary details comparing biodistribution of AAV vectors administered by injection.

[0219] 1. Microalgae

[0220] Algae are a complex, polyphyletic collection of predominantly photosynthetic organisms. These organisms include micro- and macroscopic forms. Macroalgae (seaweed) are multicellular, large-size algae, visible with the naked eye. Microalgae are microscopic single cells and include prokaryotes (e.g.. cyanobacteria), and eukaryotes, such as green algae.

[0221] Compared to photosynthetic crops, microalgae have a higher growth rate and can be cultivated on non-arable land, and also in bioreactors. Many species of microalgae can be grown year-round in industrial scale photobioreactors under controlled cultivation conditions (Adamo et al. (2021) Journal of Extracellular Vesicles 10:el2081). Algae generally are classified into eleven major phyla: Cyanophyta, Chlorophyta, Rhodophyta, Glaucophyta, Euglenophyta, Chlorarachniophyta, Charophyta, Cryptophyta, Haptophyta, Heterokontophyta, and Dinophyta (Barkia et al. (2019) Mar. Drugs 17(5) :304) . Different pigments occur in each algae group. Cyanobacteria (or Cyanophyta) contain chlorophyll-a, -d, and -f, in addition to the phycobiliproteins (proteins that capture light energy), phycocyanin, allophycocyanin, and phycoerythrin. Glaucophytes contain chlorophyll-a and harvest light via phycobiliproteins. Chiorophytes have chlorophyll-a and -b, as well as carotenoids, including P-carotene and various xanthophylls (e.g., astaxanthin, canthaxanthin, lutein, and zeaxanthin). The primary pigments of Rhodophyta (red algae) are phycoerythrin and phycocyanin, which can mask chlorophyll-a; red algae also produce a broad spectrum of carotenes and xanthophyll light-harvesting pigments (Barkia et al. (2019) Mar. Drugs 17(5):304).

[0222] Provided herein are extracellular vesicles produced by microalgae, particularly unicellular green algae, such as species of Chlorella, for use for delivery of exogenously loaded cargo to animals and plants. Species of Chlorella include, for example, Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis, Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hussii. Parachlorella species have been classified as members of the Chlorellaceae family, but have been considered to be a genus separate from Chlorella. Based on either classification, also included are MEVs from other members of the family of Chlorellaceae or other family that are Parachlorella, species, including Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hussii.

[0223] The algae are unicellular eukaryotes that typically are haploid, but can have a diploid stage of the life cycle. The algae can be cultured in bioreactors and the extracellular vesicles isolated therefrom. The resulting extracellular vesicles can be loaded by methods such as electroporation, with cargo, generally a cargo of heterologous bioactive molecules to produce compositions that contain the extracellular vesicles for administration to animals and also to plants. The compositions can be formulated for any desired route of administration, including topical, local, systemic, parenteral, and oral. These routes include oral, intravenous, subcutaneous, inhalation, mucosal, rectal, vaginal, and other suitable routes. The 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. The extracellular vesicles can be formulated as liquids, powders, including lyophilized powders, tablets, capsules, emulsions, particles, sprays, gels, ointments, creams, and other formulations. They can be used for therapeutic, diagnostic, theragnostic, cosmetic, and other uses. The extracellular vesicles can be used to treat diseases and conditions, that include cancers, inflammatory diseases, and conditions in which the immune system plays a role in the etiology or symptoms, nervous system disorders, and pathogen infections, including viral and bacterial and other pathogens. They can be used to treat dermatological diseases and conditions, lung diseases and conditions, and gastric diseases and conditions. The extracellular vesicles can be targeted to specific organs or tissues or can be locally administered.

[0224] As with extracellular vesicles (EVs) from other sources, such as mammalian EVs, microalgae EVs (MEVs) have evolved to efficiently pass genetic material and other kinds of molecules from cell to cell. They orchestrate intercellular and crosskingdom communication between cells via exchange of biologically active molecules. MEV are natural nanoparticles. They are cell-derived, so, absent synthetic cargo, and genetic modifications, there are no synthetic components; they safe, for example, there is no risk of endogenous viruses that are potentially dangerous to humans. The MEVs provided herein include all Chlor ella family MEVs, particularly Chlor ella vulgaris, a freshwater microalgae; also included among Chlorella are Parachlorella. Also included are MEVs from other members of the family of Chlorellaceae, such as Parachlorella, including Parachlorella kessleri, Parachlorella beijerinckii, and. Parachlorella hussi.

[0225] Chlorella is a unicellular haploid alga that is a natural and efficient producer of extracellular vesicles. Chlorella vulgaris has been consumed worldwide as a food supplement for decades; it is non-toxic and non-immunogenic, and can be cultured at large industrial scale at low cost. The MEVs provided herein can be directly used to protect, convey, and deliver a broad spectrum of innovative therapeutic molecules into target cells relevant to specific diseases.

[0226] As shown and described herein, the MEVs have a number of advantageous features including, for example, biodistribution patterns by route of administration, low toxicity, good pharmacokinetic profiles in vivo. They can be administered by a variety of routes including oral administration, administration to the respiratory tract, intranasally, intravenously, and to the eye by topical application and / or injection, among other routes. They traffic to specific organs, according to the route of administration, such as the intestine, the GALT, the spleen, the lungs, the liver, and back of the eye, and the brain. Based on data herein and comparison with data for other EVs and drug delivery systems, the MEVs can have longer clearance rates so that they last longer in the targeted organs, tissues, and cells than reported for other delivery systems, including mammalian EVs. It is shown herein, the MEVs can be delivered in eye drops and can traffic to the retina, thereby eliminating the need for intravitreal or subretinal or other injection. MEVs overcome natural body barriers including those in the eye, such as oral delivery, or specific lymphoid tissues delivery, or nose-to-brain delivery; and delivery to tissues in the eye) that have not been attained with liquid nano-particles and EVs of mammalian origin.

[0227] The MEVs provided herein address unmet needs. These include the ability to convey and reliably deliver therapeutic molecules specifically to the site of treatment, while avoiding premature degradation or inactivation of the therapeutic agent by the immune system or by enzymes; for treatment of diseases for which a therapeutic agent already exists but cannot be properly delivered.

[0228] As shown herein, the purified or partially -purified MEVs can be loaded by physical methods (exogenous loading; exo-loading). Exo-loading is scalable and industrializable. The MEVs can be exo-loaded with a variety of molecules, varying in size, hydrophobicity, and nature, such as siRNA, mRNA, peptides, proteins, plasmids, oligonucleotides, and small molecules. The biological activity of the exo-loaded cargo is preserved, while at the same time it is protected from degradation by enzymes and other agents present in vivo. The MEVs can deliver their cargo to recipient cells of a myriad of origins, such as microalgae, bacteria, higher plant, mammal, and human. MEVs can also deliver the cargo to the proper cell compartments, ensuring the proper expression and biological activity of cargo molecules, including those having complex biological pathways such as siRNA, mRNA, receptor-binding peptides, among others.

[0229] 2. Overview of The Eye, Diseases, Disorders, And Conditions, And Treatments

[0230] MEVs and compositions containing MEVs can be formulated for administration to the eye in any suitable form, but in particular, can be formulated as drops and can deliver the MEVs and their contents to any portion of the eye. The following discussion describes the anatomy of the eye and some exemplary disorders and therapeutics for treatments. The therapeutics can be provided in or part of MEVs and administered in drops. As shown herein, any therapeutic provided in or part of MEVs can be administered as drops and delivered to a target in the eye, particularly targets that had required injection, including intravitreal injection and / or subretinal injection.

[0231] The eye is a finely tuned optical instrument designed for vision that includes a complex of ocular structures (Figures 11A-11B), each characterized by specific anatomical and histological features. The components of the eye, their functions, related diseases / conditions, routes of administration for therapeutics, therapeutics for treatment, are discussed in sections that follow. a. The Eye and Administration of ME Vs

[0232] The eye is divided into two segments: (1) the anterior segment that contains the cornea, iris, pupil, conjunctiva, ciliary body, anterior chamber, aqueous humor, trabecular mesh work, and lens; and (2) the posterior segment that contains the vitreous humor, sclera, choroid, retina, macula, and optic nerve. There are many diseases and conditions of the eye, and pharmaceutically active agents that are used to treat such diseases and conditions. Administration can be effected by any route, such as topically using eye drops, and by injections, such as intravitreal injection. Treatment is important as some diseases eventually lead to blindness.

[0233] As shown herein, the MEVs when formulated as drops and administered to the eye, traffic to the back of the eye, and, thus, can be used to deliver therapeutics that have required injection as eyedrops. Any therapeutic for treatment of eye can be loaded into an MEV or produced in an MEV and administered to the eye by any route. In general, of interest herein, are diseases, disorders, and conditions of the back portion of the eye that have required injection for treatment. These include, for example, diseases, disorders, and conditions of the eye age-related macular degeneration, herpes stromal keratitis, glaucoma, dry eye syndrome, diabetic retinopathy, and conditions associated with ocular angiogenesis, and conditions associated with ocular hypertension. With this understanding, the following discussion describes a variety of diseases, disorders, and conditions that affect or involve the eye, and includes description of therapeutics. The MEVs can provide particular advantages for formulation for and delivery by any route. A particular advantage are formulations for administration into the eye by local or topical administration, particularly as eyedrops for topical or local administration, and for treatment of diseases, disorders, and conditions that involve the back of the eye, including tissues and structures that are localized to the back of the eye, or that span the eye and include the back of the eye. b. Structure and function of ocular components

[0234] The eye is a sensory organ that captures light and turns it into a signal carried to and interpreted by the brain (see Figures 11 A and 1 IB). The delicate tissue of the eye is contained and protected within a bony socket of the skull, an orbit, which contains seven orbital bones. The eye socket contains the eyeball, blood vessels, nerves. At the back of the eye socket is an opening to the optical canal, and at the front of the eye are located the sclera and cornea of the eyeball. Six extraocular muscles are attached to the eyeball and allow the eye to move up and down, side to side, and rotationally. The eyeball is also protected by exterior structures including the eyelashes, eyelids, and lacrimal glands. A clear membrane called the conjunctiva coats the surface of the eye and the inner surface of the eyelid to protect the eye. Tears that lubricate the eye create a tear film that contains an oil layer made by the meibomian glands, a watery layer made by the lacrimal gland, and a mucous layer made by the conjunctiva. The tear duct allows for drainage of tears from the eye.

[0235] The front of the eye contains anatomical and mechanical structures that filter and focus light toward the back of the eye including the cornea, iris, pupil, and lens. The frontmost portion of the eye has a clear dome shaped structure called the cornea. The anterior chamber is the space behind the cornea that is filled with a fluid called aqueous humor. Aqueous humor is consistently produced and drained via the drainage angle; the balance between production and drainage is necessary to maintain proper and consistent eye pressure. Behind the anterior chamber is the iris, which confers color, and the pupil, the dark center hole. The iris includes muscles that allow the pupil to dilate or constrict to thereby control the amount of light that reaches the back of the eye. The lens of the eye is directly behind the pupil and is a physical structure that focuses light on the back of the eye. The lens of the eye is surrounded by a thin membrane called the lens capsule that helps provide for the lens shape. The lens of the eye changes shape to allow the eye to properly focus on close up or distant objects. The lens is suspended from the eye wall with small fibers called zonules.

[0236] The back of the eye contains specialized tissues and cells that convert light into electrical signals that are carried to the brain. The front of the eye and back of the eye are separated by a large vitreous cavity that is filled with a gelatinous substance called vitreous humor. Light is focused through the cornea and lens and passes through the vitreous onto the retina. The retina is the light sensitive layer of cells positioned at the back of the eye near the optic nerve. The macula is a small, centralized area of the retina that is essential for central vision and viewing objects such as faces and written text, and the area surrounding the macula provides for peripheral or side vision. The specialized cells of the retina, called photoreceptors, convert light into neural signals to relay visual information. There are two types of photoreceptors, which include rods and cones. Rod cells provide for black and white perception and night vision while cone cells provide for color and central vision. c. Anatomical Parts of the Eye

[0237] Figures 11A and 11B illustrate some of the anatomical parts, structures, celltypes and features of the eye. The anatomical parts of the eye include parts such as the cornea, pupil, and iris, the anterior and posterior chambers, the ciliary body, zonules, sclera, the ora serrata, the superior and inferior rectus muscles, the optic disc, vitreous body, the retina, the fovea centralis, the optic nerve, and the central retinal artery and vein. Exemplary anatomical parts of the eye are discussed in detail below. i. Cornea

[0238] Transparent dome shaped tissue that forms the front part of the eye and covers the iris, pupil, and anterior chamber of the eye. The cornea helps refract light and contributes to the eye’s focusing power. It is a transparent and avascular structure, refracts light entering the eye and acts as a protective barrier. In includes stratified epithelium, stroma, and endothelium. The cornea is crucial for vision. Its function is impaired in conditions, such as keratitis (inflammation of the cornea) and corneal dystrophies (genetic disorders affecting corneal structure). For therapeutic interventions targeting the cornea, which is vulnerable to infections and degenerative disorders, topical administration in the form of eye drops or ointments is a feasible and typical route, which allows for direct and convenient access to the corneal surface. ii. Conjunctiva

[0239] A thin clear membrane that lines the inside of the eyelid and the sclera, or white of the eye. The conjunctiva helps produce part of the mucus that forms protective tear film that protects and lubricates the eye. The covers the anterior eye surface; it is a mucous membrane with stratified epithelium and goblet cells. Serving a protective role, it is vulnerable to inflammation (conjunctivitis). For targeted therapeutics, especially those addressing conjunctival disorders, subconjunctival injection is a possible and typical route. This approach provides site-specific delivery to the conjunctival tissues. iii. Sclera

[0240] The white of the eye that is a strong layer of tissue that serves as a protective covering around the eye extending from the cornea to the optic nerve at the back of the eye. It is a tough, fibrous outer layer rich in collagen that provides structural integrity maintaining the eye's shape. Scleritis (inflammation of the sclera) is an example of a condition impacting the sclera. Due to its structure, drug penetration via sclera is low. Subconjunctival injection or periocular administration are possible routes for therapeutic delivery to the sclera. Subconjunctival injection offers targeted delivery, overcoming the barriers posed by the sclera. iv. Iris

[0241] A thin, ring-shaped structure that regulates the diameter and size of the pupil, thus regulating the amount of light that reaches the retina. In humans and other mammals, the iris is colored, the pupil is at the center and the sclera surrounds it. It is a contractile diaphragm with pigmented layers regulating pupil size, controlling light entry, and contributing to eye color. Conditions like iritis (inflammation of the iris) highlight its susceptibility to inflammatory processes. For therapeutics targeting the iris, such as anti-inflammatory treatments, topical administration or periocular injection are possible routes. Topical administration is commonly use because of the direct accessibility of the iris and ease of administration. v. Lens

[0242] The lens is biconvex transparent structure situated between the anterior and posterior chambers of the eye. The lens refracts light to focus it onto the retina. The lens can change shape to allow focus of near and distant images / objects. The lens contains lens fibers and epithelial cells; the lens focuses light onto the retina. Cataracts, clouding of the lens, is an example of an age-related change to the lens, which impacts visual function. Therapeutics for lens-related conditions can be delivered through topical or systemic administration for indirect delivery to the lens. vi. Retina

[0243] The light sensitive layer of cells at the back of the eye contains photoreceptor cells and several layers of neurons. The retina transforms optical images into electrical signals that are relayed to the brain. The retina is a complex neural tissue with distinct layers; it transforms light into neural signals. The retina, thus, is a complex neural tissue of the back of the eye; it is a crucial functional element responsible for visual information processing by converting photons into three- dimensional images.

[0244] Retinal pathologies, include macular degeneration and diabetic retinopathy, and are among the most common causes of vision loss. Macular degeneration involves the deterioration of the macula, impacting central vision, and diabetic retinopathy results from diabetes-related damage to blood vessels in the retina. For therapeutics targeting the retina, intravitreal injection has been the only means of retinal delivery. vii. Microstructure of the retinal tissue

[0245] The structure contains ten distinct layers and orchestrates a cascade of events critical for visual perception. Ganglion cells, positioned at the innermost layer, serve as the primary output neurons, transmitting visual information to higher brain centers. Several other cell types of the retina, including horizontal, bipolar, amacrine and Muller cells, form intricate circuits for visual information processing. The outermost layers include photoreceptor cells (rods and cones), crucial for light detection, and the retinal pigment epithelium (RPE), the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells. Adjacent to the retina, Bruch’s membrane and the choroid contribute to nutrient transport and maintain the bloodretina barrier, crucial for retinal homeostasis (see, Figures 11 A and 1 IB illustrate some of the anatomical parts, structures, cell-types and features of the eye. The anatomical parts of the eye include parts such as the cornea, pupil, and iris, the anterior and posterior chambers, the ciliary body, zonules, sclera, the ora serrata, the superior and inferior rectus muscles, the optic disc, vitreous body, the retina, the fovea centralis, the optic nerve, and the central retinal artery and vein. Exemplary anatomical parts of the eye are discussed in detail below.

[0246] The central retina differs from the peripheral retina in thickness and in composition. The central retina is thicker and is packed densely with cones, while the peripheral retina is thinner and is composed primarily of rod cells. The cell layer containing the rods and cones is uniform in thickness, but differs in composition of rods and cones, with more cones located centrally and with more rods in the periphery. An area particularly rich in cones located in the center of the retina is called the macula or macula lutea. In humans, the macula has a diameter of around 5.5 mm, in which a 1.5-mm central pit is composed of closely packed cones, known as the / ovea centralis. Macular damage is related with impairment of central, high- resolution, color vision, and vision loss, among other. viii. Choroid

[0247] The choroid is a portion of the vascular layer of the eye that contains connective tissues and lies between the retina and the sclera. The choroid is responsible for providing oxygen and nourishment to the outer layer of the retina. The choroid is considered part of the uvea and with the ciliary body and iris forms the uveal tract. It is a vascular layer between the retina and sclera that nourishes the outer retina. Choroid pathologies include choroidal neovascularization that involves the abnormal growth of blood vessels in the choroid, leading to vision impairment. Intravitreal injection or suprachoroidal injection are routes for therapeutic delivery to the choroid. Suprachoroidal injection offers targeted delivery, minimizing the risk of retinal damage. As described herein, therapeutics administered by suprachoroidal injection or subretinal injection, exhibit a gradient of action throughout the retina, where it is higher in the outer layers of the retina (higher action) and decreases through the inner layers of the retina (lower of null action). The gradient differs from topical administration of MEVs because the trafficking routes differ. ix. Vitreous Humor

[0248] A clear gel-like substance that fills the interior of the eyeball and provides plasticity, strength, and structure to the eye. It fills the front of the eye, maintaining eye shape. Vitreous humor occupies the space between the lens and the retina. Vitreous-related conditions include vitreous detachment, in which the separation of the vitreous humor from the retina occurs, which can lead to floaters or flashes. For therapeutic targeting of the vitreous humor, treatment is effected via intravitreal injection, which ensures direct delivery to the vitreous cavity, optimizing therapeutic concentrations. x. Ciliary Body

[0249] A ring-shaped tissue that controls the shape of the lens of the eye and produces the aqueous humor. It lies between the posterior and anterior cavities of the eye and is connected to the lens of the eye via zonular fibers. xi. Rod Cells

[0250] A photoreceptor cell, a special type of cell found in the retina capable of phototransduction. Rod cells are more sensitive than cone cells, however, they play little role in color detection. Also, due to their increased sensitivity rod cells help provide for night vision. xii. Cone cells

[0251] Cone cells are photoreceptor cells; they occur in the retina and are capable of phototransduction. Cone cells are less sensitive to light than rod cells but allow for the perception of color as well as finer details and rapid image changes. There are three types of cone cells including S-cones, M-cones, and L-cones, each of which correspond to their sensitivities to wavelengths of light, i.e., short- wavelength, medium-wavelength, long-wavelength, respectively. xiii. Drainage angle

[0252] The drainage angle is the point of the eye where the iris and the sclera meet and where aqueous humor drains from the front portion of the eye. xiv. Eyelid

[0253] A fold of skin that covers and protects the eye from debris and from bright light. They have muscles, glands, nerves, and tissues that work together to protect and lubricate the eye, which as an upper eyelid and lower eyelid. Eyelashes are tiny hairs that stick out from the eyelids and provide an added layer of protection from dust and small particles and help keep eyes lubricated by slowing the evaporation of tears. xv. Fovea A depression at the very center of the macula is composed of closely packed cone cells, consequently, it is the point where eyesight is sharpest. The fovea is also known as the fovea centralis. xvi. Lacrimal Gland

[0254] A gland located above the eye responsible for the aqueous layer of the tear film. xvii. Macula

[0255] A centralized part of the retina at the back of the eye. This portion of the retina comprises a high concentration of photoreceptor cells and is responsible for central vision, including color vision and fine detail. xviii. Meibomian Glands

[0256] Oil glands positioned along the edges of the upper and lower eyelids. These glands are responsible for secretion of lipids that help form the oily layer of the tear film. xix. Optic Nerve

[0257] A bundle of approximately one million nerve fibers responsible for carrying all visual information from the retina to the brain. The eye’s blind spot is found at the point where the optic nerve leaves the eye, a result of the absence of photoreceptors. xx. Pupil

[0258] The hole at the center of the iris that allows light to enter the eye and hit the retina. The pupil functions as the eye’s aperture. xxi. Tear duct

[0259] Also known as the nasolacrimal duct. A narrow canal that allows for drainage of tears from the eye through the nasal bone and into the back of the nose. d. Diseases, Disorders, and Conditions of the Eye

[0260] A variety of diseases, disorders, and conditions affect the eye and the tissues surrounding the eye. All can be treated with MEVs that contain therapeutics for treating such diseases, disorders, and conditions. The MEVs can be formulated for any suitable route. It is shown and described herein that MEVs advantageously can be used to deliver therapeutics to the back of the eye by topical or local administration of eye drops, thereby eliminating the need for injection for such treatments. Diseases, disorders, and conditions include, but are not limited to, Adie’s pupil, adult strabismus, age-related macular degeneration, albinism, amblyopia, anisocoria, aphakia, arcus senilis, astigmatism, bacterial keratitis, Bell’s palsy, black eye, blepharitis, blocked tear duct, branch retinal vein occlusion (BRVO), carotid artery disease, cancer of the eye, cataracts, cellulitis, central retinal vein occlusion (CRVO), central serous chorioretinopathy, chalazion and stye, Charles Bonnet syndrome, choroidal neovascular membranes, chronic angle-closure glaucoma, coloboma, color blindness, conjunctivitis (pink eye), contact lens-related eye infections, convergence insufficiency, corneal abrasion, corneal dystrophies, corneal erosion, corneal laceration, corneal ulcer (keratitis), crossed eyes (strabismus), cytomegalovirus retinitis, dementia and the eye, detached retina, diabetes, drusen, dry eye, ectropion, endophthalmitis, entropion, excessive blinking in children, eye allergies, eye lymphoma, eyelid spasm and twitching, farsightedness, Fuchs’ dystrophy, fungal keratitis, giant cell arteritis, giant papillary conjunctivitis, glaucoma, headache and eye problems, hemangioma, herpes keratitis, herpes zoster (shingles), heterochromia, histoplasmosis, HIV / AIDS and the eye, hyperopia, hyphemia, idiopathic intracranial hypertension, iridocorneal endothelial syndrome, ischemic optic neuropathy, juvenile idiopathic arthritis uveitis, juvenile macular dystrophy, keratoconus, lattice degeneration, Leber congenital amaurosis, low vision, macular edema, macular hole, macular pucker, macular telangiectasia, Marfan syndrome, microvascular cranial nerve palsy, migraine, milia, myasthenia gravis, myopia (nearsightedness), neuropathic corneal pain, nevus, nystagmus, ocular hypertension, ocular melanoma, ocular rosacea, onchocerciasis (African River Blindness), optic neuritis, orbital fracture, photokeratitis, pigment dispersion syndrome, pinguecula and pterygium, posterior vitreous detachment, presbyopia, pseudoexfoliation syndrome, ptosis, retinal artery occlusion, retinal detachment, retinal vein occlusion, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, scleritis, Sjogren’s syndrome, sleep crust, Stargardt disease, Stickler syndrome, subconjunctival hemorrhage, thyroid eye disease (Grave’s disease), tom retina, toxoplasmosis, trachoma, trichiasis, trichotillomania, Usher syndrome, uveitis, vitamin A deficiency, vitreomacular traction, and xanthelasma. Diseases, disorders, and conditions of the eye have a variety of causes, including pathogenic, such as viral, fungal, and bacterial infections, genetic, environmental, and multifactorial causes. They vary in the tissues afflicted, disease severity, and in types of treatments available. Some diseases, disorders, and conditions of the eye are readily treated, while many rely on management of the symptoms or condition to prevent disease progression (i.e., vision loss). Described below are groups of eye diseases, disorders, and conditions and their corresponding treatments and therapeutics.

[0261] The MEVs and compositions provided herein can be loaded with therapeutics that treat such diseases, disorders, and conditions and formulated for administration to the eye. An advantage of the MEVs is that, for most, if not all applications, the MEVS can be formulated in drops and administered directly into the eye; there is no requirement for intravitreal or subretinal or other such administration. Any known therapeutic can be exogenously loaded into the MEVs. Nucleic acid and protein or polypeptide or other such therapeutics alternatively can be produced by the microalgae and produced in the microalgae and packaged in the MEVs.

[0262] The following discussion describes exemplary diseases, disorders, and conditions of the eye, and exemplary treatments and therapeutics for treatment. MEVs can be loaded with the therapeutics, exo-loaded and / or endo-loaded, depending upon the product, and formulated for administration to the eye. For treatments that require injection, the MEVs can be formulated as drops and administered to the eye. Dosage and regimen and formulation depend upon the disease, disorder, or condition. Based on disclosure and examples herein and the knowledge of their knowledge, the skilled person can select a therapeutic and formulate and administer it. Combination therapies also are within the skill in the art. Exemplary of diseases, disorders, and conditions for treatment include those the follow in this and subsequent sections and other diseases, disorders, and conditions known to those of skill in the art.

[0263] Disorders of eyelid, lacrimal system, and orbit: ectropion, lagophthalmos, blepharochalasis, ptosis, stye (an acne type infection of the sebaceous glands on or near the eyelid), xanthelasma of eyelid, dermatitis of eyelid, parasitic infestation of eyelid (in leishmaniasis, loiasis, onchocerciasis, phthiriasis), herpes viral infection, leprosy, molluscum contagiosum, tuberculosis, yaws, zoster, dacryoadenitis, epiphora, dysthyroid, conjunctivitis (infective or allergic).

[0264] Disorders of sclera, cornea, iris, and ciliary body: scleritis, keratitis, corneal ulcer, corneal abrasion, snow blindness, Thygeson's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy - cloudy morning vision, keratoconus, keratoconjunctivitis sicca, iritis, uveitis.

[0265] Disorders of lens: cataract, myopia, hypermetropia, presbyopia.

[0266] Disorders of choroid and retina: chorioretinal inflammation, focal or disseminated chorioretinitis, choroiditis, retinitis, or retinochoroiditis, cyclitis, pars planitis, Harada's disease, chorioretinal scars, macula scars of posterior pole, solar retinopathy, choroidal degeneration, atrophy, sclerosis, hereditary choroidal dystrophy, choroideremia, choroidal dystrophy, gyrate atrophy, choroidal hemorrhages, choroidal detachment, other disorders of choroid, chorioretinal disorders, chorioretinal inflammation in infectious and parasitic diseases, retinal detachments and breaks, retinoschisis, retinal vascular occlusions, other retinal disorders, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, Bull's Eye Maculopathy, epiretinal membrane, peripheral retinal degeneration, hereditary retinal dystrophy, retinitis pigmentosa, retinal hemorrhage, separation of retinal layers, central serous retinopathy, retinal detachment, macular edema, diabetic retinopathy, glaucoma, primary open-angle glaucoma, primary angle-closure glaucoma, primary normal tension glaucoma, disorders of vitreous body and globe.

[0267] Disorders of optic nerve and visual pathways: Leber's hereditary optic neuropathy, optic disc drusen, disorders of ocular muscles, binocular movement, accommodation, and refraction.

[0268] Other disorders of the ocular globe: strabismus, ophthalmoparesis, progressive external ophthalmoplegia, weakness of the external eye muscles, esotropia, exotropia, disorders of refraction and accommodation, hypermetropia, myopia, astigmatism, anisometropia, presbyopia, disorders of accommodation, internal ophthalmoplegia, amblyopia, Leber's congenital amaurosis, scotoma, color blindness, achromatopsia, nyctalopia, blindness, river blindness, microphthalmia, red eye, Argyll Robertson pupil, keratomycosis, xerophthalmia, aniridia. Ocular cancers: choroidal melanoma, retinoblastoma, diffuse large B-cell lymphoma, marginal zone lymphoma and follicular lymphoma, metastatic intraocular tumors, basal cell carcinoma of the eyelid, squamous cell carcinoma, cutaneous melanoma, sebaceous carcinoma (meibomian gland carcinoma), Merkel cell carcinoma, squamous cell carcinoma of the conjunctiva, conjunctival melanoma, orbital sarcoma, orbital and optic nerve meningiomas, adenoid cystic carcinoma, pleomorphic adenoma, other lacrimal gland carcinomas, lacrimal squamous cell carcinoma, transitional cell carcinoma of the lacrimal sac, metastatic lacrimal sac and nasolacrimal duct tumors. e. Diseases, disorders, and conditions affecting the back of the eye (posterior segment of the eye)

[0269] This following list includes various conditions affecting the vitreous humor, retina, choroid, and optic nerve within the back of the eye, which generally are treated by injection. These diseases, disorders, and conditions are particularly amenable to treatment with MEVs prepared with or loaded with therapeutics to treat each disease; the treatments can be effected by administering the MEVs formulated as ophthalmic eye drop compositions, thereby replacing treatments effected by injection.

[0270] Retinal diseases:

[0271] - Age-related macular degeneration (AMD)

[0272] - Diabetic retinopathy

[0273] - Retinal detachment

[0274] - Retinitis pigmentosa

[0275] - Retinal vascular occlusions (central retinal vein / artery occlusion)

[0276] - Retinal tears or breaks

[0277] Vitreoretinal disorders:

[0278] - Vitreous hemorrhage

[0279] - Vitreomacular traction

[0280] - Posterior vitreous detachment

[0281] Choroidal conditions:

[0282] - Choroidal neovascularization (e.g., in AMD)

[0283] - Choroiditis

[0284] - Chorioretinal scars

[0285] - Choroidal melanoma

[0286] Optic nerve disorders: - Optic neuritis

[0287] - Optic nerve atrophy

[0288] - Optic nerve head drusen

[0289] Posterior segment trauma:

[0290] - Traumatic vitreous hemorrhage

[0291] - Commotio retinae (traumatic retinal injury)

[0292] Macular disorders:

[0293] - Macular edema

[0294] - Macular hole

[0295] - Epiretinal membrane (macular pucker)

[0296] Other back-of-the-eye pathologies:

[0297] - Myopic degeneration

[0298] - Posterior uveitis

[0299] - Endophthalmitis (infection of the vitreous or aqueous humor)

[0300] - Cystoid macular edema f. Heritable Diseases, Disorders, and Conditions of the Eye

[0301] Several diseases, disorders, and conditions of the eye and retina have underlying genetic causes, such as, for example, retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis (LCS), choroideremia, Stargardt disease, X- linked Retinoschisis, and Achromatopsia, discussed below. The MEVs can deliver gene therapy products, including gene therapy, encoding nucleic acid for treatment of the diseases, and gene editing systems for modifying nucleic acid in the target locus in the eye of a disease, disorder, or condition. Gene therapy can be administered in MEVs formulated for local or topical application to the eye. i. Retinitis Pigmentosa

[0302] Retinitis pigmentosa is a genetic disease characterized by progressive loss of rod photoreceptor cells that line the retina. Symptoms of the condition include decreased night vision, loss of peripheral vision, loss of depth perception, spontaneous vision of flashes / blinking / shimmering lights, blurred vision, poor color separation, loss of central vision, and eventual blindness. ii. Usher syndrome

[0303] Usher syndrome is a genetic disease that affects vision, hearing, and sometimes balance. The vision loss in Usher syndrome is caused by retinitis pigmentosa wherein the photoreceptors of the retina degenerate. iii. Leber congenital amaurosis (LCS)

[0304] Leber congenital amaurosis is an is an autosomal recessive disorder caused by mutations in genes that allow for normal development and function of the retina. The most common genes associated with LCS include CEP290, CRB1, GUCY2D, and RPE65. The condition is characterized by vision problems including photophobia, extreme hyperopia (farsightedness), and involuntary movements of the eye. The visual impairment is typically caused by retinal dystrophy and the inability of the eye to undergo phototransduction due to disruption in the visual cycle. iv. Choroideremia

[0305] Choroideremia is an x-linked heritable retinal dystrophy that is characterized by progressive retinal deterioration including degeneration of the RPE, photoreceptors, and choriocapillaris. Mutations in the CHM gene that encodes Rab escort protein- 1; mutation or dysfunction of this protein impairs vesicular trafficking affecting the health and function of RPE cells. v. Stargardt disease

[0306] Stargardt disease is a heritable disease that causes a slow loss of central vision in both eyes. Mutations in the retinal specific protein, ABCA4, are associated with Stargardt disease. ABCA4 is a flippase, transporting materials across the membranes of photoreceptors to help in the visual cycle. vi. X-linked Retinoschisis

[0307] X-linked Retinoschisis is an inherited disease of the retina that can cause early vision loss in males. The disease is associated with mutations in the RSI gene that affect cell-cell adhesion and intercellular matrix retinal architecture. vii. Achromatopsia

[0308] A heritable retinal disease characterized by color blindness, typically monochromacy. Other symptoms of the condition include photophobia, involuntary eye movement, iris operating abnormalities, and reduced visual acuity. Several genetic mutations are associated with the condition, which mutations result in dysfunction or complete absence of cone cell activity. Identified causes include mutations in any of the following genes, CNGA3 and CNGB3, which encode cyclic nucleotide-gated ion channels; GNA2 (ACHM4), which encodes the cone cell transducin; and PCE6C and PDEH, which encode subunits of cone phosphodiesterase. g. Exemplary Treatments for Heritable Diseases, Disorders, and Conditions of the Eye

[0309] Treatments for heritable diseases, disorders, and conditions of the eye include vitamin supplements, to slow disease progression, artificial retinal implants, and intravitreal injection of small molecule drugs, intravitreal injection of gene therapy drugs. MEVs are vehicles for delivery of nucleic acids and / or encoded proteins. The microalgae can be genetically-modified to encode a therapeutic protein that is endogenously loaded into the MEVs, or the MEVs can be exogenously loaded with a therapeutic nucleic acid and / or encoded protein following purification or isolation.

[0310] Voretigene neparvovec-rzyl (Luxturna™), is the first gene therapy approved in the U.S., is an intravitreal injection of an AAV2 vector that encodes a functional copy of RPE65 cDNA (SEQ ID NO:83; encoded protein SEQ ID NO:84) with a modified Kozak sequence. This therapy can be used to treat any retinal disease resulting from a mutation in the RPE65 gene. This includes, Leber’s congenital amaurosis, retinal pigmentosa, Usher syndrome.

[0311] EA-2353, a small molecule developed by Endogena Therapeutics, is administered via intravitreal injection to treat retinitis pigmentosa. The small molecule EA-2353, for example, activates endogenous retinal stem and progenitor cells to promote their differentiation into functional photoreceptors to thereby restore or preserve vision.

[0312] OCU400 is a gene therapy drug administered by intravitreal injection of an AAV vector encoding a functional copy of NR2E3 a nuclear hormone receptor to rescue retinal degeneration (Li et al. (2021) Gene Ther. 28:223-241).

[0313] AGTC-501 is a gene therapy treatment for RPGR X-linked retinitis pigmentosa. This therapy is delivered via intravitreal injection of a AAV2 viral vector encoding a functional cDNA copy of the RPGR, retinitis pigmentosa GTPase regulator gene under control of a photoreceptor- specific promoter.

[0314] 4D-125 is a gene therapy treatment for patients with RPGR related x-linked retinitis pigmentosa. The therapy intravitreal injection of an optimized AAV vector that provides targeted delivery of an RPGR transgene insert.

[0315] CNTO 2476 are human umbilical tissue-derived cells that are subretinally administered for the treatment of advanced retinitis pigmentosa. SAR421869 is a gene therapy treatment using an equine infections anemiavirus based (EIAV) lenti viral vector expressing MY07A for subretinal delivery to patients with Usher syndrome subtype USH1B.

[0316] CEP290 is a mutated gene associated with Leber congenital amaurosis (LCA) and a gene therapy for LCA. A lentiviral vector encoding full-length CEP290 gene under control of a CMV promoter is administered by intravitreal injection.

[0317] GUCY2D is a gene therapy for the treatment of LCA in patients with a mutation in the GUCY2D gene. The gene therapy involves subretinal injection of a recombinant adeno-associated virus serotype 5 (rAAV5) to deliver a functional copy of the GUCY2D gene to the retina (Jacobsen et al. (2021) iScience 24(5): 102409).

[0318] AAV2-repl, an AAV vector encoding a functional copy of the REP1 gene, is administered by subfoveal injection to treat patients with choroideremia.

[0319] SAR422459 is a gene therapy in which an EIAV vector encoding ABCA4 for the treatment of adults Stargardt disease is administered by subretinal injection. molecule acts as an inhibitor of RPE65 to reduce chromophore biosynthesis and thereby prevent or reduce the production of toxic retinal byproducts.

[0320] STG-001 is an orally delivered small molecule inhibitor of RPB4 to block retinol transport and thereby decrease toxic retinol byproducts. STG-001 is for treatment of patients with ABCA4 mutations in Stargardt disease.

[0321] ATSN-201 is a gene therapy for male subjects with RSI -Associated XLRS. The gene therapy involves intravitreal administration of an AAV vector encoding a functional copy of the RSI gene. rAAV2tYF-CB-hRSl subretinal gene therapy injections in patients with RSI x-linked retinoschisis by administering an AAV vector encoding RSI.

[0322] CNGB3 a subretinal gene therapy injection of an AAV vector encoding CNGB3 for the treatment of achromatopsia.

[0323] Any of these therapeutic products can be loaded into MEVs formulated as drops for administration. The vectors and small molecules can be exo-loaded into the MEVs. In some instances, for gene therapy, the microalgae can encode the gene product, and the encoded protein can be packaged in vivo into the MEVs. h. Inflammatory Diseases, Disorders, and Conditions of the

[0324] Front and Outer Eye

[0325] The MEVs, which advantageously can be administered by drops in place of injections, also can be used to deliver therapeutics for treatment of other parts of the eye that have been treated by topical application.

[0326] Several diseases, disorders, and conditions affect the front and exterior parts of the eye; many such diseases, disorders, and conditions are triggered by environmental factors including trauma, irritants, and infectious agents. Others are caused by or involved dysregulation and changes in the tissues of the eye, or are due to autoimmune conditions that trigger and inflammatory response in the eye. Exemplary of such diseases, disorders, and conditions are bacterial keratitis, Bell’s palsy, blepharitis, conjunctivitis (pink eye), contact lens-related eye infections, corneal abrasion, corneal dystrophies, corneal erosion, corneal laceration, corneal ulcer, dry eye, fungal keratitis, herpes keratitis, herpes zoster (shingles), keratitis, keratoconus, neuropathic corneal pain, juvenile idiopathic arthritis uveitis, optical rosacea, onchocerciasis, scleritis, thyroid eye disease (Grave’s disease), and uveitis. i. Bacterial, Fungal, and Viral Keratitis and Contact Lens- Related Eye Infections

[0327] Bacterial, fungal, and viral keratitis are serious infections of the cornea also referred to as corneal ulcers. Infection of the cornea usually only occurs under compromised conditions including eye injury, improper contact lens usage, or people with reduced immune response. Keratitis is classified in two forms, superficial, which involves the outer layers of the cornea, and deep keratitis, which affects the deep layers of the cornea wherein scarring can occur that can affect vision. If untreated, such infections lead to scarring of the cornea and / or blindness. Bacterial agents that cause such infections include, for example, any of the following, Staphylococcus, Streptococcus, Pseudomonas, Neisseria, Corynebacterium, Chlamydia, Shigella, and Listeria. Fungal agents that cause such infections include, for example, Fusarium, Aspergillus, and Candida. Viral agents that cause such infections include herpes simplex and herpes zoster. Treatments include antibacterial, antifungal, antiviral eyedrops or oral medications, to remove the infectious agent, and also include corticosteroid eyedrops or oral medications to treat the associated inflammation. The treatment can involve scraping of the cornea to remove diseased cells. If the infection is severe or untreated, leading to vision loss, a corneal transplant can be necessary to restore vision. ii. Bell’s Palsy

[0328] Bell’s palsy is a sudden condition that affects the nerves and muscles of the face usually associated with partial paralysis of muscles on one side of the face. Bell’s palsy but is associated with viral infections, immune disorders, or restricted blood flow to a facial nerve. Due to the paralysis of the facial muscles the eyes can become dry and irritated. Treatment of the eyes involves similar treatment as with any dry eye condition, administration of lubricating eye drops to the eye. Corticosteroids and antivirals can be administered to address underlying causes. ill. Blepharitis

[0329] Blepharitis is inflammation of the eyelids characterized by red, swollen, burning or sore eyelids. The condition is caused by a variety of irritants including excess oil production, dry skin, microscopic mites, and bacterial infection. Treatments involve topical hygiene, antibiotic eyedrops or oral medicines, and / or antiinflammatory or lubricating eyedrops. iv. Conjunctivitis

[0330] Conjunctivitis is an inflammatory condition of the conjunctiva from bacteria, virus, or allergies. Symptoms include swollen red eyes; itching, burning, and / or pain of the eyes; watery eyes; photosensitivity; and sometimes mucus, pus, or yellow discharge. Treatments are generally topical such as antibiotic eye drops, eye rinses, lubricating eye drops, and oral allergy medicines. v. Corneal Abrasion, Laceration, and Erosion

[0331] Corneal abrasion is a scratch or scrape on the surface of the cornea, corneal laceration is a cut through the surface of the cornea, and corneal erosion is when the epithelial cells of the cornea loosen from the sub-epithelial layer of cells. Symptoms of abrasion and erosion conditions cause pain, irritation, tearing, blurry vision, and / or photophobia. Symptoms of a corneal laceration can additionally include severe pain, and bleeding. Treatments include wearing a patch over the affected eye; use of moisturizing eye drops (e.g., sodium chloride); use of antibiotic eye drops; pupil dilating eye drops to relieve pain; and surgery such as superficial keratectomy, to remove corneal tissue, or stromal puncture to help bind the epithelial corneal layer to the sub-epithelial layer, and corneal transplant. vi. Corneal Dystrophies

[0332] Corneal dystrophies are genetic disorders of the cornea wherein buildup of material occurs in one or more of the five layers of the cornea causing a reduction or loss of vision. Corneal dystrophies are categorized into three types, including the anterior or superficial, stromal, and posterior corneal dystrophies grouped according to the layers of the cornea that are affected. Symptoms of corneal dystrophies are similar to that of corneal abrasions, lacerations and erosions including, pain, irritation, tearing, blurring vision, photophobia. Treatments include use of moisturizing or lubricating eye drops (e.g., sodium chloride); use of antibiotic eye drops; ointments; contact lenses that protect the cornea; and surgery such as superficial keratectomy, to remove corneal tissue, or stromal puncture to help bind the epithelial corneal layer to the sub-epithelial layer, and corneal transplant. vii. Dry Eye

[0333] Dry eye is any condition in which the eyes do not produce enough tears or is improperly producing one or more layers comprising the eye’s tear film. Symptoms include dry and scratchy eyes, red eyes, excessive tear production, stinging or burning of the eyes, and strings of mucous in or around the eyes. Treatments include artificial tear eye drops, or prescription eye drops that increase tear production, tear duct plugs, or surgery to close the tear ducts. viii. Keratoconus

[0334] Keratoconus is a condition characterized by the coning of the cornea that occurs from the progressive thinning of the outer edges of the cornea. Genetic and environmental factors are involved in the development of this disease. Symptoms include blurred vision, light sensitivity, blurry vision and sudden or worsening vision. Treatment includes uses of contact lenses, and a drug-device combination product to perform corneal cross-linking (Photrexa® Viscous, Photrexa® and the KXL System). ix. Neuropathic Corneal Pain Neuropathic corneal pain is a dysfunction of the nerves of the highly innervated corneal tissue that results in sensations of burning, itch, irritation, stinging, eye-ache, pain, and photophobia. The pain can be caused by underlying inflammatory disease, neurological conditions, or as a side-effect of surgeries of the cornea or eye. x. Optical Rosacea

[0335] Optical Rosacea is an inflammatory condition of the eye that causes redness, burning and itching of the eye. The ocular condition is associated with rosacea, an inflammatory condition of the facial skin. Various factors trigger the condition including genetics, bacterial infections, environmental irritants, circulatory problems, UV exposure, stress, extreme weather conditions, alcohol, and spicy foods. Treatments include eye drops, warm compress, antibiotic pills, and protective measures such as use of sunscreen and sun protectants and use of mild pH balanced soaps. xi. Onchocerciasis

[0336] Onchocerciasis is a parasitic infection caused by the nematode Onchocerca volvulus that can lead to visual impairment and blindness. Microfilariae produced by adult female works travel through scleral and subconjunctival tissues to reach the cornea, within the cornea the microfilariae can die and release Wolbachia sp. Bacteria that induce inflammatory and immune responses resulting in a punctate keratitis in the cornea, if untreated this can cause a scarring and hardening of the cornea, leading to blindness. MF are also able to invade the retina resulting in inflammation, scarring, glaucoma, and loss of vision (Winthrop et al. (2011 ) . J Glob Infect Dis. 3(2): 151- 155). Ivermectin is one standard of treatment for the disease, however, it is unable to enter the eye due to the blood-ocular barrier. Doxycycline combined with ivermectin is also a treatment strategy. xii. Scleritis

[0337] A general inflammatory condition of the scleral tissue of the eye, or the white part of the eye. The inflammation of the sclera can be caused by a variety of factors such as autoimmune diseases, serious eye infections, trauma, and surgery of the eye. The symptoms of the condition include pain and tenderness of the eye, photophobia, and visual loss, treatment varies depending on the type of scleritis. Treatment can include: steroid eye drops, corticosteroid pills (medicine to control inflammation), nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin or ibuprofen for pain and inflammation, oral antibiotic or antiviral drugs, and drugs that weaken or modify the response of the immune system can be used with severe scleritis (immunosuppressive and immunomodulatory drugs). xiii. Thyroid Eye Disease (Grave’s Disease)

[0338] Grave’s disease is an autoimmune disorder characterized by an overactive thyroid. It affects the eye by causing inflammation of the muscles and tissue surrounding the eye that leads to bulging eyes, eyelid retraction, dry or inflamed eyes, and conjunctivitis. Treatment includes steroid administration to control inflammation and swelling and surgery to correct eyelid retraction and relieve eye pressure. Numerous other treatments have been developed. xiv. Uveitis and Juvenile Idiopathic Arthritis (JIA) Uveitis

[0339] Uveitis is an eye condition characterized by inflammation of the middle layer of the eyeball with severe cases resulting in vision loss. Symptoms include red eye, light sensitivity, blurry vision, and / or seeing floaters. The condition can be treated via corticosteroid eye drops, and in some instances sustained-release corticosteroids are delivered by intravitreal injection. JIA uveitis is best treated by early introduction of immunomodulatory therapies, methotrexate combined with anti-TNFa agents such as adalimumab, infliximab, or etanercept can be used for control intraocular inflammation. Abatacept, a CRLA-4 inhibitor, tocilizumab, another TNFa-inhibitor, and rituximab are also candidate drugs. i. Treatments of Inflammatory Diseases, Disorders, and Conditions of the Front and Outer Eye

[0340] Inflammatory conditions of the front and outer eye are generally treated by removing or addressing the underlying cause of the inflammation and irritation, removing the irritant, killing the infectious agent, or modulating the immune system. The inflammation itself can be treated using medicines and lifestyle management. In severe inflammatory diseases, disorders, and conditions surgery to remove, replace, and / or regenerate the damaged tissue can be performed. The table summarizes treatment approaches and medicines for Inflammatory Eye Conditions or Ocular Surface Diseases (adapted from Goyal S and Hamrah P. (2016) Semin Ophthalmol. 31(1-2)). j. Glaucoma and Pressure-Related Conditions of the Inner Eye and back of the eye

[0341] Conditions of the inner eye include eye conditions that can lead to damage of the optical nerve, such conditions include, iridocorneal endothelial syndrome; glaucoma, including closed-angle glaucoma and open-angle glaucoma; ocular hypertension, and pigment dispersion syndrome. i. Glaucoma

[0342] Glaucoma comprises a group of eye conditions characterized by progressive deterioration of retinal ganglion cells and damage to the optic nerve. Glaucoma is typically driven by elevated intraocular pressure, however damage to the optic nerve is possible even with normal eye pressure. Instances of glaucoma with normal eye pressure can be caused by atherosclerosis, impaired microcirculation, altered immunity, and oxidative stress. Elevated eye pressure occurs due to an imbalance in the aqueous humor entering the eye, secreted by the ciliary body, and leaving the eye, through the trabecular mesh work and uveoscleral outflow pathway. Such elevated pressure can impede blood flow to and cause damage of the optic nerve resulting in vision loss and / or blindness. Types of glaucoma with elevated eye pressure include open-angle, closed-angle glaucoma, and pigmentary glaucoma (pigment dispersion syndrome). Open-angle glaucoma, accounting for 80% of glaucoma cases, is characterized by decreased aqueous outflow that is caused by increased resistance through the trabecular meshwork of the eye. In closed-angle glaucoma the angle between the iris and cornea, the site of aqueous outflow in the eye, becomes too narrow resulting in buildup of fluid pressure. Closed-angle glaucoma is caused by disorders of the iris, the lens and retrolenticular structures. Pigment dispersion syndrome similarly blocks the drainage angle due to dislocation of pigment granules from the pigment epithelium of the iris. In late-stage closed-angle glaucoma symptoms include eye pain, headache, rainbow-colored halos around lights, low vision, blurred vision, tunnel vision, blind spots, nausea, and vomiting, and / or red eye, however, glaucoma, in general, is predominantly asymptomatic until it reaches advanced stages that result in vision loss. Regular eye exams provide the best method for early detection and prevention of vision loss (Weinreb et al. (2014) JAMA 311(18):1901-1911). ii. Iridocorneal Endothelial Syndrome

[0343] An eye condition characterized by proliferation and migration of corneal endothelial cells to the iris and iridocorneal angle. These structural changes lead to secondary angle-closure glaucoma, corneal edema, and atrophy of the iris (Das S. et al. (2023) Statpearls https: / / www.ncbi.nlm.nih.gov / books / NBK594227 / ). Treatment follows that of treatment for corneal dystrophies and for glaucoma. iii. Ocular Hypertension

[0344] A condition of elevated pressure inside the eye usually caused by impaired drainage of the aqueous humor from the front of the eye. Ocular hypertension shows no sign of optical nerve damage; it is an indicator of risk of developing glaucoma. k. Treatment of Glaucoma and Pressure-Related Conditions of the Inner Eye

[0345] Glaucoma is typically treated using eye drops that help increase drainage or decrease production of fluid such that they reduce eye pressure; the eyedrops must be continued on a daily lifelong basis. Some exemplary eye drop medications include prostaglandins, such as latanoprost (Xalatan®), travoprost (Travatan Z®), tafluprost (Zioptan®), bimatoprost (Lumigan®) and latanoprostene bunod (Vyzulta®); beta blockers, such as timolol (Betimol®, Istalol®, Timoptic®) and betaxolol (Betoptic® S); alpha-andrenergic agonists such as apraclonidine (lopidine®) and brimonidine (Alphagan P®, Qoliana®); carbonic anhydrase inhibitors such as dorzolamide and brinzolamide (Azopt®); rho-kinase inhibitors such as netarsudil (Rhopressa®); and mitotic or cholinergic agents such as pilocarpine (Isopto Carpine®). Though the eyedrops are directly administered to the eye, in addition to side-effects to the eye, there are also systemic effects that can happen due to the drug’s entry into the bloodstream such as, for example, low blood pressure, impotence, dry mouth, fatigue, high blood pressure, irregular heart rate, frequent urination, and / or tingling fingers and toes. Other methods of treatment include implant devices, laser therapy, and surgery. Implant devices are placed in each eye and provide longer duration treatment, lasting several months. Laser treatment is performed to relie ve / remove blockage and improve fluid drainage from the eye, or to destroy some of the tissue that produces the liquid, this therapy can be accompanied with the daily use of eye drops. In some instances, surgery is performed, such as to insert stents or remove tissue to increase fluid outflow from the eye.

[0346] Below are therapeutics for treatment of glaucoma and related conditions. The therapeutics can be loaded into the MEVs provided herein and formulated, generally as eye drops, for administration to the eye.

[0347]

[0348] 1. Refractive Conditions of the Eye or Conditions of the Lens of the Eye

[0349] Diseases, disorders, and conditions specific to the lens of the eye include astigmatism, cataracts, hyperopia (farsightedness), myopia (nearsightedness), and presbyopia. While these conditions may be related to physical attributes of the lens, treatments can include administration of therapeutics to ease associated symptoms or side-effects or consequences of surgical intervention. The MEVs can deliver such therapeutics. i. Astigmatism

[0350] Astigmatism is a condition of the lens or the cornea of the eye wherein the cornea or lens have an irregular curve such that light is improperly focused on the retina causing blurry, fuzzy, or distorted vision. Genetics and environmental factors can both contribute to the development of astigmatism. ii. Cataracts

[0351] Cataracts are characterized by a clouding of the lens of the eye. The development of cataracts occurs from breakdown or damage to the proteins and fibers comprising the lens resulting in the clear lens becoming hazy or cloudy. Aging, inflammation, chronic corticosteroid use, alcohol, smoking, obesity, genetics, excessive sunlight exposure can all contribute to the development of cataracts. iii. Hyperopia (Farsightedness)

[0352] Hyperopia is a refractive error of the lens in the eye wherein the eyeball is too short from the front to the back, or the shape of the cornea or the lens of the eye is misshapen resulting in light being focused behind the retina and, thus, causing close-up objects to look blurry. Genetics is the most common factor contributing to hyperopia. iv. Myopia (Nearsightedness)

[0353] Myopia is a refractive error of the lens in the eye wherein the eyeball is too long from the front to the back, or the shape of the cornea or the lens of the eye is misshapen resulting in light being focused in front of the retina and, thus, causing distant objects to look blurry. Myopia often develops or is diagnosed in childhood while there are developmental changes in the eye. Genetic factors predispose some individuals for myopia, however, environmental factors, such as excess screen use, also contribute to the development of this condition. v. Presbyopia

[0354] Presbyopia is an age-related refractive error of the lens that causes a degradation or loss of close-up vision. With age the lens of the eye becomes hardened resulting in a functional decline in the focusing accommodation of the lens. Additionally, the relative shape of the lens can also change with age, including increased thickness of the lens, and an increase in the convexity of the anterior lens. m. Treatments for Refractive Conditions of the Eye

[0355] The most common treatment for astigmatism, hyperopia (farsightedness), myopia (nearsightedness), and presbyopia is corrective lenses such as glasses or contacts. Other common treatment involves refractive laser surgery to reshape the cornea such as LASIK surgery and photorefractive keratectomy (PRK) surgery.

[0356] Additional treatments are available for children with myopia to slow or reduce myopia progression and eye growth such treatments include specialized lenses with concentric rings that provide myopic defocus to the retina, red light therapy, and low concentration atropine eye drops.

[0357] Surgery is the only available treatment for cataracts, such surgery involves removal of the clouded lens of the eye and replacement with a new artificial lens. Antioxidants and oxysterols improve and have reversed lens opacity, exemplary compounds include N-acetylcysteine amide, N-acetylcarnosine, lanosterol, and 5- cholesten-3b,25-diol (VP1-001); delivery to the lens remains an obstacle. These compounds can be introduced into MEVs for administration to the eye. n. Treatments for Diseases, Disorders, and Conditions of the Posterior Eye

[0358] Retinal diseases, disorders, and conditions of the eye, and other conditions of the posterior eye generally are treated by intravitreal injection. These diseases, disorders, and conditions include, but are not limited to, macular degeneration, including age-related macular degeneration (AMD), neovascular AMD, and geographic atrophy in AMD; diabetic retinopathy, including diabetic macular edema (DME), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR); retinal vein occlusions (RVO); endophthalmitis; uveitis; cystoid macular edema (CME); epiretinal membrane; a macular hole; retinitis pigmentosa; cytomegalovirus retinitis; retinopathy of prematurity; lattice degeneration; macular telangiectasia; optic neuritis; vitreomacular traction; cancers of the eye, such as retinoblastomas; and secondary retinal diseases, such as, choroidal neovascular membrane (CNVM). These diseases, disorders, and conditions of the posterior eye can be treated by MEVs loaded with a therapeutic(s) for each disease, disorder, or condition formulated as drops for administration to the eye. The MEV compositions do not require injection for delivery of the therapeutic to the retina or other tissue in the posterior eye. i. Age-related Macular Degeneration (AMD): pathological mechanisms, treatment options

[0359] AMD is the leading cause of blindness in developed nations and its prevalence is increasing as the population ages (Wong et al. (2014) Lancet Glob Health 2(2):el06-116). AMD is a late-onset multifactorial disease with genetic, environmental, and age-related factors affecting risk for and development of the disease. The disease is characterized central vision loss due to damage or dysfunction of the macula, a small central part of the retina with a high density of cone photoreceptors, specialized cells that are essential for central, high-acuity, color vision. A complex support system, known as the photoreceptor support system, is essential to proper photoreceptor and visual function. The photoreceptor support system contains the retinal pigment epithelium (RPE), Bruch’s membrane (BrM), and the choroidal vasculature. The RPE comprises a single layer of polarized cells that interdigitate with rod and cones cells, it maintains a blood-retina barrier that provides separation between photoreceptors and systemic circulation, delivers oxygen and metabolites to the photoreceptor cells, and is critical for recycling retinoids, compounds essential for phototransduction. The choroid is a meshwork of vasculature and capillaries that helps supply the high levels of oxygen required by photoreceptors, among the most metabolically active neurons in the body. The BrM is a laminar extracellular matrix between the choroid and the RPE that helps transport oxygen, glucose, and other metabolites to the RPE and photoreceptors, and return waste to systemic circulation. Damage or dysfunction to any part of this system contributes to aberrant RPE pigment distribution and subsequent AMD disease progression (Fritsche et al. (2014) Annu Rev Genomics Hum Genet 15:151-71).

[0360] Age-related macular degeneration (AMD), thus, is a prominent cause of vision impairment and blindness, affecting the central portion of the retina known as the macula. This degenerative disorder is categorized into two main types: dry (non- neovascular) AMD and wet (neovascular) AMD discussed below. In dry AMD, lipoproteinaceous deposits, known as drusen, accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane. Over time, these deposits lead to the separation of the RPE from Bruch’s membrane, causing RPE atrophy and subsequent degeneration of nearby photoreceptors. Wet AMD is characterized by choroidal neovascularization (CNV), where abnormal blood vessels from the choroid penetrate Bruch’s membrane, displacing the RPE and damaging photoreceptor cells. See Error! Reference source not found. A and 12B which provide a schematic representation of retinal damage in choroidal neovascularization (CNV), figures adopted from: www.allaboutvision.com / conditions / choroidal-neovascularization-cnv.

[0361] AMD classification is based on anatomical changes observed through ophthalmic imaging, which distinguishes among early, intermediate, and advanced (or late) stages. Early / intermediate forms are associated with functional changes, such as impaired dark adaptation, advanced AMD is marked by severe visual impairment and is classified into two clinical forms: neovascular AMD and geographic atrophy (GA). In neovascular (or exudative) AMD, subretinal neovascularization develops below or above the retinal pigment epithelium (RPE). These abnormal blood vessels often leak serous fluid or blood, resulting in macular edema and / or hemorrhages that can lead to rapid visual loss. Repeated disruption of retinal architecture leads to profound, irreversible vision loss. In GA due to AMD, an expanding zone of photoreceptor, RPE and choroid capillary degeneration occurs within the macula. About 1 in 6 patients with bilateral GA become severely sight impaired within 6 years from diagnosis [Chakravarthy et al. (2018) Characterizing disease burden and progression of geographic atrophy secondary to Age-related Macular Degeneration. Ophthalmology 725:842-849].

[0362] Molecular mechanisms underlying AMD involve complex interactions within the retinal structure. In wet AMD, a critical molecular event is local secretion of growth factors resulting in abnormal angiogenesis. The elevated expression of angiogenic proteins, particularly vascular endothelial growth factor (VEGF), an angiogenic protein that exerts its effects through VEGF receptor 2 (VEGFR2), occurs in the RPE cells. In response to local tissue hypoxia and stress, RPE cells upregulate VEGF expression, promoting choroidal neovascularization (CNV) - the growth of blood vessels from the choroid into the subretinal space. This vascular invasion disrupts the retinal architecture, leading to displacement of the RPE, damage to photoreceptor cells, and eventual vision impairment. VEGF promotes angiogenesis in the initial stage of CNV and further leads to increased vascular permeability; therefore, most therapeutic strategies for AMD focus on mitigating the effects of elevated VEGF levels. Anti-VEGF treatments, such as intravitreal injections of VEGF inhibitors, have been introduced in the management of wet AMD. Therapeutics, such as, for example, bevacizumab, ranibizumab and aflibercept, block local VEGF activity to inhibit abnormal angiogenesis and preserve vision.

[0363] Another therapeutic target in AMD is the complement system, a complex network of approximately 30 proteins that plays a vital role in the immune response. The complement system comprises both activating and controlling proteins that are found throughout the body in individual or complex forms. In ageing and AMD, there is an increased expression of the complement protein C3 within RPE cells and on their basal membranes, leading to complement activation, oxidative stress, and cellular dysfunction. Complement may also influence choroidal homeostasis through its effects on modulating neovascularization and immune regulation. Pegcetacoplan, a synthetic peptide inhibitor of C3, has been recently developed to reduce excessive complement activation in several conditions, including AMD. a) Dry AMD Treatments

[0364] Dry AMD, which accounts for 85-90% of AMD cases, is characterized by a thinning macula and growth of lipid protein clumps called drusen that drive geographic atrophy of the outer retinal tissue and loss of central vision. Drusen comprise lipids and various proteins including those involved in complement regulation, IMPT3, vitronectin, P-amyloid, and apolipoproteins, plus zinc and iron ions (Fritsche et al. (2014) Annu Rev Genomics Hum Genet 15:151-71). Drusen result from accumulation of unwanted and damaged proteins and lipids extruded from RPE cells in the BrM. Abnormal activity in the complement cascade, elevated cholesterol, and / or impaired macrophage function all contribute to drusen formation driving changes in RPE pigment distribution and retinal atrophy.

[0365] Dry AMD is treated via intravitreal injection of Pegcetacoplan (SYFOVRE®, Apellis Pharmaceuticals, Inc.). Pegcetacoplan is a pegylated cyclic peptide (SEQ ID NO:85; sequence: ICVWQDWGAHRCT, U.S. Patent Nos. 7,888,323, 7,989,589, 8,168,584, 9,056,076, 10,035,822, 10,125,171, 10,875,893, 11,292,815; see structure below). It targets complement protein C3 and inhibits the complement cascade.

[0366] Avacincaptad pegol (IZERVAY™) is an RNA aptamer administered via monthly intravitreal injections. Avacincaptad has the sequence (SEQ ID NO:86) cgccgcggucuc aggcgcugagucugaguuuaccugcgt or fCmGfCfCGfCmGmGfUfCfUfCmAmGmGfCGfCfUmGmAmGfUfCf'UmGm

[0367] AmGfUfUfUAfCfCfUmGfCmG-3T, where fC and fU=2' fluoro nucleotides, mG, and mA=2'-0Me nucleotides, all other nucleotides are 2'-OH) and is covalently bound to a branched PEG molecule that binds to inhibits complement protein C5, prevents cleavage of C5 into C5a and C5b thereby decreasing membrane attack complex formation (U.S. Patent Nos. 11,491,176, 11,273,171, 10,947,544, 9,617,546, 8,236,773, 7,803,931, 7,538,211, and 7,579,456). The drug reduces the inflammatory processes and reduces the rate of geographic atrophy growth, slowing the progression of AMD.

[0368] The standard method of treatment and route of administration of therapies for Dry AMD is via intravitreal injection. In dry AMD, only geographic atrophy (latestage AMD) can be treated; there is no known treatment to slow or prevent the growth of drusen. As described herein, the therapeutics can be introduced into MEVs as provided herein and administered, generally as drops, to treat dry AMD. b) Wet AMD Treatments

[0369] Wet AMD is more severe than dry AMD and can cause a rapid loss of central vision. Wet AMD is caused by the growth of new abnormal blood vessels, choroidal neovascularization (CNV), under the retina that eventually leak blood and / or other fluids and scar the macula. Choroidal neovascularization is a downstream effect of stress or damage to the RPE and the resulting immune responses that induce pro- angiogenic factors (Campagne et al. (2014) J Pathol 232(2): 151-164). Genetic studies have identified genes involved in the etiology of this condition. The early genes identified include particular variants of CFH, ARMS2, and HTRA1. The CFH variant indicates an association between overactivation of the complement pathway. Genetic studies have found multiple genes in the complement pathway that are associated with AMD including CFB, CFI, and complement components 2 and 3 (C2, and C3) (Campagne et al. (2014) J Pathol 232(2): 151-164). The genes and risk factors identified elucidate some of the factors involved in the pathophysiology of the disease including oxidative stress, lipid metabolism, extracellular matrix biology, inflammation, liberalization of the complement cascade and other immunological responses (Vyawahare et al. (2022) Cureus. 14(9):e29583)

[0370] The standard method of treatment and route of administration of therapies for Wet AMD is via intravitreal injection. Wet AMD is treated via intravitreal injection of anti-VEGF drugs, such as, for example, aflibercept (Eyela®), bevacizumab (Avastin®), and ranibizumab (Lucentis®), to reduce the growth of and slow leaking from abnormal blood vessels.

[0371] Aflibercept (Eyela®) is a recombinant protein comprising the binding domains of two vascular VEGF receptors, VEGF1 and VEGF2 thereby sequestering ligands that activate endogenous VEGF receptors; the fusion protein provides for higher affinity to VEGF than the endogenous receptors (SEQ ID NO:73, sequence: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISN ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNV GIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTK KNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0372] U.S. Patent Nos. 7,306,799, 7,531,173, 7,374,758, 7,608,261, 7,070,959, 7,374,757).

[0373] Bevacizumab (Avastin®) is a humanized monoclonal IgG antibody that binds to and inhibits VEGF-A, to thereby slow or inhibit neovascularization. It is used to treat wet AMD, diabetic eye disease, and other retinal diseases. Bevacizumab comprises heavy chain and light chain sequences as follows (SEQ ID NOs: 86 and 87, respectively).

[0374] Bevacizumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

[0375] ACEVTHQGLSSPVTKSFNRGEC; and

[0376] Bevacizumab heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYA ADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK.

[0377] The large size of monoclonal antibodies provides for difficulty crossing cell membranes; thus, the drug must be administered via intravitreal injection.

[0378] Ranibizumab (Byooviz®, Cimerli®, Lucentis®, Susvimo®) is a humanized IgGl kappa monoclonal antibody fragment that binds to VEGF-A to inhibit growth of abnormal blood vessels in wet AMD. The drug is administered via monthly intravitreal injection, or can be delivered via an implant insertion, lasting approximately 25 weeks. Ranibizumab comprises SEQ ID NOs:89 and 90, respectively (Lowe et al. (2007) Exp Eye Res. 85(4):425-430): Ranibizumab light chain sequence: DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC;

[0379] Ranibizumab heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTY AADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL.

[0380] Some small-molecules have been used to inhibit neovascularization to slow the progression of AMD. As shown, for example, end binding-3 inhibitor (EBIN), an eye drop formulation of a protein compound for treating wet AMD inhibits end binding-3 (EB3) protein to prevents neovascular leakage and choroidal neovascularization (Lee et al. (2023) Cell Reports Medicine 4(10)). Figure 19 shows the interaction of EBIN with EB3. As another example, PAN-90806 (Pan Optica) is an orally-bioavailable small molecule that binds and inhibits VEGFR2 and increases the interval between standard intravitreal injections (Khachigian et al. (2023) J Transl Med a )). ii. Diabetic Retinopathy

[0381] Diabetic retinopathy (DR) is characterized by abnormal growth and / or damage to the blood vessels at the back of the eye driven by increased levels of inflammatory cytokines in the retina. The early stage of the disease is known as non-proliferative diabetic retinopathy wherein tiny blood vessels leak and cause swelling of the retina and macula resulting in vision loss. The more advanced stage of the disease is known as proliferative diabetic retinopathy wherein neovascularization occurs. If the new blood vessels begin to leak, they block some or all vision, depending on the severity. Mild instances of DR can be treated through careful diabetes management. More severe cases are treated by intravitreal injection or surgical methods. For example, monthly intravitreal injections with anti-VEGF drugs such as, for example, Avastin®, EYELA®, and LUCENTIS®; intravitreal corticosteroid injections to the eye; pan retinal photocoagulation therapy, a laser therapy to stop growth and proliferation of new blood vessels; or vitrectomy, a surgery to remove blood from the middle eye and scar tissue from the retina. Therapies include orally delivered small-molecules, such as TTP488 that inhibit AGE, or pro-inflammatory signaling, aldolase reductase inhibitors, PKC P inhibitors, angiopoietin 2 inhibitor, NSAIDs, renin-angiotensin- aldosterone system (RAAS) blockers, and antioxidants, such therapies are proposed to be administered systematically and, thus, can have off-target effects. Other treatments include gene-therapies, which require intravitreal or intraocular injection to reach the retinal target (Sadikan et al. (2023) Pharmacol 396:3395-3406). iii. Retinal vein occlusions and treatment

[0382] Retinal vein occlusions occur when the blood vessels to the back of the eye, or the retina, become blocked, typically by a blood clot. Symptoms include any of blurred vision, loss of vision, floaters in the field of vision, sensation of pain or pressure in the eye. Treatment of this condition includes intravitreal injections to reduce macular swelling, laser surgery to shrink retinal blood vessels, and management of health conditions such as high blood pressure, diabetes, and / or glaucoma. iv. Endophthalmitis

[0383] Endophthalmitis is intraocular inflammation usually from infection of the vitreous cavity or by massive infiltration of the vitreous cavity with inflammatory cells. The condition is characterized by blurred vision, red eyes, swollen eyelid, watery eye, and light sensitivity. Treatment includes vitrectomy, intravitreal administration of antibiotics, systemic antibiotics, and / or intravitreal injection of corticosteroids. All can be introduced into MEVs, and administered as drops. v. Uveitis

[0384] Uveitis is an eye condition characterized by inflammation of the middle layer of the eyeball with severe cases resulting in vision loss. Symptoms include red eye, light sensitivity, blurry vision, and / or seeing floaters. The condition can be treated via corticosteroid eye drops, and in some instances sustained-release corticosteroids are delivered by intravitreal injection. All can be introduced into MEVs, and administered as drops. vi. Cystoid macular edema (CME)

[0385] Cystoid macular edema (CME) is characterized by the development of small fluid-filled sacs in the macula and resultant macular swelling. As with other macular conditions, swelling of the macula results in distorted central vision. Treatment can include eyedrops, intravitreal injections, administration of drugs to reduce swelling, laser surgery to repair leaking blood vessels, vitrectomy to remove macular scar tissue. Any therapeutics can be introduced into MEVs, and administered as drops. vii. Epiretinal Membrane

[0386] An epiretinal membrane is a thin sheet of fibrous tissue, usually scar tissue, that grows across the surface of the macula resulting in distortion or loss of central vision. Treatment using drops has not been effective. Treatment involves vitrectomy to remove the epiretinal membrane from the retina. viii. Macular Hole

[0387] A macular hole occurs when the vitreous is stuck to the retina and begins to shrink creating a circular opening in the macula resulting in a dark or blind spot in the central vision. Macular holes can also develop from macular swelling, or eye injury. Treatment for such condition involves a vitrectomy, a surgery to remove the vitreous that is pulling on the macula, and insertion of a gas bubble to help flatten and allow for healing of the macular hole. ix. Retinitis Pigmentosa

[0388] Retinitis pigmentosa is a genetic disease characterized by progressive loss of rod photoreceptor cells that line the retina. Symptoms of the condition include decreased night vision, loss of peripheral vision, loss of depth perception, spontaneous vision of flashes / blinking / shimmering lights, blurred vision, poor color separation, loss of central vision, and eventual blindness. Treatments include vitamin supplements, to slow disease progression, artificial retinal implants, intravitreal injection of the gene therapy voretigene neparvovec-rzyl, Luxturna®, is an AAV2 vector that encodes a copy of the RPE65 gene. It can be incorporated into MEVs for packaging of the AAV vector into MEVs for delivery in eye drops to the retina. x. Cytomegalovirus Retinitis

[0389] A viral infection of the retina usually only occurring in immunocompromised individuals. Without treatment the viral infection can destroy the retina and damage the optic nerve, also common is the development of a detached retina. To treat the condition antiviral medications such as ganciclovir are administered orally, intravascularly, or via intravitreal injection. xi. Retinopathy of Prematurity

[0390] Retinopathy of Prematurity (ROP) is disease that occurs in premature babies wherein normal eye growth or development was interrupted. The premature eye development can lead to abnormal blood vessel growth at the back of the eye and, potentially, blindness. ROP is treated by inhibiting the abnormal blood vessel growth through laser surgery or through intravitreal injection of anti-VEGF medications. xii. Lattice Degeneration

[0391] Lattice degeneration is a degenerative disease of the retinal tissue, commonly associated with retinal breaks and retinal detachment. The disease is characterized by retinal thinning, vascular fibrosis, neuronal atrophy, vitreous liquefaction, vitreoretinal adhesion, pigmentary changes, internal limiting membrane changes, and lack of basement membrane over the surface of lattice lesions and replacement with glial cells. Only retinal holes, tears, and detachments resulting from lattice degeneration are treated. xiii. Macular Telangiectasia

[0392] Macular Telangiectasia (MacTel) is disease characterized by changes in the blood vessels around the fovea, the center of the macula, that can result in loss of central vision. Two types of MacTel include Typel and Type 2 MacTel. Type 1 MacTel is characterized by the formation of aneurysms in the blood vessels of the macula resulting in vision loss due to macular edema. Type 2 MacTel is characterized by abnormal blood vessel formation around the fovea including blood vessel dilation and choroidal neovascularization. Diabetes and hypertension are risk factors in developing type 2 MacTel. Type 1 MacTel appears to be heritable and found almost entirely in males. Treatments for MacTel include laser therapy to seal leaking blood vessels, administration of steroids to reduce inflammation, or injection of anti-VEGF drugs to inhibit growth of abnormal blood vessels. xiv. Optic Neuritis

[0393] Optic neuritis is a condition wherein damage to the optic nerve occurs due to swelling or inflammation of the eye. Infections, drugs, and autoimmune conditions, for example, multiple sclerosis, neuromyelitis optica, and myelin oligodendrocyte glycoprotein antibody disorder are all associated with the development of optic neuritis. The condition is typically treated by treating inflammation and / or the source of the inflammation including administration of corticosteroids, and immunomodulating drugs. xv. Vitreomacular Traction

[0394] Vitreomacular traction is a condition of the vitreous of the eye wherein the vitreous sticks to macula, when the vitreous begins to shrink with age, it begins to pull pulls on and can cause damage to the macula. If a macular hole, macular pucker, or macular edema is seen, the condition is treated via a vitrectomy.

[0395] C. ADMINISTRATION OF THERAPEUTICS TO THE EYE

[0396] Biological drugs (biologies) for treating conditions of the back of the eye have been administered through several routes based on the targets and the nature of the therapeutic molecules. Injection is the primary manner in which therapeutics are administered for treating conditions of the back of the eye. In contrast, as described herein, providing therapeutics in MEVs allows for drop instillation or topical administration with eye drops or other formulations that are applied to the eye, obviating the need for injections or other such means and routes of administration. For treating the diverse array of conditions affecting the back of the eye, delivery of pharmaceuticals have been performed with topical, subconjunctival, suprachoroidal, subretinal, and intravitreal routes of administration, see, for example, Figure 13.

[0397] 1. Intravitreal injection for administration of the back-of-the-eye therapeutics

[0398] Administration of biologies, including large and complex molecules, generally faces challenges related to their stability, penetration through ocular barriers, and sustained therapeutic concentrations. Intravitreal injections remain the primary and most effective route for delivering biologic therapies to the posterior segment due to their ability to achieve high drug concentrations at the target site and bypass ocular barriers effectively.

[0399] Intravitreal injection is a common and effective route for administering biologies because it provides direct delivery of biologic agents, such as anti-VEGF drugs, steroids, and gene therapies, into the vitreous cavity near the affected tissues in the retina or choroid. It is a widely used route for the back-of-the-eye administration because it provides to direct delivery to the site of action, especially for diseases requiring frequent dosing or high drug concentrations at the back of the eye.

[0400] Direct injection into the vitreous humor, provides for targeted delivery to the posterior segment. Commonly used for delivering anti-VEGF drugs for AMD, diabetic retinopathy, and other conditions requiring administration directly to the retina or choroid. Intravitreal injection has been the route employed for back-of-the- eye administration of therapeutic agents, particularly in the treatment of conditions, such as age-related macular degeneration (AMD). Intravitreal injection has been required because of the barriers posed by the anatomy and physiology of the eye. Intravitreal injection is performed in a controlled environment, typically an outpatient setting, and is the method employed for treating a range of the back-of-the-eye pathologies.

[0401] Intravitreal injection provides for delivery of therapeutic agents, such as drugs, particles, and viruses, directly into the vitreous cavity, bypassing external barriers and providing a high concentration of the therapeutic agent in the vitreous humor. Once injected, the agent disperses locally, creating a reservoir for sustained release and diffusion toward the retina, independently of the choroidal blood vessels. The agent reaches and penetrates the layers of the retina, where its therapeutic effects are exerted.

[0402] Disadvantages of intravitreal injection include the discomfort and pain of injection; the procedure is performed in a controlled environment, typically an outpatient setting. Administration requires skilled personnel and has a number of further drawbacks, including the risk of ocular trauma, with the potential for retinal detachment or hemorrhage, as well as the risk of infection that can lead to severe intraocular complications. Intravitreal injections can induce transient changes in intraocular pressure, which can be a concern, especially for individuals with preexisting glaucoma or ocular hypertension. This route of drug administration typically requires topical anesthesia; but still causes discomfort and pain in most patients, which is significant psychological and discouraging burden in the long term (see, e.g., Nguyen el al. (2018) Factors associated with pain during intravitreal injection of anti-vascular endothelial growth factor, Eye 52:1544-1546). Thus, alternative feasible administration routes are needed to address the above-mentioned drawbacks and provide methods of improved safety, convenience, and efficacy for the treatment of the back-of-the-eye conditions.

[0403] Intravitreal injections have been used for treating many conditions of the eye; there, however, are risks and drawbacks, including that many conditions require frequent (monthly) injections to manage the symptoms of or prevent further deterioration of the condition. Other risks of intravitreal injections include eye pain, bleeding, retinal tear or detachment, cataracts, infection, increased intraocular pressure with damage to optic nerve, and in severe cases, loss of vision, or loss of the eye. Treatments include gene therapy for treatment of conditions of the eye. Gene therapy generally is administered via injection to the eye. Other treatments employ small molecule therapeutics, which have been administered systemically thereby posing risks for off-target effects; and intravitreal injection of mesenchymal stem-cell derived exosomes (MSC-exosomes; see, Wu et al., (2023) Pharmaceutics 15(4):1167). There remains a need for less invasive therapeutic options and methods of treatment that minimize risk and can minimize the need for in-office visits for treatment. Provided herein are MEVs that deliver therapeutics for treatment of diseases, disorders, and conditions of the eye. Advantageously, the MEVs can be administered in eye drops.

[0404] Exemplary of diseases, disorders, and conditions, and therapeutics that for treating diseases, disorders, and conditions of the posterior eye include, but are not limited to, Aflibercept (EYELA® HD), Aflibercept (EYELA®), Avacincaptad pegol (IZERVAY™), Bevacizumab (Avastin®), Brolucizumab (BEOVU®), Faricimab

[0405] (VABYSMO™), Pegcetacoplan (SYFOVRE®), Ranibizumab (LUCENTIS®),

[0406] Ranibizumab-eqm (CIMERLI®), Ranibizumab-nuna (BYOOVIZ™), as well as others discussed herein and others known to those of skill in the art. See summary table below for exemplary therapeutics.

[0407] 2. Other routes of administration that have been used for treating diseases, disorders, and conditions of the posterior (back-of-the-eye) a. Subretinal injection: Injection between the retina and the choroid, often used in experimental settings or specialized treatments for retinal diseases. This is employed in specialized cases, especially for experimental therapies or highly targeted treatments for specific retinal diseases; allows biologic agents to be delivered between the retina and choroid to address certain retinal disorders. b. Suprachoroidal injection: Injection into the suprachoroidal space, aiming to deliver drugs to the choroid, retina, and adjacent structures. An emerging technique with potential advantages in drug distribution and reducing the risk of retinal detachment. c. Periocular Administration: While less common for biologies, periocular injections, injections or placement of medication around the eye, targeting the tissues surrounding the eye, have been employed for certain conditions. Subconjunctival or retrobulbar injections of biologies is considered in specific cases where the posterior segment tissues can be reached. This includes subconjunctival or retrobulbar injections, used for delivering steroids or other agents to treat various posterior segment conditions. d. Systemic Administration: Oral medication or intravenous infusion for systemic circulation. Used where drugs have systemic effects beneficial for posterior segment conditions, such as systemic steroids for inflammatory conditions or immunosuppressive agents. Because of limited penetration through ocular barriers and potential systemic side effects, this is not a common route of administration. e. Intravenous Infusion: Rarely used for specific systemic medications that can target the posterior segment, but less common due to limited drug penetration through the blood-retinal barrier. f. Intraocular Implants: Intraocular Implants: Implantable devices can be designed to release biologic agents slowly over time, offering sustained drug delivery to the posterior segment. Devices implanted within the eye to release medication slowly over time. Limited use for sustained drug delivery to the posterior segment, providing prolonged therapeutic effects. g. Topical Administration: Eye drops, or ointments or other formulations that are applied to the surface of the eye, are used for front of the eye conditions, but are rarely, if ever, used for treating back of the eye pathologies due to barriers preventing penetration to the back of the eye. Topical delivery is of limited use because of ocular barriers, such as the cornea and blood-retinal barrier, which limit the penetration of large biological molecules delivered topically and result in impaired bioavailability of the drug to the target tissues, particularly the retina and choroid. Biological drugs also can be susceptible to degradation, limiting their stability and reducing the therapeutic effect.

[0408] 3. Gene therapy and other treatments of the back-of-the-eye conditions and their limitations

[0409] MEVs can be exogenously loaded or the microalgae genetically modified to deliver nucleic acids encoding a product or to encode gene therapy products, and, as described herein advantageously can be administered topically and traffic to the back of the eye. Other vehicles have been used for gene therapy, but all have required injection for administration. a. Mesenchymal stem cell-derived extracellular vesicles

[0410] (MSC-EVs) Extracellular vesicles from mammalian origin, more specifically mesenchymal stem cells (MSC)-derived EVs, have been reported to deliver payloads by topical administration - but only to the tissues in the front of the eye. Examples include:

[0411] • Bone-marrow-derived MSC-EVs, embedded in methylcellulose, were evaluated in a murine model of alkali-bum-induced comeal damage. The EVs were topically applied to the corneas and were shown to modulate cell death, inflammation, and angiogenetic programs in the injured tissue, thus leading to a faster recovery of comeal damage (Saccu et al.(2022) Bone marrow mesenchymal stromal / stem cell- derived extracellular vesicles promote corneal wound repair by regulating inflammation and angiogenesis, Cells 11(23):3892).

[0412] • Topical treatment on the corneal surface using nano-polymers or exosomes loaded with specific siRNA can effectively accelerate regular and diabetic comeal wound healing in vivo, with exosomes secreted by mesenchymal stem cells showing better efficacy than nano-polymers in treating corneal injury (see. Zhao et al. (2023) Accelerating corneal wound healing using exosome-mediated targeting ofNF- KB c-Rel. Inflammation and Regeneration, 43(1):6). b. Injected EVs for treatment of the back of the eye

[0413] Extracellular vesicles (exosomes) of mammalian origin, have been reported to deliver payloads to the tissues in the back of the eye but only by injection. Examples of such MSC-derived EV applications include:

[0414] • Exosomes isolated from bone marrow derived MSC have been shown to promote survival of retinal ganglion cells (RGC) and regeneration of their axons in a rat optic nerve cmsh model. Following weekly injections, intravitreally injected exosomes delivered cargo into inner retinal layers, demonstrating the use for treatment of traumatic and degenerative ocular disease (see, Mead et al, (2017) Bone marrow-derived mesenchymal stem cells-derived exosomes promote survival of retinal ganglion cells through miRNA-dependent mechanisms. Stem Cells Translational Medicine 6(4): 1273- 1285).

[0415] • Adipose mesenchymal stem cells-derived exosomes have been shown to attenuate retina degeneration of streptozotocin-induced diabetes mellitus in a rabbit model. MSCs were isolated from adipose tissue of rabbits, from which exosomes were extracted ultracentrifugation and injected intravenously, subconjunctivally, and intraocularly (Safwat et al. (2018) Adipose mesenchymal stem cells-derived exosomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits, Journal of Circulating Biomarkers 7:1849454418807827, doi: 10.1177 / 1849454418807827 eCollection 2018 Jan-Dec).

[0416] • EVs derived from MSCs have been used in a rat model of retinal ischemia, where the administration of MSC-EVs into the vitreous humor 24 h after induced ischemia significantly enhanced functional recovery, and decreased neuroinflammation and apoptosis. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia and they were present in the vitreous humor for four weeks after intravitreal administration (Mathew et al. (2019) Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion, Biomaterials 197 '.146-160 .

[0417] • MSC-derived exosomes have been also used in a retinal detachment (RD) model in which ischemia and oxygen deficiency in rat retina results in photoreceptor cell degeneration and vision decline. The exosomes were subretinally injected at the time of retinal separation; the treatment suppressed photoreceptor cell apoptosis and maintained normal retinal structure when compared to control groups (Ma et al. (2020) Therapeutic effects of mesenchymal stem cell-derived exosomes on retinal detachment. Experimental Eye Research 797:107899].

[0418] • Human EVs have been used clinically for treatment of subjects with large and refractory macular holes. EVs were isolated using sequential ultracentrifugation from MSC cultures derived from human umbilical cord tissue. Human subjects with large and long-standing idiopathic macular holes underwent vitrectomy, internal limiting membrane peeling, and intravitreal injection of EVs. Most macular holes were closed, showing the EV treatment from such stem cells can be useful for improving the visual outcomes after ocular surgery (Zhang et <z / .(2018) Effects of mesenchymal stem cells and their exosomes on the healing of large and refractory macular holes, Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologic 256(77):2041-2052).

[0419] Other types of mammalian EVs have been reported to deliver payloads to the tissues in the back of the eye by injection in the vitreous humor (intravitreal injection). RPE-derived exosomes have been tested in an animal model of retinal degeneration (RD). The experimental mice received a single subretinal injection of the exosomes and two weeks later the effects were evaluated with functional, morphological and behavior examinations. The treatment ameliorated visual function impairments and alleviated structural damages in the retina, as well as suppressed the expression levels of inflammatory factors and mitigated the oxidative damage. The findings indicate that the RPE-derived exosomes have therapeutic effects against the visual impairments and photoreceptor death (see, Wang et al. (2021) RPE-derived exosomes rescue the photoreceptors during retina degeneration: an intraocular approach to deliver exosomes into the subretinal space. Drug Delivery 28(1 ):218- 228).

[0420] There are no examples that show that any EVs reach the back of the eye following non-invasive administration. It has been understood that rapid clearance of EVs of mammalian origin occurs following topical application (see, Mansoor et al. (2019) Current trends and future perspective of mesenchymal stem cells and exosomes in corneal diseases. International Journal of Molecular Sciences 20(12f.285 >). c. Other Delivery Systems

[0421] Ongoing research explores innovative delivery systems (such as nanoparticles, liposomes, microparticles or exosomes) to encapsulate and transport biologies effectively to the posterior segment while overcoming ocular barriers. i) Lipid nanoparticles (LNPs)

[0422] LNPs have been reported to deliver pay loads to the tissues in the back of the eye by injection in the vitreous humor (intravitreal injection). A recent example includes LNP-based gene therapy approach that aimed to deliver reporter-encoding mRNA in vivo with subretinal and intravitreal injections. Cell-specific protein expression in various cell types of mouse retina was achieved, including the retinal pigment epithelium (RPE), Muller glia, the optic nerve head, and the trabecular meshwork, depending on route of administration [Ryals et al. (2020). The effects of PEGylation on LNP based mRNA delivery to the eye. PloS One, 15(10), e0241006]. ii) Viral vectors

[0423] Engineered viral vectors, particularly adeno-associated virus (AAV)-based vectors have bene used to deliver payloads to the tissues in the back of the eye by injection in the vitreous humor (intravitreal injection). The examples include, AAV vectors discussed above, and the following:

[0424] • ADVM-022 by Adverum Biotechnologies, an in-office intravitreal gene therapy to treat neovascular age-related macular degeneration (nAMD) and diabetic macular edema. ADVM-022 aims to treat nAMD through a single intravitreal injection utilizing the AAV.7m8 capsid to deliver a codon-optimized cDNA expressing an aflibercept-like protein (Grishanin et al. (2019) Preclinical evaluation of ADVM-022, a novel gene therapy approach to treating wet age-related, macular degeneration. Mol Ther. 27:118-29).

[0425] • RGX-314 (REGENXBIO Inc.) uses an AAV8 associated gene therapy for the treatment of nAMD. RGX-314 expresses a monoclonal antibody fragment similar to ranibizumab, a humanized monoclonal antibody fragment that binds to human VEGF-A to suppress choroidal neovascularization. RGX-314 uses subretinal or suprachoroidal delivery for stable anti- VEGF antibody production [Siddiqui et al. (2020) Gene therapy for neovascular AMD An update on ongoing clinical trials. Retinal Physician, 17:36-39].

[0426] • HMR59 by Hemera Biosciences is an AAV2 vector-based gene therapy expressing sCD59 administered intravitreally 7 days after a single intravitreal injection of anti- VEGF treatment. CD59 is found on the surface of RPE cells, and since an overactivation of the complement system is believed to be one of the pathological pathways leading to nAMD, HMR59 aims to upregulate CD59 expression on RPE to protect against the complement cascade suspected in causing macular neovascularization.

[0427] D. OCULAR BARRIERS LIMITING THE APPLICABILITY OF TOPICAL EYE TREATMENTS (DROP INSTILLATIONS)

[0428] Topical administration of eye drops is a non-invasive method for delivering medications for anterior segment conditions such as conjunctivitis or glaucoma. As discussed above, treatment of back of the eye pathologies is effected via injection. Ocular barriers, such as the cornea and blood-retinal barrier, and barriers described below, collectively limit the amount of drug that can reach the back of the eye following topical administration. While drug formulations and delivery systems are designed to enhance penetration and efficacy, eye drops for treating conditions - Ill - affecting the back of the eye have not been developed. Hence, other routes, such as intravitreal injections, are required for targeted delivery to the posterior segment of the eye.

[0429] Ocular barriers limit the effectiveness of topical of administration. These include the following.

[0430] 1. Corneal barrier: The cornea acts as the primary barrier for topical drugs. Its outermost layer, the epithelium, is designed to protect the eye and restrict the penetration of foreign substances, including drugs, to maintain optical clarity. Only a small fraction of the administered drug can penetrate the cornea due to its low permeability to larger molecules like biologies.

[0431] 2. Conjunctival and scleral barriers: The conjunctiva, a mucous membrane covering the eye's sclera, also presents a barrier to drug penetration. Both the conjunctiva and sclera have a relatively low permeability to larger molecules, limiting the transport of drugs to the posterior segment.

[0432] 3. Tear film and clearance: The tear film rapidly clears and dilutes topically applied medications, reducing their contact time with the ocular surface and limiting drug absorption. Frequent blinking and tear production wash away the drug before it can penetrate deeper into the eye.

[0433] 4. Blood-Retinal Barrier: Even if a small amount of drug manages to penetrate through the cornea and reach the anterior chamber, there's the challenge of crossing the blood-retinal barrier to reach the back of the eye. This barrier, formed by tight junctions between retinal capillary endothelial cells, restricts the passage of many drugs, especially larger molecules like biologies, from reaching the retina and choroid.

[0434] 5. Enzymatic degradation: Enzymes present in tears and ocular tissues can metabolize drugs, reducing their bioavailability and therapeutic efficacy. This is particularly relevant to labile molecules, including RNAs and proteins.

[0435] 6. Nasolacrimal drainage system: Systemic absorption via the tear (nasolacrimal) duct can limit the amount of drug available for the back of the eye. This drainage can lead to systemic side effects of topically administered ocular drug formulations. E. EXTRACELLULAR VESICLES

[0436] 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 able to cross barriers such as the plasma (or cytoplasmic) membrane and the blood / brain barrier, and provide for the horizontal transfer of their functional contents (z.e., proteins, lipids, RNA molecules, and circulating DNA) from a donor to a recipient cell (Kuruvinashetti et al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357). EVs also are naturally stable in various biological fluids, immunologically inert, and can exhibit organ- specific targeting abilities (Picciotto et al. (2021) Biomater. Sci., 9(8):2917-2930, doi:10.1039 / d0bm01696a).

[0437] EVs contain endogenous lipids, nucleic acids, and proteins. Although results differ due to variations in isolation techniques and methods of analyzing the data, EVs generally contain proteins associated with the plasma membrane, cytosol and those involved in lipid metabolism (see, e.g., Doyle and Wang (2019) Cells 8(7):727). Proteins involved in the biogenesis of EVs (e.g., components of the ESCRTs), EV formation and release (e.g., RAB27A, RABI IB, and ARF6), signal transduction, and antigen presentation, as well as tetraspanins, occur in EVs (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312, doi: 10.1007 / sl0571-016-0366-z). EVs are enriched for cholesterol, sphingomyelin, glyco sphingolipids, and phosphatidylserine (Kuruvinashetti et al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357). Although a small number of studies have identified some genomic and mitochondrial DNA in EVs, EVs primarily are enriched with endogenous small RNAs. Studies have identified mRNAs, miRNAs, rRNAs, long and short non-coding RNA, tRNA fragments, piwi-interacting RNA, vault RNA, and Y RNA in EVs. Most of the RNA that naturally occurs in EVs is -200 nucleotides long (with a small portion up to 4 kb) and thus it is fragmented, although circular RNAs also have been shown to be enriched and stable in EVs. RNA in EVs is protected from RNase digestion in the extracellular environment by the lipid bilayer (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312, doi: 10.1007 / sl0571-016-0366-z). The Exocarta, Vesiclepedia, and EVpedia databases are publicly available and provide data on the protein, nucleic acid, and lipid content of EVs (generally EVs from mammalian origin, such as human origin), as well as the isolation and purification procedures used, from EV studies (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312).

[0438] EVs are used by cells to mediate several physiological processes or affect various pathological conditions associated with the activation of an immune response or the spread of disease or infection, and also constitute cross-species communication and are in all kingdoms of life. Sources of EVs include mammalian cells, bacteria, bovine milk, and plants (Adamo et al. (2021) J. Extracell. Vesicles 10:el2081). Although plants and algae possess a cell wall outside their plasma membrane, which could be a physical barrier for the release of EVs, plants and algae release EVs (Picciotto et al. (2021) Biomater. Sci., 9(8):2917-2930, doi:10.1039 / d0bm01696a).

[0439] 1. Types of Extracellular Vesicles (EVs) a. Exosomes

[0440] There are three primary subtypes of EVs; they are classified based on their biogenesis, mode of release, size, content, and function: microvesicles (MVs), exosomes, and apoptotic bodies (Doyle and Wang (2019) Cells 8(7):727). Exosomes, or intraluminal vesicles (ILVs) generally are 30-150 nm in diameter and are released through multivesicular bodies (MVBs) in the endosomal pathway. In the endosomal pathway, early endosomes form by inward budding of the plasma membrane and can transform into late endosomes, which accumulate ILVs by inward budding of the endosomal membrane. Late endosomes which contain a number of small vesicles are called MVBs. MVBs either fuse with the lysosome and are degraded, or the plasma membrane which releases the ILVs as exosomes into the extracellular space. The endosomal sorting complexes required for transport (ESCRT) pathway regulates MVB transportation and exosome formation and is reported to be the primary driver of exosome biogenesis, although other mechanisms of exosome biogenesis exist, including those mediated by the sphingolipid ceramide, which can facilitate membrane invagination, or proteins in the tetraspanin family. The ESCRT accessory proteins Alix, TSG101, HSC70 and HSP90P are often referred to as exosomal marker proteins (Doyle and Wang (2019) Cells 8(7):727).

[0441] Exosomes are released into the extracellular space by the fusion of the MVB limiting membrane with the plasma membrane. A number of proteins are involved in the release of exosomes, including Rab GTPases, diacylglycerol kinase a, and SNARE proteins (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312).

[0442] Exosomes have been candidates for drug delivery systems: they have a long circulating half-life; exosomes are tolerated by the human body and can penetrate cell membranes and target specific cell types; and they can be loaded with genetic material, a protein, or a small molecule (Doyle and Wang (2019) Cells 8(7):727). b. Microvesicles

[0443] Microvesicles (MVs, or ectosomes) form by outward budding, or pinching, of the cell’s plasma membrane, and have a diameter of 100 nm to 1 pm. The formation of MVs involves cytoskeleton components, such as actin and microtubules, molecular motors such as kinesins and myosins, and fusion machinery such as SNAREs and tethering factors. The physiological state and microenvironment of the donor cell effects the number of MVs produced, and the physiological state and microenvironment of the recipient cell effects the number of MVs consumed. MVs also have a number of marker proteins, including cytosolic and plasma membrane associated proteins, as well as cytoskeletal proteins, heat shock proteins, integrins, and proteins containing post-translational modifications, although there are no known specific markers to distinguish MVs from exosomes. Like exosomes, MVs can be loaded with cargo (such as proteins, nucleic acids, and lipids) for delivery to another cell, thereby altering the recipient cell’s functions (Doyle and Wang (2019) Cells c. Apoptotic Bodies

[0444] Apoptotic bodies are released by dying cells into the extracellular space, and have a diameter from 50 nm to 5000 nm. Apoptotic bodies are formed when the cell’s plasma membrane separates from the cytoskeleton due to increased hydrostatic pressure after the cell contracts. Unlike exosomes and MVs, apoptotic bodies contain intact organelles, chromatin, and small amounts of glycosylated proteins (Doyle and Wang (2019) Cells 8(7):727).

[0445] 2. Uptake of EVs

[0446] Cells internalize EVs by fusion with the plasma membrane, or more commonly by endocytosis (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312). Uptake via endocytosis can be through several types of endocytotic processes, and different processes have been described in different cell types: clathrin- dependent endocytosis and phagocytosis have been described in neurons, macropinocytosis in microglia, phagocytosis and receptor-mediated endocytosis in dendritic cells, caveolin-mediated endocytosis in epithelial cells, and cholesterol- and lipid raft-dependent endocytosis in tumor cells. Blocking heparin sulfate proteoglycans (HSPGs) on the plasma membrane with heparin reduces the uptake of EVs in cell culture, as does blocking the scavenger receptor type B-l (SR-B1) with a synthetic nanoparticle mimic of HDL, which suggests a role for HSPGs and SR-B 1 in EV uptake (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312).

[0447] Fusion of EVs with the plasma membrane also is a method of uptake and requires low pH conditions; treatment of EVs with the combination of a pH- sensitive fusogenic peptide with cationic lipids resulted in increased cellular uptake of exosomes and the cytosolic release of cargo within the 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), so that EVs for delivering therapeutic payloads to tumor cells can enter cells through fusion with the plasma membrane.

[0448] Like cells, EVs have extracellular receptors and ligands on the outside and cytoplasmic proteins and nucleic acid on the inside, and thus communicate with cells in different ways. EVs bind to the cell surface, undergo endocytosis, and / or fuse with the plasma membrane, and release their cargos in the extracellular space. If entering by endocytosis, the EV cargo must escape the degradative pathway; late endosomes can fuse with lysosomes or the plasma membrane, so cargo must exit before it is degraded in a lysosome or re-released through the fusion of MVBs with the plasma membrane. EVs containing cargo, including mRNAs and non-coding RNAs, 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).

[0449] 3. General Methods for Isolating EVs a. Ultracentrifugation

[0450] Ultracentrifugation methods are used to isolate exosomes; alternative methods also have been developed. Due to the complex nature of the biological fluids from which exosomes are isolated, the overlap in physiochemical and biochemical properties between exosomes and other types of EVs, and the heterogeneity among exosomes, isolation methods can result in complex mixtures of EVs and other components of the extracellular space. Differential ultracentrifugation depends on the initial sedimentation of larger and denser particles from the extracellular matrix, and results in an enrichment of exosomes, but not a complete separation of exosomes from other components in 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 (typically made of sucrose or iodixanol) in the centrifuge tube. Density gradient centrifugation effectively separates EVs from protein aggregates and non-membranous particles but has low exosome recovery, although purity can be improved by coupling differential ultracentrifugation with types of density gradient centrifugation, such as rate-zonal centrifugation or isopycnic centrifugation (Doyle and Wang (2019) Cells 8(7):727). b. Size-Based Techniques

[0451] There are a number of size-based techniques for isolating exosomes (Doyle and Wang (2019) Cells 8(7):727). Ultrafiltration separates particles based on the size and molecular weight cut-off of the membrane, whereby particles larger than the molecular weight cut-off of the membrane are retained, and particles smaller than the molecular weight cut-off of the membrane are passed through into the filtrate; low isolation efficiency can occur however if the filter becomes clogged and vesicles become trapped. The ExoMir™ Kit (Bioo Scientific; Austin, TX) is a commercially available kit in which two membranes (200 nm and 20 nm) are placed into a syringe and a sample (typically pre-treated with centrifugation and proteinase K) is passed through the syringe; the larger vesicles remain above the first 200 nm filter, the smallest vesicles are passed through the syringe and discarded, and the vesicles between 20 and 200 nm remain between the two filters in the syringe. Sequential filtration also relies on a series of filtration steps to isolate exosomes (Doyle and Wang (2019) Cells 8(7):727).

[0452] Size Exclusion Chromatography (SEC), often used in parallel with ultracentrifugation methods (in which the exosome pellet obtained from ultracentrifugation is resuspended and further purified using SEC), of exosomes is similar to using SEC to separate proteins. In SEC, a column is packed with a porous stationary phase in which small particles can penetrate and thus elute after larger particles. Typically, SEC methods require several hours of run time; however, the qEV Exosome Isolation Kit (iZON Science, New Zealand) allows for rapid and precise exosome isolation by SEC within 15 minutes (Doyle and Wang (2019) Cells Flow Field-Flow Fractionation (FFFF), a sample injected into a chamber is subjected to parabolic flow as it is pushed down the chamber, in addition to a flow perpendicular to the parabolic flow, a crossflow, to separate particles in the sample. Larger particles are more affected by the crossflow and are pushed toward the walls of the chamber, which have a slower parabolic flow, and smaller particles remain in the center. Smaller particles elute earlier, and larger particles later, in FFFF (Doyle and Wang (2019) Cells 8(7):727).

[0453] In Hydrostatic Filtration Dialysis (HFD), hydrostatic pressure forces a sample through a dialysis tube with a membrane having a molecular weight cut off of 1000 kDa. The result is that small solutes are able to pass through the tube, but larger particles, including exosomes and EVs, remain in the tube and can then be further separated using, for example, ultracentrifugation (Doyle and Wang (2019) Cells c. Immunoaffinity Capture-Based Techniques

[0454] Immunoaffinity capture-based techniques can isolate exosomes based on expression of an antigen on the surface of the exosome, and allow for the isolation of exosomes derived from a particular source. In these methods, an antibody specific for a target antigen can be attached to a plate (e.g., in Enzyme-Linked Immunosorbent Assay, ELISA), magnetic beads (e.g., in magneto -immunoprecipitation), resins and microfluidic devices; these surfaces are then exposed to the exosome sample, resulting in the immobilization of the exosomes expressing the antigen. This assay requires that the protein / antigen for isolating the exosomes be expressed on the surface of the exosomes, and its specificity is limited by the specificity of the antibody that is used, often resulting in a lower yield but higher purity of isolated exosomes. These methods also can be used to separate exosomes within mixed populations of EVs. Immunoaffinity capture-based techniques often are used after ultracentrifugation or ultrafiltration (Doyle and Wang (2019) Cells 8(7):727). d. Exosome Precipitation Methods for precipitation of exomes include precipitation by polyethylene glycol (PEG) and lectin. In PEG precipitation, the PEG polymer ties-up the water molecules, allowing the other particles, including exosomes, to precipitate out of solution. PEG precipitation is quick and is not limited to the starting volume of solution, but lacks selectivity, as other EVs, extracellular proteins, and protein aggregates are precipitated with EVs. Sample pretreatment using filtration and / or ultracentrifugation can improve exosome yield. Commercially available kits for isolating exosomes using precipitation include, for example, ExoQuick® (System Biosciences, Palo Alto, CA) and the Invitrogen™ Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA). Alternatively, lectin precipitation can be used, typically after ultracentrifugation, whereby lectins bind to carbohydrates on the surface of exosomes, altering their solubility and leading to their precipitation out of solution (Doyle and Wang (2019) Cells 8(7):727). e. Microfluidic Based Isolation Techniques

[0455] Microfluidic based techniques isolate exosomes based on their physical and biochemical properties simultaneously, and are rapid, efficient, and require small starting volumes. In acoustic nanofilter, a matrix containing EVs and other cellular components is injected into a chamber and exposed to ultrasound waves. The particles respond differently to the radiation forces exerted by the waves, depending on their size and density; large particles experience stronger forces and migrate faster toward the pressure nodes. The immuno-based microfluidic isolation technique is similar to that of an ELISA, although, unlike ELISAs, it does not require prior ultrafiltration or ultracentrifugation of exosomes (Doyle and Wang (2019) Cells 8(7):727). The ExoChip (Kanwar et al. (2014) Lab Chip. 14(11): 1891-1900) and ExoSearch Chip (Zhao et al. (2016) Lab Chip. 16(3):489-496) have been developed to isolate exosomes using microfluidic technology.

[0456] 4. Microalgae and Microalgae-Derived Extracellular Vesicles (MEVs)

[0457] Taxonomy and classification of microalgae can vary. According to some schemes there are seven (7) divisions of microalgae: Euglenophyta (Euglenoids), Chrysophyta (Golden-brown algae and Diatoms), Pyrrophyta (Fire algae), Chlorophyta (Green algae), Rhodophyta (Red algae), Paeophyta (Brown algae), and Xanthophyta (Yellow-green algae). Of interest herein are photosynthetic microalgae, such as the species Chlorella and Chlamydomonas . The methods and uses described herein use MEVs generally from green algae. Exemplary of such algae are Chlamydomonas and Chlorella, which belong to the classes Chlorophyceae and Trebouxiophyceae, respectively.

[0458] Microalgae are bioresources for the production of EVs for use in nanomedicine and other fields. The mechanism of secretion of EVs from microalgae is known in relation to primary and motile cilia / flagella (Picciotto et al. (2021) Biomater. Sci., 9(8):2917-2930, doi:10.1039 / d0bm01696a). Chlamydomonas flagella are devoid of MVBs, thus, ciliary EVs shed from Chlamydomonas are classified as ectosomes. Studies have shown the shedding of ectosomes from flagellar and ciliary tips of the chiorophyte Chlamydomonas reinhardtii. EVs also have been observed along the length of the cilium in Chlamydomonas. Membrane budding and ciliary EV formation are mediated by components of the endosomal sorting complex required for transport (ESCRT), which are found in isolated ciliary transition zones, ciliary membranes, and ciliary EVs in Chlamydomonas and can act as sensors of membrane curvature. The formation of ciliary EVs also can occur when ciliary membrane trafficking is disrupted or during ciliary resorption (Wang and Barr (2018) Essays Biochem. 62(2):205-213). Ciliary ectosomes from Chlamydomonas contain a lytic enzyme that digests the mother cell wall and is required for the release of daughter cells. 7 iSS-null mutants that do not have flagella were unable to be released from the mother cell, and the addition of ciliary ectosomes from wild-type cells rescued the phenotype, suggesting a role for the flagella and intraflagellar transport (IFT) machinery in EV production (Wang and Barr (2016) Cell Mol. Neurobiol. 36(3):449- 457).

[0459] EVs have been extracted from algal cells using ultra-centrifugation (Kuruvinashetti et al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357). In accord with this method, algal cells are cultured; the cultured algal cells are collected and centrifuged; the supernatant is collected (and further centrifuged); a sucrose solution is added to the supernatant; and the algal supernatant with the sucrose solution is ultra-centrifuged; because of the sucrose solution, the high-density EVs settle at the bottom of the ultra-centrifugation tube and can be collected using a pipette. Extracted algal EVs can be characterized in size and concentration using Nanoparticle Tracking Analysis (NTA). Studies using this method have isolated green algal EVs that range in size from 25-200 nm, with a concentration of 0.89E8 to 0.94E8 particles / mL (Kuruvinashetti et al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357).

[0460] An ultra-centrifugation protocol also can be used to isolate EVs from marine microalgae grown under various conditions; NTA showed that the nano-particles have a size distribution between 100 and 200 nm, and western blotting of proteins confirmed the presence of EV markers (VES4US, Extracellular vesicles from a natural source for tailor-made nanomaterials, 2020). Subsequent studies have identified microalgal small EVs (sEVs) isolated from the marine photosynthetic microalgal chiorophyte Tetraselmis chuii. termed nanoalgosomes. The production of nanoalgosomes is an evolutionarily conserved trait within microalgal strains as similar results were obtained using sEVs isolated from batch cultures of two other microalgae species, the chiorophyte Dunaliella tertiolecta, and the dinoflagellate Amphidinium sp. The nanoalgosomes were isolated using differential centrifugation (dUC) and tangential flow filtration (TFF), as well as gradient ultracentrifugation, which was used to further purify samples enriched for small EVs by TFF or dUC. The isolated nanoalgosomes were shown to share characteristics of EVs from other sources. The EV yield (measured by sEV protein content and sEV number) from dUC and TFF was consistent with reported numbers of isolated EVs, around 109EV particles / pg EV proteins. 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 consistent size distributions, with the size that appeared the most frequently from DLS (DLS mode) around 70 nm. Compared to exosomes derived from mammalian cells, which have a density of 1.15-1.19 g / mol, nanoalgosomes had a slightly lower density of 1.13 g / mol. Electron microscopy revealed that the nanoalgosomes are spherical, heterogeneous in size and shape, and possess a lipid-bilayer structure. Compared to the microvesicles (or large EVs, lEVs) and lysates, the sEVs were enriched for three of the four target protein biomarkers (Alix, enolase, HSP70 and P-actin). DLS measurements indicated that the nanoalgosomes were resistant to changes in pH and stable in human blood plasma. The tumorigenic MDA-MB-231 breast cancer cell line, the non-tumorigenic 1-7 HB2 cell line, and the human hepatocarcinoma Hep G2 cell line did not show cytotoxic or genotoxic effects after nanoalgosome treatment. Furthermore, the nanoalgosome were taken up by the MDA-MB-231 and 1-7 HB2 cell lines (Adamo et al. (2021) J. Extracell. Vesicles 10:el2081).

[0461] EVs have been isolated from at least eighteen microalgae strains from the main microalgal lineages (Ankistrodesmus sp., Brachiomonas sp., Chlamydomonas reinhardtii, Dunaliella tertiolecta, Tetraselmis chuii, Chloromonas sp., Rhodella violacea, Kirchneriella sp., Pediastrum sp., Nannochloropsis sp., Cyanophora paradoxa, Cryptomonas pyrenoidifera, Phaeodactylum tricomutum, Phaeothamnion sp., Diacronema sp., Isochrysis galbana, Stauroneis sp., and Amphidinium sp.) and have been studied. Studied strains include strains with a variety of features such as saltwater and freshwater inhabitants, small and large sized cells, colonial and single cells, and species with sequenced genomes.

[0462] MEVs can be isolated using a differential ultracentrifugation protocol and characterized following the International Society for Extracellular Vesicles (IS EV) guidelines. All strains tested showed the presence of MEVs in the culture medium. EV-producing microalgae strains were established based on the EV protein content, the expression of EV protein markers (e.g., Alix, Hsp70, enolase, and P-actin), the total scatting signal (measured by dynamic light scattering, DLS) or total particle number (measured by NTA), and the sEV average size and size range. These EV- producing strains include Cyanophora paradoxa, Tetraselmis chuii, Amphidinium sp., Rhodella violacea, Diacronema sp., Dunaliella tertiolecta, Phaeodactylum tricomutum, Pediastrum sp., and Phaeothamnion sp. (Picciotto et al. (2021) Biomater. Sci., 9(8):2917-2930, doi:10.1039 / d0bm01696a). The data for Cyanophora paradoxa showed ~2xl09sEV particles per mL of microalgal-conditioned media, with strong positive signals for EV markers, and a size distribution with a mode of 130 + 5 nm, in agreement with data from plant-derived vesicles. Cytotoxicity and genotoxicity studies showed that sEVs isolated from Cyanophora paradoxa, a freshwater Glaucophyte, did not show toxicity on the tumorigenic MDA-MB-231 breast cancer or C2C12 myoblast cell lines, neither over time nor at different concentrations, nor did MDA-MB-231 cells treated with the sEVs show morphological nuclear changes associated with apoptotic events (Picciotto et al. (2021) Biomater. Sci., 9(8):2917-2930, doi:10.1039 / d0bm01696a).

[0463] EVs also have been isolated from Synechocystis sp. PCC6803 (a cyanobacterium), Chlamydomonas reinhardtii (a green microalgae), Euglena gracilis (an euglenophyte), and Haematococcus pluvialis (a chiorophyte) in work done by Zhao et al., who also performed RNomic and proteomic analyses in EVs isolated from C. reinhardtii at different stages of cell growth and under different types of abiotic stress (Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / vl). EVs were isolated using differential ultracentrifugation and filtration, and the resuspension was shown to contain membrane structures with small clumps of particles 110-120 nm in diameter, in line with the reported diameter of exosomes and small MVs, although there were differences in diameters between the species of microalgae. Specifically, EVs from C. reinhardtii had diameters between 37-710 nm, with an average particle diameter of 120.1 nm. Synechocystis-denved EVs had diameters between 24-450 nm, with an average particle size of 94.68 nm. Despite the presence of a cell wall, Chlamydomonas cells were able to uptake EVs, as shown by the presence of EVs labeled with a fluorescent lipophilic dye inside microalgal cells. Thus, microalgal EVs can be absorbed by recipient cells. Non-coding RNAs were detected in microalgal EVs at different growth stages and treatment (biotic stress, nitrogen depletion, and nitrogen recovery), and proteomic analyses identified many flagellar-associated membrane proteins in microalgal EVs (Zhao et al. (2020) doi:10.21203 / rs.3.rs- 38027 / vl).

[0464] These studies show that microalgae produce EVs that can be isolated using traditional or standard methods; microalgal-derived EVs are similar in size and concentration, and exhibit similar markers compared to EVs isolated from other species; EVs isolated from microalgae do not show cytotoxic or genotoxic effects in vitro', and that microalgal-derived EVs can be taken up by cells.

[0465] It has been shown that EVs from mammalian origin can deliver cargo to a target cell and thus have therapeutic use for delivery of a variety of cargos for use in treating a number of diseases or conditions; this has not been shown for in general for MEVs. Mammalian EVs, except for bovine milk EVs, however, cannot be administered orally because they do not survive the harsh conditions of the stomach. For example, small molecules such as hydrophobic and hydrophilic drugs can be injected into exosomes, or macromolecular proteins and nucleic acids can be embedded into the exosomes. The nucleic acids can include those encoding a gene of interest. Specific targeting ligands, imaging probes, and covalent linkage could be attached to the exosome surface and tracked using NTA, fluorescence, or by bioluminescence.

[0466] Besides a mention in a publication that microalgae EVs possibly can be used to deliver a drug of interest to a targeted cell, tissue, or organ (Kuruvinashetti el al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357), there is no published evidence nor technical descriptions for use of MEVs for delivery for treatment of mammalian disease, disorders, or conditions. There are no publications or technical descriptions describing how knowledge for application of EV technology to microalgae-derived extracellular vesicles, nor whether it is possible to do so, nor how to do so. Prior studies have not considered Chlorella species, nor have the prior studies assessed biodistribution and related properties of the MEVs in general. Hence methods, such as methods of oral delivery, exemplified herein with Chlorella, can employ MEVs from other microalgae.

[0467] As described and shown herein, however microalgal EVs have a number of advantages over the use of existing drug delivery systems, such as, exosomes derived from mesenchymal stem cells, gold nanoparticles, liposomes and other plant- and animal-derived EVs. Mesenchymal stem cells are a commonly used source of exosomes, and exosomes derived from mesenchymal stem cells are used in drug delivery, for example, anti-cancer vaccines, because they have enhanced passive targeting (a method of preparing a drug carrier system so that it remains circulating in the blood stream). Mesenchymal stem cell derived EVs possess the ability to passively target due to their small size, indigenous nature, and their ability to cross biological barriers. Mesenchymal stem cells, however, have limited secretion of exosomes, and scaling up production of exosomes is difficult due to the need to optimize purification, increase the homogeneity of exosomes, and establish efficient transfection strategies. Nanoparticles can lead to toxicity and current techniques for synthesizing nanoparticles limit their ability to scale for manufacturing purposes. Nanoparticle and liposome-based drug delivery methods also can lead to the formation of a teratoma (a tumor comprised of several different types of tissue). Liposome-based drug delivery methods have been further shown to be less efficient for internalization into a specific cell, tissue, or organ, compared to exosomes. Plant- derived EVs, such as those from curcumin, ginger, grapefruit, and lemon, have been used for drug delivery, but their extraction process and use in treatment has not yet been optimized. The production of EVs from agricultural products, such as fruits and milk, is economically impractical and need 3-4 months to grow, compared to algal EVs, which can be grown anywhere and within a few days. Algal EVs avoid phagocytosis or degradation by macrophages and circulate for prolonged times in vivo, and have low immunogenicity. Algal EVs also have a lower risk of teratoma formation. Algae, thus, provide a source from which pure, well-characterized EVs of high quality can be obtained (Kuruvinashetti et al. (2020) IEEE 20thInternational Conference on Nanotechnology 354-357). Kuruvinashetti et al. does not describe the use of Chlorella species as a source of EVs, nor its advantages as a source. Prior art does not describe the biodistribution of MEVs per se, nor the implications thereof for administration of MEVs with drugs directed to particular organs, tissues, or systems.

[0468] 5. Green algae - Chlorella species

[0469] Previous studies and consideration of EVs have not focused on nor assessed Chlorella species as sources of EVs. Chlorella and the resulting EVs have advantages for growth, manipulation, and administration of drugs that other species and EVs do not provide. Green algae belong to phylum Chlorophyta, and encompass a diverse group of photosynthetic eukaryotes. Green algae include unicellular and multicellular organisms. Algae originally included in the genus Chlorella are among the most widely distributed and frequently encountered algae in freshwater. These algae exist in aqueous environments and on land. They are typically small (~2 to 10 pm in diameter), unicellular, spherical in shape, non-motile, and contain a single chloroplast, and some have a rigid cell wall (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Molecular analyses have separated Chlorella species into two classes of chiorophytes: the Trebouxiophyceae, which contains the true Chlorella', and the Chlorophyceae. For use herein, Chlorella species include any that can be or that are used as food complement or that can be consumed by humans or other animals, such as livestock. Exemplary species include, but are not limited to, the species: Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis.

[0470] 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 Trebouxiophyceae include most of the known green algal endo symbionts, living in lichens, unicellular eukaryotes, plants, and animals (for example mussels and hydra). For example, Chlorella variabilis NC64A is a hereditary photosynthetic endosymbiont (or photobiont) of Paramecium bursaria, a unicellular protozoan, and NC64A also is a host for a family of large double- stranded DNA viruses that are occur in freshwater (Blanc et al. (2010) Plant Cell 22(9):2943-2955). a. Life Cycle

[0471] In unicellular organisms, such as microalgae, life cycle is the same as the cell cycle. Chlorella is a haploid organism that reproduces asexually by auto sporulation. The cell cycle and proliferation of Chlorella vulgaris has been investigated using flow cytometric analysis of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)- stained algal cells by Rioboo et al. (2009) Aquatic Toxicology, 94(3):229- 237, doi:10.1016 / j.aquatox.2009.07.009. Their results indicate that, as generally described for microalgae, the growth of C. vulgaris mother cells takes place during light periods, whereas cytoplasmic division and liberation of daughter cells takes place during dark periods. C. vulgaris also shows a distinct light / dark cycle, marked by an increase in cell size, cell complexity, and autofluorescence during periods of light, measured over a 96-hour period. A monoparametric histogram of CFSE-stained C. vulgaris cells showing only one peak of daughter cells indicates that each mother cell undergoes only one division cycle in 96 hours; the cytoplasmic division was further shown to take place during periods of darkness. Thus, the strain of C. vulgaris used exhibits three life cycle phases: 1) growth of mother cells, 2) cell division, and 3) liberation of daughter cells. C. vulgaris cells grew during 2 light periods and began to divide during following dark period; cell division occurs once the mother cells are double the size of daughter cells. Furthermore, C. vulgaris cells exposed to the herbicide terbutryn need a longer growth period in order to reach a large enough cell size to divide. This suggests there is a critical threshold size needed for C. vulgaris to complete the growth phase and begin the division phase, and that this critical threshold can control the progression of the G1 phase of the C. vulgaris cell cycle. Finally, this study demonstrates that the intensity of the peak of CFSE-fluorescence of mother cells is four times greater than that of the daughter cells, indicating that 4 daughter cells are produced from each mother cell. Thus, C. vulgaris cells undergo a first mitosis followed by cytoplasmic division, and then two other simultaneous mitoses, which result in the liberation of 4 daughter cells (see e.g., Rioboo et al. (2009) Aquatic Toxicology, 94(3):229-237, doi:10.1016 / j.aquatox.2009.07.009). b. Genomic Analyses of Chlorella Species

[0472] Although species of Chlorella are reported to be non-motile and lack a sexual cycle, genomic analyses of Chlorella variabilis NC64A (NC64A) and Chlorella vulgaris 211 / 1 IP (211 / 1 IP) reveal the presence of genes involved in sexual reproduction and motility (Blanc et al. (2010) Plant Cell 22(9):2943-2955; Cecchin et al. (2019) Plant J. 100(6): 1289-1305). The NC64A nuclear genome (GenBank Accession No. ADIC00000000.1) is 46.2 Mb, and composed of 12 chromosomes. The meiosis-specific proteins dosage suppressor of MCkl DMC1, homologous-pairing proteins HOP1 and HOP2, meiotic recombination protein MER3, meiotic nuclear division protein MND1, and mutS homolog protein MSH4 are encoded in NC64A; these genes also occur in most of the other sequenced chiorophyte algal species. Nineteen homologs of the Chlamydomonas gametolysin proteins, which promote disassembly of the gametic cells walls and allow gamete fusion, also were identified in NC64A. Additionally, an ortholog of the Chlamydomonas GCS1 protein, which is essential for cell fusion, occurs in NC64A (Blanc et al. (2010) Plant Cell 22(9):2943- 2955). The primary genes involved in meiosis also occur in the Chlorella vulgaris 211 / 1 IP 40 Mb genome (GenBank Accession No. SIDB00000000), in addition to the gene encoding gametolysin (g3347), and a gene encoding a protein that contains a domain with a putative GCS1 / HAP2 function (Cecchin et al. (2019) Plant J. 100(6): 1289-1305). Thus, although Chlorella species have been observed only in the haploid phase, the presence of meiosis genes indicates that the life cycle of Chlorella could include a diploid phase.

[0473] Similarly, while flagella have not been observed in NC64A, orthologs of the Chlamydomonas flagellar proteins were identified in the NC64A genome, including orthologs to the intraflagellar transport (IFT) proteins IFT52, IFT57, and IFT88, kinesin-2 motor protein FLA8, the kinesin-associated protein KAP, and proteins involved in the axonemal outer dynein arm (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0474] Sequencing of three Chlorella sorokiniana strains, strain 1228, UTEX 1230, and DOE 1412, reveals the presence of sex- and flagella-related genes (Hovde et al. (2018) Algal Research 35:449-461). The genome of several other Chlorella species has been sequenced: Chlorella protothecoides sp. 0710 (Gao et al. (2014) BMC Genomics 15(1):582; GenBank Accession No. APJOOOOOOOOO); Chlorella sorokiniana UTEX 1602 (GenBank Accession No. LHPG00000000) and Chlorella sp. strain SAG 241.80 (Micractinium conductrix', GenBank Accession No. LHPF00000000) (Arriola et al. (2018) Plant J. 93(3):566-586); and the Chlorella vulgaris strains UTEX 395 (Guarnieri et al. (2018) Front. Bioeng. Biotechnol. 6:37; GenBank Accession No. LDKB00000000), UMT-M1 (Teh et al. (2019) Data Brief 27:104680; GenBank Accession No. VJNP00000000), UTEX 259 (GenBank Accession No. VATW00000000) and NJ-7 (Wang et al. (2020) Mol. Biol. Evol. 37(3):849-863; GenBank Accession No. VATV00000000). c. Commercial and Biotechnological Uses of Chlorella

[0475] The commercial cultivation of microalgae for food purposes began with the production of Chlorella vulgaris in Japan and Taiwan in the 1960s. Dried biomass products from Arthrospira and Chlorella are included in dietary supplements due to reports of high protein content, nutritive value, and health benefits. For example, Chlorella extracts have been shown to lower cholesterol and have antioxidant, antibacterial, and antitumor activities. Production of high yields of Chlorella is routine, and, as detailed herein, MEVs can be isolated from the cell culture medium. For its use as a pharmaceutical, it is known that ingestion of Chlorella is non-toxic and non-immunogenic in humans.

[0476] Chlorella has been used in a variety of biotechnology applications, including biofuels, sequestering CO2, producing molecules of high economic value, or removing heavy metals from wastewaters (Blanc et al. (2010) Plant Cell 22(9):2943- 2955). Chlorella species show metabolic flexibility in response to environmental perturbations, and are capable of using nutrients, such as organic carbon and minerals, directly from wastewater for growth. Among microalgae, Chlorella species have higher photosynthetic efficiency over other photosynthetic organisms. Additionally, Chlorella vulgaris is able to grow either in autotrophic, heterotrophic or mixotrophic conditions (Zuniga et al. (2016) Plant Physiol. 172(l):589-602).

[0477] Chlorella species also can be genetically modified by Agrobacterium- mediated transformation. A study by Cha et al. developed a method to genetically transform Chlorella vulgaris using the Agrobacterium tumefaciens strain LBA4404, and the presence of gene fragments in 30% of the transgenic lines, compared to the wild-type non-infected Chlorella, indicates the T-DNA was integrated into the Chlorella genome (Cha et al. (2012) World J. Microbiol. Biotechnol. 28:1771-1779). d. Chlorella ME Vs

[0478] As described herein, Chlorella species, such as C. vulgaris, are advantageous species for the production of EVs, referred to herein as MEVs, for use for delivery of biomolecules and small molecules for many applications, including therapeutic, diagnostic, and cosmetic uses. Of particular interest herein are MEVs produced by Chlorella species. Chlorella EVs have not been exploited as sources of MEVs for exogenous loading of biomolecular products or small molecule drugs or diagnostic agents. Chlorella, as a source of EVs for such applications, provides numerous advantages. Chlorella is a haploid organism, which means that specific and targeted variants can be produced by genetic engineering; it readily can be genetically modified or loaded to produce or contain biologically active molecules and small molecules. Stable cell lines can be produced, including stable producers of encoded products. They are defined products, and, when exogenously loaded, the resulting compositions contain EVs that contain the same cargo.

[0479] Detailed genetic maps can be obtained, and correlations between genotype and phenotype can be established. Chlorella genomes have been fully sequenced, so the structure and function of various genes can be known. Phylogenetically, Chlorella is at the very crossroads between higher plants and microalgae. As such, Chlorella shares with higher plants a significant (and useful) number of molecular biological and metabolic features, but still is a unicellular haploid microalga. Exemplary of molecular biological features shared with eukaryotes is the intracellular machinery that involves the dicer enzyme 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 nutrients or factors of animal origin.

[0480] With respect to use of its EVs as therapeutics, Chlorella species are not toxic. For example, tablets made from Chlorella vulgaris biomass (i.e., compressed whole Chlorella cells) have been consumed regularly for years by the public worldwide as a dietary supplement, without constraints related to toxicity or immunogenicity. Japan is the world leader in the consumption of Chlorella biomass. It also is used, for example, in Japan, for medical treatments because it has shown to have immunomodulatory properties and purported anti-cancer activities, for use for antiaging applications, such as for cardiovascular diseases, hypertension and cataracts; it reduces the risk of atherosclerosis and stimulates the synthesis of collagen for the skin.

[0481] Chlorella cells naturally produce extracellular vesicles (EVs) that respond to the ‘standard specifications’ of better known EVs (such as mammalian EVs). EVs from plant origin bear a number of features that make them more promising / convenient than synthetic nanoparticles or semisynthetic EVs, for use as a drug delivery system in humans. These include, for example, higher stability, lower toxicity, and lower immunogenicity. Being as close as plants as it is, Chlorella provides a source of EVs with similar characteristics to plant EVs. At the same time, mass production of Chlorella in large scale is easier and cheaper than for higher plants. The glycosylation pattern of membrane proteins in Chlorella is similar / identical to the glycosylation pattern present in higher plants.

[0482] The size of the Chlorella MEVs ranges between about or between 50 nm and 200m, with an average size of about 130 nm. The morphology resembles plant and mammalian exosomes. For use for administration, the size distribution can be rendered more uniform by separating the MEVs by size and selecting those of a size of interest, which can vary depending upon the intended use and route of administration.

[0483] F. EXOGENOUSLY LOADED MICROALGAE EXTRACELLULAR VESICLES (MEVs), CARGO, AND TARGETS

[0484] Targets and cargo (see discussions below) include any known to those of skill in the art. Sections F and G and examples below describe the biodistribution of MEVs following administration by various routes, and the implications, uses and methods for targeting or treating particular diseases, disorders, and conditions, and for formulating and administering the MEVs.

[0485] 1. Isolation of MEVs

[0486] Methods for isolation are discussed in the sections above and detailed in the Examples.

[0487] 2. MEV Loading and Cargos

[0488] The MEVs can be loaded with any desired cargo (also referred to as a payload), including, but not limited to, nucleic acid molecules, including, for example RNAi, plasmids, anti-sense nucleic acids, nucleic acids encoding the RNAi or antisense nucleic acid, detectable marker proteins and tags, small molecule drugs, gene editing systems, and others, and combinations thereof. The MEVs can deliver therapeutic molecules, can serve as vaccines, and can be used in human and other animal health, agricultural applications, gene therapy applications, including delivery genes, modification of genes with gene editing systems, and gene silencing nucleic acids, cosmetic applications, dermatological applications, diagnostic applications, industrial uses, and others. The MEVs can deliver nutrients, or regulators of gene pathways to produce a beneficial product, and can be used to deliver gene editing systems, such as CRISPR / Cas and to effect gene editing. The MEVs can be used to deliver gene therapy vectors, such as, but not limited to, adeno-associated (AAV) virus vectors, adenovirus vectors, vaccinia virus-derived vectors, and others, and products.

[0489] Diseases and conditions that can be treated include any known to those of skill 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 disease, such as hepatitis, HIV, corona viruses, including SARS-Cov-2, CNS diseases, ocular diseases, and liver diseases. As discussed, delivered cargo includes protein products, such as antibodies and antigenbinding forms thereof, RNA products, such as, but not limited to, siRNA, miRNA (micro-RNA), IncRNA (long non-coding RNA), saRNA (small activating RNA), shRNA, and mRNA, nucleic acid encoding the products, such as plasmids, nucleic acid products such as DNA encoding anti-sense oligonucleotides and also the antisense oligonucleotides, and small molecule drugs.

[0490] The MEVs can carry cargos that include reporter genes and proteins and other detectable products, such as, for example, a fluorescent protein, such as, but not limited to an enhanced green fluorescent protein (EGFP; SEQ ID NO: 10), a luciferase gene (SEQ ID NO: 11), luxA (SEQ ID NO:8), luxB (SEQ ID NO:9), and the Lux operon (luxCDABE and luxABCDE; SEQ ID NO: 12).

[0491] Other cargos can target genes or products involved in diseases, such as, but not limited to, Peptidyl-prolyl cis-trans isomerase FKBP4 or FKBP52 (SEQ ID NO:1); gamma-aminobutyric acid type B receptor subunit 1 (GABBR1; SEQ ID NOG); oncogenes such as MYCN or NMYC (SEQ ID NO:38), RAS (H-RAS, N-RAS, and K-RAS see SEQ ID NOs:39, 40, and 41, respectively), BCL2 (SEQ ID NO:43), and PLK1 (SEQ ID NO:44). Genes involved in diseases, such as oncogenes, and checkpoints, can be modulated by cargo that encodes a product that inhibits or agonizes expression of a gene, or inhibits or agonizes a gene product. Exemplary of such modulators, are RNAi-type modulators, such as for example, siRNAs, miRNAs, shRNAs, anti-sense oligonucleotides (ASOs), peptides and / or tetratricopeptides. For example, siRNAs and ASOs targeting EGFP (SEQ ID NOs:5 and 6), firefly luciferase (SEQ ID NOG), MYCN (SEQ ID NOs: 13-19), RAS (SEQ ID NOs:20-27), BCL2 (SEQ ID NOs:29-31), and PLK1 (SEQ ID NOs:32-35), and microRNA-34A, which targets MYC and BCL2 (SEQ ID NO:28), are exemplified herein.

[0492] Gene silencing using RNA interference, including siRNAs and microRNAs, can be used to silence developmental genes, such as, for example, adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth / differentiation factors and their receptors, and neurotransmitters and their receptors; oncogenes; tumor suppressor genes; enzymes; genes associated with a pathological condition; genes associated with autoimmune diseases; anti-angiogenic genes; angiogenic genes; immunomodulator genes; genes associated with alcohol metabolism and liver function; genes associated with neurological disease; genes associated with tumorigenesis or cell transformation; and genes associated with metabolic diseases and disorders (see, e.g., WO 2009 / 082606, JP 2014-240428A, WO 2011 / 072292A2, WO 2010 / 141724, and WO 2020 / 097540). These types of products can be delivered in or encoded in MEVs to activate genes or pathways or to provide therapeutic effects. Certain cytokines can be used to treat diseases / disorders, such as certain cancers, in which immune suppression plays a role.

[0493] Extracellular vesicles and exosomes also can be used to transfer therapeutic agents such as nucleic acids, such as microRNA, mRNA, tRNA, rRNA, siRNA, regulatory RNA, non-coding and encoding RNA, DNA fragments, and DNA plasmids (see, e.g., CN105821081A and CN110699382A); nucleotides or amino acids comprising a detectable moiety or a toxin or that disrupts transcription or translation, respectively; polypeptides (e.g., enzymes); lipids; carbohydrates; and small molecules e.g., small molecule drugs and toxins) (see, U.S. Patent No. 10,195,290). Nonlimiting examples of proteins that can be encoded for by the nucleic acid cargo molecule include, but are not limited to: antibodies, intrabodies, single chain variable fragments, affibodies, enzymes, transporters, tumor suppressors, viral or bacterial inhibitors, cell component proteins, DNA and / or RNA binding proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transporters, nucleotide binding proteins, heat shock proteins, CRISPR- associated proteins, cytokines, cytokine receptors, caspases and any combination and / or derivatives thereof (see, e.g., AU2018365299).

[0494] For example, as summarized in the table below, a cocktail of three siRNA oligonucleotides targeting human MYCN with two thymidine residues (dTdT) at the 3'-end of the sequence (purchased from B-Bridge International Inc. (Sunnyvale, CA)) can be used. The anti-MYCN siRNA (siMYCN) and negative control siRNA (nontarget control pool) (siNeg) (both ON-TARGETplus siRNA, Dharmacon, Cambridge, UK) were used (see Ref 1). Exemplary target oncogenes and exemplary sequences of siRNA (see, also, SEQ ID NOs: 13-35, respectively) are provided in the table below.

[0495] 1 Nara et al. (2007) Int. J. Oncol. 30(5): 1189-1196; Silencing of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification

[0496] 2 Maeshima et al. (2020) Nucleic Acid Ther. 30(4):237-248; MYCN Silencing by RNAi Induces Neurogenesis and Suppresses Proliferation in Models of Neuroblastoma with Resistance to Retinoic Acid

[0497] 3 Veas-Perez de Tudela et al. (2010) J. Neurochem. 113(4):819-825; Human neuroblastoma cells with MYCN amplification are selectively resistant to oxidative stress by transcriptionally up-regulating glutamate cysteine ligase

[0498] 4 Watson et al. (1991) Cancer Res. 51(15):3996-4000; Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer

[0499] 5 Yoshikawa et al. (2019) Mol. Ther. Methods Clin. Dev. 13:290-302; Anti-cancer Effects of a Chemically Modified miR-143 on Bladder Cancer by Either Systemic or Intravesical Treatment

[0500] 6 Tsujino et al. (2019) Cancer Sci. 110(7):2189-2199; MicroRNA- 143 / Musashi- 2 / KRAS cascade contributes positively to carcinogenesis in human bladder cancer

[0501] 7 Tirella et al. (2019) Int. J. Pharm. 561:114-123; CD44 targeted delivery of siRNA by using HA-decorated nanotechnologies for KRAS silencing in cancer treatment 8 Nakada et al. (2001) Pancreatology 1(4):314-319; Antisense oligonucleotides specific to mutated K-ras genes inhibit invasiveness of human pancreatic cancer cell lines

[0502] 9 Adams et al. (2015) Expert Opin. Ther. Targets 20(6):737-753; The Tumor- Suppressive and Potential Therapeutic Functions of miR-34a in Epithelial Carcinomas

[0503] 10 Poeck et al. (2008) Nat. Med. 14(11): 1256- 1263; 5'-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma

[0504] 11 Szegedi et al. (2008) Pathol. Oncol. Res. 14(3):275-279; Bcl-2 Antisense Oligonucleotide Inhibits the Proliferation of Childhood Leukemia / lymphoma Cells of the B-cell Lineage

[0505] 12 Ripoil et al. (2018) RSC Adv. 8:20758-20763; Co-delivery of anti-PLK-1 siRNA and camptothecin by nanometric polydiacetylenic micelles results in a synergistic cell killing

[0506] 13 Liu et al. (2012) BMC Cancer 12(1):519-529; MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

[0507] 14 Spankuch et al. (2008) Neoplasia 10(3):223-234; Downregulation of Plkl expression by receptor- mediated uptake of antisense oligonucleotide-loaded nanoparticles

[0508] 3. Reporter genes, reporter proteins, and / or modulators thereof can be delivered in the ME Vs.

[0509] Reporter proteins

[0510] Target sequences, in the form of siRNAs, miRNAs, anti-sense oligonucleotides (ASOs), peptides and / or tetratricopeptides, to modulate (inhibition or stimulation) of each of the marker genes, such as a GEP protein, a eukaryotic luciferase, or a prokaryotic Luciferase, such as: Lux operon (luxCDABE) and lux operon (luxABCDE), can be used, for example for diagnostics and gene expression assessments (SEQ ID NOs:5-6, 7, and 62-65, respectively):

[0511] Cargo generally are agents that are for treating diseases, disorders, and conditions involving the eye or that can be treated by delivering agents into the eye, particularly the back of the eye. Other exemplary cargo can include chemotherapeutic agents, which include but are not limited to alkylating agents such as thiotepa and cyclophosphamide (available under the trademark CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (sold under the trademark Fareston®); anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiopho sphoramide and trimethylol melamine; folic acid replenisher such as folinic acid; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes, e.g., paclitaxel (such as paclitaxel sold under the trademark TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (such as Tomudex); additional chemotherapeutics including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defosfamide; demecolcine; diaziquone; difluoromethylomithine (DFMO); eflomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2 "-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine (such as vinorelbine tartrate sold under the trademark Navelbine®); Novantrone; teniposide; daunomycin; aminopterin; capecitabine (sold, for example, as Xeloda®); ibandronate; CPT-11; retinoic acid; esperamycins; capecitabine; and topoisomerase inhibitors such as irinotecan. Pharmaceutically acceptable salts, acids, or derivatives of any of the above also can be used.

[0512] Chemotherapeutic agents include prodrugs, which include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate- containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxy acetamide-containing prodrugs or optionally substituted phenylacetamide- containing prodrugs, 5 -fluorocytosine and other 5 -fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.

[0513] Other cargo includes, for example, anti- angiogenic agents. Anti- angiogenic agents can be a small molecule or protein, such as an antibody, Fc fusion, and cytokine, that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. Examples of anti- angiogenic agents include but are not limited to antibodies that bind to Vascular Endothelial Growth Factor (VEGF) or that bind to VEGF-R, RNA-based therapeutics that reduce levels of VEGF or VEGF-R expression, VEGF-toxin fusions, Regeneron's VEGF-trap, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, BeneFin, bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, Combretastatin A-4, endostatin (collagen XVIII fragment), famesyl transferase inhibitors, fibronectin fragment, GRO-beta, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon inducible protein 10 (IP- 10), interleukin- 12, kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (e.g., TIMPs), 2- methoxyestradiol, MMI 270 (CGS 27023A), plasminogen activator inhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDa fragment, proliferin -related protein (PRP), PTK 787 / ZK 222594, retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin- 1 (TSP-1), TNP470, transforming growth factor beta (TGF-P), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

[0514] Other cargo includes tyrosine kinase inhibitors, which include, but are not limited to quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3- d) pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino) phthalimide); tyrphostins containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tyrphostins (U.S. Pat. No. 5,804,396); PTK- 787 (Novartis / Schering A G); pan-ErbB inhibitors such as Cl-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (STI571, Gleevec®; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); Cl-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); INC-1 Cl l (ImClone); or as described in any of the following patent publications: U.S. Pat. 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 (Iressa®, ZD 1839, AstraZeneca), and OSL774 (Tarceva®, OSI Pharmaceutic als / Genentech) .

[0515] Other cargo includes immunomodulatory agents that increase or decrease production of one or more cytokines, up-or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells. Examples of immunomodulatory agents include but are not limited to non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketorolac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g., glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, triamcinolone, azulfidine eicosanoids such as prostaglandins, thromboxanes, and leukotrienes; as well as topical steroids such as anthralin, calcipotriene, clobetasol, and tazarotene); cytokines such as TGFp, IFNa, IFNP, IFNy, IE-2, IE-4, IL- 10; cytokine, chemokine, or receptor antagonists including antibodies, soluble receptors, and receptor-Fc fusions, B7, CCR2, CCR5, CD2, 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, eotaxin, Fas, ICAM, IFNa, IFNP, IFNy, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGFp, TNFa, TNFp, TNF-R1, T-cell receptor, including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (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, brequinar, Bromocryptine, cyclophosphamide, cyclosporine A, D- penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamides (e.g., leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasalazine.

[0516] Other cargo includes cytokines which include, but are not limited to lymphokines, monokines, and traditional polypeptide hormones. Included 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); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; Mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM- CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNE-alpha or TNE-beta; and other polypeptide factors including LIE and kit ligand (KL).

[0517] Other exemplary cargo includes cytokines and other agents that stimulate cells of the immune system and enhance desired effector function. Lor example, agents that stimulate NK cells include IL-2; agents that stimulate macrophages include but are not limited to C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine. Cargo includes agents that stimulate neutrophils, such as, for example, G-CSE and GM-CSE. Additional agents include, but are not limited to, interferon gamma, IL-3, and IL-7.

[0518] Cargo includes antibiotics, for treatment of infections, particularly for hard-to- treat bacterial infections, including urinary tract infection, respiratory infections, particularly Pseudomonas aeruginosa or Staphylococcus aureus infections in subjects with cystic fibrosis, and sinus infections, which can be treated by local administration, such as by inhalation of aerosols containing the MEVs. The antibiotic treatments for pulmonary infections in subjects with cystic fibrosis can be combined with gene therapy using the same or different MEVs that comprise nucleic acid, DNA or RNA, encoding the cystic fibrosis transmembrane conductance regulator (CETR) protein or providing a gene editing system to correct the defect in CETR protein.

[0519] Antibiotics that can be loaded as cargo in the MEVs include but are not limited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), aminocyclitols (e.g., spectinomycin), amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin), carbapenems (e.g., imipenem, meropenem, and panipenem); cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxime, cefixime, cephalexin, and cephradine), cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan); lincosamides e.g., clindamycin and lincomycin); macrolide (e.g., azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, and tobramycin), monobactams (e.g., aztreonam, carumonam, and tigemonam); mupirocin;

[0520] Oxacephems (e.g., flomoxef, latamoxef, and moxalactam); penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamecillin, penethamate hydriodide, penicillin o-benethamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penimepicycline, and phenethicillin potassium); polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin); quinolones (amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin); rifampin; streptogramins (e.g., quinupristin, and dalfopristin); sulfonamides (sulfanilamide, and sulfamethoxazole); and tetracyclines (chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, Duramycin®, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

[0521] Cargo also includes anti-fungal agents, which include, but are not limited to, amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, terconazole, and tioconazole. In some examples, cargo-loaded MEVs described herein are administered with one or more antiviral agents, including but not limited to protease inhibitors, reverse transcriptase inhibitors, and others, including type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, amprenavir, clevudine, enfuvirtide, entecavir, foscamet, ganciclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine.

[0522] In all instances, the form of the cargo includes proteins, and also, nucleic acid encoding the proteins, such as the plasmids, and also, mRNA. The nucleic acids can be operably linked to regulatory elements that are recognized in the particular subject, such as a mammal, in which they are to be delivered.

[0523] 4. Generation of Payload-Loaded MEVs

[0524] As shown herein, the isolated Chlorella can be loaded with cargo for delivery to humans by any suitable route, including but not limited to intravenous, oral, topical, mucosal, inhalation, and any other routes known to those of skill in the art for delivery of vehicles, such as lipid nanoparticles, vectors, therapeutic bacteria, and therapeutic viruses. Upon administration, the MEVs are taken up by cells. Any cargo presently delivered in vectors, bacteria, exosomes, nanoparticles, and other such delivery vehicles can be loaded into the MEVs provided herein. The loaded cargo can be selected so that it only is expressed or produced in targeted cells, such as in instances in which the cargo is a plasmid encoding a therapeutic product. Transcription regulatory signals can be selected so that the encoded product is expressed in targeted cells. For example, for expression in the liver, the encoded product can be expressed under control of a liver-specific promoter, or the product can be targeted to a receptor or target expressed in targeted cells, such as in tumors or in the tumor microenvironment. Loading methods, described above, and in the Examples below, include, but are not limited to: a. Electroporation b. Sonication c. Extrusion d. Surfactants e. Other Methods known to those of skill in the art for introducing exosomes into cells.

[0525] 5. Exemplary Cargo and Exemplary Uses of the Exogenously Loaded MEVs a. Cargo

[0526] As described above, the MEVs are loaded with cargo that can be used for any purpose of interest, including any for which other delivery vehicles are used. These uses include delivery of mRNA, such as mRNA encoding corona virus spike proteins and modified spike proteins to improve the immune response to the viruses, RNAi, such as siRNA, and anti-sense RNA, or anti-sense DNA (ASO), to silence genes, such as bacterial and viral pathogen virulence genes, antibiotic resistance genes, antimicrobial resistance genes, genes that suppress the immune system, tumor genes, such as oncogenes, and host factors for viral infection, such as targeting angiotensinconverting enzyme-2 (ACE2), transmembrane protein serine 2 (TMPRSS2), and other such genes. The cargo also can include any therapeutic antibodies. Therapeutic antibodies, include, but are not limited to, anti-cancer antibodies, antibodies to treat autoimmune or inflammatory disease, antibodies to treat transplant rejection, antibodies to treat graft-versus-host-disease (GVHD), and antibodies to treat infectious diseases.

[0527] 1) RNA Cargo

[0528] The mechanism of RNA interference or RNAi was originally described as a process of sequence- specific silencing of gene expression in the nematode Caenorhabditis elegans (Fire et al. (1998) Nature 391(6669):806-l 1 ; Fire and Mello, 2006 Nobel Prize in Medicine awarded to Andrew Fire and Craig Mello). The process of small RNAs targeting (and silencing) messenger RNAs involves a particular RNAi machinery (including silencing factors, such as DICER and ARGONAUTE).

[0529] In the plant kingdom, RNAi is involved in antiviral defense mechanisms, and in defense mechanisms against phytopathogenic fungi and oomycetes. Small regulatory RNAs can be active in silencing genes inside bacterial cells, which lack the said RNAi machinery. The silencing activity of siRNA has been demonstrated to be inter-kingdom (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).

[0530] RNAi-mediated regulation of gene expression has been exploited for several years in the field of biotechnology to confer resistance to viruses (Baulcombe (2015) Current Opinion in Plant Biology 26:141-146). The inter-kingdom RNAi has been used to characterize the function of genes of eukaryotic pathogens and / or eukaryotic parasites as well as to induce protection against these organisms.

[0531] In Drosophila and Caenorhabdilis. RNAi plays a crucial role in antiviral defense by directly targeting viral RNAs via the small RNAs produced by the host in response to viruses. Recent work has shown that plant EVs naturally loaded (loaded by the plant cells producing the EVs) with small RNAs, from human edible plants, can modify the composition of the human gut microbiota and oral microbiota by silencing the expression of specific genes in certain commensal bacteria (Teng et al. (2018) Cell Host & Microbes 24:637-652; Sundaram et al. (2019) iScience 21:308- 327).

[0532] Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA- based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The siRNAs and miRNAs have a role in gene regulation, and serve as targets for drug discovery and development. Compared with conventional small therapeutic molecules, siRNAs and miRNAs offer the potential to be highly potent and able to act on “non-druggable” targets (for example, proteins which lack an enzymatic function); RNAi can be designed to target and / or affect expression of any gene of interest.

[0533] 2) Antibody Cargo

[0534] Examples of anti-cancer antibodies and other antibodies, include, but are not limited to, anti-17-IA cell surface antigen antibodies such as the antibody sold or provided under the trademark Panorex® (edrecolomab); anti-4-lBB antibodies; anti- 4Dc antibodies; anti-A33 antibodies such as A33 and CDP-833; anti-al integrin antibodies such as natalizumab; anti-a4p7 integrin antibodies such as LDP-02; anti- aVpi integrin antibodies such as F-200, M-200, and SJ-749; anti-aVp3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, and Vitaxin®; anti-complement factor 5 (C5) antibodies such as 5G1.1; anti-CA125 antibodies such as sold or provided under the trademark OvaRex® (oregovomab); anti-CD3 antibodies such as those sold or provided under the trademark Nuvion® (visilizumab) and Rexomab™; anti-CD4 antibodies such as IDEC-151, MDX-CD4, 0KT4A; 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.vl6, 2H7.vl 14, 2H7.vl 15, the product sold or provided under the trademark Bexxar® (tositumomab), the antibody sold or provided under the trademark Rituxan® (rituximab), and the antibody sold or provided under the trademark Zevalin® (Ibritumomab tiuxetan); anti-CD22 antibodies such as the those sold or provided under the following generic names, tradenames, or tra...

Claims

What is claimed is:

1. A microalgae extracellular vesicle (MEV), comprising heterologous cargo, wherein: the heterologous cargo comprises a therapeutic or comprises nucleic acid encoding a therapeutic for treatment of a disease, disorder, or condition of the eye; and the disease, disorder, or condition involves one or more of choroidal cells, retinal pigment epithelium (RPE) cells, the retina, and photoreceptor cells.

2. The MEV of claim 1, wherein the disease, disorder, or condition is one or more of an age-related macular degeneration (AMD), a retinal degenerative disease, disorder, or condition, or a disease, disorder, or condition involving choroidal neovascularization, an inheritable disease, disorder, or condition involving the retina, and an ocular hypertension.

3. The MEV of claim 1 or claim 2, wherein the disease, disorder, or condition is selected from one or more of an age-related macular degeneration (AMD), diabetic retinopathy, retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion (RVO), ocular hypertension, and optic neuropathy glaucoma disease.

4. The MEV of claim 1 or claim 2, wherein the disease, disorder, or condition is wet AMD, geographic atrophy, diabetic retinopathy, macular edema, retinitis pigmentosa, or glaucoma.

5. The method, MEV, or composition of any of claims 1-4, wherein the disease, disorder, or condition can be treated by delivery of a therapeutic to the ciliary body and / or to the choroid.

6. The MEV of any of claims 1-5, wherein therapeutic is for treatment of a disease, disorder, or condition that can be treated by delivery of the MEV or therapeutic to the ciliary body and / or to the choroid.

7. The MEV of any of claims 1-6, wherein the cargo is a therapeutic that inhibits a VEGFR or inhibits VEGF.

8. An MEV of any of claims 1-6, wherein the cargo comprises a VEGF inhibitor, or VEGFR inhibitor, or an inhibitor of a complement pathway protein.

9. The MEV of claim 8, wherein the cargo inhibits complement protein 3 (C3) or complement protein 5 (C5).

10. The MEV of any of claims 1-9, wherein the cargo comprises the therapeutic or the cargo comprises nucleic acid encoding the therapeutic protein or the cargo is RNA.

11. The MEV of any of claims 1-10, wherein the cargo comprises a detectable product or a reporter gene.

12. The MEV of claim 11, wherein the cargo is a fluorescent protein or nucleic acid encoding the fluorescent protein.

13. The MEV of claim 12, wherein the fluorescent protein or encoding nucleic acid is selected from among the enhanced green fluorescent protein (EGFP) of SEQ ID NO:63 or a variant thereof that is fluorescent and has at least 95% sequence identity to SEQ ID NO:63; mRNA encoding EGFP of SEQ ID NO:64 or a fluorescent variant thereof having at least 95% sequence identity to the protein encoded by SEQ ID NO:64, a luciferase or encoding nucleic acid of SEQ ID NO:65 or an enzymatically active variant thereof having at least 95% sequence identity to SEQ ID NO:65.

14. The MEV of any of claims 1-13, wherein the cargo comprises siRNA targeting HIF-la (SEQ ID NOs:66 and 67), siRNA targeting VEGF (SEQ ID NOs:68 and 69), FKBPL (FK506 Binding Protein-Like) peptide (SEQ ID NO:72), aflibercept (a.k.a. VEGF Trap-Eye) protein (SEQ ID NO:73) or its encoding mRNA (SEQ ID NO:74 or 75), Norrin protein (SEQ ID NO:76), siRNA targeting SRPK1 (SEQ ID NOs:77, 78, and 79), pBCB-23-0041-CDS-EGFP protein (SEQ ID NO:80), pBCB- 23-0070-CDS-aflibercept-l(SEQ ID NO: 81), pBCB-23-0065-CDS-Aflibercept-2 (SEQ ID NO:82), or variants thereof having at least 95% sequence identity thereto or to the encoded protein.

15. The MEV of any of claims 1-14, wherein the cargo is selected from the following table for treatment of the disease, disorder, or condition as follows:

16. An MEV of any of claims 1-15, comprising cargo selected from among a prostaglandin (latanoprost, travoprost, bimatoprost), a beta-blocker (timolol, carteolol, levobunolol, betaxolol, nebivolol), a parasympathomimetic (pilocarpine), alpha2-agonists (apraclonidine, brimonidine), a carbonic anhydrase inhibitor (CAI) (brinzolamide, dorzolamide), and an anti-oxidant (nordihydroguaiaretic acid, mesonordihydroguaiaretic (masoprocol) ) .

17. An MEV of any of claims 1-15, comprising cargo selected from among Voretigene neparvovec-rzyl, EA-2353, OCU400, AGTC-501, 4D-125, CNTO 2476, SAR421869, CEP290, GUCY2D, AAV2-repl, SAR422459, Emixustat HC1, STG- 001, ATSN-201, rAAV2tYF-CB-hRSl, CNGB3.

18. An MEV of any of claims 1-17, comprising cargo selected from among Bevacizumab (Avastin®), nibizumab, aflibercept (Eyela®), ranibizumab, Avacincaptad pegol, and Pegcetacoplan.

19. An MEV of any of claims 1-18, comprising nucleic acid or nucleic acid encoding a polypeptide or a polypeptide of any of SEQ IDS NOs: 73, 74, and 83- 95 or comprising a plasmid of any of SEQ ID NOs:80-82.

20. The MEV of any of claims 1-19, wherein the disease, disorder, or condition involves the choroid-retina region.

21. The MEV of any of claims 1-20, wherein: a) the cargo comprises DNA or RNA that encodes a therapeutic product or double-stranded RNA; or b) the cargo comprises mRNA or modified mRNA; or c) the cargo comprises a gene editing system; or d) the cargo comprises a plasmid; or e) the cargo comprises a viral vector; or f) the cargo comprises a therapeutic or diagnostic protein or polypeptide; or g) the cargo comprises a protein complex; or h) the cargo comprises a vaccine; or i) the cargo comprises or encodes a protein that is an antibody or antigenbinding fragment thereof; or j) the cargo is a product that stimulates the immune system of a subject treated with the composition; or k) the cargo is a cosmeceutical or a cosmetic or cosmetically active product; or l) the cargo comprises a small molecule bioactive molecule; or m) the cargo is a chemotherapeutic agent; or n) the cargo is an immunotherapeutic agent; or o) the cargo comprises a diagnostic marker or detectable product; or p) the cargo comprises a prodrug; or q) the cargo comprises an antibody, or an scFv or another antigen-binding fragment of an antibody, or a bi-specific antibody; or r) the cargo comprises a peptide or a polypeptide; or s) the cargo comprises a siRNA, or a miRNA, or another active small RNA; or t) the cargo comprises a IncRNA.

22. The MEV of any of claims 1-21, wherein the cargo comprises a diagnostic reporter marker that comprises a luciferase or nucleic acid encoding aluciferase, a fluorescent protein or nucleic acid encoding a fluorescent protein, or a luciferase operon; and the reporter detects a marker indicative of a disease, disorder, or condition of the eye.

23. The MEV of claim 22, wherein the marker is a cell surface marker or an enzyme.

24. The MEV of any of claims 1-23, wherein the cargo comprises siRNA or other inhibitory RNA targeting HIF-la (SEQ ID NOs:66 and 67), siRNA targeting VEGF (SEQ ID NOs:68 and 69), FKBPL (FK506 Binding Protein-Like) peptide (SEQ ID NO:72), Aflibercept (a.k.a. VEGF Trap-Eye) protein (SEQ ID NO:73) or its encoding mRNA (e.g., SEQ ID NO:74 or 75), Norrin protein (SEQ ID NO:76), siRNA targeting SRPK1 (SEQ ID NOs:77, 78 and 79), 1.99.25 antibody, and / or the F4L5.13 antibody, or plasmid encoding Aflibercept (SEQ ID NO: 81 or 82), or plasmid encoding eGFP (SEQ ID NO:82).

25. The MEV of any of claims 1-24, wherein: the cargo is endogenously (endo-loaded) by genetically modified microalgae; and the cargo comprises nucleic acid or a polypeptide.

26. The MEV of any of claims 1-24, wherein the cargo is exogenously- loaded (exo-loaded).

27. The MEV of any of claims 1-26, wherein the microalgae is the microalgae is a species of the family Chlorellaceae.

28. The MEV of any of claims 1-26 wherein the microalgae is the microalgae is a species of the genus Chlor ella or Parachlorella.

29. The MEV of claim 28, wherein the genus is Chlor ella and is selected from among Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis.

30. The MEV of claim 29, wherein the Chlorella is Chlorella vulgaris.

31. The MEV of claim 28 or claim 29, wherein the microalgae is a species of Parachlorella selected from among Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hussii.

32. A composition, comprising the MEV of any of claims 1-31, wherein the composition is formulated for administration to the eye.

33. The composition of any of claims 1-32 that is a pharmaceutical composition comprising a pharmaceutically acceptable vehicle for administration to the eye.

34. A composition, comprising the MEV of any of claims 1-33, wherein the composition is formulated for administration to the by eye drop instillation on the eye surface, or intraocular injection, intravitreal injection, supra-choroidal injection, or subretinal injection.

35. The composition of claim 33 or claim 34, wherein the composition is formulated for intraocular injection, intravitreal injection, supra-choroidal injection, or subretinal injection.

36. The composition of claim 33 or claim 34, wherein the composition is formulated as eyedrops.

37. The composition of any of claims 32-36, comprising phosphate buffered saline (PBS).

38. The composition of any of claims 32-37, wherein the composition is formulated as a suspension or as an emulsion.

39. The composition of claim 38, wherein the composition is an emulsion that is a microemulsion or is a nanoemulsion.

40. The MEV or composition of any of claims 1-39 for use for treating a disease, disorder, or condition of the eye.

41. The MEV or composition of claim 40, wherein the disease, disorder, or condition is a disease, disorder, or condition of the back of the eye.

42. A method of treatment of a disease, disorder, or condition of the eye, comprising administering an MEV or composition of any of claims 1-39.

43. The method of claim 42, wherein the MEV or composition is administered topically to the eye or is injected by subretinal or intravitreal injection.

44. The method of claim 42 or claim 43, wherein the disease, disorder, or condition is a disease, disorder, or condition of the back of the eye.

45. The method, MEV, or composition of any of claims 1-44, wherein: the disease, disorder, or condition is a disease, disorder, or condition of the choroid-retina region; and the MEVs are for topical administration or are administered topically.

46. The method, MEV, or composition of any of claims 1-45, wherein the MEVs traffic to the ciliary body and then to the choroid.

47. The method, MEV, or composition of any of claims 1-46, wherein the disease, disorder, or condition of the eye or involving the eye is selected from among Adie’s pupil, adult strabismus, age-related macular degeneration (AMD), albinism, amblyopia, anisocoria, aphakia, arcus senilis, astigmatism, bacterial keratitis, Bell’s palsy, black eye, blepharitis, blocked tear duct, branch retinal vein occlusion (BRVO), carotid artery disease, cancer of the eye, cataracts, cellulitis, central retinal vein occlusion (CRVO), central serous chorioretinopathy, chalazia and stye, Charles Bonnet syndrome, choroidal neovascular membranes, chronic angle-closure glaucoma, coloboma, color blindness, conjunctivitis (pink eye), contact lens-related eye infections, convergence insufficiency, corneal abrasion, corneal dystrophies, corneal erosion, corneal laceration, corneal ulcer (keratitis), crossed eyes (strabismus), cytomegalovirus retinitis, dementia and the eye, detached retina, diabetes, drusen, dry eye, ectropion, endophthalmitis, entropion, excessive blinking in children, eye allergies, eye lymphoma, eyelid spasm and twitching, farsightedness, Fuchs’ dystrophy, fungal keratitis, giant cell arteritis, giant papillary conjunctivitis, glaucoma, headache and eye problems, hemangioma, herpes keratitis, herpes zoster (shingles), heterochromia, histoplasmosis, HIV / AIDS and the eye, hyperopia, hyphemia, idiopathic intracranial hypertension, iridocorneal endothelial syndrome, ischemic optic neuropathy, juvenile idiopathic arthritis uveitis, juvenile macular dystrophy, keratoconus, lattice degeneration, Leber congenital amaurosis, low vision, macular edema, macular hole, macular pucker, macular telangiectasia, Marfan syndrome, microvascular cranial nerve palsy, migraine, milia, myasthenia gravis, myopia (nearsightedness), neuropathic corneal pain, nevus, nystagmus, ocular hypertension, ocular melanoma, ocular rosacea, onchocerciasis (African River Blindness), optic neuritis, orbital fracture, photokeratitis, pigment dispersion syndrome, pinguecula and pterygium, posterior vitreous detachment, presbyopia, pseudoexfoliation syndrome, ptosis, retinal artery occlusion, retinal detachment, retinal vein occlusion, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, scleritis, Sjogren’s syndrome, sleep crust, Stargardt disease, Stickler syndrome, subconjunctival hemorrhage, thyroid eye disease (Grave’s disease), tomretina, toxoplasmosis, trachoma, trichiasis, trichotillomania, Usher syndrome, uveitis, vitamin A deficiency, vitreomacular traction, and xanthelasma.

48. The method, MEV, or composition of any of claims 1-47, wherein the disease, disorder, or condition is AMD or a diabetic retinopathy or a disease, disorder, or condition involving the photoreceptors.

49. The method, MEV, or composition of any of claims 1-48, wherein the disease, disorder, or condition is treated with an inhibitor of a VEGF receptor or an inhibitor of VEGF.

50. The method, MEV, or composition of any of claims 1-49, wherein the disease, disorder, or condition is AMD.

51. The method, MEV, or composition of claim 50, wherein the disease, disorder, or condition is wet AMD.

52. The method, MEV, or composition of any of claims 1-51, wherein the disease, disorder, or condition can be treated by a therapeutic delivered to a cell or tissue in the eye that can accessed by delivery of the MEV or therapeutic to the ciliary body and / or to the choroid.