Platforms, compositions, and methods for therapeutics delivery

Abstract

SOLUTION: Described herein are platforms for generating extracellular vesicles. Described herein are compositions of extracellular vesicles. Also described herein are methods of using the extracellular vesicles for therapeutics delivery.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 864,566, filed June 21, 2019, and U.S. Provisional Patent Application No. 62 / 875,001, filed July 17, 2019, the entireties of which are incorporated herein by reference.

[0002] Citation by reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. This application cites "Riazifar M, Mohammadi MR, Pone EJ, et al. Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders. ACS Nano. 2019;13(6):6670-6688. doi:10.1021 / acsnano.9b01004," the entire contents of which are incorporated by reference herein. [Background technology]

[0003] Effective means of delivering therapeutic agents to target cells are one of the cornerstones of modern medicine, and extracellular vesicles have been explored and utilized as carriers for delivering therapeutic agents to target cells. Summary of the Invention

[0004] Current methods for delivering therapeutic agents via extracellular vesicles have drawbacks. Extracellular vesicles may be heterogeneous in terms of size, number, membrane properties, or stability. The amount of therapeutic agent deliverable by extracellular vesicles may be uneven or unpredictable, resulting in ineffective treatment. In some instances, the yield of extracellular vesicles may be inadequate. In addition, targeting extracellular vesicles to target cells in vivo remains difficult, as the majority of circulating extracellular vesicles accumulate in the liver, spleen, and kidneys. Therefore, there remains a need for compositions and pharmaceutical compositions containing extracellular vesicles for delivering sufficient amounts of therapeutic agents to target cells. There also remains a need for platforms and methods for producing extracellular vesicles for delivering sufficient amounts of therapeutic agents to target cells. There also remains a need for methods of using extracellular vesicles to deliver therapeutic agents and to treat diseases or conditions.

[0005] Aspects of the present disclosure include compositions comprising extracellular vesicles, the extracellular vesicles comprising an immune checkpoint moiety comprising VISTA, PD-L1, CTLA-4, or any combination thereof, and a transmembrane moiety comprising CD63, the CD63 comprising three transmembrane domains, wherein the immune checkpoint moiety is attached to the transmembrane moiety. In some embodiments, the immune checkpoint moiety is attached to an extracellular loop of the CD63 to generate a modified CD63. In some embodiments, the extracellular loop is the large extracellular loop or the second extracellular loop of the modified CD63. In some embodiments, the modified CD63 comprises an amino acid sequence at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 6-17. In some embodiments, the modified CD63 comprises an amino acid sequence at least 95% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 6-17. In some embodiments, the modified CD63 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 6-17. A composition comprising an extracellular vesicle, the extracellular vesicle comprising an immune checkpoint moiety comprising PD-L1 and a transmembrane moiety comprising lactadherin, wherein the immune checkpoint moiety is conjugated to the transmembrane moiety. A composition comprising an extracellular vesicle, the extracellular vesicle comprising an immune checkpoint moiety comprising a V domain Ig suppressor of T cell activation (VISTA), PD-L1, CTLA-4, or any combination thereof, and a transmembrane moiety comprising glycosylphosphatidylinositol (GPI), wherein the immune checkpoint moiety is conjugated to the transmembrane moiety. A composition comprising an extracellular vesicle, the extracellular vesicle comprising at least one of an immune checkpoint moiety and a transmembrane moiety. In some embodiments, the immune checkpoint moiety is encapsulated in the extracellular vesicle. In some embodiments, the immune checkpoint moiety is expressed on the surface of the extracellular vesicle. In some embodiments, the immune checkpoint moiety is secreted by the extracellular vesicle. In some embodiments, the immune checkpoint moiety is complexed with the transmembrane moiety. In some embodiments, the immune checkpoint moiety isIn some embodiments, the immune checkpoint moiety is covalently linked to a transmembrane moiety. In some embodiments, the immune checkpoint moiety is selected from the group consisting of VISTA, PD-L1, CTLA-4, PD-L2, B7-(CD80), B7-2(CD86), B7-H3(CD276), B7-H2, B7-H4(VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin 3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47 , stabilin-1 (Clever-1), neuropilin 1, neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRPα (CD172a), or a combination thereof. In some embodiments, the immune checkpoint moiety comprises VISTA, PD-L1, CTLA-4, or a combination thereof. In some embodiments, the immune checkpoint moiety comprises PD-L1. In some embodiments, the transmembrane moiety is selected from the group consisting of 14-3-3 protein zeta / delta, 4-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase / AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAM10, alkaline phosphatase, alpha-enolase, alpha-synuclein, aminopeptidase N, amyloid beta A4 / APP, Annexin 5A, Annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, β-actin (ACTB), β-amyloid 42, caveolin 1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24, CD26 / DPP4, CD29 / ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudin, claudin 1, cofilin-1,Complement fixing proteins CD55 and CD59, cytosolic heat shock protein 90α, cytosolic heat shock protein 90β, EBV LMP1, EBV LMP2A, EF-1α-1, EF2, EFGR EGFR VIII, emmprin / CD147, enolase 1α (ENO1), EPCAM, ERBB2, tetraspanins (CD9, CD63, and CD81), fatty acid synthase, fetuin A, flotillin-1, flotillin-2, fructose bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5' nucleotidase, GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 and HSP90), heparan sulfate proteoglycans, heparinase, heterotrimeric G protein, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax, huntingtin, ICAM1, integrin, lactadherin, LAMP1 / 2, leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosomal-associated membrane glycoprotein 1, lysosomal-associated membrane glycoprotein 2, MHC class I, MHC class II, MUC1, multidrug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP / Alix, PECAM1, phosphoglycerate kinase, placental prion protein, prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap 1B, resistin, sonic hedgehog (SHH), surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein, TYRP-2, vacuolar sorting protein 35, or zeta polypeptide (YWHAZ). In some embodiments, the transmembrane portion comprises lactadherin. In some embodiments, the transmembrane portion comprises LAMP2, or a variant or fragment thereof,The LAMP2 is at least 70% identical to the peptide sequence of SEQ ID NO: 4. In some embodiments, the transmembrane portion comprises CD63, or a variant or fragment thereof, wherein the CD63 is at least 70% identical to the peptide sequence of SEQ ID NO: 5. In some embodiments, the CD63 is a modified CD63. In some embodiments, the modified CD63 is a truncated CD63. In some embodiments, the modified CD63 is modified to include at least one additional CD63 transmembrane domain. In some embodiments, the modified CD63 includes one transmembrane domain. In some embodiments, the modified CD63 includes two transmembrane domains. In some embodiments, the modified CD63 includes three transmembrane domains. In some embodiments, the modified CD63 includes four transmembrane domains. In some embodiments, the modified CD63 includes five transmembrane domains. In some embodiments, the immune checkpoint moiety forms a complex with the modified CD63 at an extracellular loop of the modified CD63. In some embodiments, the immune checkpoint moiety forms a complex with the modified CD63 at the large extracellular loop of the modified CD63. In some embodiments, the composition further comprises a targeting moiety. In some embodiments, the targeting moiety comprises a peptide that targets a cytokine. In some embodiments, the targeting moiety comprises a peptide that targets a cancer cell marker. In some embodiments, the composition further comprises a fusogenic moiety. In some embodiments, the fusogenic moiety comprises a viral fusogenic moiety. In some embodiments, the fusogenic moiety comprises a mammalian fusogenic moiety. In some embodiments, the composition further comprises an immune evasion moiety. In some embodiments, the immune evasion moiety comprises CD47. The composition does not comprise enucleated cells. In some embodiments, the extracellular vesicles comprise exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles. In some embodiments, the extracellular vesicles areIn some embodiments, the extracellular vesicles comprise exosomes. In some embodiments, the extracellular vesicles comprise a plurality of immune checkpoint moieties. In some embodiments, the extracellular vesicles comprise at least 10,000 units of immune checkpoint moieties per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 9,000 units of immune checkpoint moieties per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 8,000 units of immune checkpoint moieties per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 7,000 units of immune checkpoint moieties per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 6,000 units of immune checkpoint moieties per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 5,000 units of immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm. In some embodiments, the extracellular vesicles comprise at least 3,000 units of immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm. In some embodiments, the extracellular vesicles comprise at least 2,500 units of immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm. In some embodiments, the extracellular vesicles comprise at least 2,000 units of immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm. In some embodiments, the extracellular vesicles comprise at least 1,500 units of immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm. In some embodiments, the composition comprises a plurality of exosomes. In some embodiments, the composition comprises a therapeutically effective amount of exosomes. In some embodiments, the composition further comprises at least 10^6 exosomes. In some embodiments, the composition further comprises at least 10^7 exosomes. In some embodiments, the composition further comprises at least 10^8 exosomes. In some embodiments, the composition further comprisesIn some embodiments, the composition further comprises at least 1 μg of exosomes. In some embodiments, the composition further comprises at least 10 μg of exosomes. In some embodiments, the composition further comprises at least 20 μg of exosomes. In some embodiments, the composition further comprises at least 50 μg of exosomes. In some embodiments, the composition further comprises at least In some embodiments, the composition further comprises 100 μg of exosomes. In some embodiments, the composition further comprises at least 150 μg of exosomes. In some embodiments, the composition further comprises at least 200 μg of exosomes. In some embodiments, the composition further comprises at least 250 μg of exosomes. In some embodiments, the composition further comprises at least 500 μg of exosomes. In some embodiments, the composition further comprises at least 750 μg of exosomes. In some embodiments, the composition further comprises at least 1 mg of exosomes. In some embodiments, the composition further comprises at least 2 mg of exosomes. In some embodiments, the composition further comprises at least 3 mg of exosomes. In some embodiments, the composition further comprises at least 4 mg of exosomes. In some embodiments, the composition further comprises at least 5 mg of exosomes. In some embodiments, the composition further comprises at least 6 mg of exosomes. In some embodiments, the composition further comprises at least 7 mg of exosomes. In some embodiments, the composition further comprises at least 100 mg of exosomes. In some embodiments, the composition further comprises at least 200 mg of exosomes. In some embodiments, the composition further comprises at least 300 mg of exosomes. In some embodiments, the composition further comprises at least 400 mg of exosomes. In some embodiments, the composition further comprises at least 500 mg of exosomes. In some embodiments, the composition further comprises at least 600 mg of exosomes. In some embodiments, the composition further comprises at least 700 mg of exosomes. In some embodiments, the composition is derived from cells. In some embodiments, the composition is stored frozen. In some embodiments, the composition is lyophilized. In some embodiments, the composition is stable at 37°C for 24 hours. In some embodiments, the composition is stable at 37°C for 48 hours.In some embodiments, the composition is stable at 37° C. for 72 hours.

[0006] Other aspects of the present disclosure include cells configured to produce extracellular vesicles or exosomes of any one of the embodiments described herein. In some embodiments, the cells are stem cells. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells. In some embodiments, the cells are mesenchymal stem cells. In some embodiments, the cells are genetically modified cells. In some embodiments, the cells are genetically modified to produce extracellular vesicles or exosomes described in any one of the embodiments described herein.

[0007] Another aspect of the present disclosure includes a method for purifying extracellular vesicles configured to express one or more immune checkpoint moieties, the method comprising: obtaining a heterogeneous population of extracellular vesicles; exposing the heterogeneous population of extracellular vesicles to a detection assay solution containing a detection moiety to form complexes with the immune checkpoint moiety; detecting a signal resulting from the complex formed between the immune checkpoint moiety and the detection moiety, wherein the intensity of the signal is proportional to the units of the expressed immune checkpoint moiety; and isolating a subpopulation of the extracellular vesicles based on the intensity of the signal. In some embodiments, the detection moiety comprises an antibody. In some embodiments, the detection moiety comprises an anti-VISTA antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof. In some embodiments, the detection moiety comprises a ligand of the immune checkpoint moiety. In some embodiments, the detection assay solution further comprises a peptide configured to bind to the immune checkpoint moiety. In some embodiments, the peptide configured to bind to the immune checkpoint moiety comprises PD-1, CD80, CD86, or a combination thereof.

[0008] Other aspects of the present disclosure include a pharmaceutical composition comprising any one of the compositions of the embodiments described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises at least one additional active agent. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration, intraocular administration, intravitreal administration, retinal administration, intravenous administration, intramuscular administration, intraventricular administration, intracerebral administration, intracerebellar administration, intraventricular administration, intraparenchymal administration, subcutaneous administration, or a combination thereof.

[0009] Another aspect of the present disclosure includes a method of treating an autoimmune disease, the method comprising administering a pharmaceutical composition of any one of the embodiments described herein. In some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, celiac disease, Crohn's disease, Graves' disease, juvenile arthritis, chronic Lyme disease, optic neuritis, psoriatic arthritis, scleritis, scleroderma, ulcerative colitis (UC), uveitis, inflammatory eye disease, vitiligo, COPD, complications from organ transplantation, or graft-versus-host disease. In some embodiments, the autoimmune disease is rheumatoid arthritis.

[0010] Another aspect of the present disclosure includes a method of suppressing CD8+CD25+ cells in a patient in need thereof, the method comprising administering a composition of any one of the embodiments described herein.

[0011] Another aspect of the present disclosure includes a kit comprising the pharmaceutical composition of any one of the embodiments described herein.

[0012] Another aspect of the present disclosure includes a platform that includes components for producing the composition of any one of the embodiments described herein.

[0013] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 illustrates proteomic analysis of human MSC-derived exosomes detecting programmed cell death ligand-1 (PD-L1). [Figure 2] FIG. 1 illustrates the experimental design for PD-L1 blockade to determine how blockade of PD-L1 affects CD8+ T cells. [Figure 3] Figure 1 illustrates that the presence of blocking PD-L1 antibody abolished the effect of PD-L1 on CD25+CD8+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the disclosure described herein may be utilized in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of the claims and equivalents thereof be covered thereby.

[0016] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "before," "after," "lastly," and "finally," is meant to be exemplary and not limiting of the scope of the embodiments disclosed herein.

[0017] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0018] As used herein, the phrases "at least one," "one or more," and "and / or" are both conjunctive and discriminative in operation. For example, the phrases "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" mean A alone, B alone, C alone, A and B, A and C, B and C, or A, B, and C, respectively.

[0019] All systems, methods, software, and platforms described herein are modular and not limited to sequential processes, and therefore terms such as "first" and "second" do not necessarily indicate an order of priority, importance, or operation.

[0020] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations per implementation for a given value. When particular values ​​are described in this application and in the claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0021] The terms "increased" or "increase" are used herein to generally mean an increase by a statistically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a baseline value, e.g., an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100% compared to a baseline value, standard, or control, or any increase between 10 and 100%. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more compared to a baseline value.

[0022] The terms "decreased" or "decreasing" are used herein to generally mean a statistically significant decrease. In some embodiments, "decreased" or "decreasing" refers to a decrease of at least 10% compared to a reference value, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to a 100% decrease compared to a reference value (e.g., a nonexistent or undetectable value compared to a reference value), or any decrease between 10% and 100%. In the context of a marker or symptom, these terms refer to a statistically significant decrease in such value. This decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to a value within the normal range for an individual without a given disease.

[0023] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or limit situations characterized by the supervision (e.g., continuous or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, janitor, or hospice worker).

[0024] As used herein, "cell" generally refers to a biological cell. A cell can be a structural, functional, and / or biological unit of a living organism. A cell may be from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, fodder, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, bryophytes, liverworts, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.), and the like. C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., cells from yeast, mushrooms), animal cells, invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), mammalian-derived cells (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells are not derived from a natural organism (e.g., the cells may be synthesized, in which case they are referred to as artificial cells).

[0025] The term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules, including these. The term nucleotide, as used herein, may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled using well-known techniques. Labeling may also be achieved using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include, but are not limited to, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, California; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Indiana; and Boehringer Ingelheim, Indianapolis, Indiana. Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Mannheim, and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, and Oregon Green available from Molecular Probes, Eugene, Oregon. 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP,Examples of suitable biotinylated dNTPs include Texas Red-5-dUTP and Texas Red-12-dUTP. Nucleotides may also be labeled or marked by chemical modification. The chemically modified single nucleotide may be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0026] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to polymeric forms of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides, or their analogs, in single-, double-, or multi-stranded form. Polynucleotides can be extracellular or intracellular. Polynucleotides can exist in a cell-free environment. Polynucleotides can be genes or fragments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to sugars), thiols, nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.The sequence of nucleotides may be interrupted by non-nucleotide components.

[0027] The term "transfection" or "transfected" generally refers to the introduction of nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule may be a gene sequence encoding an entire protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0028] The term "expression" or "expressing" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and encoded polypeptide may collectively be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. "Upregulated," with respect to expression, generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its wild-type state level, while "downregulated" generally refers to a decrease in the level of expression of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its wild-type state level.

[0029] The term "gene," as used herein, refers to a portion of nucleic acid (also referred to as a "coding sequence" or "coding region") that encodes a particular protein or RNA, optionally along with associated control regions such as a promoter, operator, and terminator, which may be located upstream or downstream of the coding sequence. The term "gene" is to be interpreted broadly and may encompass mRNA, cDNA, cRNA, and genomic DNA forms of a gene. In some applications, the term "gene" encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region encompasses an "open reading frame" that encodes a polypeptide. In some applications of the term, a "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some embodiments, a gene does not encode a polypeptide, e.g., a ribosomal RNA gene (rRNA) or a transfer RNA (tRNA) gene. In some embodiments, the term "gene" includes, in addition to the transcribed sequence, non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. The term "gene" can encompass mRNA, cDNA, and genomic forms of a gene.

[0030] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein in reference to a polymer of amino acid residues. A protein refers to a full-length polypeptide as translated from a coding open reading frame or processed to its mature form, while a polypeptide or peptide can refer to a degradation or processing fragment of a protein that uniquely or identically maps to a particular protein. A polypeptide can be a single linear polymeric chain of amino acids joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. A polypeptide can be modified, for example, by the addition of carbohydrates or phosphorylation. A protein can comprise one or more polypeptides.

[0031] As used herein, the term "fragment," or synonyms thereof, may refer to a portion of a protein that is less than the full length of the protein in length, and optionally maintains the function of the protein. Furthermore, when this portion of the protein is blasted against the protein, the portion of the protein sequence may, for example, align with at least 80% identity to a portion of the protein sequence.

[0032] The terms "complement," "complements," "complementary," and "complementarity," as used herein, generally refer to a sequence that is perfectly complementary to and hybridizable with a given sequence. In some cases, a sequence that hybridizes with a given nucleic acid is referred to as the "complement" or "reverse complement" of the given molecule when the sequence of bases across a given region is capable of complementary binding with the sequence of its binding partner, for example, forming AT, AU, GC, and GU base pairs. Generally, a first sequence that can hybridize to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of sequences is preferred over hybridization with non-target sequences during a hybridization reaction (e.g., conditions that are more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art). Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as 25% to 100% complementarity, including at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.Sequence identity, such as for assessing the percentage of complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner, available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, for example, the BLAST alignment tool, available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner, available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0033] The term "percent (%) identity," as used herein, generally refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The percent identity of two sequences can be calculated by aligning the test sequence with the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0034] The term " mismatch " generally refers to the lack of complementarity between two nucleotides when aligned.The complementary bases of DNA are AT and GC.The complementary bases of RNA are AU and GC.Therefore, mismatch occurs when two oligonucleotide sequences are aligned at one or more nucleotide positions where A does not pair with T or G does not pair with C in DNA, or A does not pair with U or G does not pair with C in RNA.

[0035] As used herein, the term "in vivo" can be used to describe events that occur in the body of a subject.

[0036] As used herein, the term "ex vivo" can be used to describe events that occur outside of a subject's body. An "ex vivo" assay cannot be performed on a subject. Rather, it can be performed on a sample separate from the subject. Ex vivo can be used to describe events that occur in intact cells outside of a subject's body.

[0037] As used herein, the term "in vitro" can be used to describe events that occur when a container is included to hold a test reagent, such that the material is separated from the living biological source organism from which it is obtained. In vitro assays can include cell-based assays in which living or dead cells are utilized. In vitro assays can also include cell-free assays in which intact cells are not utilized.

[0038] "Treating" or "treatment" can refer to both therapeutic and prophylactic (prophylactic or preventative) measures, whereby a targeted disease or disorder is prevented or delayed (alleviated) in a subject. Those in need of treatment include those already suffering from the disorder, as well as those prone to developing the disorder or those in whom the disorder is to be prevented. Therapeutic benefit can refer to the eradication or amelioration of the symptoms being treated or the underlying disorder. Therapeutic benefit can also be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be affected by the underlying disorder. Prophylactic benefit can include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. In a prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may receive treatment even if a diagnosis of the disease cannot be made.

[0039] The terms "effective amount" and "therapeutically effective amount," as used interchangeably herein, generally refer to the amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), comprising a system of the present disclosure, sufficient to produce a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay the onset of, prevent the progression of, or reduce or alleviate at least one symptom of a disorder treated by a method of the present disclosure.

[0040] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A compound may be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. A compound may further be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit-risk ratio. Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 5th Edition”, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005, Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0041] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical components, such as a diluent or carrier. A pharmaceutical composition can facilitate administration of a compound to an organism. Various techniques for administering a compound exist in the art, including, but not limited to, oral administration, injection, aerosol administration, parenteral administration, and topical administration.

[0042] The present disclosure is directed to mesenchymal stem cell exosomes engineered with PD-L1, a novel cell-free therapeutic agent for inducing tolerance in autoimmune diseases and transplantation. Extracellular vesicles, such as exosomes and microvesicles (also known as shedding vesicles), deliver bioactive molecules that affect the extracellular environment and the immune system. It has been shown that exosomal PD-L1 has the same membrane topology as cell-surface PD-L1, with its extracellular domain exposed on the surface of exosomes. Exosomal PD-L1 binds to PD-1 in a concentration-dependent manner, and this interaction can be disrupted by PD-L1-blocking antibodies. High levels of exosomal PD-L1 may reflect T cell "exhaustion" to a stage where T cells cannot be further restored by anti-PD-1 treatment. Furthermore, increased exosomal PD-L1 in response to IFN-γ can enable tumor cells to adaptively inactivate CD8 T cells.

[0043] Mesenchymal stem cells are first isolated from healthy individuals. Once expanded, these cells may be stored for further use. These cells can then be engineered with PD-L1. To do this, the PDL1 gene can be fused with the lactadherin gene, allowing the PD-L1 molecule to be inserted externally into exosomes. Upon confirming successful engineering, these cells are grown in a bioreactor, and the conditioned medium can be harvested to isolate exosomes. In exosome isolation, tangential flow filtration is used to harvest and purify exosomes. These exosomes have the ability to suppress the immune system of specific CD8+ and CD25+ effector cells. These effector cells are important in the rejection process in hospitals and transplants for all autoimmune diseases. Once these exosomes are produced in a cGMP facility and pass all potency and other requirements, they can be used to treat patients. In some embodiments, mesenchymal stem cell exosomes can be engineered with PD-L1 to enhance their suppressive activity. In some embodiments, PD-L1 engineered mesenchymal stem cell exosomes may be a treatment for all autoimmune diseases, including type 1 diabetes, multiple sclerosis, COPD, lupus, inflammatory bowel disease, rheumatoid arthritis, or psoriasis, and transplantation. In some embodiments, the function of PD-L1 engineered mesenchymal stem cell exosomes may be increased when engineered regulatory T cells are cultured under hypoxic conditions. In some embodiments, PD-L1 engineered mesenchymal stem cell exosomes may have the ability to suppress an activated autoimmune system, specifically a patient's CD8+CD25+ cells. In some embodiments, using the platform described herein, any other cells or exosomes, as well as mesenchymal stem cells and mesenchymal stem cell-derived exosomes, may be engineered to contain PD-L1. In some embodiments, PD-L1 engineered mesenchymal stem cell exosomes can be prepared in a cGMP facility.In some embodiments, PD-L1 engineered mesenchymal stem cell exosomes may be considered clinical grade exosomes for treating all autoimmune diseases and patients who have received any organ as a transplant procedure.

[0044] This disclosure relates to mesenchymal stem cell exosomes engineered with VISTA / PD-L1 / CTLA-4, a novel cell-free therapeutic agent that induces tolerance in autoimmune diseases and transplantation. Extracellular vesicles, such as exosomes and microvesicles (also known as shedding vesicles), carry bioactive molecules that affect the extracellular environment and the immune system. It has been shown that exosomal PD-L1 has the same membrane topology as cell-surface PD-L1, with its extracellular domain exposed on the surface of exosomes. Exosomal PD-L1 binds to PD-1 in a concentration-dependent manner, and this interaction can be disrupted by PD-L1-blocking antibodies. High levels of exosomal PD-L1 may reflect T cell "exhaustion" to a stage where T cells cannot be further restored by anti-PD-1 treatment. Furthermore, increased exosomal PD-L1 in response to IFN-γ can enable tumor cells to adaptively inactivate CD8 T cells. V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T cell function expressed in hematopoietic cells. VISTA levels are elevated in the tumor microenvironment, where blocking these levels can enhance antitumor immune responses in mice. VISTA is predominantly expressed in the hematopoietic compartment, with expression highest in the myeloid lineage. VISTA-Ig suppressed T cell proliferation and blunted the production of T cell cytokines and activation markers. VISTA is an inhibitory immune checkpoint molecule that suppresses CD4+ and CD8+ T cell activation when expressed on antigen-presenting cells. VISTA critically regulates inflammatory responses mediated by DCs and IL-17-producing TCRγδ+ and CD4+ Th17 T cells after TLR7 stimulation. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed on regulatory T cells but is only upregulated on conventional T cells after activation, a phenomenon particularly prominent in cancer. CTLA4 acts as an "off" switch upon binding to CD80 or CD86 on the surface of antigen-presenting cells.

[0045] In some embodiments, mesenchymal stem cells can first be isolated from a healthy individual. Once expanded, these cells may be stored for further use. These cells can then be engineered with VISTA / PD-L1 / CTLA-4 alone or in combination. The following immune checkpoints can be used instead of or in combination with VISTA, PD-L1, and CTLA-4: PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), galectin-9, galectin-3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (nectin-2, CD112), FGL-1, PECAM -1, TSG-6, CD47, stabilin-1 (Clever-1), neuropilin-1, neuropilin-2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, or SIRPα (CD172a).

[0046] To this end, one or a combination of the immune checkpoint genes described herein can be fused to a lactadherin gene, a GPI protein, or other proteins or linkers. This allows the checkpoint protein to be inserted externally into exosomes. Upon confirming successful completion of the procedure, the cells can be grown in a bioreactor, and the conditioned medium can be harvested to isolate exosomes. For exosome isolation, tangential flow filtration can be used to harvest and purify exosomes. These exosomes have the ability to suppress specific CD4+ / CD25+, i.e., CD8+CD25+, effector cells, or dendritic cells and B cells, acting as immune suppressors. This suppression depends on the combination of checkpoint molecules on the surface of the exosome. These effector cells are crucial in the rejection process in hospitals and transplants for all autoimmune diseases. Once produced in a cGMP facility and passed all potency and other requirements, these exosomes can be used to treat patients. In some embodiments, mesenchymal stem cell exosomes can be engineered with checkpoint molecules to enhance their suppressive activity. The checkpoints can be the combination of VISTA / PD-L1 / CTLA-4, or any one alone. In some embodiments, the checkpoints can be a combination of immune checkpoint genes described herein. In some embodiments, checkpoint-decorated mesenchymal stem cell exosomes can be a treatment for all autoimmune diseases and transplants, including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, celiac disease, Crohn's disease, Graves' disease, juvenile arthritis, Lyme disease, optic neuritis, psoriatic arthritis, scleritis, scleroderma, ulcerative colitis (UC), uveitis, inflammatory eye disease, vitiligo, COPD, or organ transplantation. In some embodiments, any other cells or exosomes can be engineered with immune checkpoints (similar to mesenchymal stem cells and their human fibroblast exosomes) using the platform described herein.In some embodiments, checkpoint molecule-decorated exosomes can be manufactured as injections, eye drops, nebulizers or sprays, creams, and topical ointments or any other form that is approved for pharmaceutical use. 30 In some embodiments, these exosomes can be prepared in a cGMP facility and may be considered clinical-grade exosomes for treating all autoimmune diseases and patients who have received any organs as transplant procedures.

[0047] I. Composition Some embodiments described herein provide compositions comprising extracellular vesicles produced from the platforms and methods described herein. In some embodiments, the extracellular vesicles are membrane-bound particles secreted by cells. In some embodiments, the extracellular vesicles are membrane-bound particles produced in vitro. In some embodiments, the extracellular vesicles are membrane-bound particles produced ex vivo. In some embodiments, the extracellular vesicles are membrane-bound particles produced without the use of cells. In some embodiments, the extracellular vesicles are exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomes, enveloped viruses, exomers, or other very large extracellular vesicles. In some embodiments, the extracellular vesicles are exosomes.

[0048] In some instances, the extracellular vesicles comprise a diameter of about 1 nm to about 10,000 nm.In some examples, the extracellular vesicles may be between about 1 nm and about 5 nm, about 1 nm and about 10 nm, about 1 nm and about 20 nm, about 1 nm and about 50 nm, about 1 nm and about 100 nm, about 1 nm and about 200 nm, about 1 nm and about 500 nm, about 1 nm and about 1,000 nm, about 1 nm and about 2,000 nm, about 1 nm and about 5,000 nm, about 1 nm and about 10,000 nm, about 5 nm and about 10 nm, about 5 nm and about 20 nm, about 5 nm and about 50 nm, about 5 nm and about 100 nm, about 5 nm and about 200 nm, about 5 nm and about 500 nm, about 5 nm and about 1,000 nm, about 5 nm and about 2 ,000nm, about 5nm to about 5,000nm, about 5nm to about 10,000nm, about 10nm to about 20nm, about 10nm to about 50nm, Approximately 10nm to approximately 100nm, approximately 10nm to approximately 200nm, approximately 10nm to approximately 500nm, approximately 10nm to approximately 1,000nm, approximately 10nm to approximately 2,000nm, about 10nm to about 5,000nm, about 10nm to about 10,000nm, about 20nm to about 50nm, about 20nm to about 100 nm, approximately 20 nm to approximately 200 nm, approximately 20 nm to approximately 500 nm, approximately 20 nm to approximately 1,000 nm, approximately 20 nm to approximately 2,000 nm, approximately 2 0nm to approx. 5,000nm, approx. 20nm to approx. 10,000nm, approx. 50nm to approx. 100nm, approx. 50nm to approx. 200nm, approx. 50nm ~500nm, approximately 50nm ~ approximately 1,000nm, approximately 50nm ~ approximately 2,000nm, approximately 50nm ~ approximately 5,000nm, approximately 50nm ~ approximately 1 0,000nm, about 100nm to about 200nm, about 100nm to about 500nm, about 100nm to about 1,000nm, about 100nm to about 2 ,000nm, approx. 100nm ~ approx. 5,000nm, approx. 100nm ~ approx. 10,000nm, approx. 200nm ~ approx. 500nm, approx. 200nm ~ and a diameter of about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about 10,000 nm.In some examples, the extracellular vesicles comprise a diameter of about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some examples, the extracellular vesicles comprise a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about 5,000 nm. In some examples, the extracellular vesicles comprise a diameter of at most about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

[0049] In some embodiments, the extracellular vesicles comprise a diameter of at least about 1 nm to about 10,000 nm.In some embodiments, the extracellular vesicles are at least about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm, 5nm to about 2,000nm, about 5nm to about 5,000nm, about 5nm to about 10,000nm, about 10nm to about 20nm, about 10nm to about 50nm, about 10nm to about 100nm, about 10nm to about 200nm, about 10nm to about 500nm, about 10nm to about 1,000nm, about 1 0nm to approx. 2,000nm, approx. 10nm to approx. 5,000nm, approx. 10nm to approx. 10,000nm, approx. 20nm to approx. 50nm, approx. 20nm ~about 100nm, about 20nm to about 200nm, about 20nm to about 500nm, about 20nm to about 1,000nm, about 20nm to about 2,000nm m, about 20nm to about 5,000nm, about 20nm to about 10,000nm, about 50nm to about 100nm, about 50nm to about 200nm, about 5 0nm to approx. 500nm, approx. 50nm to approx. 1,000nm, approx. 50nm to approx. 2,000nm, approx. 50nm to approx. 5,000nm, approx. 50nm ~about 10,000nm, about 100nm to about 200nm, about 100nm to about 500nm, about 100nm to about 1,000nm, about 100nm ~approx. 2,000nm, approx. 100nm ~ approx. 5,000nm, approx. 100nm ~ approx. 10,000nm, approx. 200nm ~ approx. 500nm, approx. 200n The diameters include about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about 10,000 nm.In some embodiments, the extracellular vesicles comprise a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some embodiments, the extracellular vesicles comprise a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about 5,000 nm. In some embodiments, the extracellular vesicles comprise a diameter of at least up to about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

[0050] In some embodiments, the composition comprises a heterogeneous population of extracellular vesicles, in some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of about 1 nm to about 10,000 nm.In some embodiments, the heterogeneous population of extracellular vesicles ranges from about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm, 5nm to about 2,000nm, about 5nm to about 5,000nm, about 5nm to about 10,000nm, about 10nm to about 20nm, about 10nm to about 50nm, about 10nm to about 100nm, about 10nm to about 200nm, about 10nm to about 500nm, about 10nm to about 1,000nm, about 1 0nm to approx. 2,000nm, approx. 10nm to approx. 5,000nm, approx. 10nm to approx. 10,000nm, approx. 20nm to approx. 50nm, approx. 20nm ~about 100nm, about 20nm to about 200nm, about 20nm to about 500nm, about 20nm to about 1,000nm, about 20nm to about 2,000nm m, about 20nm to about 5,000nm, about 20nm to about 10,000nm, about 50nm to about 100nm, about 50nm to about 200nm, about 5 0nm to approx. 500nm, approx. 50nm to approx. 1,000nm, approx. 50nm to approx. 2,000nm, approx. 50nm to approx. 5,000nm, approx. 50nm ~about 10,000nm, about 100nm to about 200nm, about 100nm to about 500nm, about 100nm to about 1,000nm, about 100nm ~approx. 2,000nm, approx. 100nm ~ approx. 5,000nm, approx. 100nm ~ approx. 10,000nm, approx. 200nm ~ approx. 500nm, approx. 200n The diameters include about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about 10,000 nm.In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about 5,000 nm. In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of at most about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

[0051] In some embodiments, the heterogeneous population of extracellular vesicles comprises diameters of at least about 1 nm to about 10,000 nm.In some embodiments, the heterogeneous population of extracellular vesicles has a size of at least about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm m, about 5nm to about 2,000nm, about 5nm to about 5,000nm, about 5nm to about 10,000nm, about 10nm to about 20nm, about 10n m ~ about 50nm, about 10nm - about 100nm, about 10nm - about 200nm, about 10nm - about 500nm, about 10nm - about 1,000nm, Approximately 10nm to approximately 2,000nm, approximately 10nm to approximately 5,000nm, approximately 10nm to approximately 10,000nm, approximately 20nm to approximately 50nm, approximately 20 nm ~ approx. 100 nm, approx. 20 nm ~ approx. 200 nm, approx. 20 nm ~ approx. 500 nm, approx. 20 nm ~ approx. 1,000 nm, approx. 20 nm ~ approx. 2,00 nm 0nm, about 20nm to about 5,000nm, about 20nm to about 10,000nm, about 50nm to about 100nm, about 50nm to about 200nm, Approximately 50nm to approximately 500nm, approximately 50nm to approximately 1,000nm, approximately 50nm to approximately 2,000nm, approximately 50nm to approximately 5,000nm, approximately 50n m ~ approx. 10,000nm, approx. 100nm ~ approx. 200nm, approx. 100nm ~ approx. 500nm, approx. 100nm ~ approx. 1,000nm, approx. 100n m ~ approx. 2,000 nm, approx. 100 nm ~ approx. 5,000 nm, approx. 100 nm ~ approx. 10,000 nm, approx. 200 nm ~ approx. 500 nm, approx. 200 nm to about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about 10,000 nm.In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about 5,000 nm. In some embodiments, the heterogeneous population of extracellular vesicles comprises a diameter of at least up to about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

[0052] In some embodiments, the composition comprises a homogenous population of extracellular vesicles. In some embodiments, the homogenous population of extracellular vesicles comprises a diameter of about 10 nm to about 150 nm. In some embodiments, the homogenous population of extracellular vesicles comprises a diameter of about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 10 nm to about 100 nm, about 10 nm to about 110 nm, about 10 nm to about 120 nm, about 10 nm to about 150 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, or about 30 nm to about 80 nm, approximately 30 nm to approximately 90 nm, approximately 30 nm to approximately 100 nm, approximately 30 nm to approximately 110 nm, approximately 30 nm to approximately 120 nm, approximately 30 nm to approximately 150 nm, approximately 40 nm to approximately 50 nm, approximately 40 nm to approximately 60 nm, approximately 40 nm to approximately 70 nm, approximately 40 nm ~about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 40nm to about 110nm, about 40nm to about 120nm, about 40nm to about 150nm, about 50nm to about 60nm, about 50nm to about 70nm, about 50nm to about 80nm, about 5 0nm to about 90nm, about 50nm to about 100nm, about 50nm to about 110nm, about 50nm to about 120nm, about 50nm to about 150nm, about 60nm to about 70nm, about 60nm to about 80nm, about 60nm to about 90nm, about 60nm to about 100 nm, about 60nm to about 110nm, about 60nm to about 120nm, about 60nm to about 150nm, about 70nm to about 80nm, about 70nm to about 90nm, about 70nm to about 100nm, about 70nm to about 110nm, about 70nm to about 120nm, about 70nm The diameter may be from about 100 nm to about 150 nm, from about 80 nm to about 90 nm, from about 80 nm to about 100 nm, from about 80 nm to about 110 nm, from about 80 nm to about 120 nm, from about 80 nm to about 150 nm, from about 90 nm to about 100 nm, from about 90 nm to about 110 nm, from about 90 nm to about 120 nm, from about 90 nm to about 150 nm, from about 100 nm to about 110 nm, from about 100 nm to about 120 nm, from about 100 nm to about 150 nm, from about 110 nm to about 120 nm, from about 110 nm to about 150 nm, or from about 120 nm to about 150 nm.In some embodiments, the homogenous population of extracellular vesicles comprises a diameter of about 10 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, or about 150 nm. In some embodiments, the homogenous population of extracellular vesicles comprises a minimum diameter of about 10 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. In some embodiments, the homogenous population of extracellular vesicles comprises a maximum diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, or about 150 nm.

[0053] Immune checkpoint part In some embodiments, compositions comprising extracellular vesicles comprising immune checkpoint moieties are described herein. In some embodiments, the extracellular vesicles comprise multiple immune checkpoint moieties, where the immune checkpoint moieties may be the same or different. In some embodiments, the immune checkpoint moiety is encapsulated in the extracellular vesicle. In some embodiments, the immune checkpoint moiety is expressed on the surface of the extracellular vesicle. In some embodiments, the immune checkpoint moiety is secreted by the extracellular vesicle. In some embodiments, the immune checkpoint moiety is encapsulated in the extracellular vesicle. In some embodiments, the immune checkpoint moiety is expressed on the surface of the extracellular vesicle, secreted by the extracellular vesicle, delivered to a target cell or target microenvironment by the extracellular vesicle, or a combination thereof. In some embodiments, the immune checkpoint moiety comprises a therapeutic property for treating a disease or disorder. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, a composition comprising extracellular vesicles comprising an immune checkpoint moiety can be administered to a subject to treat the disease or disorder.

[0054] In some embodiments, the immune checkpoint moiety is VISTA, PD-L1, CTLA-4, PD-L2, B7-(CD80), B7-2(CD86), B7-H3(CD276), B7-H2, B7-H3, B7-H4(VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9, Galectin 3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG- In some embodiments, the immune checkpoint moiety comprises a polypeptide comprising a peptide sequence encoding VISTA, a variant thereof, or a fragment thereof. In some embodiments, the immune checkpoint moiety comprises a peptide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1. In some embodiments, the immune checkpoint moiety comprises a peptide sequence 100% identical to SEQ ID NO: 1 (Table 1). In some embodiments, the immune checkpoint moiety comprises a peptide sequence encoding PD-L1, a variant thereof, or a fragment thereof. In some embodiments, the immune checkpoint moiety comprises a peptide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2. In some embodiments, the immune checkpoint moiety comprises a peptide sequence 100% identical to SEQ ID NO: 2 (Table 1). In some embodiments, the immune checkpoint moiety comprises a peptide sequence encoding CTLA-4, a variant thereof, or a fragment thereof.In some embodiments, the immune checkpoint portion comprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the immune checkpoint portion comprises a peptide sequence that is 100% identical to SEQ ID NO: 3 (Table 1).

[0055] [Table 1]

[0056] In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide comprises an mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. Optionally, the heterologous polynucleotide comprises an mRNA. In some embodiments, the heterologous polynucleotide is selected from the group consisting of VISTA, PD-L1, CTLA-4, PD-L2, B7-(CD80), B7-2(CD86), B7-H3(CD276), B7-H2, B7-H3, B7-H4(VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9, Galectin 3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM- In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding VISTA. In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding PD-L1. In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding CTLA-4.

[0057] In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding a cytokine. In some embodiments, the immune checkpoint moiety comprises a polypeptide comprising a peptide sequence of a cytokine. Exemplary cytokines that can be used as immune checkpoint moieties include 4-1BBL, acylated stimulatory protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 , CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L / CD178, Cerberus (protein), chemokine, CLCF1, CNTF, Colony-stimulating factor, common b chain (CD131), common g chain (CD132), CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCR3, CXCR4, CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM 19A5, Flt-3L, FMS-like tyrosine kinase 3 ligand, Foxp3, GATA-3, GcMAF, G-CSF, GITRL, GM-CSF, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, IFNA1, IFNA1 0, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5 / IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-α, IFN-β, IFN-γ, IFNω / IFNW1, IL-1, IL-10, IL-10family, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17 family, IL-17A-F, IL-18, IL-18BP, IL-19, IL-1A, IL-1B, IL-1F10, IL- 1F3 / IL-1RA, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1-like, IL-1RA, IL-1RL2, IL-1α, IL-1β, IL-2, IL-20, IL-21, IL-22, IL-23, IL-24, IL-28A , IL-28B, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-8 / CXCL8, IL-9, inflammasome, interferome, interferon, interferon beta-1a, interferon beta-1b, interferon gamma, type I interferon, type II interferon, type III interferon, interferon, interleukin, interleukin-1 receptor antagonist, interleukin-8, IRF4, leptin, leukemia inhibitory factor (LIF), leukocyte-stimulating factor, LIGHT, LTA / TNFB, LT-β, ​​lymphokine, lymphotoxin, lymphotoxin α, lymphotoxin β, macrophage colony-stimulating factor, macrophage inflammatory protein, macrophage-activating factor, M-CSF, MHC class III, various hematopoietins, monokines, MSP, myokine, myonectin, nicotinamide phosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L, platelet factor 4, promethazine Examples of such inhibitors include gapoietin, RANKL, SCF, STAT3, STAT4, STAT6, stromal cell-derived factor 1, TALL-1, TBX21, TGF-α, TGF-β, TGF-β1, TGF-β2, TGF-β3, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-α, TNF-β, Tpo, TRAIL, TRANCE, TWEAK, vascular endothelial growth inhibitor, XCL1, or XCL2.

[0058] In some embodiments, the immune checkpoint moiety can be complexed with a transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be non-covalently complexed with a transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be covalently complexed with a transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and a transmembrane moiety. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and a fragment of a transmembrane moiety. In some embodiments, the N-terminus of the immune checkpoint moiety can be fused to a transmembrane moiety. In some embodiments, the C-terminus of the immune checkpoint moiety can be fused to a transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be fused to and adjacent to a transmembrane moiety at both the N-terminus and C-terminus of the immune checkpoint moiety. For example, the immune checkpoint moiety can be inserted into the transmembrane moiety as part of a fusion peptide, where the N-terminus of the fusion peptide comprises a fragment of the transmembrane moiety, followed by the immune checkpoint moiety (or a variant or fragment thereof), and the C-terminus of the fusion peptide comprises another fragment of the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises a fusion peptide, where the immune checkpoint moiety is fused to the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises an immune checkpoint moiety that forms a complex with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises an immune checkpoint moiety that forms a non-covalent complex with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises an immune checkpoint moiety that forms a covalent complex with the transmembrane moiety.

[0059] In some embodiments, the extracellular vesicles comprise a plurality of immune checkpoint moieties described herein. In some embodiments, a plurality of immune checkpoint moieties are encapsulated in the extracellular vesicles. In some embodiments, the extracellular vesicles encapsulate at least 1, 10, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or more units of the immune checkpoint moiety. In some embodiments, the extracellular vesicles deliver the encapsulated immune checkpoint moiety to a target cell or a target microenvironment.

[0060] In some embodiments, the extracellular vesicles secrete a plurality of immune checkpoint moieties described herein. In some embodiments, the extracellular vesicles secrete at least 1, 10, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or more units of the immune checkpoint moiety. In some embodiments, the extracellular vesicles secrete the encapsulated immune checkpoint moiety into a target cell or target microenvironment.

[0061] In some embodiments, multiple immune checkpoint moieties are expressed on the surface of the extracellular vesicle. In some embodiments, multiple immune checkpoint moieties are expressed as part of a fusion peptide comprising an immune checkpoint moiety and a transmembrane moiety. In some examples, the extracellular vesicle comprising the immune checkpoint moiety expressed on the surface of the extracellular vesicle contacts a target cell or target environment.

[0062] In some embodiments, the number of units of an immune checkpoint moiety that can be expressed on the surface of an extracellular vesicle is limited by a theoretical maximum determined by the ratio between the dimensions of the extracellular vesicle and the dimensions of the expressed immune checkpoint moiety or expressed fusion peptide that comprises the immune checkpoint moiety. In some embodiments, the platforms and methods described herein can produce and select extracellular vesicles that express a number of units of an immune checkpoint moiety that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum number of units of an immune checkpoint moiety that can be expressed on the surface of an extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles that express a number of immune checkpoint moiety units that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum number of immune checkpoint moiety units that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles that express a number of immune checkpoint moiety units on the surface of the extracellular vesicles, wherein the number of immune checkpoint moiety units is at least 30% of the theoretical maximum number of immune checkpoint moiety units that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, wherein the number of units of the immune checkpoint moiety is at least 70% of the theoretical maximum number of units of the immune checkpoint moiety that can be expressed on the surface of the extracellular vesicle.In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of immune checkpoint moieties on the surface of the extracellular vesicles, wherein the number of units of immune checkpoint moieties is at least 75% of the theoretical maximum number of units of immune checkpoint moieties that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of immune checkpoint moieties on the surface of the extracellular vesicles, wherein the number of units of immune checkpoint moieties is at least 80% of the theoretical maximum number of units of immune checkpoint moieties that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of immune checkpoint moieties on the surface of the extracellular vesicles, wherein the number of units of immune checkpoint moieties is at least 85% of the theoretical maximum number of units of immune checkpoint moieties that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of an immune checkpoint moiety on the surface of the extracellular vesicles, wherein the number of units of the immune checkpoint moiety is at least 90% of the theoretical maximum number of units of the immune checkpoint moiety that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of an immune checkpoint moiety on the surface of the extracellular vesicles, wherein the number of units of the immune checkpoint moiety is at least 95% of the theoretical maximum number of units of the immune checkpoint moiety that can be expressed on the surface of the extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select heterogeneous populations of extracellular vesicles expressing a number of units of an immune checkpoint moiety on the surface of the extracellular vesicles, wherein the number of units of the immune checkpoint moiety is at least 95% of the theoretical maximum number of units of the immune checkpoint moiety that can be expressed on the surface of the extracellular vesicles.

[0063] In some embodiments, each extracellular vesicle expresses a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, and this number of units can be from about 5 units to about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, and this number of units can be from about 5 units to about 10 units, from about 5 units to about 50 units, from about 5 units to about 100 units, from about 5 units to about 500 units, from about 5 units to about 1,000 units, from about 5 units to about 5,000 units, from about 5 units to about 10,000 units, from about 5 units to about 50,000 units, from about 5 units to about 100,000 units, or about 5 units. ~approximately 500,000 units, approximately 5 units to approximately 1,000,000 units, approximately 10 units to approximately 50 units, approximately 10 units to approximately 100 units, approximately 10 units to approximately 500 units, approximately 10 units to approximately 1,000 units, approximately 10 units to approximately 5,000 units, approximately 10 units to approximately 10,000 units, approximately 10 units to approximately 50,000 units, approximately 10 units to approximately 100,000 units, approximately 10 units to approximately 500,000 units, approximately 10 units to approximately 1,000,000 units 0 units, approximately 50 units to approximately 100 units, approximately 50 units to approximately 500 units, approximately 50 units to approximately 1,000 units, approximately 50 units to approximately 5,000 units, approximately 50 units to approximately 10,000 units, approximately 50 units to approximately 50,000 units, approximately 50 units to approximately 100,000 units, approximately 50 units to approximately 500,000 units, approximately 50 units to approximately 1,000,000 units, approximately 100 units to approximately 500 units, approximately 100 units to approximately 1,000 units, approximately 1 00 units to approximately 5,000 units, approximately 100 units to approximately 10,000 units, approximately 100 units to approximately 50,000 units, approximately 100 units to approximately 100,000 units, approximately 100 units to approximately 500,000 units, approximately 100 units to approximately 1,000,000 units, approximately 500 units to approximately 1,000 units, approximately 500 units to approximately 5,000 units, approximately 500 units to approximately 10,000 units, approximately 500 units to approximately 50,000 units, approximately 500 units to approximately 100,000 units, approximately 500 units to approximately 500,000 units, approximately 500 units to approximately 1,000,000 units, approximately 1,000 units to approximately 5,000 units, approximately 1,000 units to approximately 10,000 units, approximately 1,000 units to approximately 50,000 units, approximately 1,000 units to approximately 100,000 units, approximately 1,000 units to approximately 500,000 units, approximately 1,000 units to approximately 1,000,000 units, approximately 5,000 units to approximately 10,000 units, approximately 5,000 units to approximately 50,000 units, approximately 5,000 units to approximately 100,000 units, approximately 5,000 units to approximately 500,000 units, approximately 5 The amount may be from about 1,000 units to about 1,000,000 units, from about 10,000 units to about 50,000 units, from about 10,000 units to about 100,000 units, from about 10,000 units to about 500,000 units, from about 10,000 units to about 1,000,000 units, from about 50,000 units to about 100,000 units, from about 50,000 units to about 500,000 units, from about 50,000 units to about 1,000,000 units, from about 100,000 units to about 500,000 units, from about 100,000 units to about 1,000,000 units, or from about 500,000 units to about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, which may be about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, which may be a minimum of about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, or about 500,In some embodiments, each extracellular vesicle expresses a number of units of an immune checkpoint moiety on the surface of the extracellular vesicle, which may be up to about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units.

[0064] In some embodiments, the compositions described herein comprise a heterogeneous population of extracellular vesicles expressing any number of units of an immune checkpoint moiety described herein on the surface of the extracellular vesicles. In some embodiments, the compositions described herein comprise a homogenous population of extracellular vesicles expressing a range of numbers of units of an immune checkpoint moiety on the surface of the extracellular vesicles. In some embodiments, the homogenous population of extracellular vesicles expresses from about 5 units to about 1,000,000 units of an immune checkpoint moiety. In some embodiments, the homogenous population of extracellular vesicles expresses from about 5 units to about 10 units, from about 5 units to about 50 units, from about 5 units to about 100 units, from about 5 units to about 500 units, from about 5 units to about 1,000 units, from about 5 units to about 5,000 units, from about 5 units to about 10,000 units, from about 5 units to about 50,000 units, from about 5 units to about 100,000 units, or from about 5 units to about 500,000 units, approximately 5 units to approximately 1,000,000 units, approximately 10 units to approximately 50 units, approximately 10 units to approximately 100 units, approximately 10 units to approximately 500 units, approximately 10 units to approximately 1,000 units, approximately 10 units to approximately 5,000 units, approximately 10 units to approximately 10,000 units, approximately 10 units to approximately 50,000 units, approximately 10 units to approximately 100,000 units, approximately 10 units 50 units to approximately 500,000 units, approximately 10 units to approximately 1,000,000 units, approximately 50 units to approximately 100 units, approximately 50 units to approximately 500 units, approximately 50 units to approximately 1,000 units, approximately 50 units to approximately 5,000 units, approximately 50 units to approximately 10,000 units, approximately 50 units to approximately 50,000 units, approximately 50 units to approximately 100,000 units, approximately 50 units to approximately 500,000 Units, approximately 50 units to approximately 1,000,000 units, approximately 100 units to approximately 500 units, approximately 100 units to approximately 1,000 units, approximately 100 units to approximately 5,000 units, approximately 100 units to approximately 10,000 units, approximately 100 units to approximately 50,000 units, approximately 100 units to approximately 100,000 units, approximately 100 units to approximately 500,000 units, approximately 100 units to approximately 1,000,000 units, approximately 500 units to approximately 1,000 units, approximately 500 units to approximately 5,000 units, approximately 500 units to approximately 10,000 units, approximately 500 units to approximately 50,000 units, approximately 500 units to approximately 100,000 units, approximately 500 units to approximately 500,000 units, approximately 500 units to approximately 1,000,000 units, approximately 1,000 units to approximately 5,000 units Knit, approximately 1,000 units to approximately 10,000 units, approximately 1,000 units to approximately 50,000 units, approximately 1,000 units to approximately 100,000 units, approximately 1,000 units to approximately 500,000 units, approximately 1,000 units to approximately 1,000,000 units, approximately 5,000 units to approximately 10,000 units, approximately 5,000 units to approximately 50,000 units, approximately 5,000 units Knits to approximately 100,000 units, approximately 5,000 units to approximately 500,000 units, approximately 5,000 units to approximately 1,000,000 units, approximately 10,000 units to approximately 50,000 units, approximately 10,000 units to approximately 100,000 units, approximately 10,000 units to approximately 500,000 units, approximately 10,000 units to approximately 1,000,000 units, approximately 50,000 units In some embodiments, the homogenous population of extracellular vesicles express at least about 5 units, about 10 units, about 50 units, about 100 units, about 50,000 units, about 500,000 units, about 50,000 units, about 1,000,000 units, about 100,000 units, about 500,000 units, about 100,000 units, about 1,000,000 units, or about 500,000 units, about 1,000,000 units of the immune checkpoint moiety. In some embodiments, the homogenous population of extracellular vesicles has a minimum of about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,In some embodiments, the homogenous population of extracellular vesicles expresses at least about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units of the immune checkpoint moiety.

[0065] In some embodiments, the homogenous population of extracellular vesicles expresses a number of units of an immune checkpoint moiety of at most about 5 units to about 1,000,000 units. In some embodiments, the homogenous population of extracellular vesicles expresses a number of units of an immune checkpoint moiety of at most about 5 units to about 10 units, about 5 units to about 50 units, about 5 units to about 100 units, about 5 units to about 500 units, about 5 units to about 1,000 units, about 5 units to about 5,000 units, about 5 units to about 10,000 units, about 5 units to about 50,000 units, about 5 units to about 100,000 units, about 5 units to about 500,000 units, about 5 units to about 1,000,000 units, about 10 Units to about 50 units, about 10 units to about 100 units, about 10 units to about 500 units, about 10 units to about 1,000 units, about 10 units to about 5,000 units, about 10 units to about 10,000 units, about 10 units to about 50,000 units, about 10 units to about 100,000 units, about 10 units to about 500,000 units, about 10 units to about 1,000,000 units, about 50 units to about 100 units, about 50 units to about 500 units, about 50 units 1,000 units, approx. 50 units to approx. 5,000 units, approx. 50 units to approx. 10,000 units, approx. 50 units to approx. 50,000 units, approx. 50 units to approx. 100,000 units, approx. 50 units to approx. 500,000 units, approx. 50 units to approx. 1,000,000 units, approx. 100 units to approx. 500 units, approx. 100 units to approx. 1,000 units, approx. 100 units to approx. 5,000 units, approx. 100 units to approx. 10,000 units, approx. 100 units to approx. 50 ,000 units, approximately 100 units to approximately 100,000 units, approximately 100 units to approximately 500,000 units, approximately 100 units to approximately 1,000,000 units, approximately 500 units to approximately 1,000 units, approximately 500 units to approximately 5,000 units, approximately 500 units to approximately 10,000 units, approximately 500 units to approximately 50,000 units, approximately 500 units to approximately 100,000 units, approximately 500 units to approximately 500,000 units, approximately 500 units to approximately 1,000,000 units, approximately 1,000 units to approximately 5,000 units, approximately 1,000 units to approximately 10,000 units, approximately 1,000 units to approximately 50,000 units, approximately 1,000 units to approximately 100,000 units, approximately 1,000 units to approximately 500,000 units, approximately 1,000 units to approximately 1,000,000 units, approximately 5,000 units to approximately 10,000 units, approximately 5,000 units to approximately 50,000 units, approximately 5,000 units to approximately 100,000 units, approximately 5,000 units to approximately 500,000 units, approximately 5,000 units to approximately 1,000,000 units, approximately 10,000 units to approximately 5 The antibody expresses about 0,000 units, about 10,000 units to about 100,000 units, about 10,000 units to about 500,000 units, about 10,000 units to about 1,000,000 units, about 50,000 units to about 100,000 units, about 50,000 units to about 500,000 units, about 50,000 units to about 1,000,000 units, about 100,000 units to about 500,000 units, about 100,000 units to about 1,000,000 units, or about 500,000 units to about 1,000,000 units. In some embodiments, a homogenous population of extracellular vesicles expresses at most about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units of an immune checkpoint moiety. In some embodiments, a homogenous population of extracellular vesicles expresses at most a minimum of about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, or about 500,000 units of an immune checkpoint moiety. In some embodiments, the homogenous population of extracellular vesicles has a maximum of at most about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,The antibody expresses about 1,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units of the immune checkpoint moiety.

[0066] In some embodiments, the homogenous population of extracellular vesicles expresses a number of units of an immune checkpoint moiety of about 1,000 units to about 10,000 units. In some embodiments, the homogenous population of extracellular vesicles expresses a number of units of an immune checkpoint moiety of about 1,000 units to about 1,500 units, about 1,000 units to about 2,000 units, about 1,000 units to about 2,500 units, about 1,000 units to about 3,000 units, about 1,000 units to about 4,000 units, about 1,000 units to about 5,000 units, about 1,000 units to about 6,000 units, about 1,000 units to about 7,000 units, about 1,000 units to about 8,000 units, about 1,000 units, or about 1,000 units. Approximately 1,500 units to approximately 9,000 units, approximately 1,000 units to approximately 10,000 units, approximately 1,500 units to approximately 2,000 units, approximately 1,500 units to approximately 2,500 units, approximately 1,500 units to approximately 3,000 units, approximately 1,500 units to approximately 4,000 units, approximately 1,500 units to approximately 5,000 units, approximately 1,500 units to approximately 6,000 units, approximately 1,500 units to approximately 7,000 units, approximately 1,500 units to approximately 8,000 units, approximately 1,500 units to approximately 9,000 units Knit, approximately 1,500 units to approximately 10,000 units, approximately 2,000 units to approximately 2,500 units, approximately 2,000 units to approximately 3,000 units, approximately 2,000 units to approximately 4,000 units, approximately 2,000 units to approximately 5,000 units, approximately 2,000 units to approximately 6,000 units, approximately 2,000 units to approximately 7,000 units, approximately 2,000 units to approximately 8,000 units, approximately 2,000 units to approximately 9,000 units, approximately 2,000 units to approximately 10,000 units, approximately 2,5 00 units to approximately 3,000 units, approximately 2,500 units to approximately 4,000 units, approximately 2,500 units to approximately 5,000 units, approximately 2,500 units to approximately 6,000 units, approximately 2,500 units to approximately 7,000 units, approximately 2,500 units to approximately 8,000 units, approximately 2,500 units to approximately 9,000 units, approximately 2,500 units to approximately 10,000 units, approximately 3,000 units to approximately 4,000 units, approximately 3,000 units to approximately 5,000 units, approximately 3,000 units to approximately 6,000 units, approximately 3,000 units to approximately 7,000 units, approximately 3,000 units to approximately 8,000 units, approximately 3,000 units to approximately 9,000 units, approximately 3,000 units to approximately 10,000 units, approximately 4,000 units to approximately 5,000 units, approximately 4,000 units to approximately 6,000 units, approximately 4,000 units to approximately 7,000 units, approximately 4,000 units to approximately 8,000 units, approximately 4,000 units to approximately 9,000 units, approximately 4,000 units to approximately 10,000 units, approximately 5,000 units to approximately 6,000 units, approximately 5,000 units to approximately 7,000 units, approximately 5,000 units to approximately 8,000 units, The number of units of the immune checkpoint moiety expressed is about 5,000 units to about 9,000 units, about 5,000 units to about 10,000 units, about 6,000 units to about 7,000 units, about 6,000 units to about 8,000 units, about 6,000 units to about 9,000 units, about 6,000 units to about 10,000 units, about 7,000 units to about 8,000 units, about 7,000 units to about 9,000 units, about 7,000 units to about 10,000 units, about 8,000 units to about 9,000 units, about 8,000 units to about 10,000 units, or about 9,000 units to about 10,000 units. In some embodiments, the homogenous population of extracellular vesicles express about 1,000 units, about 1,500 units, about 2,000 units, about 2,500 units, about 3,000 units, about 4,000 units, about 5,000 units, about 6,000 units, about 7,000 units, about 8,000 units, about 9,000 units, or about 10,000 units of the immune checkpoint moiety. In some embodiments, the homogenous population of extracellular vesicles express a minimum of about 1,000 units, about 1,500 units, about 2,000 units, about 2,500 units, about 3,000 units, about 4,000 units, about 5,000 units, about 6,000 units, about 7,000 units, about 8,000 units, or about 9,000 units of the immune checkpoint moiety. In some embodiments, the homogenous population of extracellular vesicles comprises up to about 1,500 units, about 2,The antibody expresses about 1,000 units, about 2,500 units, about 3,000 units, about 4,000 units, about 5,000 units, about 6,000 units, about 7,000 units, about 8,000 units, about 9,000 units, or about 10,000 units of the immune checkpoint moiety.

[0067] Transmembrane part In some embodiments, compositions are described herein that include extracellular vesicles containing a transmembrane portion. In some embodiments, the transmembrane portion comprises a full-length protein, a variant thereof, or a fragment thereof. In some embodiments, the transmembrane portion comprises 14-3-3 protein zeta / delta, 4-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase / AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAM10, alkaline phosphatase, alpha-enolase, alpha-synuclein, aminopeptidase N, amyloid beta A4 / APP, annexin 5A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, β-actin (ACTB), β-amyloid 42, caveolin 1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24, CD26 / DPP4, CD29 / ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73, CD81, CD82, CD9, CD90, claudins, claudin 1, cofilin-1, complement-fixing proteins CD55 and CD59, cytosolic heat shock protein 90α, cytosolic heat shock protein 90β, EBV LMP1, EBV LMP2A, EF-1α-1, EF2, EFGR, EGFR VIII, emmprin / CD147, enolase 1α (ENO1), EPCAM, ERBB2, tetraspanins (CD9, CD63, and CD81), fatty acid synthase, fetuin A, flotillin-1, flotillin-2, fructose bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5' nucleotidase, GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 and HSP90), heparan sulfate proteoglycan, heparinase, heterotrimeric G protein, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax, huntingtin, ICAM1, integrin, lactadherin, LAMP1 / 2, LAMP2b,Leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosomal-associated membrane glycoprotein 1, lysosomal-associated membrane glycoprotein 2, MHC class I, MHC class II, MUC1, multidrug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP / Alix, PECAM1, phosphoglycerate kinase, placental prion protein, prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap 1B, resistin, sonic hedgehog (SHH), survivin, syndecan-1, syndecan-4, syntenin-1, transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein, TYRP-2, vacuolar sorting protein 35, or zeta polypeptide (YWHAZ). In some embodiments, the transmembrane portion comprises lactadherin. In some embodiments, the transmembrane portion comprises the C1C2 domain of lactadherin. In some embodiments, the transmembrane portion comprises LAMP2. In some embodiments, the transmembrane portion comprises LAMP-like domain 1 of LAMP2. In some embodiments, the transmembrane portion comprises LAMP2b. In some embodiments, the transmembrane portion comprises a peptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4 (Table 2). In some embodiments, the transmembrane portion comprises a peptide 100% identical to SEQ ID NO: 4. In some embodiments, the transmembrane portion comprises a glycosylphosphatidylinositol (GPI) protein. In some embodiments, the transmembrane portion comprises a glycan portion of GPI. In some embodiments, the transmembrane portion comprises a lipid portion of GPI. Optionally, the transmembrane portion comprises CD63. In some embodiments, the transmembrane portion comprises a peptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5 (Table 2). In some embodiments, the transmembrane portion isThe transmembrane moiety comprises a peptide 100% identical to SEQ ID NO:5. In some embodiments, the transmembrane moiety comprises a modified CD63. In some embodiments, the modified CD63 comprises at least one CD63 transmembrane domain. The transmembrane domain may be CD63 transmembrane domain 1 (TM1), CD63 transmembrane domain 2 (TM2), CD63 transmembrane domain 3 (TM3), CD63 transmembrane domain 4 (TM4), or any combination thereof. In some embodiments, the modified CD63 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CD63 transmembrane domains. In some embodiments, the modified CD63 comprises one transmembrane domain. In some embodiments, the modified CD63 comprises two transmembrane domains. In some embodiments, the modified CD63 comprises three transmembrane domains. In some embodiments, the modified CD63 comprises four transmembrane domains. In some embodiments, the modified CD63 comprises five transmembrane domains. In some embodiments, the modified CD63 comprises six transmembrane domains. In some embodiments, the modified CD63 comprises seven transmembrane domains. In some embodiments, the modified CD63 comprises eight transmembrane domains. In some embodiments, the modified CD63 comprises nine transmembrane domains. In some embodiments, the modified CD63 comprises ten transmembrane domains. In some embodiments, the modified CD63 may be a truncated CD63 in which at least one transmembrane domain has been removed. In some embodiments, the modified CD63 may be a truncation of CD63 and the addition of at least one transmembrane domain of CD63. For example, a modified CD63 may be truncated at the N-terminus to remove TM1 and further include an additional TM3 and TM4, resulting in a modified CD63 comprising transmembrane domains in the following order: CD63, TM2, TM3, TM4, TM3, TM4.

[0068] [Table 2]

[0069] In some embodiments, the modified CD63 comprises an insertion or replacement of a non-transmembrane domain (either a cytoplasmic loop or an extracellular loop) of the modified CD63. In some embodiments, the modified CD63 comprises a cytoplasmic loop inserted or replaced with an immune checkpoint moiety. In some embodiments, the modified CD63 comprises an extracellular loop inserted or replaced with an immune checkpoint moiety. For example, a polypeptide comprising an immune checkpoint moiety can be inserted into an extracellular loop of the modified CD63. Alternatively, a polypeptide comprising an immune checkpoint moiety can replace a fragment of an extracellular loop of the modified CD63. In some embodiments, the immune checkpoint moiety can be fused to a truncated version of the modified CD63. In some embodiments, the immune checkpoint moiety can be fused to the N-terminus of the modified CD63, where the modified CD63 is truncated at the N-terminus to remove at least one of the transmembrane domains and / or at least one of the non-transmembrane domains. In some embodiments, the immune checkpoint moiety can be fused to the N-terminus of a modified CD63, where the modified CD63 is truncated at the N-terminus to remove at least one of the transmembrane domains and / or at least one of the non-transmembrane domains.

[0070] In some embodiments, the immune checkpoint moiety can be fused to a transmembrane moiety, such as CD63, via a linker, hi some embodiments, the linker is a linker peptide. In addition to connecting the transmembrane segments, linker peptides may also provide many other functions, such as maintaining cooperative interdomain interactions and maintaining biological activity (Gokhale RS, Khosla C. Role of linkers in communication between protein modules. Curr Opin Chem Biol. 2000; 4: 22-27; Ikebe M, Kambara T, Stafford WF, Sata M, Katayama E, Ikebe RA hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. J Biol Chem. 1998; 273: 17702-17707; and Chen XY, Zaro J, and Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 2014; 65, 1357-1369, all of which are cited herein). Linker peptides can be grouped into small, medium, and large linkers, with average lengths of up to 4.5±0.7, 9.1±2.4, and 21.0±7.6 residues, respectively, although all examples within these three bounding sets are contemplated. In some embodiments, the linker peptide comprises 5-200 amino acids. In some embodiments, the linker peptide comprises 5-25 amino acids. In some embodiments, the linker peptide is cleavable (e.g., a linker peptide comprising a peptide sequence recognized and cleavable by Tev protease).

[0071] SEQ ID NOs: 6-17 exemplify various configurations of the immune checkpoint moiety PD-L1 fused to the transmembrane moiety CD63 (Table 3).

[0072] [Table 3-1]

[0073] [Table 3-2]

[0074] [Table 3-3]

[0075] [Table 3-4]

[0076] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 6. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 6 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0077] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 7. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 7 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0078] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 8. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 8 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0079] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 9. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 9 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0080] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 10. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 10 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0081] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 11. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 11 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0082] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 12. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 12 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0083] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 13. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 13 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0084] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 14. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 14 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0085] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 15. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 15 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0086] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 16. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 16. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 16 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0087] In some embodiments, the extracellular vesicles described herein express a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17. In some embodiments, the extracellular vesicles described herein express a polypeptide that is 100% identical to SEQ ID NO: 17. In some embodiments, the expressed polypeptide corresponding to SEQ ID NO: 17 is partially embedded in the membrane of the extracellular vesicle and partially expressed on the surface of the extracellular vesicle.

[0088] In some embodiments, the transmembrane portion can be complexed with an immune checkpoint moiety described herein. In some embodiments, the transmembrane portion can be non-covalently complexed with an immune checkpoint moiety described herein. In some embodiments, the transmembrane portion can be covalently complexed with an immune checkpoint moiety described herein. In some embodiments, the transmembrane portion can be fused to an immune checkpoint moiety described herein at the N-terminus of the transmembrane portion. In some embodiments, the transmembrane portion can be fused to an immune checkpoint moiety described herein at the C-terminus of the transmembrane portion.

[0089] In some embodiments, the immune checkpoint moiety comprises a therapeutic property for treating a disease or disorder. In some embodiments, the immune checkpoint moiety comprises a therapeutic property for treating an autoimmune disease as described herein. In some embodiments, the immune checkpoint moiety targets and modulates the activity of an immune cell. In some embodiments, the immune cell can be a T cell, including a cytotoxic T cell, a natural killer T cell, a regulatory T cell, and a T helper cell.

[0090] targeting part Some embodiments described herein include extracellular vesicles containing a targeting moiety. In some embodiments, the targeting moiety may be expressed on the surface of the extracellular vesicle. In some embodiments, the targeting moiety may be secreted by the extracellular vesicle. The localization of the extracellular vehicle containing the targeting moiety in the target cell or target environment is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold higher than that of the extracellular vesicle lacking the targeting moiety. In some embodiments, the targeting moiety includes EBV glycoprotein 350, which targets CD19+ B cells. In some embodiments, the targeting moiety includes Lamp2b, which targets acetylcholine receptors on neurons. In some embodiments, the targeting moiety includes the C1C2 domain of lactadherin, which targets immune cells or blood cells. In some embodiments, the targeting moiety includes EGFR or PDGFR, which targets cells expressing EGFR. In some embodiments, the targeting moiety comprises a GPI-anchored membrane protein.

[0091] In some embodiments, the targeting moiety may target a cell surface protein or a protein secreted by the target cell. Non-limiting examples of cell surface or secreted proteins include any one of the chemokines described herein.

[0092] Pharmaceutical Composition Described herein are pharmaceutical compositions comprising the compositions described herein. In some embodiments, the pharmaceutical composition comprises both a composition comprising extracellular vesicles and cells secreting the extracellular vesicles. As used herein, a pharmaceutical composition refers to a mixture of a therapeutic agent comprising extracellular vesicles with other chemical components (i.e., pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, fillers, suspending agents, flavorings, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, humectants, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. Optionally, the composition comprises two or more therapeutic agents (e.g., one or more therapeutic agents and one or more additional agents) as discussed herein. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a therapeutic agent described herein is administered in a pharmaceutical composition to a mammal suffering from a disease, disorder, or condition to be treated, such as an autoimmune disease. In some embodiments, the mammal is a human. Therapeutically effective amounts may vary depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents described herein can be used alone or in combination with one or more other therapeutic agents as components of mixtures.

[0093] The pharmaceutical formulations described herein are administered to a subject by a suitable route of administration, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, oral mucosal, inhalation, or intraperitoneal.The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multiparticulate formulations, and immediate mixing and controlled release formulations.

[0094] Pharmaceutical compositions containing the therapeutic agent may be manufactured in conventional manner, such as by way of example only, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, compressing processes.

[0095] Pharmaceutical compositions may include at least one therapeutic agent as an active ingredient in free acid or free base form, or in a pharmaceutically acceptable salt form. Additionally, the methods and pharmaceutical compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, as well as active metabolites of the foregoing compounds having the same type of activity. In some embodiments, the therapeutic agent exists in an unsolvated form or a solvated form with a pharmaceutically acceptable solvent, such as water or ethanol. Solvated forms of the therapeutic agent are also considered to be disclosed herein.

[0096] In some embodiments, therapeutic agents exist as tautomers. All tautomers are included within the scope of the agents provided herein. It is therefore understood that therapeutic agents or their salts may exhibit the phenomenon of tautomerism, whereby two chemical compounds can be readily interconverted by exchanging a hydrogen atom between two atoms to form a covalent bond to one of them. Tautomeric compounds may be considered different isomeric forms of the same compound because they exist in equilibrium with one another.

[0097] In some embodiments, therapeutic agents exist as enantiomers, diastereomers, or other stereoisomeric forms. The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms, as well as mixtures thereof.

[0098] In some embodiments, the therapeutic agents described herein may be prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, depending on the situation, the prodrug may be easier to administer than the parent drug. A prodrug may be bioavailable, for example, by oral administration, while the parent drug is not. A prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A non-limiting example of a prodrug is a therapeutic agent described herein. The therapeutic agent is administered as an ester ("prodrug") to facilitate transport across cell membranes, where aqueous solubility would impair mobility, but is then metabolically hydrolyzed to the active carboxylic acid moiety once inside the cell, where aqueous solubility is beneficial. A further example of a prodrug may be a short-chain peptide (polyamino acid) linked to an acid group that reveals the active moiety upon metabolism of the peptide. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to the biologically, pharmaceutically, or therapeutically active form of the therapeutic agent. In certain embodiments, the prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically, or therapeutically active form of the therapeutic agent.

[0099] Prodrug forms of therapeutic agents, where the prodrug is metabolized in vivo to produce a drug as specified herein, are included within the scope of the claims. Prodrug forms of therapeutic agents described herein, where the prodrug is metabolized in vivo to produce a drug as specified herein, are included within the scope of the claims. In some cases, some of the therapeutic agents described herein may be prodrugs to another derivative or active compound. In some embodiments described herein, hydrazones are metabolized in vivo to produce a therapeutic agent.

[0100] In some embodiments, the compositions provided herein contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridium chloride.

[0101] In some embodiments, the formulations described herein benefit from antioxidants, metal chelators, thiol-containing compounds, and other general stabilizing agents. Examples of such stabilizers include (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) heparinoids such as pentosan polysulfate, (m) divalent cations such as magnesium or zinc, or (n) combinations thereof.

[0102] The pharmaceutical compositions described herein can be formulated into any suitable dosage form, including, but not limited to, oral aqueous dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, fast-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed-release formulations, extended-release formulations, pulsed-release formulations, multiparticulate formulations, and mixtures of immediate-release and controlled-release formulations. In one aspect, the therapeutic agent as described herein, for example, a therapeutic agent, is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders that are reconstituted into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, cremophor, and the like), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. In some cases, it may be desirable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents delaying absorption, such as aluminum monostearate or gelatin.

[0103] For intravenous injection, infusion, or infusion, the therapeutic agents described herein are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or buffered saline. For oral mucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0104] Parenteral injection may require bolus injection or continuous infusion. Injectable preparations may be provided in unit dosage form, for example, in ampoules or multi-dose containers, with additional preservatives. The pharmaceutical compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous solvent, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable solvent, for example, pyrogen-free distilled water, before use.

[0105] For inhalation administration, the therapeutic agent is formulated for use as an aerosol, mist, or powder. The pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray dispensed from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. By way of example only, capsules and cartridges, such as gelatin, used in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base, such as lactose or starch.

[0106] Representative intranasal formulations are described, for example, in U.S. Patent Nos. 4,476,116, 5,116,817, and 6,391,452. Formulations containing therapeutic agents are prepared as solutions in saline, utilizing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. See, e.g., Ansel, H.C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably, these compositions and formulations are prepared with suitable, non-toxic, pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the art in the preparation of nasal dosage forms, some of which can be found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005. The selection of an appropriate carrier will depend on the exact nature of the desired nasal dosage form, e.g., solution, suspension, ointment, or gel. Nasal dosage forms generally contain a large amount of water in addition to the active ingredient.Other components, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffering agents, and other stabilizers and solubilizers, may also optionally exist in small amounts.Preferably, nasal dosage forms should be isotonic with nasal secretions.

[0107] Oral pharmaceutical preparations can be prepared by mixing one or more solid excipients with one or more therapeutic agents described herein, optionally milling the resulting mixture, and processing the granular mixture, after adding suitable additives as needed, to obtain tablets or dragee cores. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose; or other excipients, such as polyvinylpyrrolidone (PVP or povidone) and calcium phosphate. If necessary, disintegrants, such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt of alginic acid, such as sodium alginate, are added. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic doses.

[0108] In some embodiments, the pharmaceutical formulation of the therapeutic agent is in the form of a capsule, including push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. Capsules can be prepared, for example, by placing a large mixture of the therapeutic agent formulation inside the capsule. In some embodiments, the formulation (non-aqueous suspensions and solutions) is placed in a soft gelatin capsule. In other embodiments, the formulation is placed in a standard gelatin or non-gelatin capsule, such as a capsule containing HPMC. In other embodiments, the formulation is placed in a sprinkle capsule. The capsule is swallowed whole or opened and the contents are sprinkled on food before eating.

[0109] All formulations for oral administration are in dosage forms suitable for such administration. In one aspect, solid oral dosage forms are prepared by mixing a therapeutic agent with one or more carrier materials, such as antioxidants, flavoring agents, and binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage forms disclosed herein are in the form of tablets (including suspension tablets, fast-dissolving tablets, bite-disintegration tablets, rapid-disintegration tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, bioerodible dosage forms, controlled-release formulations, pulsed-release dosage forms, multiparticulate dosage forms, beads, pellets, or granules. In other embodiments, the pharmaceutical formulation is in the form of a powder. Compressed tablets are solid dosage forms prepared by compression of bulk mixtures of the above-mentioned formulations. In various embodiments, the tablets contain one or more flavoring agents. In other embodiments, the tablet comprises a film surrounding the final compressed tablet. In some embodiments, the film coating can slow the release of the therapeutic agent from the formulation. In other embodiments, the film coating aids patient compliance (e.g., Opadry® coating or sugar coating). Film coatings, including Opadry®, typically comprise about 1% to about 3% by weight of the tablet. In some embodiments, solid dosage forms, such as tablets, effervescent tablets, and capsules, are prepared by blending particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, individual unit doses comprise a film coating. These formulations are manufactured by conventional formulation techniques.

[0110] In another aspect, the dosage form comprises a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Exemplary materials include, but are not limited to, pH adjusters, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Exemplary useful microencapsulation materials include hydroxypropyl cellulose ethers (HPC), such as Klucel® or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methylcellulose ethers (HPMC), such as Seppifilm-LC, Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers, such as Methocel®-A, hydroxypropyl methylcellulose acetate stearate Aqoat (HF-LS, HF-LG, HF-MS), Metolose®, ethylcellulose (EC) and mixtures thereof, such as E461, Ethocel®, Aqualon®-EC, Surelease®, Opadry®, and the like. Polyvinyl alcohol (PVA) such as AMB, hydroxyethylcellulose such as Natrosol®, carboxymethylcellulose and salts of carboxymethylcellulose (CMC), such as Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol copolymers such as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycol, modified food starch, acrylic polymers, acrylic polymers with Eudragit® EPO, Eudragit® L30D-55, Eudragit® FS 30DThese include, but are not limited to, mixtures with cellulose ethers such as Eudragit® L100-55, Eudragit® L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5, Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.

[0111] The dosage form of the liquid formulation for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable oral aqueous dispersions, emulsions, solutions, elixirs, gels, and syrups. See, for example, Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to the therapeutic agent, the liquid dosage form optionally contains additives such as (a) disintegrants, (b) dispersants, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further contains a crystal formation inhibitor.

[0112] In some embodiments, the pharmaceutical formulations described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, usually in the form of droplets. Typically, emulsions are created by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to an excess of water without the need for any external mechanical dispersion or stirring. The advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or an aqueous phase is optionally added immediately prior to administration, ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS improve the bioavailability of hydrophobic active ingredients. Methods for producing self-emulsifying dosage forms include, but are not limited to, US Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.

[0113] Buccal dosage forms containing therapeutic agents can be administered using various dosage forms known in the art.For example, such formulations include, but are not limited to, U.S. Patent Nos. 4,229,447, 4,596,795, 4,755,386 and 5,739,136.In addition, the buccal dosage forms described herein can further comprise a biodegradable (hydrolyzable) polymer carrier, which also serves to attach the dosage form to the buccal mucosa.For buccal or sublingual administration, the composition can be in the form of tablets, lozenges or gels formulated in a conventional manner.

[0114] For intravenous injections, the therapeutic agent is formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or buffered saline. For oral mucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, suitable formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[0115] Parenteral injections optionally require bolus injection or continuous infusion. Injectable formulations are optionally provided in unit dosage form, e.g., in ampoules or multi-dose containers, with added preservatives. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection as a sterile suspension, aqueous solution, or emulsion in an oily or aqueous solvent, and contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of agents that modulate the activity of the carotid body in aqueous solution form. Additionally, suspensions of agents that modulate the activity of the carotid body are optionally prepared as suitable, e.g., oily injection suspensions.

[0116] Conventional formulation techniques include, for example, one or a combination of the following methods: (1) dry blending, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Worcester coating), tangential coating, top spraying, tabletting, extrusion, and the like.

[0117] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol, and the like.

[0118] Suitable fillers for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0119] Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starches such as corn starch or potato starch, pregelatinized starch, or sodium starch glycolate, celluloses such as methylcrystalline cellulose, methylcellulose, microcrystalline cellulose, croscarmellose, or cross-linked celluloses such as cross-linked sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, or cross-linked croscarmellose, cross-linked starches such as sodium starch glycolate, cross-linked polymers such as crospovidone, cross-linked polyvinylpyrrolidone, alginates such as alginic acid or salts of alginic acid such as sodium alginate, gums such as agar, guar, locust bean, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, and the like.

[0120] Binders impart cohesiveness to solid oral dosage formulations. In powder-filled capsule formulations, binders aid in the formation of fillable plugs in soft or hard shell capsules, and in tablet formulations, they ensure that the tablet remains intact after compression and help ensure a uniform mix prior to the compression or filling step. Suitable materials for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose, microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonite, gelatin, polyvinylpyrrolidone / vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, sugars such as sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, lactose, natural or synthetic gums such as acacia, tragacanth, ghatti gum, isapol husk mucilage, starch, polyvinylpyrrolidone, larch arabogalactan, polyethylene glycol, waxes, sodium alginate, and the like.

[0121] Binder levels of 20-70% are commonly used in powder-filled gelatin capsule formulations. Binder levels in tablet formulations vary depending on whether the formulation is direct compression, wet granulation, roller compaction, or the use of other excipients such as fillers that can act as modest binders themselves. Binder levels of up to 70% are common in tablet formulations.

[0122] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, magnesium stearate, zinc stearate, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol, or methoxypolyethylene glycol such as Carbowax®, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium, or sodium lauryl sulfate.

[0123] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and cyclodextrins), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins, and the like.

[0124] Suitable humectants for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS, and the like.

[0125] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, poloxamers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, such as Pluronic® (BASF), and the like.

[0126] Suitable suspending agents for use in the solid dosage forms described herein include polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, polyvinylpyrrolidone K30, polyethylene glycol (e.g., polyethylene glycol having a molecular weight of about 300 to about 6000, about 3350 to about 4000, or about 7000 to about 5400), vinylpyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, polysorbate 8, 0, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum, acacia gum, guar gum, xanthan including xanthan gum, sugars, cellulosics such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone.

[0127] Suitable antioxidants for use in the solid dosage forms described herein include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

[0128] It should be understood that there is considerable overlap between the additives used in the solid dosage forms described herein.Therefore, the additives listed above are merely examples of the types of additives that can be included in the solid dosage forms of the pharmaceutical compositions described herein, and should not be construed as limiting.The amount of such additives can be easily determined by those skilled in the art according to the specific properties desired.

[0129] In various embodiments, particles of the therapeutic agent and one or more excipients are dry mixed and compressed into a mass, such as a tablet, that has sufficient hardness to provide a pharmaceutical composition that substantially disintegrates in less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, or less than about 60 minutes after oral administration, thereby releasing the formulation into gastrointestinal fluids.

[0130] In other embodiments, powders containing a therapeutic agent are formulated to include one or more pharmaceutical excipients and flavorings. Such powders are prepared, for example, by mixing a therapeutic agent and, optionally, a pharmaceutical excipient to form a bulk blend composition. Additional embodiments also include a suspending agent and / or a wetting agent. This bulk blend is then uniformly subdivided into units in unit-dose or multi-dose packages.

[0131] In yet another embodiment, effervescent powders are also prepared. Effervescent salts are used to disperse medications in water for oral administration.

[0132] In some embodiments, pharmaceutical dosage forms are formulated to provide controlled release of a therapeutic agent. Controlled release refers to the release of a therapeutic agent from a dosage form into which it is incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, extended release, pulsed release, and delayed release profiles. In contrast to immediate-release compositions, controlled-release compositions allow for drug delivery to a subject over an extended period of time according to a predetermined profile. Such release rates provide therapeutically effective levels of the drug over an extended period of time, thereby providing a longer-term pharmacological response while minimizing side effects compared to conventional rapid-release dosage forms. Such extended response offers many unique benefits not achieved by corresponding short-acting immediate-release preparations.

[0133] In some embodiments, the solid dosage forms described herein are formulated as enterically coated delayed-release oral dosage forms, i.e., oral dosage forms of pharmaceutical compositions as described herein, utilizing an enteric coating that affects release in the small or large intestine. In one aspect, the enterically coated dosage forms are compressed, molded, or extruded tablets / molds (coated or uncoated) that themselves contain coated or uncoated granules, powders, pellets, beads, or particles of the active ingredient and / or other composition ingredients. In one aspect, the enterically coated oral dosage form is in the form of a capsule containing pellets, beads, or granules, which contain a coated or uncoated therapeutic agent.

[0134] Any coating must be applied thick enough that the entire coating is insoluble in gastrointestinal fluids below a pH of about 5 but soluble at pH 5 or above. Coatings are typically selected from shellac (which dissolves in media with a pH greater than 7) or acrylic polymers (examples of suitable acrylic polymers include methacrylic acid copolymers and ammonium methacrylate copolymers). Eudragit series E, L, S, RL, RS, and NE (Rohm Pharma) are available solubilized in organic solvents, aqueous dispersions, or dry powders. Eudragit series RL, NE, and RS are insoluble in the gastrointestinal tract but permeable and are primarily used for colonic targeting. Eudragit series E dissolves in the stomach. Eudragit series L, L-30D, and S are insoluble in the stomach but dissolve in the intestine. Polyvinyl acetate phthalate (PVAP) dissolves at a pH greater than 5 and has low permeability to water vapor and gastric fluids. Conventional coating techniques such as spray coating or pan coating are used to apply the coating. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until it reaches the desired site of topical delivery, which is the intestinal tract.

[0135] In other embodiments, the formulations described herein are delivered using a pulsatile dosage form. Pulsatile dosage forms can provide one or more immediate-release pulses at predetermined time points or at specific sites after an adjusted delay time. Exemplary pulsatile dosage forms and methods for their manufacture are disclosed in U.S. Patent Nos. 5,011,692, 5,017,381, 5,229,135, 5,840,329, and 5,837,284. In one embodiment, the pulsatile dosage form comprises at least two groups of particles (i.e., multiparticulates), each containing a formulation described herein. The first group of particles provides a substantially immediate dose of the therapeutic agent after ingestion by a mammal. The first group of particles may be uncoated or may include a coating and / or sealant. In one aspect, the second group of particles comprises coated particles. The coating on the second group of particles provides a delay of about 2 to about 7 hours after ingestion before the release of the second dose. Suitable coatings for pharmaceutical compositions are described herein or known in the art.

[0136] In some embodiments, a pharmaceutical formulation is provided that includes particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which, when mixed with water, results in a substantially uniform suspension.

[0137] In some embodiments, particles formulated for controlled release are incorporated into a gel, patch, or wound dressing.

[0138] In one aspect, the liquid formulation for oral administration and / or topical administration as a cleanser is in the form of an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable oral aqueous dispersions, emulsions, solutions, elixirs, gels, and syrups. See, for example, Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to particles of the therapeutic agent, the liquid dosage form contains additives such as (a) disintegrants, (b) dispersants, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion may further contain a crystallization inhibitor.

[0139] In some embodiments, the liquid formulation also contains inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers. Exemplary emulsifiers include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, sodium lauryl sulfate, docusate sodium, cholesterol, cholesterol esters, taurocholic acid, phosphatidylcholine, oils such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, or mixtures of these substances.

[0140] Additionally, the pharmaceutical compositions optionally contain pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, hydrochloric acid, etc., bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, trishydroxymethylaminomethane, etc., and buffers such as citrate / dextrose, sodium bicarbonate, ammonium chloride, etc. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0141] In addition, the pharmaceutical composition optionally contains one or more salts in an amount necessary to bring the osmolality of the pharmaceutical composition into an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0142] Other pharmaceutical compositions optionally contain one or more preservatives that inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridium chloride.

[0143] In one embodiment, the aqueous suspensions and dispersions described herein remain homogeneous as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905) for at least 4 hours. In one embodiment, the aqueous suspensions are resuspended into a homogeneous suspension by physical agitation lasting less than 1 minute. In another embodiment, agitation is not required to maintain a homogeneous aqueous dispersion.

[0144] Examples of disintegrants used in aqueous suspensions and dispersions include, but are not limited to, starches, for example, natural starches such as corn starch or potato starch, pregelatinized starch or sodium starch glycolate, celluloses such as methylcrystalline cellulose, methylcellulose, croscarmellose, or cross-linked celluloses, for example, cross-linked sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, or cross-linked croscarmellose, cross-linked starches such as sodium starch glycolate, cross-linked polymers such as crospovidone, cross-linked polyvinylpyrrolidone, alginates such as alginic acid or salts of alginic acid such as sodium alginate, gums such as agar, guar, locust bean, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combined starches, and the like.

[0145] In some embodiments, dispersing agents suitable for the aqueous suspensions and dispersions described herein include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone, and carbohydrate-based dispersing agents such as hydroxypropyl cellulose and hydroxypropyl cellulose ethers, hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate, hydroxypropylmethyl-cellulose acetate stearate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone / vinyl acetate copolymer, 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers, and poloxamines. In other embodiments, the dispersing agent is selected from the group that does not include one of the following agents: hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP), hydroxypropyl cellulose and hydroxypropyl cellulose ethers, hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate, hydroxypropylmethyl-cellulose acetate stearate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), 4-(1,1,3,3-tetramethylbutyl)-phenol polymers with ethylene oxide and formaldehyde, poloxamers, or poloxamines.

[0146] Suitable wetting agents for aqueous suspensions and dispersions described herein include, but are not limited to, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g., commercially available Tweens such as Tween 20 and Tween 80), polyethylene glycol, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodium taurocholate, simethicone, phosphatidylcholine, and the like.

[0147] Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methylparaben and propylparaben), benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. Preservatives, as used herein, are incorporated into the dosage form at concentrations sufficient to inhibit microbial growth.

[0148] Suitable viscosity enhancing agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Plasdon® S-630, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof. The concentration of the viscosity enhancing agent will depend on the agent selected and the desired viscosity.

[0149] Examples of sweeteners suitable for the aqueous suspensions or dispersions described herein include, for example, acacia syrup, acesulfame K, alitame, aspartame, chocolate, cinnamon, citrus, cocoa, cyclamate, dextrose, fructose, ginger, glycyrrhetinate, licorice (licorice) syrup, monoammonium glycyrrhizinate (MagnaSweet®), malitol, mannitol, menthol, neohesperidin DC, neotame, Prosweet® Powder, saccharin, sorbitol, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, sucralose, tagatose, thaumatin, vanilla, xylitol, or any combination thereof.

[0150] In some embodiments, the therapeutic agent is prepared as a transdermal dosage form. In some embodiments, the transdermal formulations described herein comprise at least three components: (1) a therapeutic agent, (2) a penetration enhancer, and (3) an optional aqueous adjuvant. In some embodiments, the transdermal formulation may include additional compounds, such as, but not limited to, a gelling agent, a cream, or an ointment base. In some embodiments, the transdermal formulation is provided as a patch or wound dressing. In some embodiments, the transdermal formulation further comprises a woven or nonwoven backing to facilitate absorption and prevent removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein can be maintained in a saturated or supersaturated state to facilitate distribution across the skin.

[0151] In one aspect, formulations suitable for transdermal administration of the therapeutic agents described herein utilize transdermal delivery devices and transdermal delivery patches and can be lipid-soluble emulsions or buffered aqueous solutions dissolved and / or dispersed in a polymer or adhesive. In one aspect, such patches are constructed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents. Additionally, transdermal delivery of the therapeutic agents described herein can be achieved by iontophoretic patches, etc. In one aspect, the transdermal patch provides controlled delivery of the therapeutic agent. In one aspect, the transdermal device is in the form of a bandage comprising a backing member, a reservoir containing the therapeutic agent, optionally with a carrier, optionally a rate-limiting barrier for delivering the therapeutic agent to the host's skin at a controlled and predetermined rate over an extended period of time, and a means for securing the device to the skin.

[0152] In further embodiments, the topical formulation comprises a gel formulation (e.g., a gel patch that adheres to the skin). In some such embodiments, the gel composition comprises any polymer that forms a gel upon contact with the body (e.g., gel formulations comprising hyaluronic acid, Pluronic® polymers, poly(lactic-co-glycolic acid) (PLGA)-based polymers, etc.). In some composition forms, the formulation comprises a low-melting wax, such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter, which is melted first. Optionally, the formulation further comprises a moisturizer.

[0153] In certain embodiments, delivery systems for medical therapeutic agents may be utilized, such as, for example, liposomes or emulsions. In certain embodiments, the compositions provided herein may also include a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0154] In some embodiments, the therapeutic agents described herein may be administered topically and may be formulated into a variety of topically administrable compositions such as solutions, suspensions, lotions, gels, pastes, medicated sticks, pain relievers, creams, or ointments. Such medicinal therapeutic agents may include solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

[0155] Nasal aerosol formulations are aqueous solutions designed to be administered to the nasal passages as a whole in drops or sprays. Nasal solutions are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range may also be used, similar to nasal secretions. Antimicrobial agents or preservatives may also be included in the formulation.

[0156] Inhalants and aerosol formulations for inhaled medications may be designed to deliver the drug or drug combination to the subject's respiratory tree when administered via the nasal or oral respiratory route. Inhalation solutions can be administered, for example, by a nebulizer. Inhalants or insufflations containing finely divided or liquid drugs can be delivered to the respiratory system, for example, as medicinal aerosols of a solution or suspension of the drug or drug combination in a propellant to aid distribution. Propellants can be liquefied gases, including fluorocarbons, such as halogenated carbons, e.g., fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[0157] Halocarbon propellants may include fluorocarbon propellants in which all hydrogens are replaced by fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced by chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Useful hydrocarbon propellants include, for example, propane, isobutane, n-butane, pentane, isopentane, and neopentane. Mixtures of hydrocarbons can also be used as propellants. Ether propellants include, for example, dimethyl ether, as well as ethers. Aerosol formulations may also contain more than one propellant. For example, aerosol formulations may contain more than one propellant from the same class, such as two or more fluorocarbons, or more than one, two, or three propellants from different classes, such as a fluorohydrocarbon and a hydrocarbon. The pharmaceutical compositions of the present disclosure may also be dispensed with a compressed gas, such as an inert gas, such as carbon dioxide, nitrous oxide, or nitrogen.

[0158] The aerosol formulation may also contain other ingredients, such as ethanol, isopropanol, propylene glycol, as well as surfactants or other ingredients such as oils and detergents, which may serve to stabilize the formulation and / or lubricate the valve elements.

[0159] Aerosol formulations can be packaged under pressure and may be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid preparations. For example, aerosol formulation solutions may contain a solution of a drug, such as a transporter, carrier, or ion channel inhibitor, in a (substantially) pure propellant or as a mixture of a propellant and a solvent. The solvent can be used to dissolve the drug and / or slow the evaporation of the propellant. Solvents may include, for example, water, ethanol, and glycol. Any suitable solvent combination can be used, optionally combined with preservatives, antioxidants, and / or other aerosol components.

[0160] The aerosol formulation may be a dispersion or a suspension. The aerosol formulation suspension may comprise a suspension of a drug or a drug combination, such as a transporter, a carrier, an ion channel inhibitor, and a dispersing agent. The dispersing agent may comprise, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil. The aerosol formulation suspension may further comprise a lubricant, a preservative, an antioxidant, and / or other aerosol components.

[0161] Aerosol formulations may also be formulated as emulsions. Aerosol formulation emulsions may contain, for example, alcohol (e.g., ethanol), surfactants, water, and propellants, as well as drugs or drug combinations, such as transporters, carriers, and ion channels. The surfactants used may be nonionic, anionic, or cationic. One example of an aerosol formulation emulsion contains, for example, ethanol, surfactants, water, and propellants. Another example of an aerosol formulation emulsion contains, for example, vegetable oil, glyceryl monostearate, and propane.

[0162] kit In some embodiments, disclosed herein are kits for using the compositions described herein. In some embodiments, the kits disclosed herein can be used to treat a disease or disorder in a subject, select a subject for treatment, and / or monitor the treatments disclosed herein. In some embodiments, the kits include compositions described herein that can be used to perform the methods described herein. The kits are a collection of materials or components, including at least one of the compositions. Thus, in some embodiments, the kits contain compositions, including pharmaceutical compositions, for treating autoimmune diseases.

[0163] In some examples, the kits described herein include components for selecting a homogenous population of extracellular vesicles. In some embodiments, the kits include components for analyzing the number of units of an immune checkpoint moiety expressed on the surface of extracellular vesicles. In some embodiments, the kits include components for performing analyses such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, qPCR, etc. The exact nature of the components included in the kit depends on its intended purpose. For example, some embodiments are configured for treating a disease or disorder disclosed herein (e.g., an autoimmune disease) in a subject. In some embodiments, the kits are specifically configured for treating a mammalian subject. In some embodiments, the kits are specifically configured for treating a human subject.

[0164] Instructions for use may be included in the kit. Optionally, the kit further includes other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting tools, measuring tools, bandages, or other useful paraphernalia. The materials or components assembled in the kit can be provided to medical professionals and stored in a convenient and appropriate manner to preserve their operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form, and may be provided at room temperature, refrigerated temperature, or frozen temperature. The components are generally contained in suitable packaging. As used herein, the phrase "packaging" refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging material is preferably constructed by well-known methods to provide a sterile, contaminant-free environment. The packaging materials used in the kit are those conventionally used in gene expression assays and performing procedures. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, capable of holding individual kit components. Thus, for example, the package may be a glass vial or a pre-filled syringe used to contain an appropriate amount of the pharmaceutical composition. The packaging material has an exterior label indicating the contents and / or purpose of the kit and its component parts.

[0165] II. Platform In some embodiments herein, a platform for producing the extracellular vesicles described herein is described. In some embodiments, the platform conforms to Good Manufacturing Practice (GMP) standards. In some embodiments, a composition comprising the extracellular vesicles is produced in accordance with Good Manufacturing Practice (GMP). In some embodiments, the composition comprises a pathogen level that is nearly free of pathogens. In some embodiments, the composition has an impurity level that is nearly free of impurities. In some embodiments, the composition comprises low immunogenicity.

[0166] In some embodiments, the compositions described herein are produced and isolated by hypotonic treatment and centrifugation. In some embodiments, extracellular vesicles are isolated from mesenchymal stem cells (MSCs) that express extracellular vesicles primarily using hypotonic treatment to rupture the MSCs and release the extracellular vesicles. In some examples, MSCs are resuspended in a hypotonic solution to induce cell swelling. In some embodiments, the platform includes a phase-contrast microscope for monitoring cell swelling. In some embodiments, the platform includes a homogenizer for rupturing the swollen cells and releasing the extracellular vesicles. In some embodiments, the platform includes a means for separating the ruptured cells in a gradient (e.g., a sucrose gradient) to isolate the extracellular vesicles. In some embodiments, the platform includes other components for generating extracellular vesicles by other techniques for lysing MSCs, such as mild sonication, freeze-thawing, French press, or needle-passaging. In some embodiments, the platform includes a centrifuge for isolating a fraction containing extracellular vesicles by centrifugation. In some embodiments, the platform comprises a means for separating a fraction containing extracellular vesicles by flotation in a discontinuous sucrose density gradient.

[0167] In some embodiments, the platform includes a means for producing extracellular vesicles by extrusion. In some embodiments, the extrusion process separates and isolates extracellular vesicles based on their size or diameter. An exemplary extrusion process includes the use of membranes with varying pore sizes. The membranes can separate extracellular vesicles based on their size or diameter from a solution containing ruptured MSCs. The extracellular vesicles may be further isolated and reduced in size by continued extrusion with increasingly smaller membrane pore sizes ranging from 150 nm to 10 nm. Upon completion of the final extrusion, the extracellular vesicles may be pelleted by centrifugation. In some embodiments, the platform includes components for sonication, extrusion, high pressure / homogenization, microfluidic manipulation, or detergent dialysis.

[0168] In some embodiments, the platform includes a component for determining the number of units of immune checkpoint moiety per extracellular vesicle.

[0169] III. Method Extracellular vesicle generation In some embodiments, methods for producing the compositions described herein using the platforms described herein are described. In some embodiments, extracellular vesicles may be isolated from cells or secreted by cells. For example, extracellular vesicles can be produced by lysing cells to release extracellular vesicles. In some cases, cells may secrete extracellular vesicles, which can then be isolated.

[0170] In some embodiments, the cells for producing extracellular vesicles can be derived from cell lines, stem cells, primary cells, or differentiated cells. In some embodiments, the donor cells for extracellular vesicles can be selected from the group consisting of human embryonic fibroblasts (HEF), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipose stromal cells, endothelial cells, enucleated cells, neural stem cells, immature dendritic cells, and immune cells, bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPCs), intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, umbilical cord stem cells, progenitor cells, muscle progenitor cells, myoblasts, cardiac myoblasts, neural progenitor cells, glial progenitor cells, neuronal progenitor cells, or hepatoblasts.

[0171] In some embodiments, the cells for producing extracellular vesicles may be genetically modified cells, in which case the genetic modification moiety is introduced into the modified cells. In some embodiments, at least one heterologous polynucleotide encoding a transgene is introduced into the modified cells. In some embodiments, the heterologous polynucleotide encodes any one of the immune checkpoint moieties described herein. In some embodiments, the heterologous polynucleotide encodes any one of the targeting moieties described herein. In some embodiments, the heterologous polynucleotide encodes any one of the transmembrane moieties described herein. In some embodiments, the heterologous polynucleotide encodes any one of the fusion peptides described herein. In some embodiments, the heterologous polynucleotide encodes any one of the immune evasion moieties described herein. In some embodiments, the heterologous polynucleotide may be integrated into the chromosome of the modified cell. In some embodiments, the heterologous polynucleotide is not integrated into the chromosome of the modified cell.

[0172] In some embodiments, the genetic modification moiety regulates expression of the heterologous polynucleotide. In some embodiments, the genetic modification moiety increases expression of the heterologous polynucleotide. In some embodiments, the genetic modification moiety comprises a CRISPR-Cas polypeptide. In some embodiments, the genetic modification moiety can be, for example, a Class 1 CRISPR-associated (Cas) polypeptide, a Class 2 Cas polypeptide, a Type I Cas polypeptide, a Type II Cas polypeptide, a Type III Cas polypeptide, a Type IV Cas polypeptide, a Type V Cas polypeptide, and a Type VI CRISPR-associated RNA-binding protein, or a functional fragment thereof. Suitable Cas polypeptides for use in the present disclosure include Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, CaslOd, CasF, CasG, CasH, Cs yl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cul966, and derivatives, mutants, or any fragments thereof. In some embodiments, Cas13 may include, but is not limited to, Cas13a, Cas13b, Cas13c, and Cas13d (e.g., CasRx). CRISPR / Cas may be DNA and / or RNA cleaving or may exhibit reduced cleavage activity. The genetic modification portion may be configured to form a complex with at least one heterologous RNA polynucleotide.Optionally, the genetic modification moiety may be fused to a transcriptional activator or a transcriptional repressor.

[0173] Any suitable nuclease (eg, endonuclease) can be used as the genetic modification moiety. Suitable nucleases include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides, zinc finger nucleases (ZFNs), nuclease activator-like effector nucleases (TALENs), meganucleases, RNA-binding proteins (RBPs), CRISPR-associated RNA-binding proteins, recombinases, flippases, transposases, Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and extreme halophilic Argonaute (Natronobacterium gregoryi Argonaute (NgAgo), adenosine deaminase acting on RNA (ADAR), CIRT, PUF, homing endonuclease, or any functional fragment thereof, any derivative, and mutant thereof, and any fragment thereof, but are not limited to these.

[0174] Genetic modification moieties as disclosed herein can be attached (e.g., linked or fused) to additional peptide sequences that are not involved in regulating gene expression, such as linker sequences, targeting sequences, etc. The term "targeting sequence," as used herein, refers to a nucleotide sequence and corresponding amino acid sequence that encodes a targeting polypeptide that mediates the localization (or retention) of a protein to a subcellular location, such as the cell membrane or the membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome, or other organelle. For example, a targeting sequence can direct a protein (e.g., a receptor polypeptide or an adapter polypeptide) to the nucleus using a nuclear localization signal (NLS), the outside of the cell's nucleus (e.g., the cytoplasm) using a nuclear export signal (NES), mitochondria using a mitochondrial targeting signal, the endoplasmic reticulum (ER) using an ER retention signal, peroxisomes using a peroxisomal targeting signal, the cell membrane using a membrane localization signal, or a combination thereof.

[0175] The genetically modified portion as disclosed herein may be part of a fusion construct (e.g., a fusion protein). As used herein, "fusion" may refer to a protein and / or nucleic acid comprising one or more non-native sequences (e.g., moieties). A fusion may comprise one or more of the same non-native sequences. A fusion may comprise one or more different non-native sequences. A fusion may be chimeric. A fusion may comprise a nucleic acid affinity tag. A fusion may comprise a barcode. A fusion may comprise a peptide affinity tag. A fusion may provide subcellular localization of a polypeptide directed to a site (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). A fusion may provide a non-native sequence (e.g., an affinity tag) that can be used for tracking or purification. The fusion can be a small molecule such as biotin, or a dye such as an Alexa fluor dye, Cyanine3 dye, or Cyanine5 dye.

[0176] A fusion may refer to any protein that has a functional effect. For example, the fusion protein can comprise a methyltransferase activity, a demethylase activity, a dismutase activity, an alkylating activity, a depurinating activity, an oxidizing activity, a pyrimidine dimer forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity (e.g., a reverse transcriptase activity), a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, a remodeling activity, a protease activity, an oxidoreductase activity, a transferase activity, a hydrolase activity, a lyase activity, an isomerase activity, a synthase activity, a synthetase activity, or a demyristoylating activity. The effector protein may modify a genomic locus. The fusion protein may be a fusion in a Cas protein. The fusion protein may be a non-native sequence in a Cas protein.

[0177] In some embodiments, the genetic modification portion can be fused to one or more transcriptional repressor domains, activator domains, epigenetic domains, recombinase domains, transposase domains, flippase domains, nickase domains, or any combination thereof. The activator domain can include one or more tandem activation domains located at the carboxyl terminus of the protein. Optionally, the genetic modification portion can include one or more tandem repressor domains located at the carboxyl terminus of the protein. Exemplary activation domains include, but are not limited to, GAL4, herpes simplex activation domain VP16, VP64 (tetramer of herpes simplex activation domain VP16), NF-κB p65 subunit, and Epstein-Barr virus R transactivator (Rta), as described in Chavez et al., Nat Methods, 2015, 12(4):326-328. Exemplary repressor domains include, but are not limited to, the KRAB (Kruppel-associated box) domain of Kox1, the Mad mSIN3-interacting domain (SID), and the ERF repressor domain (ERD), as described in Chavez et al., Nat Methods, 2015, 12(4):326-328. In some embodiments, the genetically modified portion comprises one or more tandem repressor domains located at the amino terminus of the protein.

[0178] In some embodiments, the nucleases disclosed herein can be proteins lacking nucleic acid cleavage activity. Optionally, the Cas protein is a death Cas protein. The death Cas protein can be a protein lacking nucleic acid cleavage activity. The Cas protein can include a modified form of a wild-type Cas protein. The modified form of a wild-type Cas protein can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the Cas protein. For example, the modified form of a Cas protein can have less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of a wild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified form of a Cas protein can also lack substantial nucleic acid cleavage activity. When a Cas protein is in a modified form lacking substantial nucleic acid cleavage activity, it can be referred to as enzymatically inactive and / or "dead" (abbreviated "d"). A death Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some embodiments, the death Cas protein is a death Cas9 protein.

[0179] In some embodiments, a dCas (e.g., dCas9) polypeptide can associate with a single guide RNA (sgRNA) to activate or suppress transcription of a target DNA. The sgRNA can be introduced into a cell expressing an engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs that target the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell.

[0180] In some embodiments, the genetic modification portion can include a catalytically inactive Cas polypeptide, in which case the nuclease activity of the Cas polypeptide is eliminated or substantially eliminated.

[0181] In some examples, the genetic modification portion may include catalytically inactivated Cas9 (dCas9), any derivative, mutant, or any fragment thereof.

[0182] In some examples, the genetic modification portion may include catalytically inactivated Cas12 (dCas12), any derivative, mutant, or any fragment thereof.

[0183] In some examples, the genetic modification portion may include a catalytically inactivated Cas13 (dCas13), a derivative, mutant, or any fragment thereof.

[0184] In some embodiments, the genetic modification portion can form a complex with at least one heterologous polynucleotide as described herein. In some embodiments, the at least one heterologous polynucleotide can be either a heterologous DNA polynucleotide or a heterologous RNA polynucleotide. In some embodiments, the genetic modification portion can form a complex with at least one heterologous RNA polynucleotide. In some embodiments, the complex formation with at least one heterologous RNA polynucleotide targets the genetic modification portion to the heterologous polynucleotide portion.

[0185] Optionally, the compositions and methods described herein comprise at least one heterologous polynucleotide. Optionally, the compositions and methods described herein comprise multiple heterologous nucleic acids. In some embodiments, the polynucleotide may be deoxyribonucleic acid (DNA). Optionally, the DNA sequence may be single-stranded or double-stranded. In some embodiments, at least one heterologous nucleic acid polynucleotide may be ribonucleic acid (RNA).

[0186] In some embodiments, the genetic modification portion can form a complex with at least one heterologous RNA polynucleotide. The at least one heterologous RNA polynucleotide may include a nucleic acid targeting region comprising a sequence complementary to the nucleic acid sequence of a heterologous polynucleotide encoding any one of the portions described herein for targeting specificity dependent on the genetic modification portion. In some embodiments, the at least one heterologous RNA polynucleotide may be a guide nucleic acid (or guide RNA) comprising two separate nucleic acid molecules, which may be referred to as a dual guide nucleic acid, or a guide nucleic acid comprising a single nucleic acid molecule, which may be referred to as a single guide nucleic acid (e.g., sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a transcription-activating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA but lacking a tracrRNA. In some embodiments, the guide nucleic acid is a dual guide nucleic acid comprising an unfused crRNA and a tracrRNA. An exemplary dual guide nucleic acid may comprise a crRNA-like molecule and a tracrRNA-like molecule. An exemplary single guide nucleic acid may comprise a crRNA-like molecule. An exemplary single guide nucleic acid may include a fused crRNA-like molecule and a tracrRNA-like molecule.

[0187] The crRNA may comprise a nucleic acid targeting segment (e.g., a spacer region) of the guide nucleic acid and a stretch of nucleotides capable of forming one half of a double-stranded duplex of the Cas protein binding segment of the guide nucleic acid.

[0188] The tracrRNA may include a nucleotide stretch that forms the other half of the double-stranded duplex of the Cas protein-binding segment of the gRNA. The nucleotide stretch of the crRNA may be complementary to and hybridize with the nucleotide stretch of the tracrRNA to form the double-stranded duplex of the Cas protein-binding domain of the guide nucleic acid.

[0189] The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The crRNA may further provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., a protospacer). The sequence of the crRNA or tracrRNA molecule, including the spacer region, can be designed to be specific for the species in which the guide nucleic acid is used.

[0190] In some embodiments, the nucleic acid targeting region of the guide nucleic acid may be 18 to 72 nucleotides in length. The nucleic acid targeting region of the guide nucleic acid (e.g., the spacer region) may have a length of about 12 nucleotides to about 100 nucleotides. For example, the nucleic acid targeting region of the guide nucleic acid (e.g., the spacer region) may have a length of about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, about 12 nt to about 30 nt, about 12 nt to about 25 nt, about 12 nt to about 20 nt, about 12 nt to about 19 nt, about 12 nt to about 18 nt, about 12 nt to about 17 nt, about 12 nt to about 16 nt, or about 12 nt to about 15 nt. Alternatively, the DNA targeting segment can have a length of about 18 nt to about 20 nt, about 18 nt to about 25 nt, about 18 nt to about 30 nt, about 18 nt to about 35 nt, about 18 nt to about 40 nt, about 18 nt to about 45 nt, about 18 nt to about 50 nt, about 18 nt to about 60 nt, about 18 nt to about 70 nt, about 18 nt to about 80 nt, about 18 nt to about 90 nt, about 18 nt to about 100 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, about 20 nt to about 60 nt, about 20 nt to about 70 nt, about 20 nt to about 80 nt, about 20 nt to about 90 nt, or about 20 nt to about 100 nt. The length of a nucleic acid targeting region can be a minimum of 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The length of a nucleic acid targeting region (e.g., a spacer region) can be a maximum of 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides.

[0191] In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 20 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 19 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 18 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 17 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 16 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 21 nucleotides in length. In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 22 nucleotides in length.

[0192] The nucleotide sequence of the guide nucleic acid complementary to the nucleotide sequence of the target nucleic acid (target sequence) may have a length of, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt. The nucleotide sequence of the guide nucleic acid complementary to the nucleotide sequence of the target nucleic acid (target sequence) may have a length of, for example, from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, or from about 19 nt to about It may have a length of about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt.

[0193] The protospacer sequence of a targeted polynucleotide can be identified by identifying a PAM within the region of interest and selecting a region upstream or downstream of the PAM of the desired size as the protospacer. The corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

[0194] Spacer sequences can be identified using a computer program (e.g., machine-readable code) that may use variables such as predicted melting temperature, secondary structure formation, and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency, methylation status, presence of SNPs, etc.

[0195] The percentage of complementarity between the nucleic acid targeting sequence (e.g., the spacer sequence of at least one heterologous polypeptide as disclosed herein) and the target nucleic acid (e.g., the protospacer sequence of a heterologous polynucleotide encoding any one of the moieties described herein) is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%. The percentage of complementarity between the nucleic acid targeting sequence and the target nucleic acid can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% over approximately 20 adjacent nucleotides.

[0196] The Cas protein-binding segment of a guide nucleic acid may comprise two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to each other. These two stretches of nucleotides (e.g., crRNA and tracrRNA) can be covalently linked by intervening nucleotides (e.g., a linker in the case of a single guide nucleic acid). The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to each other can hybridize to form a double-stranded RNA duplex or hairpin of the Cas protein-binding segment, resulting in a stem-loop structure. The crRNA and tracrRNA can be covalently linked via the 3' end of the crRNA and the 5' end of the tracrRNA. Alternatively, the tracrRNA and crRNA can be covalently linked via the 5' end of the tracrRNA and the 3' end of the crRNA.

[0197] The Cas protein-binding segment of the guide nucleic acid may have a length of about 10 nucleotides to about 100 nucleotides, e.g., about 10 nucleotides (nt) to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. For example, the Cas protein-binding segment of the guide nucleic acid may have a length of about 15 nucleotides (nt) to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt.

[0198] The dsRNA duplex of the protein-binding segment of the Cas guide nucleic acid can have a length of about 6 base pairs (bp) to about 50 bp. For example, the dsRNA duplex of the protein-binding segment can have a length of about 6 bp to about 40 bp, about 6 bp to about 30 bp, about 6 bp to about 25 bp, about 6 bp to about 20 bp, about 6 bp to about 15 bp, about 8 bp to about 40 bp, about 8 bp to about 30 bp, about 8 bp to about 25 bp, about 8 bp to about 20 bp, or about 8 bp to about 15 bp. For example, the dsRNA duplex of the protein-binding segment may have a length of about 8 bp to about 10 bp, about 10 bp to about 15 bp, about 15 bp to about 18 bp, about 18 bp to about 20 bp, about 20 bp to about 25 bp, about 25 bp to about 30 bp, about 30 bp to about 35 bp, about 35 bp to about 40 bp, or about 40 bp to about 50 bp.

[0199] In some embodiments, the dsRNA duplex of the Cas protein-binding segment may have a length of 36 base pairs. The percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment is at least about 60%. For example, the percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. Optionally, the percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment is 100%.

[0200] The linker (e.g., connecting the crRNA and tracrRNA in a single guide nucleic acid) can have a length of about 3 nucleotides to about 100 nucleotides. For example, the linker can have a length of about 3 nucleotides (nt) to about 90 nt, about 3 nucleotides (nt) to about 80 nt, about 3 nucleotides (nt) to about 70 nt, about 3 nucleotides (nt) to about 60 nt, about 3 nucleotides (nt) to about 50 nt, about 3 nucleotides (nt) to about 40 nt, about 3 nucleotides (nt) to about 30 nt, about 3 nucleotides (nt) to about 20 nt, or about 3 nucleotides (nt) to about 10 nt. For example, the linker may have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. In some embodiments, the linker of the DNA-targeting RNA is 4 nt.

[0201] Guide nucleic acids of the present disclosure may contain modifications or sequences that provide additional desirable characteristics (e.g., modified or modulated stability, intracellular targeting, tracking by fluorescent labels, binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, a 5' cap (7-methylguanylate cap (m7G)), a 3' polyadenylation tail (3' poly(A) tail), a riboswitch sequence (e.g., that allows for regulation of stability and / or regulation of accessibility by proteins and / or protein complexes), a stability regulatory sequence, a sequence that forms a dsRNA duplex (hairpin), a modification or sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplast), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, and combinations thereof).

[0202] A guide nucleic acid may contain one or more modifications (e.g., base modifications, backbone modifications) to provide a nucleic acid with novel or enhanced characteristics (e.g., improved stability). A guide nucleic acid may contain a nucleic acid affinity tag. A nucleoside may be a base-sugar combination. The base portion of a nucleotide may be a heterocyclic base. Purines and pyrimidines are the two most common classes of such heterocyclic bases. A nucleotide may be a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming a guide nucleic acid, the phosphate groups can be covalently linked to adjacent nucleosides to form a linear polymeric compound. The ends of this linear polymeric compound may then be further linked to form a circular compound. However, linear compounds may also be suitable. Additionally, linear compounds may have internal nucleotide base complementarity and therefore fold in a manner to produce a fully or partially double-stranded compound. Furthermore, within a guide nucleic acid, the phosphate groups are often referred to as forming the internucleoside backbone of the guide nucleic acid. The linkage or backbone of the guide nucleic acid may be a 3' to 5' phosphodiester linkage.

[0203] The guide nucleic acid can comprise a modified backbone and / or a modified internucleoside linkage. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0204] Suitable modified guide nucleic acid backbones incorporating a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidites including 3'-amino phosphoramidites and aminoalkyl phosphoramidites, phosphorodiamidates, thionophosphoramidites, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and other alkylphosphonates such as boranophosphates with normal 3'-5' linkages, 2'-5' linkage analogs, and reverse polarity, where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages. Suitable guide nucleic acids with reverse polarity may contain a single 3'-3' linkage at the 3'-most internucleotide linkage (such as a single reverse nucleoside residue with a missing nucleobase or a hydroxyl group in its place). Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms may also be included.

[0205] The guide nucleic acid may contain one or more phosphorothioate and / or heteroatom internucleoside linkages, specifically, -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (methylene (methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (where the natural phosphodiester internucleotide linkage is represented as -OP(=O)(OH)-O-CH2-).

[0206] The guide nucleic acid may include a morpholino backbone structure. For example, the nucleic acid may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages replace phosphodiester linkages.

[0207] Guide nucleic acids may comprise polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, which may include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl backbones, thioformacetyl backbones, methyleneformacetyl backbones, thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino backbones, methylenehydrazino backbones, sulfonate backbones, sulfonamide backbones, amide backbones, and others having mixtures of N, O, S, and CH2 moieties.

[0208] The guide nucleic acid may include a nucleic acid mimic. The term "mimetic" may be intended to include a polynucleotide in which only the furanose ring or both the furanose ring and the nucleotide linkage are replaced with a non-furanose group; replacement of only the furanose ring may also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleotides may be retained and directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, giving the PNA an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0209] Guide nucleic acids may contain linked morpholino units (morpholino nucleic acids), in which a heterocyclic base is attached to a morpholino ring. Linking groups can link morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds have fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides may be nonionic mimics of guide nucleic acids. Various compounds within the morpholino class can be linked using different linking groups. Another class of polynucleotide mimics is sometimes referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of the native complex. Further modifications may include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is attached to the 4'-carbon atom of the sugar ring, resulting in a 2'-C,4'-C-oxymethylene linkage, resulting in a bicyclic sugar moiety. This linkage may be methylene (-CH2-), a group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs may exhibit very high dual thermal stability (Tm = +3 to +10°C) with complementary nucleic acids, stability toward 3'-exonucleolytic degradation, and good solubility properties.

[0210] A guide nucleic acid may contain one or more substituted sugar moieties. Suitable polynucleotides may contain sugar substituents selected from OH, F, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl, or C2-C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Sugar substituents may be selected from C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intervening agent, group for improving the pharmacokinetic properties of the guide nucleic acid, or group for improving the pharmacodynamic properties of the guide nucleic acid, and other substituents with similar properties. Suitable modifications may include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-O-CH2CHOCH3, an alkoxyalkoxy group, also known as 2'-MOE). Further suitable modifications may include 2'-dimethylaminooxyethoxy, (O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE), and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), 2'-O-CH2-O-CH2N(CH3)2.

[0211] Other suitable sugar substituents may include methoxy (-O-CH), aminopropoxy (-O-CH-CH-NH), allyl (-CH-CH=CH), -O-allyl (-O-CH-CH=CH), and fluoro (F). The 2'-sugar substituent may be at the arabino (up) or ribo (down) position. A suitable 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions in the oligomeric compound, specifically the 3' position of the sugar on the 3'-terminal nucleoside or in 2'-5' linked nucleotides, as well as the 5' position of the 5'-terminal nucleotide. Oligomeric compounds may also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0212] A guide nucleic acid may further include nucleobase (or "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and alkynyl derivatives of other pyrimidine bases, 6-azouracil, cytosine, and cytosine. These may include cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(4,5-b)indol-2-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), and pyridoindole cytidine (H-pyrido(3',2';4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0213] Heterocyclic base moieties may include those in which the purine or pyrimidine base is replaced with other heterocyclic compounds, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nucleobases may be useful for increasing the binding affinity of polynucleotide compounds. These may include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions can increase nucleic acid duplex stability by 0.6-1.2°C and may be a suitable base substitution (e.g., when combined with a 2'-O-methoxyethyl sugar modification).

[0214] Modification of the guide nucleic acid may include chemically linking one or more moieties or conjugates to the guide nucleic acid that can enhance the activity, cellular distribution, or cellular uptake of the guide nucleic acid. These moieties or conjugates may include conjugate groups covalently attached to functional groups such as primary or secondary hydroxyl groups. Conjugate groups may include, but are not limited to, intervening agents, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups capable of enhancing the pharmacokinetic properties of oligomers. Conjugate groups may include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups capable of enhancing pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of nucleic acids. Conjugate moieties may include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers (e.g., hexyl-S-tritylthio), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), polyamine or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0215] In some embodiments, a heterologous RNA polynucleotide comprising a crRNA is described herein. In some embodiments, the cRNA comprises a sequence that is complementary to a heterologous polynucleotide encoding any one of the portions described herein.

[0216] In some embodiments, the crRNA comprises 5 nt to 100 nt. In some embodiments, the crRNA comprises 5 nt to 6 nt, 5 nt to 7 nt, 5 nt to 8 nt, 5 nt to 9 nt, 5 nt to 10 nt, 5 nt to 15 nt, 5 nt to 20 nt, 5 nt to 25 nt, 5 nt to 50 nt, 5 nt to 100 nt, 6 nt to 7 nt, 6 nt to 8 nt, 6 nt to 9 nt, 6 nt to 10 nt, 6 nt to 15 nt, 6 nt to 20 nt, 6 nt to 25 nt, 6 nt to 50 nt, 6 nt to 100 nt, 7 nt to 8 nt, 7 nt to 9 nt, 7 nt to 10 nt, 7 nt to 15 nt, 7 nt to 20 nt, 7 nt to 25 nt, 7 nt to 50 nt, 7 nt to 100 nt, 8 nt to 9 nt, 8 nt 10nt, 8nt, 15nt, 8nt, 20nt, 8nt, 25nt, 8nt, 50nt, 8nt, 100nt, 9nt, 10nt, 9nt, 15nt, 10nt, 20nt, 10nt, 25nt, 10nt, 50nt, 10nt, 100nt, 15nt, 20nt, 15nt, 25nt, 15nt, 50nt, 15nt, 100nt, 20nt, 25nt, 20nt, 50nt, 20nt, 100nt, 25nt, 50nt, 25nt, 100nt, or 50nt, 100nt. In some embodiments, the crRNA contains 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In some embodiments, the crRNA contains a minimum of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, or 50 nt. In some embodiments, the crRNA contains a maximum of 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In some embodiments, the crRNA contains at least 5 nt to 100 nt.In some embodiments, the crRNA comprises at least 5nt to 6nt, 5nt to 7nt, 5nt to 8nt, 5nt to 9nt, 5nt to 10nt, 5nt to 15nt, 5nt to 20nt, 5nt to 25nt, 5nt to 50nt, 5nt to 100nt, 6nt to 7nt, 6nt to 8nt, 6nt to 9nt, 6nt to 10nt, 6nt to 15nt, 6nt to 20nt, 6nt to 25nt, 6nt to 50nt, 6nt to 100nt, 7nt to 8nt, 7nt to 9nt, 7nt to 10nt, 7nt to 15nt, 7nt to 20nt, 7nt to 25nt, 7nt to 50nt, 7nt to 100nt, 8nt to 9 ... nt to 10nt, 8nt to 15nt, 8nt to 20nt, 8nt to 25nt, 8nt to 50nt, 8nt to 100nt, 9nt to 10nt, 9nt to 15nt, 9nt to 20nt, 9nt to 25nt, 9nt to 50nt, 9nt to 100nt, 10nt to 15nt, 10nt to 20nt, 10nt to 25nt, 10nt to 50nt, 10nt to 100nt, 15nt to 20nt, 15nt to 25nt, 15nt to 50nt, 15nt to 100nt, 20nt to 25nt, 20nt to 50nt, 20nt to 100nt, 25nt to 50nt, 25nt to 100nt, or 50nt to 100nt. In some embodiments, the crRNA contains at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In some embodiments, the crRNA contains at least a minimum of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, or 50 nt. In some embodiments, the crRNA contains at least a maximum of 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In some embodiments, the crRNA contains at most 5 nt to 100 nt.In some embodiments, the crRNA comprises at most 5nt to 6nt, 5nt to 7nt, 5nt to 8nt, 5nt to 9nt, 5nt to 10nt, 5nt to 15nt, 5nt to 20nt, 5nt to 25nt, 5nt to 50nt, 5nt to 100nt, 6nt to 7nt, 6nt to 8nt, 6nt to 9nt, 6nt to 10nt, 6nt to 15nt, 6nt to 20nt, 6nt to 25nt, 6nt to 50nt, 6nt to 100nt, 7nt to 8nt, 7nt to 9nt, 7nt to 10nt, 7nt to 15nt, 7nt to 20nt, 7nt to 25nt, 7nt to 50nt, 7nt to 100nt, 8nt to 9 ... nt to 10nt, 8nt to 15nt, 8nt to 20nt, 8nt to 25nt, 8nt to 50nt, 8nt to 100nt, 9nt to 10nt, 9nt to 15nt, 9nt to 20nt, 9nt to 25nt, 9nt to 50nt, 9nt to 100nt, 10nt to 15nt, 10nt to 20nt, 10nt to 25nt, 10nt to 50nt, 10nt to 100nt, 15nt to 20nt, 15nt to 25nt, 15nt to 50nt, 15nt to 100nt, 20nt to 25nt, 20nt to 50nt, 20nt to 100nt, 25nt to 50nt, 25nt to 100nt, or 50nt to 100nt. In some embodiments, the crRNA contains at most 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In some embodiments, the crRNA contains at most a minimum of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, or 50 nt. In some embodiments, the crRNA contains at most a maximum of 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt.

[0217] In some embodiments, the genetic modification portion and heterologous polynucleotide can be delivered to cells using an expression vector. In terms of the expression vector, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, the expression vector can be transferred to the host cell by physical, chemical, or biological means. In some embodiments, the genetic modification portion and heterologous polynucleotide can be delivered to cells by physical methods, such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, or electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are suitable for the methods herein (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for introducing a polynucleotide into a host cell is calcium phosphate transfection. In some embodiments, the genetic modification portion and heterologous polynucleotide can be delivered to cells by biological methods, such as the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. In some embodiments, other viral vectors are derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some examples, retroviral vectors include gamma retroviral vectors, such as vectors derived from the Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or murine stem cell virus (MSCV) genomes.In some examples, retroviral vectors further include lentiviral vectors, such as those derived from the human immunodeficiency virus (HIV) genome. In some examples, the AAV vector includes the AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some examples, the viral vector is a chimeric viral vector containing viral segments from two or more viruses. In further examples, the viral vector is a recombinant viral vector. In some embodiments, the genetically modified moiety and heterologous polynucleotide can be delivered to cells by chemical means, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other state-of-the-art methods for targeted delivery of nucleic acids are also available, such as delivery of polynucleotides via targeted nanoparticles or other suitable submicron-sized delivery systems. In some embodiments, the genetically modified moiety and heterologous polynucleotide can be delivered to cells via non-viral delivery systems. The non-viral delivery system can be a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (ex vivo, in vivo, or in vitro). In other aspects, the nucleic acid is associated with a lipid. In some embodiments, the lipid-associated nucleic acid is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule associated with the liposome and the oligonucleotide therapeutic, entrapped within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained in a lipid as a suspension, contained in or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, in some embodiments, these compositions exist as micelles in a bilayer structure or with a "collapsed" structure.Alternatively, they may simply be scattered throughout the solution, potentially resulting in aggregates that are not uniform in size or shape. Lipids are fatty substances that, in some embodiments, are naturally occurring or synthetic. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds encompassing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use are obtained from commercial sources. For example, in some embodiments, dimyristyl phosphatidylcholine ("DMPC") is obtained from Sigma, St. Louis, Mo. In some embodiments, dicetyl phosphate ("DCP") is obtained from K&K Laboratories (Plainview, NY). In some embodiments, cholesterol ("Choi") is obtained from Calbiochem-Behring. Dimyristyl phosphatidylglycerol ("DMPG") and other lipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform / methanol are often stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid solutions formed by the formation of enclosed lipid bilayers or aggregates. Liposomes are often characterized by a porous structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes are separated into multiple lipid layers by the aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures different from the normal porous structure in solution are also included. For example, in some embodiments, lipids exhibit a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.In some embodiments, the genetically modified portion and the heterologous polynucleotide can be delivered to a cell, encapsulated, and delivered to a cell via an extracellular vesicle. The extracellular vesicle can be any membrane-bound particle. In some embodiments, the extracellular vesicle can be any membrane-bound particle secreted by at least one cell. In some embodiments, the extracellular vesicle is a membrane-bound particle synthesized in vitro. In some embodiments, the extracellular vesicle is a membrane-bound particle synthesized without the use of cells. In some embodiments, the extracellular vesicle can be an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exosome, an enveloped virus, an exomer, or other very large extracellular vesicle.

[0218] Identification and isolation of a homogeneous population of extracellular vesicles In some embodiments herein, methods are described that utilize the platforms described herein to generate compositions comprising a homogenous population of extracellular vesicles. In some embodiments, the methods identify and isolate homogenous populations of extracellular vesicles based on the dimensions (e.g., diameter or size) of the extracellular vesicles. In some embodiments, the methods identify and isolate homogenous populations of extracellular vesicles based on the mass of the extracellular vesicles. In some embodiments, the methods identify and isolate homogenous populations of extracellular vesicles based on the number of units of an immune checkpoint moiety encapsulated, secreted, or expressed on the surface of the extracellular vesicles. In some embodiments, the methods identify and isolate homogenous populations of extracellular vesicles based on a combination of the dimensions and the number of units of an immune checkpoint moiety encapsulated, secreted, or expressed on the surface of the extracellular vesicles. In some embodiments, the methods identify and isolate homogenous populations of extracellular vesicles based on the number of units of an immune checkpoint moiety expressed on the surface of the extracellular vesicles.

[0219] In some embodiments, the method for identifying and isolating a homogenous population of extracellular vesicles includes performing differential ultracentrifugation to isolate a homogenous population of extracellular vesicles based on density. In some embodiments, the method includes performing filtration or ultrafiltration to isolate a homogenous population of extracellular vesicles based on weight or size. In some embodiments, the method includes performing HPLC. In some embodiments, the method includes performing sedimentation of the extracellular vesicles, in which a water-excluding polymer, such as polyethylene glycol (PEG), binds to water molecules, preventing most soluble components from leaving the solution. Thus, the sediment containing the extracellular vesicles can be isolated by either low-speed centrifugation or filtration. In some embodiments, the method includes performing affinity-based capture by capturing the extracellular vesicles by immunoaffinity. Examples of proteins or epitopes displayed on the surface of extracellular vesicles include CD9, CD63, CD81, Alix, caveolin-1, CD41, CD4, flotillin, Rab5, HSC70, and Lamp-3. In some embodiments, the methods include performing microfluidics-based isolation methods on extracellular vesicles to isolate homogenous populations of extracellular vesicles based on size, density, and immunoaffinity, and innovative sorting mechanisms such as acoustic, electrophoretic, and electromagnetic manipulation are feasible. The use of such devices is expected to significantly reduce sample volume, reagent consumption, and isolation time.

[0220] In some embodiments, methods for identifying and isolating a homogenous population of extracellular vesicles include a step based on the number of immune checkpoint moieties expressed on the surface of the extracellular vesicles. In some embodiments, the method includes an immunoassay in which an antibody that recognizes and binds to the immune checkpoint moiety is used. In some embodiments, the antibody is conjugated to a detectable label. In some embodiments, a signal detected from an antibody that recognizes and binds to the immune checkpoint moiety correlates with the number of immune checkpoint moieties expressed on the surface of the extracellular vesicles. Exemplary detectable moieties include enzymatic moieties (e.g., horseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase, etc.), fluorescent dyes, luminescent moieties, emissive moieties, colorimetric labels, colored latex particles or nanoparticles, and metal-conjugated moieties, such as metal nanolayers, metal nanoparticles, and metal nanoshell-conjugated moieties. In some embodiments, the detectable moiety is directly or indirectly tagged for colorimetric assays (e.g., for detection of HRP or β-galactosidase activity), visual inspection using a light microscope, immunofluorescence microscopy, confocal microscopy with flow cytometry (FACS), autoradiography electron microscopy, immunostaining, or subcellular fractionation.

[0221] In some embodiments, the method for identifying and isolating a homogenous population of extracellular vesicles includes identifying and isolating a homogenous population of extracellular vesicles based on both the diameter and the number of units of an immune checkpoint moiety expressed on the surface of the extracellular vesicles. For example, the method identifies and isolates a homogenous population of extracellular vesicles with diameters of about 50 nm and about 2000 units expressed on the surface of the extracellular vesicles. In some embodiments, the method identifies and isolates a homogenous population of extracellular vesicles with diameters of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, or more, and 500 units, 1000 units, 1500 units, 2000 units, 2500 units, or more expressed on the surface of the extracellular vesicles. A homogenous population of extracellular vesicles having a size of 1000, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, or more is identified and isolated.

[0222] treatment In some embodiments, disclosed herein are methods for treating a disease or disorder in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition or pharmaceutical composition described herein. In some embodiments, the disease or disorder is an autoimmune disease, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, celiac disease, Crohn's disease, Graves' disease, juvenile arthritis, chronic Lyme disease, optic neuritis, psoriatic arthritis, scleritis, scleroderma, ulcerative colitis (UC), uveitis, inflammatory eye disease, vitiligo, COPD, complications from organ transplantation, or graft-versus-host disease.

[0223] In some embodiments, the method comprises contacting a cell with a composition or pharmaceutical composition described herein, wherein upon contacting, the immune checkpoint moiety is delivered to the target cell. In some embodiments, the immune checkpoint moiety modulates the immune response of the target cell. In some embodiments, the contacting occurs in vivo, ex vivo, or in vitro. In some embodiments, the composition or pharmaceutical composition can be administered directly to a subject.

[0224] In some embodiments, the composition or pharmaceutical composition can be administered alone to a subject (e.g., stand-alone treatment). In some embodiments, the composition is administered in combination with an additional agent. In some embodiments, the composition is a first-line treatment for a disease or disorder. In some embodiments, the composition is a second-, third-, or fourth-line treatment for an autoimmune disease.

[0225] Generally, the methods disclosed herein involve administering the composition orally. However, in some examples, the methods involve administering the composition via intraperitoneal injection. In some examples, the methods involve administering the composition in the form of a rectal suppository. In some examples, the methods involve administering the composition intravenously (i.v.). It is further contemplated that the compositions disclosed herein can be administered via other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, or any other suitable parenteral administration. In some embodiments, the route for local delivery near the site of injury or inflammation preferably overlies the systemic route. The route, dose, time point, and duration of therapeutic administration can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of acute, chronic, or both symptoms of a disease or disorder.

[0226] The dosages and doses of the compositions herein effective for preventing or treating an autoimmune disease are determined by the observed beneficial response associated with the autoimmune disease or condition, or the symptoms of the autoimmune disease. In some examples, the beneficial response includes a reduction in autoimmune disease symptoms. Additional beneficial responses include the prevention, alleviation, prevention, or cure of the autoimmune disease. If the composition is not therapeutically effective or does not provide sufficient relief of the disease or condition, or the symptoms of the disease or condition, the dosage and / or route of administration can be modified, or additional agents can be administered to the subject along with the composition. In some embodiments, the patient is discontinued from a second treatment regimen (e.g., the dosage is gradually reduced) once the composition regimen is initiated.

[0227] The appropriate dosage and dose for administration to a subject will depend on factors including, but not limited to, the particular composition, the state and severity of the disease, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined depending on the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the disease being treated, and the subject being treated.

[0228] In some embodiments, the composition is administered once every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years. The effective dose range can be adjusted based on the subject's response to treatment. Some administration routes require higher concentrations of the therapeutic agent to be effective than other routes.

[0229] In certain embodiments where the patient's condition does not improve, at the physician's discretion, administration of the composition is chronic, i.e., administered for the patient's lifetime to improve or otherwise control or limit the patient's disease or symptoms. In certain embodiments where the patient's condition improves, the dosage of the administered composition may be temporarily reduced or temporarily discontinued for a period of time (drug holiday). In certain embodiments, the drug holiday may be between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or longer than 28 days. The dosage reduction during the drug holiday period may be 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the dosage of the administered drug may be temporarily reduced or temporarily discontinued for a period of time ("drug diversion"). In certain embodiments, the duration of drug diversion may be between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or longer than 28 days. Dose reductions during drug diversion range from 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After an appropriate period of time, the patient is optionally returned to the normal dosing schedule.

[0230] In some embodiments, once the patient's symptoms have improved, a maintenance dose is administered as needed. Thereafter, in certain embodiments, the dosage and / or frequency is reduced, depending on the symptoms, to a level at which improvement in the disease, disorder, or condition is maintained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon recurrence of symptoms.

[0231] Toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining the LD50 and ED50. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD50 to ED50. In certain embodiments, data obtained from cell culture assays and animal studies are used to formulate a therapeutically effective daily dose range and / or therapeutically effective unit dose for use in mammals, including humans. In some embodiments, the daily dose of the compositions described herein lies within a range of circulating concentrations that include the ED50 with minimal toxicity. In certain embodiments, the daily dose range and / or unit dose will vary within this range depending on the dosage form employed and the route of administration utilized.

[0232] The composition can be used alone or in combination with an additional agent. Optionally, the "additional agent" as used herein is administered alone. The composition and the additional agent can be administered together or sequentially. The combined therapeutic agent can be administered on the same day, or can be administered one or more days, weeks, months, or years apart. Examples of additional agents include other immune modulators, such as antibodies targeting cytokines or small molecules.

[0233] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein are available for practicing the present invention. It is therefore contemplated that the present invention shall cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. [Example]

[0234] The following illustrative examples are representative of embodiments of the stimuli, systems, and methods described herein and are not intended to be limiting in any way. [Example]

[0235] Mesenchymal stem cells (MSCs) and their corresponding extracellular vesicles (EVs) contain PD-L1. The presence of PD-L1 in MSCs and their corresponding extracellular vesicles was confirmed by Western blot. As shown in Figure 1, a clear band of 45 kDa was detected on MSCs and their corresponding exosomes. Starvation medium was used as a negative control, and island exosomes were used as a positive control. To normalize these results, the same analysis was performed on MSC EVs obtained from three different umbilical cord MSC donors.

[0236] MSC lysates and MSC EV samples were prepared by sonication. The amount of MSC lysates and MSC EV samples corresponded to 25 μg of protein. These samples were analyzed using gradient precast polyacrylamide gels (Mini-PROTEAN; Bio-Rad Laboratories, Hercules, CA, USA). The samples were then transferred to nitrocellulose membranes, which were blocked with 5% blotting-grade blocker non-fat dry milk (Bio-Rad Laboratories) in Tris-buffered saline (TBS) for 2 hours. The membranes were then incubated overnight at 4°C with purified anti-human CD274 (B7-H1, PD-L1) antibody (Biolegend, cat#329701) in 0.25% blotting-grade blocker non-fat dry milk in TBS-Tween (TBST). The membranes were then washed three times with TBST for 10 minutes. The secondary antibody, ECL anti-mouse IgG F(ab')2 fragment conjugated to horseradish peroxidase, was diluted in 0.25% blotting-grade Blocker Non-Fat Dry Milk in TBST and incubated with the membrane for 1.5 hours. Finally, the membrane was analyzed using ECL Prime Western Blotting Detection (GE Healthcare) and a VersaDoc 4000 MP (Bio-Rad Laboratories). [Example]

[0237] MSC EVs suppress CD25+CD8+ immune cells. Significant suppression of CD25+CD8+ immune cells was observed in human peripheral blood mononuclear cells (PBMCs) stimulated with IL-2 (500 U / ml) for 6 days. This suppression was also observed to be reduced in the experimental group in which PBMCs were also exposed to a PD-L1 blocking antibody. The experimental scheme is shown in Figure 2.

[0238] Representative flow cytometry plots (Figure 3) show that the suppressive activity of MSC EVs against the CD8+CD25+ population was significantly reduced in the presence of PD-L1 blockade. Furthermore, the control group containing an isotype-modulating antibody exhibited similar suppressive activity to MSC EVs alone. These results suggest that the suppressive activity of MSC EVs is primarily due to the presence of PD-L1. [Example]

[0239] MSCs were genetically modified to produce more exosomal PD-L1 than wild-type MSCs. MSCs are harvested from the umbilical cords of healthy donors. After harvesting, these MSCs are cultured for further experiments. Transmembrane CD63 is targeted for genetic modification using known techniques (e.g., CRISPR / Cas9). The extracellular domain of PD-L1 is fused to CD63. Various specific subsets of CD63 amino acid sequences were identified as particularly favorable regions for PD-L1 fusion: immediately following RDKVMSE, immediately following NNNFRQQ, and immediately following YPKNNHT. These CD63 portions were found in the large extracellular loop (LEL) of CD63. The extracellular domain of PD-L1 is fused to CD63 using one, two, three, or more G4S linkers.

[0240] After genetic modification of MSCs, the genetically modified MSCs were cultured. Samples of genetically modified MSC lysate and genetically modified MSC EVs were prepared according to the protocol in Example 1. These samples were analyzed by Western blot to test for the presence of PD-L1. A clear band measuring 45 kDa was detected in the genetically modified MSCs and their corresponding exosomes. The color of these bands was darker than in the Western blot analysis in Example 1, indicating that the genetically modified MSCs and their corresponding exosomes produced more PD-L1 than wild-type MSCs and EVs produced by wild-type MSCs. Starvation medium was used as a negative control, and island exosomes were used as a positive control. [Example]

[0241] MSCs are genetically modified to produce exosomal immune checkpoint inhibitors. Using the method of Example 3, VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), galectin-9, galectin-3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, and stabilin-1 (Clever-1) were detected. , neuropilin 1, neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRPα (CD172a), or a combination thereof.

[0242] Western blot analysis will be performed on genetically modified MSCs and their corresponding EVs to test for the presence of selected immune checkpoint inhibitors. [Example]

[0243] Generation / purification of genetically modified potent MSCs and their corresponding EVs The genetically modified MSCs and their corresponding EVs of Example 3 are analyzed to generate sufficient MSCs and their corresponding EVs with increased CD25+CD8+ immune cell suppression compared to the results of Example 2.

[0244] The genetically modified MSCs of Example 3 and their corresponding EVs are prepared in a solution containing an anti-PD-L1 antibody conjugated with a detectable label and soluble PD-1. The soluble PD-1 binds to exosomal PD-L1. Because soluble PD-1 and exosomal PD-L1 interact on MSCs and their corresponding EVs, the anti-PD-L1 antibody conjugated with a detectable label selectively binds to MSCs and EVs in a sample that expresses higher amounts of exosomal PD-L1 compared to MSCs and EVs, where the soluble PD-1 present in the sample occupies the corresponding exosomal PD-L1.

[0245] Using this analysis, potent MSCs and EVs are generated / purified from the sample. [Example]

[0246] Genetically modified potent MSCs and their corresponding EVs exhibit improved suppression of CD25+CD8+ immune cells. The potent MSCs and EVs of Example 5 are analyzed in the experiments of Example 2. The experimental group containing potent MSCs and EVs shows significantly increased CD25+CD8+ immune cell suppression compared to the results of Figure 3. [Example]

[0247] Genetically modified potent MSCs and their corresponding EVs for the treatment of rheumatoid arthritis A pharmaceutical composition containing the potent EV of Examples 5 and 6 and a pharmaceutically acceptable excipient is prepared to treat patients with rheumatoid arthritis. The pharmaceutical composition contains approximately 10 6 ~about 10^ 8 EVs, or about 1 μg to about 700 mg of EVs.

[0248] The pharmaceutical composition is administered to the site of inflammation in patients with rheumatoid arthritis. The increased suppression of CD25+CD8+ immune cells by EVs is sufficient to treat rheumatoid arthritis. [Example]

[0249] Genetically modified potent MSCs and their corresponding EVs for the treatment of graft-versus-host disease in patients undergoing kidney transplantation. A pharmaceutical composition containing the potent EV of Examples 5 and 6 and a pharmaceutically acceptable excipient is prepared to treat graft-versus-host disease in patients undergoing kidney transplantation. This pharmaceutical composition is effective in treating approximately 10 6 ~about 10^ 8 EVs, or about 1 μg to about 700 mg of EVs.

[0250] The pharmaceutical composition is administered to the site of inflammation in patients with graft-versus-host disease in kidney transplant recipients. The increased suppression of CD25+CD8+ immune cells by EVs is sufficient to treat graft-versus-host disease in kidney transplant recipients.

[0251] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail can be made therein without departing from the true scope of the present disclosure. For example, any of the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A composition comprising extracellular vesicles, the extracellular vesicles comprising: a. an immune checkpoint moiety comprising VISTA, PD-L1, CTLA-4, or any combination thereof; b. a transmembrane portion comprising CD63, wherein the CD63 comprises three transmembrane domains; Including, The composition, wherein the immune checkpoint moiety is linked to the transmembrane moiety.

2. 2. The composition of claim 1, wherein the immune checkpoint moiety is attached to an extracellular loop of the CD63 to generate a modified CD63.

3. The composition of claim 1 or 2, wherein the extracellular loop is the large extracellular loop or the second extracellular loop of the modified CD63.

4. The composition of any one of claims 1 to 3, wherein the modified CD63 comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 17.

5. The composition of any one of claims 1 to 4, wherein the modified CD63 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 17.

6. The composition of any one of claims 1 to 5, wherein the modified CD63 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 17.

7. A composition comprising extracellular vesicles, the extracellular vesicles comprising: a. an immune checkpoint moiety comprising PD-L1; and b. A transmembrane segment containing lactadherin; Including, The composition, wherein the immune checkpoint moiety is linked to the transmembrane moiety.

8. A composition comprising extracellular vesicles, the extracellular vesicles comprising: a. an immune checkpoint moiety comprising V domain Ig suppressor of T cell activation (VISTA), PD-L1, CTLA-4, or any combination thereof; b. A transmembrane segment containing glycosylphosphatidylinositol (GPI) Including, The composition, wherein the immune checkpoint moiety is linked to the transmembrane moiety.

9. A composition comprising extracellular vesicles, the extracellular vesicles comprising: a. an immune checkpoint moiety, and b. Transmembrane part A composition comprising at least one of:

10. 10. The composition of any one of claims 1 to 9, wherein the immune checkpoint moiety is encapsulated in an extracellular vesicle.

11. 11. The composition of any one of claims 1 to 10, wherein the immune checkpoint moiety is expressed on the surface of an extracellular vesicle.

12. 12. The composition of any one of claims 1 to 11, wherein the immune checkpoint moiety is secreted by an extracellular vesicle.

13. 13. The composition of any one of claims 1 to 12, wherein the immune checkpoint moiety is complexed with the transmembrane moiety.

14. 14. The composition of any one of claims 1 to 13, wherein the immune checkpoint moiety is covalently linked to the transmembrane moiety.

15. The immune checkpoint moiety is selected from the group consisting of VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), galectin-9, galectin-3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, stabilin-1 (Clever- 15. The composition of any one of claims 1 to 14, comprising a marker selected from the group consisting of: neuropilin 1, neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRPα (CD172a), or a combination thereof.

16. 16. The composition of any one of claims 1 to 15, wherein the immune checkpoint moiety comprises VISTA, PD-L1, CTLA-4, or a combination thereof.

17. 17. The composition of any one of claims 1 to 16, wherein the immune checkpoint moiety comprises PD-L1.

18. The transmembrane portion may be selected from the group consisting of 14-3-3 protein zeta / delta, 4-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase / AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAM10, alkaline phosphatase, α-enolase, α-synuclein, aminopeptidase N, and amyloid beta. A4 / APP, annexin 5A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, β-actin (ACTB), β-amyloid 42, caveolin 1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24, CD26 / DPP4, CD29 / ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudins, claudin 1, cofilin-1, complement binding proteins CD55 and CD59, cytosolic heat shock protein 90α, cytosolic heat shock protein 90β, EBV LMP1, EBV LMP2A, EF-1α-1, EF2, EFGR EGFR VIII, emmprin / CD147, enolase 1α (ENO1), EPCAM, ERBB2, tetraspanins (CD9, CD63, and CD81), fatty acid synthase, fetuin A, flotillin-1, flotillin-2, fructose bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5' nucleotidase, GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 and HSP90), heparan sulfate proteoglycan, heparinase, heterotrimeric G protein, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1Tax, huntingtin, ICAM1, integrin, lactadherin, LAMP1 / 2, leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associated membrane glycoprotein 1, lysosome-associated membrane glycoprotein 2, MHC class I, MHC class II, MUC1, multidrug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP / Alix, PECAM1, phosphoglycerate kinase, placental prion protein, prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap 18. The composition of any one of claims 1 to 17, wherein the protein is selected from the group consisting of: 1B, resistin, sonic hedgehog (SHH), surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein, TYRP-2, vacuolar sorting protein 35, or zeta polypeptide (YWHAZ).

19. 19. The composition of any one of claims 1 to 18, wherein the transmembrane moiety comprises lactadherin.

20. 20. The composition of any one of claims 1 to 19, wherein the transmembrane portion comprises LAMP2, or a variant or fragment thereof, wherein the LAMP2 is at least 70% identical to the peptide sequence of SEQ ID NO:

4.

21. 21. The composition of any one of claims 1 to 20, wherein the transmembrane portion comprises CD63, or a variant or fragment thereof, which is at least 70% identical to the peptide sequence of SEQ ID NO:

5.

22. The composition of claim 21 , wherein the CD63 is a modified CD63.

23. The composition of claim 22, wherein the modified CD63 is a truncated CD63.

24. The composition of claim 22, wherein the modified CD63 is modified to contain at least one additional CD63 transmembrane domain.

25. The composition of claim 22, wherein the modified CD63 comprises one transmembrane domain.

26. The composition of claim 22, wherein the modified CD63 comprises two transmembrane domains.

27. The composition of claim 22, wherein the modified CD63 comprises three transmembrane domains.

28. The composition of claim 22, wherein the modified CD63 comprises four transmembrane domains.

29. The composition of claim 22, wherein the modified CD63 comprises five transmembrane domains.

30. 30. The composition of any one of claims 1 to 29, wherein the immune checkpoint moiety forms a complex with the modified CD63 at an extracellular loop of the modified CD63.

31. 31. The composition of any one of claims 1 to 30, wherein the immune checkpoint moiety forms a complex with the modified CD63 at the large extracellular loop of the modified CD63.

32. 32. The composition of any one of claims 1 to 31, further comprising a targeting moiety.

33. 33. The composition of claim 32, wherein the targeting moiety comprises a peptide that targets a cytokine.

34. 34. The composition of claim 32 or 33, wherein the targeting moiety comprises a peptide that targets a cancer cell marker.

35. 19. The composition of any one of claims 1 to 18, further comprising a fusogenic moiety.

36. 36. The composition of claim 35, wherein the fusogenic moiety is a viral fusogenic moiety.

37. 37. The composition of claim 35 or 36, wherein the fusogenic moiety comprises a mammalian fusogenic moiety.

38. 38. The composition of any one of claims 1 to 37, further comprising an immune evasion moiety.

39. 39. The composition of claim 38, wherein the immune evasion moiety comprises CD47.

40. 40. The composition of any one of claims 1 to 39, which does not contain enucleated cells.

41. 41. The composition of any one of claims 1 to 40, wherein the extracellular vesicles comprise exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomes, enveloped viruses, exomers, or other very large extracellular vesicles.

42. 42. The composition of any one of claims 1 to 41, wherein the extracellular vesicles comprise exosomes.

43. 43. The composition of any one of claims 1 to 42, wherein the extracellular vesicle comprises multiple immune checkpoint moieties.

44. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 10,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

45. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 9,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

46. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 8,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

47. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 7,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

48. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 6,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

49. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 5,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 100 nm.

50. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 3,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm.

51. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 2,500 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm.

52. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 2,000 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm.

53. 44. The composition of claim 43, wherein the extracellular vesicles comprise at least 1,500 units of an immune checkpoint moiety per extracellular vesicle and comprise a diameter of 50 nm.

54. 54. The composition of any one of claims 1 to 53, comprising a plurality of exosomes.

55. 55. The composition of any one of claims 1 to 54, comprising a therapeutically effective amount of exosomes.

56. 56. The composition of claim 54 or 55, comprising at least 10^6 exosomes.

57. 56. The composition of claim 54 or 55, comprising at least 10^7 exosomes.

58. 56. The composition of claim 54 or 55, comprising at least 10^8 exosomes.

59. 56. The composition of claim 54 or 55, comprising at least 1 μg of exosomes.

60. 56. The composition of claim 54 or 55, comprising at least 10 μg of exosomes.

61. 56. The composition of claim 54 or 55, comprising at least 20 μg of exosomes.

62. 56. The composition of claim 54 or 55, comprising at least 50 μg of exosomes.

63. 56. The composition of claim 54 or 55, comprising at least 100 μg of exosomes.

64. 56. The composition of claim 54 or 55, comprising at least 150 μg of exosomes.

65. 56. The composition of claim 54 or 55, comprising at least 200 μg of exosomes.

66. 56. The composition of claim 54 or 55, comprising at least 250 μg of exosomes.

67. 56. The composition of claim 54 or 55, comprising at least 500 μg of exosomes.

68. 56. The composition of claim 54 or 55, comprising at least 750 μg of exosomes.

69. 56. The composition of claim 54 or 55, comprising at least 1 mg of exosomes.

70. 56. The composition of claim 54 or 55, comprising at least 2 mg of exosomes.

71. 56. The composition of claim 54 or 55, comprising at least 3 mg of exosomes.

72. 56. The composition of claim 54 or 55, comprising at least 4 mg of exosomes.

73. 56. The composition of claim 54 or 55, comprising at least 5 mg of exosomes.

74. 56. The composition of claim 54 or 55, comprising at least 6 mg of exosomes.

75. 56. The composition of claim 54 or 55, comprising at least 7 mg of exosomes.

76. 56. The composition of claim 54 or 55, comprising at least 100 mg of exosomes.

77. 56. The composition of claim 54 or 55, comprising at least 200 mg of exosomes.

78. 56. The composition of claim 54 or 55, comprising at least 300 mg of exosomes.

79. 56. The composition of claim 54 or 55, comprising at least 400 mg of exosomes.

80. 56. The composition of claim 54 or 55, comprising at least 500 mg of exosomes.

81. 56. The composition of claim 54 or 55, comprising at least 600 mg of exosomes.

82. 56. The composition of claim 54 or 55, comprising at least 700 mg of exosomes.

83. 83. The composition of any one of claims 1 to 82, which is derived from a cell.

84. 84. The composition of any one of claims 1 to 83, which is stored frozen.

85. 85. The composition of any one of claims 1 to 84, which is lyophilized.

86. 86. The composition of any one of claims 1 to 85, which is stable at 37°C for 24 hours.

87. 87. The composition of any one of claims 1 to 86, which is stable at 37°C for 48 hours.

88. 88. The composition of any one of claims 1 to 87, which is stable at 37°C for 72 hours.

89. 89. A cell configured to produce the extracellular vesicle of any one of claims 1 to 88.

90. 90. The cell of claim 89, which is a stem cell.

91. 90. The cell of claim 89, which is a human cell.

92. 90. The cell of claim 89, which is a non-human cell.

93. 93. A cell according to any one of claims 89 to 92, which is a mesenchymal stem cell.

94. 94. The cell of any one of claims 89 to 93, which is a genetically modified cell.

95. 1. A method for purifying extracellular vesicles configured to express one or more immune checkpoint moieties, comprising: a. Obtaining a heterogeneous population of extracellular vesicles; b. Exposing the heterogeneous population of extracellular vesicles to a detection assay solution containing a detection moiety to form complexes with the immune checkpoint moiety; c. detecting a signal resulting from a complex formed between the immune checkpoint moiety and the detection moiety, wherein the intensity of the signal is proportional to the units of the expressed immune checkpoint moiety; d. isolating a subpopulation of the extracellular vesicles based on the intensity of the signal; A method comprising:

96. 96. The method of claim 95, wherein the detection moiety comprises an antibody.

97. 97. The method of claim 96, wherein the detection moiety comprises an anti-VISTA antibody, an anti-PD-L1 antibody, an anti-CTLA-4, or a combination thereof.

98. 96. The method of Claim 95, wherein the detection moiety comprises a ligand of the immune checkpoint moiety.

99. 96. The method of Claim 95, wherein the detection assay solution further comprises a peptide configured to bind to the immune checkpoint moiety.

100. 100. The method of claim 99, wherein the peptide configured to bind to the immune checkpoint moiety comprises PD-1, CD80, CD86, or a combination thereof.

101. 89. A pharmaceutical composition comprising the composition of any one of claims 1 to 88.

102. 102. The pharmaceutical composition of claim 101, comprising a pharmaceutically acceptable carrier.

103. 102. The pharmaceutical composition of claim 101, comprising at least one additional active agent.

104. 102. The pharmaceutical composition of claim 101, formulated for intrathecal administration, intraocular administration, intravitreal administration, retinal administration, intravenous administration, intramuscular administration, intraventricular administration, intracerebral administration, intracerebellar administration, intraventricular administration, intraparenchymal administration, subcutaneous administration, or a combination thereof.

105. 105. A method for treating an autoimmune disease, comprising administering a pharmaceutical composition according to any one of claims 101 to 104.

106. The method of claim 105, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease, celiac disease, Crohn's disease, Graves' disease, juvenile arthritis, chronic Lyme disease, optic neuritis, psoriatic arthritis, scleritis, scleroderma, ulcerative colitis (UC), uveitis, inflammatory eye disease, vitiligo, COPD, complications from organ transplantation, or graft-versus-host disease.

107. 107. The method of claim 106, wherein the autoimmune disease is rheumatoid arthritis.

108. 90. A method of suppressing CD8+CD25+ cells in a patient in need thereof, comprising administering a composition of any one of claims 1 to 88.

109. 105. A kit comprising the pharmaceutical composition of any one of claims 101 to 104.

110. 89. A platform comprising components for producing the composition of any one of claims 1 to 88.

Images