Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
  • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
  • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/36—Lipids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/999—Small molecules not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells
  • C12N2510/02—Cells for production

Definitions

• the present disclosure relates to production of extracellular vesicles, e.g., exosomes, by producer cells, wherein the producer cells are contacted with (i) a cholesterol biosynthetic pathway inhibitor and (ii) cholesterol.
• Extracellular vesicles in particular exosomes, have been gaining interest as a new modality capable of an efficient delivery of various payloads to cells of all types within a living organism.
• technology gaps have emerged between the current state of the art for producing exosomes and the capabilities necessary to support large scale clinical and commercial manufacturing.
• considerable attempts have been focused on sustaining growth and productivity of the producer cell line in vitro, however, maximizing exosome yield remains a challenge. Therefore, novel methods for efficient, low-cost and reliable high titer production of extracellular vesicles are needed.
• Some aspects of the present disclosure are directed to a method of increasing a number of extracellular vesicles (EVs) produced from producer cells, comprising contacting the producer cells with (i) a cholesterol biosynthetic pathway inhibitor and (ii) cholesterol.
• EVs extracellular vesicles
• Some aspects of the present disclosure are directed to a method of producing extracellular vesicles (EVs) from producer cells, comprising contacting the producer cells with (i) a cholesterol biosynthetic pathway inhibitor and (ii) cholesterol.
• EVs extracellular vesicles
• the producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 8 days.
• the EVs produced by the producer cells have an increased yield compared to EVs produced by producer cells not contacted with a cholesterol biosynthetic pathway inhibitor.
• the yield is increased at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, or at least about 30 fold.
• the yield is increased about 1.5 fold to about 30 fold, about 1.5 fold to about 25 fold, about 1.5 fold to about 20 fold, about 1.5 fold to about 15 fold, about 1.5 fold to about 10 fold, about 1.5 fold to about 5 fold, about 2 fold to about 30 fold, about 2 fold to about 25 fold, about 2 fold to about 20 fold, about 2 fold to about 15 fold, about 2 fold to about 10 fold, about 2 fold to about 5 fold, about 2.5 fold to about 30 fold, about 2.5 fold to about 25 fold, about 2.5 fold to about 20 fold, about 2.5 fold to about 15 fold, about 2.5 fold to about 10 fold, or about 2.5 fold to about 5 fold.
• the yield is increased about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold.
• the cholesterol biosynthetic pathway inhibitor comprises a statin, a cariprazine, a PROTAC, AY9944, or BM15766.
• the statin comprises atorvastatin, lovastatin, pitavastatin, pravastatin, fluvastatin, cerivastatin, rosuvastatin, simvastatin, or combinations thereof.
• the statin is contacted at a concentration of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, or about 20 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, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220
• the statin is contacted at a concentration of between about 0.1 nM to about 100 nM, about 0.1 nM to about 90 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 70 nM, about 0.1 nM to about 60 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about 0.1 nM to about 30 nM, about 0.1 nM to about 20 nM, 0.1 nM to about 10 nM, or about 1 nM to about 20 nM, about 1 nM to about 10 nM, about InM to about 5 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM
• the cholesterol biosynthesis pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 10 nM. In some aspects, the cholesterol biosynthesis pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 15 nM. In some aspects, the cholesterol biosynthesis pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 20 nM.
• the cholesterol biosynthesis pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 10 nM. In some aspects, the cholesterol biosynthesis pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 25 nM. In some aspects, the cholesterol biosynthesis pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 50 nM.
• the producer cells are contacted with cholesterol at a concentration of about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 6
• the producer cells are contacted with cholesterol at a concentration of about 0.1 pM to about 100 pM, about 0.1 pM to about 75 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 40 pM, about 0.1 pM to about 30 pM, about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, 1 pM to about 30 pM, or about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 2 pM to about 3 pM, about 20 pM to about 30 pM, about 25 pM to about 30 pM, or about 20 pM to about 25 pM.
• the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 2.5 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 25 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 2.5 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 25 pM.
• the producer cells have increased viability compared to producer cells (i) contacted with the cholesterol biosynthetic pathway inhibitor and (ii) not contacted with cholesterol. Tn some aspects, the producer cells have increased viability compared to producer cells (i) not contacted with the cholesterol biosynthetic pathway inhibitor and (ii) not contacted with cholesterol.
• the viability is increased by at least about 10%, at least about 20%, at least about 25%, 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%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%.
• the EVs produced by the producer cells have decreased cholesterol content per EV compared to EVs produced by producer cells not contacted with the cholesterol biosynthetic pathway inhibitor.
• the cholesterol content per EV is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
• the cholesterol content per EV is reduced by about 1% to about 80%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50%.
• DMSO dimethyl sulfoxide
• the producer cells are contacted with DMSO at a concentration of about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.
• the producer cells are contacted with cholesterol at a concentration of about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.05% to about 2%, about 0.05% to about 1.5%, about 0.05% to about 1%, about 0.05% to about .5%, 0.1% to about 2%, or about 0.1% to about 1.5%, about 0.1% to about 1%, or about 0.1% to about 0.5%.
• the producer cells are contacted with DMSO at a concentration of about 0.1%.
• the EVs produced by the producer cells do not have a difference in average size distribution compared to EVs produced by producer cells not contacted with the cholesterol biosynthetic pathway inhibitor.
• the producer cells are mammalian cells.
• the producer cells are HEK293 cells, HEK293S cells, HEK293SF cells, Chinese Hamster Ovary (CHO) cells, mesenchymal stem cells (MSCs), B J human foreskin fibroblast cells, fHDF fibroblast cells, AGE.HN® neuronal precursor cells, CAP® amniocyte cells, adipose mesenchymal stem cells, RPTEC/TERT1 cells, dendritic cells, macrophages, B cells, mast cells, neutrophils, Kupffer- Browicz cells, PER.C6 cells, Induced pluripotent stem cells (iPSCs), or C2C12 cells.
• the producer cells are stem cells.
• the EVs further comprise a scaffold moiety.
• the scaffold moiety comprises a Scaffold X.
• Scaffold X is selected from the group consisting of prostaglandin F2 receptor negative regulator (the PTGFRN protein); basigin (the BSG protein); immunoglobulin superfamily member 2 (the IGSF2 protein); immunoglobulin superfamily member 3 (the IGSF3 protein); immunoglobulin superfamily member 8 (the IGSF8 protein); integrin beta-1 (the ITGB1 protein); integrin alpha-4 (the ITGA4 protein); 4F2 cellsurface antigen heavy chain (the SLC3A2 protein); a class of ATP transporter proteins (the ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins), and any combination thereof.
• the scaffold moiety is a PTGFRN protein.
• the scaffold moiety comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 1.
• the scaffold moiety comprises a Scaffold Y.
• the Scaffold Y is selected from the group consisting of myristoylated alanine rich Protein Kinase C substrate (the MARCKS protein); myristoylated alanine rich Protein Kinase C substrate like 1 (the MARCKSL1 protein); brain acid soluble protein 1 (the BASP1 protein), and any combination thereof.
• the EV further comprises at least a therapeutic agent linked to a scaffold moiety. In some aspects, the EV further comprises at least a therapeutic agent.
• the therapeutic agent comprises a cytokine, a small molecule, a growth factor, an antigen, an antisense oligonucleotide, an siRNA, an shRNA, a miRNA, a dsDNA, a IncRNA, a PROTAC, an adjuvant, an immune modulator, or any combination thereof.
• the therapeutic agent is IL-12.
• the therapeutic agent comprises an IL-2 polypeptide.
• the therapeutic agent is a STING agonist.
• the therapeutic agent is an antisense oligonucleotide.
• the antisense oligonucleotide targets Kras, STAT3, Nras, STAT6, CEBP/b, NLRP3, or any combination thereof.
• Some aspects of the present disclosure are directed to producer cells for use in a method disclosed herein. Some aspects of the present disclosure are directed to producer cells prepared by a method disclosed herein.
• Some aspects of the present disclosure are directed to extracellular vesicles produced by a method disclosed herein or producer cells disclosed herein. Some aspects of the present disclosure are directed to a bioreactor comprising producer cells disclosed herein or extracellular vesicles disclosed herein.
• Some aspects of the present disclosure are directed to a method of treating or preventing a disease or a condition in a subject in need thereof comprising administering extracellular vesicles disclosed herein. Some aspects of the present disclosure are directed to the use of extracellular vesicles disclosed herein to treat or prevent a disease or condition in a subject in need thereof. Some aspects of the present disclosure are directed to extracellular vesicles disclosed herein for treating or preventing a disease or condition in a subject in need thereof.
• FIG. 1 is a line graph illustrating producer cell viability (%) following culture for up to 6 days in control medium or medium supplemented with an increasing concentration of rosuvastatin.
• FIGs. 2A-2B are line graphs illustrating producer cell proliferation (FIG. 2A) and EV production as measured by secreted luciferase activity (FIG. 2B) following culture in medium comprising cholesterol ("CH”), simvastatin (“SIM”), or both for up to 6 days.
• FIGs. 3A-3B are line graphs illustrating producer cell proliferation (FIG. 2A) and viability (FIG. 2B) following culture in medium comprising DMSO, cholesterol ("CH”), simvastatin (“SIM”), or both cholesterol and simvastatin for up to 15 days.
• FIGs. 4A-4B are line graphs illustrating EV production as measured by secreted luciferase activity following culture in medium comprising DMSO or cholesterol (“CHOL”) (FIG. 4A) or a combination of cholesterol (“CHOL”) simvastatin (“SIM”) (FIG. 4B), as compared to untreated controls, or both cholesterol and simvastatin for up to 14 days.
• DMSO or cholesterol
• CHOL cholesterol
• SIM simvastatin
• the present disclosure is directed to methods of increasing a number of extracellular vesicles (EVs) produced from producer cells, comprising (i) inhibiting a biosynthetic pathway in the producer cells and (ii) contacting the producer cells with cholesterol.
• the biosynthetic pathway is inhibited by contacting the producer cell with a cholesterol biosynthetic pathway inhibitor (e.g., a statin).
• a cholesterol biosynthetic pathway inhibitor e.g., a statin
• the present disclosure is directed to methods of producing extracellular vesicles (EVs) produced from producer cells, comprising (i) inhibiting a biosynthetic pathway in the producer cells and (ii) contacting the producer cells with cholesterol.
• the biosynthetic pathway is inhibited by contacting the producer cell with a cholesterol biosynthetic pathway inhibitor (e.g., a statin).
• a cholesterol biosynthetic pathway inhibitor e.g., a statin
• a or “an” entity refers to one or more of that entity; for example, "a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
• the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
• the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a negative limitation.
• Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.
• the term "about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some aspects, the term used herein means within 5% of the referenced amount, e.g., about 50% is understood to encompass a range of values from 47.5% to 52.5%.
• extracellular vesicle refers to a cell-derived vesicle comprising a membrane that encloses an internal space.
• Extracellular vesicles comprise all membrane-bound vesicles (e.g., exosomes, nanovesicles) that have a smaller diameter than the cell from which they are derived.
• extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular payload either within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and/or spanning the membrane.
• the payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
• an extracellular vesicle comprises a scaffold moiety.
• extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).
• Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells. In some aspects, the extracellular vesicles are produced by cells that express one or more transgene products.
• the percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• the comparison of sequences and determination of percent sequence identity between two sequences may be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences.
• One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S.
• HMGCR 3-hydroxy-3-methylglutaryl-coenzyme A reductase
• HMG Co-A reductase HMGCR
• HMGCR is suppressed by cholesterol derived from the internalization and degradation of low-density lipoprotein (LDL) via the LDL receptor.
• LDL low-density lipoprotein
• HMGCR gene refers to any transcript, genomic DNA, pre-mRNA, or mRNA.
• HMGCR protein refers to HMGCR isoform 1, HMGCR isoform 2, or HMGCR isoform 3, disclosed above, as well as variants and mutants thereof.
• HMGCR protein also encompasses any fragment or variant of any of the isoforms disclosed herein that has at least one function of the wild type HMGCR protein.
• a reduction in level of HMGCR gene in a modified cell refers to a decrease in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and/or mRNA that is capable of encoding a functional HMGCR protein, e.g., wild type HMGCR protein, compared to a reference (e.g., untreated) cell.
• a reduction in HMGCR protein can refer to changes resulting in the expression of a functional HMGCR protein, e.g., wild type HMGCR protein, including but not limited to changes (e.g., mutations or post-translational modifications) that cause a loss of function (partial or complete), or to the activity of molecules that bind to functional sites of HMGCR altering, e.g., its enzyme activity.
• sterol regulatory element binding factor 2 refers to a transcription factor that is the master regulator of cholesterol synthesis, activating expression of genes such as HMG-CoA reductase (HMGCR), HMG-CoA synthase (HMGCS), and mevalonate kinase (MVK).
• HMGCR HMG-CoA reductase
• HMGCS HMG-CoA synthase
• MVK mevalonate kinase
• the SREBF2 protein is encoded by the human SREBF2 gene, which is located on chromosome 15 (bases 82,031,470 to 82,089,580; NCBI Reference Sequence: NC_000081.7).
• the SREBF2 protein has two isoforms produced by alternative splicing. The sequences are shown in Table 2 below.
• SREBF2 is also known as lo, nu, SRE, nuc, SREB, SREBP, bHLHd, lopl3, SREBP2, bHLHd2, SREBP-2, AI608257, and SREBP2gc.
• reduced gene and/or protein function refers both to reduction in physical levels (e.g., less gene sequence due to edition from the genome, or less protein due a decrease in protein expression) and to reduction in function.
• a reduction in level of SREBF2 gene can refer to a decrease in gene function, e.g., due to the introduction of a mutation introducing a stop codon or a frame shift, to an epigenetic modification that would alter transcription, or to a mutation or other change on a promoter gene or another gene that regulates SREBF2 expression.
• a reduction in level of SREBF2 gene in a modified cell refers to a decrease in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and/or mRNA that is capable of encoding a functional SREBF2 protein, e.g., wild type SREBF2 protein, compared to a reference (e.g., untreated) cell.
• a reduction in SREBF2 protein can refer to changes resulting in the expression of a functional SREBF2 protein, e.g., wild type SREBF2 protein, including but not limited to changes (e.g., mutations or post-translational modifications) that cause a loss of function (partial or complete), or to the activity of molecules that bind to functional sites of SREBF2 altering, e.g., its transcriptional activating activity.
• a functional SREBF2 protein e.g., wild type SREBF2 protein, including but not limited to changes (e.g., mutations or post-translational modifications) that cause a loss of function (partial or complete), or to the activity of molecules that bind to functional sites of SREBF2 altering, e.g., its transcriptional activating activity.
• free IL- 12 moiety means an IL- 12 moiety that is not associated with an extracellular vesicle, but otherwise identical to the IL-12 moiety associated with the extracellular vesicle. Especially when compared to an extracellular vesicle associated with an IL- 12 moiety, the free IL- 12 moiety is the same IL- 12 moiety associated with the extracellular vesicle.
• the amount of the free IL-12 moiety compared to the IL-12 moiety associated with the extracellular vesicle is the same as the amount of the IL- 12 moiety associated with the EV.
• the term “ligand” refers to a molecule that binds to a receptor and modulates the receptor to produce a biological response. Modulation can be activation, deactivation, blocking, or damping of the biological response mediated by the receptor.
• Receptors can be modulated by either an endogenous or an exogenous ligand.
• Non-limiting examples of endogenous ligands include antibodies and peptides.
• Non-limiting examples of exogenous agonist include drugs, small molecules, and cyclic dinucleotides.
• the ligand can be a full, partial, or inverse ligand.
• the term “pharmaceutical composition” refers to one or more of the compounds described herein, such as, e.g., an EV mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically-acceptable carriers and excipients.
• a pharmaceutical composition is to facilitate administration of preparations of EVs to a subject.
• excipient or “carrier” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
• administration refers to introducing a composition, such as an EV, or agent into a subject and includes concurrent and sequential introduction of a composition or agent.
• the introduction of a composition or agent into a subject is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intra-arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly or topically.
• Administration includes self- administration and the administration by another.
• a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
• the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
• the compositions and methods described herein are applicable to both human therapy and veterinary applications.
• the subject is a mammal, and in other aspects the subject is a human.
• a “mammalian subject” includes all mammals, including without limitation, humans, domestic animals (e.g, dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like).
• perfusion culture refers to cell culturing methods or cell cultures in which cells are continuously fed with fresh media and spent media is continuously removed while keeping the cells in the culture vessel.
• culture vessels for perfusion culture comprise cell retention devices, such as capillary fibers or membranes.
• perfusion bioreactors with cell retention devices include bioreactors, such as the N-terminal production vessel, such as a stirred-tank bioreactor, connected to a cell retention device, such as hollow fiber filters or an acoustic cell separator.
• the perfusion culture is single-cell perfusion culture.
• the perfusion culture comprises use of a single-cell suspension perfusion bioreactor wherein individual cells are isolated for addition of fresh medium and removal of spent medium.
• the perfusion culture comprises separating cells from spent medium by centrifugation.
• fed-batch refers to cell culturing methods or cell cultures wherein cells remain in the culturing vessel until harvesting of the cells, in which an initial culture medium is added to an initial cell culture and additional feed medium is added to prevent nutrient depletion.
• feed medium is added once during the culturing process.
• the feed medium is added multiple times during the culturing process.
• fed-batch culture can include constantly-fed batch culture and exponential -fed batch culture.
• Treat,” “treatment,” or “treating,” as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
• the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
• the term “treating”, or “treatment” means inducing an immune response in a subject against an antigen.
• the disease or condition is a cancer.
• Prevent refers to decreasing or reducing the occurrence or severity of a particular outcome. In some aspects, preventing an outcome is achieved through prophylactic treatment. In some aspects, an EV, e.g., an exosome, comprising a cytokine, e.g., an IL-12 moiety, described herein is administered to a subject prophylactically.
• an EV e.g., an exosome, comprising a cytokine, e.g., an IL-12 moiety, described herein is administered to a subject prophylactically.
• genetic and/or pharmacological inhibition of a cholesterol synthesis pathway in producer cells increased the yield of harvested EVs compared to producer cells that did not have a genetic and/or pharmacological inhibition of a cholesterol synthesis pathway. This increase in EV yield comes at the cost of producer cell viability.
• Applicant has surprisingly discovered that inhibiting cholesterol biosynthesis in the producer cells while also contacting the producer cells with cholesterol restores cell viability and maintains increased EV production, relative to control cells.
• the producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 8 days. In some aspects, producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 9 days. In some aspects, producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 10 days. In some aspects, producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 11 days. In some aspects, producer cells are contacted with (i) the cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 12 days.
• the yield is increased at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, or at least about 30 fold.
• the EV yield is increased at least about 3 fold relative EVs produced by producer cells not contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, the EV yield is increased at least about 3.5 fold relative EVs produced by producer cells not contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, the EV yield is increased at least about
• the yield is increased about 2 fold to about 30 fold, about 2 fold to about 25 fold, about 2 fold to about 20 fold, about 2 fold to about 15 fold, about 12 fold to about 10 fold, about 2 fold to about 5 fold, about 5 fold to about 30 fold, about 5 fold to about 25 fold, about 5 fold to about 20 fold, about 5 fold to about 15 fold, about 5 fold to about 10 fold, about 10 fold to about 30 fold, about 10 fold to about 25 fold, about 10 fold to about 20 fold, about 10 fold to about 15 fold, about 15 fold to about 30 fold, about 15 fold to about 25 fold, about 15 fold to about 20 fold, about 20 fold to about 30 fold, about 20 fold to about 25 fold, or about 25 fold to about 30 fold. In some aspects, the yield is increased about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold.
• the viability of the producer cells is comparable to the viability of producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor and/or cholesterol.
• Cholesterol biosynthesis can be inhibited in the producer cell using any methods.
• the producer cell is contacted with a cholesterol biosynthetic pathway inhibitor.
• the cholesterol biosynthetic pathway inhibitor comprises a statin, a cariprazine, a PROTAC, AY9944, or BM 15766.
• the cholesterol biosynthetic pathway inhibitor comprises Meglutol, Clinofibrate, Hesperetin 7-O-glucoside, Cmpd 81 (also named HMG499, and related structures, described in Jiang et al., Nature Communications 9, 5138 (2016), which is herein incorporated by reference in its entirety), or Monacolin J.
• the expression of SM qualene monooxygenase or its protein can be inhibited or reduced.
• the expression of AC ATI acetyl-CoA acetyltransferase 1 or its protein can be inhibited or reduced.
• the expression of ACAT2 acetyl- CoA acetyltransferase 2) or its protein can be inhibited or reduced.
• the expression of HMGCS2 (3 -hydroxy-3 -methylglutaryl-CoA synthase 2) or its protein can be inhibited or reduced.
• the expression of MVK mevalonate kinase or its protein can be inhibited or reduced.
• the expression of FDFT1 farnesyl-diphosphate farnesyltransferase 1 or its protein can be inhibited or reduced.
• the expression of SQLE qualene epoxidase or its protein can be inhibited or reduced.
• the expression of LSS lanosterol synthase or its protein can be inhibited or reduced.
• the expression of DHCR24 24-dehydrochol esterol reductase
• the expression of CYP51A1 cytochrome P450 family 51 subfamily A polypeptide 1 or its protein can be inhibited or reduced.
• TM7SF2 transmembrane 7 superfamily member 2
• FAXDC2 C5orf4 fatty acid hydroxylase domain containing 2
• MSM01 SC4MOL methylsterol monooxygenase
• NSDHL NSD(P) dependent steroid dehydrogenase-like
• EBP emopamil binding protein (sterol isomerase) or its protein can be inhibited or reduced.
• HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7 or its protein
• HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7
• DHCR7 7-dehydrocholesterol reductase
• CEL carboxyl ester lipase
• LIPA lipase A, lysosomal acid
• SOAT1 sterol O-acetyltransferase 1
• SOAT1 sterol O-acetyltransferase 1
• the expression of SREBF1 (sterol binding element transcription factor 1) or its protein can be inhibited or reduced.
• the expression of SREBF2 (sterol binding element transcription factor 2) or its protein can be inhibited or reduced.
• the expression of SCAP (SREBF chaperone) can be inhibited or reduced.
• the expression of MBTPS1 (SIP membrane bound transcription factor peptidase site 1) or its protein can be inhibited or reduced.
• MBTPS2 S2P, membrane bound transcription factor peptidase site 2) or its protein can be inhibited or reduced.
• the expression of INSIG 1 (insulin induced gene 1) or its protein can be inhibited or reduced.
• the expression of INSIG2 insulin induced gene 2 or its protein can be inhibited or reduced.
• the expression of AMFR GP78, autocrine motility factor receptor E3 ubiquitin protein ligase
• the expression of NR1H3 LXRA, nuclear receptor subfamily 1 group H member 3 or its protein can be inhibited or reduced.
• the expression of NR1H2 LXRB, nuclear receptor subfamily 1 group H member 2) or its protein can be inhibited or reduced.
• the expression of RXRA retinoid X receptor alpha
• RXRB retinoid X receptor beta
• MYLIP myosin regulatory light chain interacting protein
• the methods of the present disclosure comprise modifying the producer cells to exhibit a reduced gene and/or protein function in a cholesterol biosynthetic pathway of the producer cells. In some aspects, the methods of the present disclosure comprise culturing the producer cells, which exhibit a reduced gene and/or protein function in a cholesterol biosynthetic pathway. In some aspects, the reduced gene and/or protein in a cholesterol biosynthesis pathway comprises HMGCR gene and/or HMGCR protein.
• HMGCR gene levels e.g, presence/absence of the entire gene or a portion thereof, or gene function
• HMGCR protein levels e.g, presence/absence of the HMGCR protein or fragments thereof, or quantification or protein function
• HMGCR protein levels can be measured by various methods known in the art.
• the HMGCR gene expression is reduced about 2 to about 20 fold. In some aspects, the HMGCR gene expression is reduced about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold.
• the gene and/or protein function in a cholesterol biosynthetic pathway is reduced at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10%, at least about 5%, or at least about 1%. In some aspects, the gene and/ protein function in a cholesterol biosynthetic pathway is reduced at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, or at least about 30%.
• the agent capable of reducing the gene and/or protein function in a cholesterol biosynthetic pathway comprises a gene editing technology.
• the gene editing technology comprises a shRNA, siRNA, miRNA, antisense oligonucleotides, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof.
• the gene editing technology comprises siRNA.
• the methods of the present disclosure comprise modifying the producer cells to exhibit a reduced gene and/or protein function in a cholesterol biosynthetic pathway of the producer cells.
• the methods of the present disclosure comprise culturing the producer cells, which exhibit a reduced gene and/or protein function in a cholesterol biosynthetic pathway.
• the reduced gene and/or protein in a cholesterol biosynthesis pathway comprises SREBF2 gene and/or SREBF2 protein.
• SREBF2 gene levels e.g., presence/absence of the entire gene or a portion thereof, or gene function
• SREBF2 protein levels e.g., presence/absence of the SREBF2 protein or fragments thereof, or quantification or protein function
• SREBF2 protein levels can be measured by various methods known in the art.
• the reduced expression levels of SREBF2 gene and/or expression or functional levels of SREBF2 protein in the producer cells can increase the yield of the EVs produced by the producer cells compared to EVs produced by producer cells that do not have a reduced expression levels of SREBF2 gene and/or expression levels of SREBF2 protein.
• the yield is increased at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, or at least about 30 fold.
• the yield is increased about 2 fold to about 30 fold, about 2 fold to about 25 fold, about 2 fold to about 20 fold, about 2 fold to about 15 fold, about 2 fold to about 10 fold, about 2 fold to about 5 fold, about 5 fold to about 30 fold, about 5 fold to about 25 fold, about 5 fold to about 20 fold, about 5 fold to about 15 fold, about 5 fold to about 10 fold, about 10 fold to about 30 fold, about 10 fold to about 25 fold, about 10 fold to about 20 fold, about 10 fold to about 15 fold, about 15 fold to about 30 fold, about 15 fold to about 25 fold, about 15 fold to about 20 fold, about 20 fold to about 30 fold, about 20 fold to about 25 fold, or about 25 fold to about 30 fold.
• the yield is increased about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold.
• the SREBF2 gene expression is reduced by about 2 fold to about 30 fold. In some aspects, the SREBF2 gene expression is reduced about 2 fold, about 3, fold, about
• the gene and/or protein function in a cholesterol biosynthetic pathway is reduced at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10%, at least about 5%, or at least about 1%. In some aspects, the gene and/or protein function in a cholesterol biosynthetic pathway is reduced at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, or at least about 30%.
• the reduced expression levels of HMGCR gene and/or expression or functional levels of HMGCR protein in the producer cells and/or the contacting with cholesterol can increase the yield of the EVs produced by the producer cells compared to EVs produced by a producer cells that do not have a reduced expression levels of HMGCR gene and/or expression levels of HMGCR protein or that were not contacted with cholesterol.
• the yield is increased at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about
• the yield is increased about 2 fold to about 30 fold, about 2 fold to about 25 fold, about 2 fold to about 20 fold, about 2 fold to about 15 fold, about 12 fold to about 10 fold, about 2 fold to about
• the yield is increased about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold.
• the modifying of the methods described herein comprises contacting the producer cells with an agent capable of reducing the SREBF2 gene and/or SREBF2 protein function.
• the agent comprises a Betulin (See Tang et al., CellMetab. 2011 Jan 5;13(l):44-56, which is herein incorporated by reference in its entirety).
• the agent comprises Fatostatin.
• the agent comprises Pseudoprotodioscin.
• the agent comprises oligomeric amyloid P42.
• the agent comprises luteolin.
• the agent comprises clofibrate.
• the modifying comprises contacting the producer cells with an agent capable of reducing the gene and/or protein function in a cholesterol biosynthetic pathway.
• the producer cells are modified prior to the culturing by contacting the producer cells with an agent capable of reducing the gene and/or protein function in a cholesterol biosynthetic pathway.
• the agent capable of reducing the gene and/or protein function in a cholesterol biosynthetic pathway comprises a gene editing technology.
• the gene editing technology comprises a shRNA, siRNA, miRNA, antisense oligonucleotides, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof.
• the gene editing technology comprises siRNA.
• one or more protein inhibitors can be used in the methods of increasing a number of extracellular vesicles produced from producer cells and methods of producing extracellular vesicles of the present disclosure.
• the protein inhibitor tool that can be used in the present disclosure comprises a proteolysis targeting chimera (PROTAC).
• PROTACs are heterobifunctional small molecules with three chemical elements: a ligand binding to a target protein, a ligand binding to E3 ubiquitin ligase, and a linker for conjugating these two ligands.
• PROTAC is a chemical knockdown strategy that degrades the target protein through the ubiquitin-proteasome system.
• PROTACs are catalytic in their mode of action, which can promote target protein degradation at low exposures.
• traditional small molecule inhibitors usually inhibit the enzymatic activity of the target, while PROTACs affect not only the enzymatic activity of the protein but also non- enzymatic activity by degrading the entire protein.
• the PROTAC comprises a statin (e.g., an HMGCR ligand) linked to E3 ubiquitin ligase ligand (e.g., VHL, CRBN, cIAPs, and MDM2), as described in Luo et al., Acta Pharmaceutica Sinica B, 2021 ;11(5): 1300-1314, which is herein incorporated by reference in its entirety.
• the PROTAC degrades HMCGR as described in Li et al., J. Med. Chem. 2020, 63, 9, 4908-4928, which is herein incorporated by reference in its entirety.
• the protein inhibitor comprises cariprazine.
• statins work by competitively blocking the active site of the first and key rate-limiting enzyme in the mevalonate pathway (e.g., cholesterol biosynthetic pathway), HMG-CoA reductase (HMGCR). Inhibition of this site prevents substrate access, thereby blocking the conversion of HMG-CoA to mevalonic acid.
• mevalonate pathway e.g., cholesterol biosynthetic pathway
• HMGCR HMG-CoA reductase
• statins The active component of statins is a modified 3, 5-dihydroxy glutaric acid moiety, which is structurally similar to the endogenous substrate, HMG-CoA, and the mevaldyl CoA transition state intermediate. This active site binds to and inhibits HMG-CoA reductase activity in a stereoselective process that requires the statin to have a 3R,5R configuration.
• statins arise from the ring that is attached to the active moiety, which can be a partially reduced naphthalene (lovastatin, simvastatin, pravastatin), a pyrrole (atorvastatin), an indole (fluvastatin), a pyrimidine (rosuvastatin), a pyridine (cerivastatin), or a quinoline (pitavastatin).
• the substituents on the ring define the solubility and pharmacological properties of the statin.
• statin Hydrophilicity (pravastatin and rosuvastatin) originates from the common active site plus other polar substituents, whereas lipophilicity (atorvastatin, lovastatin, fluvastatin, pitavastatin, simvastatin, and cerivastatin) arises because of the addition of nonpolar substituents.
• the statin can be selected from atorvastatin, lovastatin, pitavastatin, pravastatin, fluvastatin, cerivastatin, rosuvastatin, mevastatin, simvastatin, or combinations thereof.
• the statin comprises simvastatin.
• the statin comprises lovastatin, simvastatin, and/or pravastatin. In some aspects, the statin comprises atorvastatin. In some aspects, the statin comprises fluvastatin. In some aspects, the statin comprises rosuvastatin. In some aspects, the statin comprises cerivastatin. In some aspects, the statin comprises pitavastatin. In some aspects, the stating comprises pravastatin and/or rosuvastatin. In some aspects, the statin comprises atorvastatin, lovastatin, fluvastatin, pitavastatin, simvastatin, and/or cerivastatin.
• the statin e.g., simvastatin
• the producer cells at a concentration of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, or about 20 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, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about
• the statin is contacted at a concentration of between about 0.1 nM to about 100 nM, about 0.1 nM to about 90 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 70 nM, about 0.1 nM to about 60 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about 0.1 nM to about 30 nM, about 0.1 nM to about 20 nM, 0.1 nM to about 10 nM, or about 1 nM to about 20 nM, about 1 nM to about 10 nM, about InM to about 5 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to
• the statin e.g., simvastatin
• the statin is contacted with the producer cells at a concentration of about 1 nM to about 10 nM. In some aspects, the statin (e.g., simvastatin) is contacted with the producer cells at a concentration of about 10 nM to about 20 nM. In some aspects, the statin (e.g., simvastatin) is contacted with the producer cells at a concentration of about 10 nM to about 15 nM. In some aspects, the statin (e.g., simvastatin) is contacted with the producer cells at a concentration of about 15 nM to about 20 nM.
• the producer cell is contacted with about 10 nM simvastatin. In some aspects, the producer cell is contacted with about 15 nM simvastatin. In some aspects, the producer cell is contacted with about 20 nM simvastatin. In some aspects, the producer cell is contacted with about 25 nM simvastatin. In some aspects, the producer cell is contacted with about 30 nM simvastatin. In some aspects, the producer cell is contacted with about 35 nM simvastatin. In some aspects, the producer cell is contacted with about 40 nM simvastatin. In some aspects, the producer cell is contacted with about 45 nM simvastatin. In some aspects, the producer cell is contacted with about 50 nM simvastatin.
• the producer cell is contacted with about 10 nM rosuvastatin. In some aspects, the producer cell is contacted with about 15 nM rosuvastatin. In some aspects, the producer cell is contacted with about 20 nM rosuvastatin. In some aspects, the producer cell is contacted with about 25 nM rosuvastatin. In some aspects, the producer cell is contacted with about 30 nM rosuvastatin. In some aspects, the producer cell is contacted with about 35 nM rosuvastatin. In some aspects, the producer cell is contacted with about 40 nM rosuvastatin. In some aspects, the producer cell is contacted with about 45 nM rosuvastatin. In some aspects, the producer cell is contacted with about 50 nM rosuvastatin.
• the statin is contacted with the producer cells prior to harvesting EVs. In some aspects, the statin is contacted with the producer cells about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours prior to harvesting the EVs.
• the statin is contacted with the producer cells about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, about 132 hours, about 144 hours, about 156 hours, about 168 hours, about 180 hours, about 192 hours, about 204 hours, about 216 hours, about 228 hours, or about 240 hours.
• the statin is contacted with the producer cells between about 10 days to about 15 days, between about 10 days to about 20 days, between about 10 days to about 25 days, between about 10 days to about 30 days, between about 10 days to about 35 days, or between about 10 days to about 40 days prior to harvesting the EVs.
• statin is contacted with the producer cells while EVs are harvested. In some aspects, the statin is contacted with the producer cells over the duration of a continuous (e.g., perfusion) cell culture process that spans about 30 days, about 35 days, or about 40 days.
• a continuous cell culture process that spans about 30 days, about 35 days, or about 40 days.
• the producer cells are modified prior to the culturing by contacting the producer cells with an agent capable of reducing the gene and/or protein function in a cholesterol biosynthetic pathway.
• the agent is contacted with the producer cells at a concentration of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, or about 20 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, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about
• the statin is contacted at a concentration of between about 0.1 nM to about 100 nM, about 0.1 nM to about 90 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 70 nM, about 0.1 nM to about 60 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about 0.
• cholesterol biosynthesis is inhibited by genetically modifying the producer cell.
• One or more gene editing tools can be used in the methods of increasing a number of extracellular vesicles from producer cells and production and methods of producing extracellular vesicles of the present disclosure.
• the gene editing tool that can be used in the present disclosure comprises a CRISPR/Cas system.
• CRISPR/Cas systems can employ, for example, a Cas9 nuclease, which in some instances, is codon-optimized for the desired cell type in which it is to be expressed (e.g., CHO cells, e.g., MSC cells).
• CRISPR/Cas systems use Cas nucleases, e.g., Cas9 nucleases, that are targeted to a genomic site by complexing with a synthetic guide RNA (gRNA) that hybridizes to a target DNA sequence immediately preceding an NGG motif recognized by the Cas nuclease, e.g., Cas9.
• gRNA synthetic guide RNA
• a modified version of a Cas nuclease (e.g., Cas9) can be used that will lead to a single stranded nick as opposed to a double stranded break. Additional fusions with other enzymes can lead to site-specific base editing in the absence of a double stranded break.
• a unique capability of the CRISPR/Cas9 system is the ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more gRNAs (e.g., at least one, two, three, four, five, six, seven, eight, nine or ten gRNAs).
• Such systems can also employ a guide RNA (gRNA) that comprises two separate molecules.
• gRNA guide RNA
• the two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or “activator-RNA” or “tracrRNA” or “scaffold”) molecule.
• a crRNA comprises both the DNA-targeting segment (single stranded) of the gRNA and a stretch of nucleotides that forms one half of a double stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA.
• a corresponding tracrRNA comprises a stretch of nucleotides that forms the other half of the dsRNA duplex of the proteinbinding segment of the gRNA.
• a stretch of nucleotides of a crRNA is complementary to and hybridizes with a stretch of nucleotides of a tracrRNA to form the dsRNA duplex of the proteinbinding domain of the gRNA.
• each crRNA can be said to have a corresponding tracrRNA.
• the crRNA additionally provides the single stranded DNA-targeting segment.
• a gRNA comprises a sequence that hybridizes to a target sequence and a tracrRNA.
• a crRNA and a tracrRNA hybridize to form a gRNA. If used for modification within a cell, the exact sequence and/or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used (e.g., humans).
• Naturally-occurring genes encoding the three elements are typically organized in operon(s).
• Naturally-occurring CRISPR RN As differ depending on the Cas9 system and organism but often contain a targeting segment of between 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides (see, e.g., WO2014/131833).
• DR direct repeats
• the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long.
• the 3' located DR is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.
• a CRISPR system used herein can further employ a fused crRNA- tracrRNA construct (i.e., a single transcript) that functions with the codon-optimized Cas9.
• This single RNA is often referred to as a guide RNA or gRNA or single guide RNA, or sgRNA.
• the crRNA portion is identified as the "target sequence" for the given recognition site and the tracrRNA is often referred to as the "scaffold.”
• a short DNA fragment containing the target sequence is inserted into a guide RNA expression plasmid.
• the gRNA expression plasmid comprises the target sequence (in some aspects around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells.
• the scaffold a form of the tracrRNA sequence
• a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells.
• Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid.
• the gRNA expression cassette and the Cas9 expression cassette are then introduced into the cell. See, for example, Mali P et al., (2013) Science 2013 Feb. 15; 339(6121):823-6; Jinek M et al. , Science 2012 Aug. 17; 337(6096):816-21; Hwang W Y etal., NatBiotechnolZQV March; 31(3):227-9; Jiang W etal., Nat Biotechnol 2013 March; 31 (3):233-9; Cronican etal., ACS Chem. Biol. 5(8):747-52 (2010); and Cong L et al., Science 2013 Feb.
• the Cas9 nuclease can be provided in the form of a protein.
• the Cas9 protein can be provided in the form of a complex with the gRNA.
• the Cas9 nuclease can be provided in the form of a nucleic acid encoding the protein.
• the nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA.
• mRNA messenger RNA
• the gRNA can be provided in the form of RNA.
• the gRNA can be provided in the form of DNA encoding the RNA.
• the gRNA can be provided in the form of separate crRNA and tracrRNA molecules, or separate DNA molecules encoding the crRNA and tracrRNA, respectively.
• two separate Cas proteins .g., nickases
• nickases specific for a target site on each strand of dsDNA
• the overhanging ends created by contacting a nucleic acid with two nickases specific for target sites on both strands of dsDNA can be either 5' or 3' overhanging ends.
• a first nickase can create a single strand break on the first strand of dsDNA
• a second nickase can create a single strand break on the second strand of dsDNA such that overhanging sequences are created.
• the target sites of each nickase creating the single strand break can be selected such that the overhanging end sequences created are complementary to overhanging end sequences on a different nucleic acid molecule.
• the complementary overhanging ends of the two different nucleic acid molecules can be annealed by the methods disclosed herein.
• the target site of the nickase on the first strand is different from the target site of the nickase on the second strand.
• the expression of HMGCR and/or SREBF2 gene, and the HMGCR and/or SREBF2 protein encoded thereof is reduced by contacting the cell with a CRISPR (e.g., CRISPR-Cas9 system) that is, e.g., specific to the HMGCR and/or SREBF2 gene.
• a CRISPR e.g., CRISPR-Cas9 system
• gene editing using CRISPR reduces (e.g., HMGCR and/or SREBF2') gene levels at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% with respect to HMGCR and/or SREBF2 gene levels observed in a reference cell (e.g., a corresponding cell that has not been subjected to gene editing using CRISPR).
• HMGCR and/or SREBF2 gene levels can be measured using any technique known in the art, e.g., by digital droplet PCR
• the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. For example, for ZFPs in which the finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger binding domain; a nine- nucleotide target sequence is bound by a three-finger binding domain, etc.
• RNAi Interference RNA
• shRNAs or “short hairpin RNA” molecules
• the double-stranded region is typically about 19 nucleotides to about 29 nucleotides in length on each side of the stem, and the loop region is typically about three to about ten nucleotides in length (and 3'- or 5'-terminal singlestranded overhanging nucleotides are optional).
• shRNAs can be cloned into plasmids or in nonreplicating recombinant viral vectors to be introduced intracellularly and result in the integration of the shRNA-encoding sequence into the genome. As such, an shRNA can provide stable and consistent repression of endogenous target gene translation and expression.
• a gene editing tool useful for the present disclosure comprises one or more siRNAs.
• siRNAs refer to double stranded RNA molecules typically about 21-23 nucleotides in length.
• the siRNA associates with a multi protein complex called the RNA-induced silencing complex (RISC), during which the "passenger” sense strand is enzymatically cleaved.
• RISC RNA-induced silencing complex
• the antisense "guide” strand contained in the activated RISC guides the RISC to the corresponding mRNA because of sequence homology and the same nuclease cuts the target mRNA, resulting in specific gene silencing.
• ASOs can induce the degradation of the HMGCR and/or SREBF2 mRNA and thereby, reduce the expression of HMGCR and/or SREBF2 protein.
• Some aspects of the present disclosure are directed to methods of producing EVs from producer cells, comprising inhibiting a cholesterol biosynthesis pathway (e.g., by contacting the producer cells with a statin) and contacting the producer cells with cholesterol.
• the producer cells are contacted with (i) a statin and (ii) cholesterol.
• the producer cells are contacted with the statin prior to being contacted with the cholesterol.
• the producer cells are contacted with the statin after being contacted with the cholesterol.
• the producer cells are contacted with the statin and the cholesterol concurrently.
• the producer cells are cultured in a medium comprising a statin and cholesterol. In some aspects, the producer cells are cultured in the medium for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days. In some aspects, the producer cells are cultured in the medium for more than 6 days.
• the producer cells are cultured in the medium for more than 7 days. In some aspects, the producer cells are cultured in the medium for more than 8 days. In some aspects, the producer cells are cultured in the medium for more than 9 days. In some aspects, the producer cells are cultured in the medium for more than 10 days. In some aspects, the producer cells are cultured in the medium for more than 10 days. In some aspects, the producer cells are cultured in the medium for more than 11 days. In some aspects, the producer cells are cultured in the medium for more than 12 days. In some aspects, the producer cells are cultured in the medium for more than 13 days. In some aspects, the producer cells are cultured in the medium for more than 14 days. In some aspects, the producer cells are cultured in the medium for more than 15 days.
• the producer cells are cultured in the medium for more than 16 days. In some aspects, the producer cells are cultured in the medium for more than 17 days. In some aspects, the producer cells are cultured in the medium for more than 18 days. In some aspects, the producer cells are cultured in the medium for more than 19 days. In some aspects, the producer cells are cultured in the medium for more than 20 days. In some aspects, the producer cells are cultured in the medium for more than 21 days.
• the producer cells are contacted with cholesterol at a concentration of about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 6
• the producer cells are contacted with cholesterol at a concentration of about 0.1 pM to about 100 pM, about 0.1 pM to about 75 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 40 pM, about 0.1 pM to about 30 pM, about 0.1 pM to about 25 pM, about 0.1 pM to about 20 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, 1 pM to about 30 pM, or about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 2 pM to about 3 pM, about 20 pM to about 30 pM, about 25 pM to about 30 pM, or about 20 pM to about 25 pM
• the producer cells are contacted with cholesterol at a concentration of about 2.5 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 5 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 6 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 7 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 8 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 9 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 10 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 15 pM.
• the producer cells are contacted with cholesterol at a concentration of about 20 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 25 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 30 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 35 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 40 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 45 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 50 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 60 pM.
• the producer cells are contacted with cholesterol at a concentration of about 70 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 80 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 90 pM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 100 pM.
• the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 2.5 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 5 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 10 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 15 pM.
• the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 20 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 25 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 30 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 35 pM.
• the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 40 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 45 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 50 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 60 pM.
• the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 70 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 80 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 90 pM. In some aspects, the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 100 pM.
• the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 2.5 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 5 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 10 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 15 pM.
• the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 20 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 25 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 30 pM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and (ii) cholesterol at a concentration of about 35 pM.
• the cholesterol is added to the culture medium.
• the cholesterol is produced by a cell in the culture.
• the cholesterol is produced by the producer cell.
• the cholesterol is a synthetic cholesterol.
• DMSO dimethyl sulfoxide
• the producer cells are cultured in a medium comprising DMSO. In some aspects, the producer cells are cultured in a medium comprising DMSO and cholesterol. In some aspects, the producer cells are cultured in a medium comprising DMSO and a statin. In some aspects, the producer cells are cultured in a medium comprising (i) DMSO, (ii) a statin, and (iii) cholesterol.
• the producer cells are cultured in the medium for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days.
• the producer cells are cultured in the medium for more than 6 days. In some aspects, the producer cells are cultured in the medium for more than 7 days.
• the producer cells are cultured in the medium for more than 8 days. In some aspects, the producer cells are cultured in the medium for more than 9 days. In some aspects, the producer cells are cultured in the medium for more than 10 days. In some aspects, the producer cells are cultured in the medium for more than 10 days. In some aspects, the producer cells are cultured in the medium for more than 11 days. In some aspects, the producer cells are cultured in the medium for more than 12 days. In some aspects, the producer cells are cultured in the medium for more than 13 days. In some aspects, the producer cells are cultured in the medium for more than 14 days. In some aspects, the producer cells are cultured in the medium for more than 15 days. In some aspects, the producer cells are cultured in the medium for more than 16 days.
• the producer cells are cultured in the medium for more than 17 days. In some aspects, the producer cells are cultured in the medium for more than 18 days. In some aspects, the producer cells are cultured in the medium for more than 19 days. In some aspects, the producer cells are cultured in the medium for more than 20 days. In some aspects, the producer cells are cultured in the medium for more than 21 days.
• the producer cells are contacted with DMSO at a concentration of about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.
• the producer cells are contacted with cholesterol at a concentration of about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.05% to about 2%, about 0.05% to about 1.5%, about 0.05% to about 1%, about 0.05% to about .5%, 0.1% to about 2%, or about 0.1% to about 1.5%, about 0.1% to about 1%, or about 0.1% to about 0.5%.
• the producer cells are contacted with DMSO at a concentration of about 0.1%.
• EVs e.g., exosomes.
• the methods improve EV, e.g., exosome production yields.
• the EVs produced by the producer cells have decreased cholesterol content per EV compared to EVs produced by producer cells that the gene and/or protein function in a cholesterol biosynthetic pathway is not reduced, e.g., an EV produced by producer cells not contacted with a statin disclosed herein.
• the cholesterol content per EV is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
• the cholesterol content per EV is reduced by about 1% to about 80%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50%.
• the EVs produced by the producer cells have a cholesterol content per EV that is similar to or the same as EVs produced by producer cells that the gene and/or protein function in a cholesterol biosynthetic pathway is not reduced, e.g., an EV produced by producer cells not contacted with a statin disclosed herein.
• an EV, e.g., exosome, of the present disclosure has a diameter between about 20-290 nm, 20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160 nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 20-50 nm, 20-40 nm, 20-30 nm, 30-300 nm, 30-290 nm, 30-280 nm, 30-270 nm, 30-260 nm, 30- 250 nm, 30-240 nm,
• an EV, e.g., exosome, of the present disclosure comprises a bilipid membrane ("EV, e.g., exosome, membrane”), comprising an interior (luminal) surface and an exterior surface.
• the interior (luminal) surface faces the inner core (i.e., lumen) of the EV, e.g., exosome.
• the exterior surface can be in contact with the endosome, the multivesicular bodies, or the membrane/cytoplasm of a producer cell or a target cell.
• the EV, e.g., exosome, membrane comprises lipids and fatty acids.
• the EV, e.g., exosome, membrane comprises an inner leaflet and an outer leaflet.
• the composition of the inner and outer leaflet can be determined by transbilayer distribution assays known in the art, see, e.g., Kuypers et al., Biohim Biophys Acta 1985 819: 170.
• the composition of the outer leaflet is between approximately 70-90% choline phospholipids, between approximately 0-15% acidic phospholipids, and between approximately 5-30% phosphatidylethanolamine.
• the composition of the inner leaflet is between approximately 15-40% choline phospholipids, between approximately 10-50% acidic phospholipids, and between approximately 30-60% phosphatidylethanolamine.
• the EV, e.g., exosome comprises between about 40% to about 60% cholesterol.
• the EVs produced by the producer cells have a decreased cholesterol content per EV compared to EVs produced by producer cells that the gene and/or protein function in a cholesterol biosynthetic pathway is not reduced.
• the EV e.g., exosome, membrane comprises one or more polysaccharide, such as glycan.
• the EV e.g., exosome
• the EV comprises an IL- 12 moiety, wherein the IL-12 moiety is linked to the EV via a scaffold moiety, either on the exterior surface of the EV or on the luminal surface of the EV.
• the EV e.g., exosome, of the present disclosure comprises an IL- 12 moiety in the lumen of the EV.
• the EV comprises an IL-12 moiety on the exterior surface of the EV, optionally linked via a first scaffold moiety (e.g., Scaffold X).
• the EV comprises an IL-12 moiety on the luminal surface of the EV, optionally linked via a scaffold moiety (e.g., Scaffold X or Scaffold Y).
• One or more scaffold moieties can be used to anchor an IL- 12 moiety to the EV of the present disclosure.
• the IL-12 moiety is linked to the scaffold moiety.
• the EV comprises more than one scaffold moiety.
• the IL- 12 is linked to a first scaffold moiety and a second moiety (e.g., a second polypeptide or a polynucleotide) is linked to a second scaffold moiety.
• the first scaffold moiety and the second scaffold moiety are the same type of scaffold moiety, e.g., the first and second scaffold moi eties are both a Scaffold X protein.
• the first scaffold moiety and the second scaffold moiety are different types of scaffold moiety, e.g., the first scaffold moiety is a Scaffold Y protein and the second scaffold moiety is a Scaffold X protein.
• the first scaffold moiety is a Scaffold Y, disclosed herein.
• the first scaffold moiety is a Scaffold X, disclosed herein.
• the second scaffold moiety is a Scaffold Y, disclosed herein.
• the second scaffold moiety is a Scaffold X, disclosed herein.
• the EV comprises one or more scaffold moi eties, which are capable of anchoring, e.g., n IL- 12 moiety, to the EV, e.g., exosome, (e.g., either on the luminal surface or on the exterior surface).
• the scaffold moiety is a polypeptide ("scaffold protein").
• the scaffold protein comprises an exosome protein or a fragment thereof.
• scaffold moieties are non-polypeptide moieties.
• scaffold proteins include various membrane proteins, such as transmembrane proteins, integral proteins and peripheral proteins, enriched on the exosome membranes.
• a scaffold moiety (e.g., scaffold protein) comprises Scaffold X.
• a scaffold moiety (e.g., exosome protein) comprises Scaffold Y.
• a scaffold moiety (e.g., exosome protein) comprises both a Scaffold X and a Scaffold Y.
• the IL-12 moiety is linked to a scaffold moiety (e.g., Scaffold X) on the exterior surface of the EV. In some aspects, the IL-12 moiety is linked to a scaffold moiety (e.g., Scaffold X) on the luminal surface of the EV. In some aspects, the IL-12 moiety is linked to a scaffold moiety (e.g., Scaffold Y) on the luminal surface of the EV.
• a scaffold moiety e.g., Scaffold X
• EVs e.g., exosomes
• their membrane compositions can be modified by changing the protein, lipid, or glycan content of the membrane.
• the surface-engineered EVs e.g., exosomes
• the surface-engineered EVs are generated by chemical and/or physical methods, such as PEG-induced fusion and/or ultrasonic fusion.
• the surface-engineered EVs, e.g., exosomes are generated by genetic engineering.
• EVs, e.g., exosomes, produced from a genetically-modified producer cell or a progeny of the genetically-modified cell can contain modified membrane compositions.
• surface- engineered EVs e.g., exosomes
• have scaffold moiety e.g., exosome protein, e.g., Scaffold X
• higher or lower density e.g., higher number
• surface (e.g., Scaffold X)-engineered EVs can be produced from a cell (e.g., HEK293 cells) transformed with an exogenous sequence encoding a scaffold moiety (e.g., exosome proteins, e.g., Scaffold X) or a variant or a fragment thereof.
• EVs including scaffold moiety expressed from the exogenous sequence can include modified membrane compositions.
• scaffold moiety modified to have enhanced affinity to a binding agent can be used for generating surface-engineered EV that can be purified using the binding agent.
• Scaffold moi eties modified to be more effectively targeted to EVs and/or membranes can be used.
• Scaffold moieties modified to comprise a minimal fragment required for specific and effective targeting to exosome membranes can be also used.
• Scaffold moieties can be engineered to be expressed as a fusion molecule, e.g., fusion molecule of Scaffold X to an IL-12 moiety.
• the fusion molecule can comprise a scaffold moiety disclosed herein (e.g., Scaffold X, e.g., PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof) linked to an IL-12 moiety.
• the surface (e.g., Scaffold X)-engineered EVs described herein demonstrate superior characteristics compared to EVs known in the art.
• surface (e.g., Scaffold X)-engineered contain modified proteins more highly enriched on their surface than naturally occurring EVs or the EVs produced using conventional exosome proteins.
• the surface (e.g., Scaffold X)-engineered EVs of the present disclosure can have greater, more specific, or more controlled biological activity compared to naturally occurring EVs or the EVs produced using conventional exosome proteins.
• the Scaffold X comprises Prostaglandin F2 receptor negative regulator (the PTGFRN polypeptide).
• the PTGFRN protein can be also referred to as CD9 partner 1 (CD9P-1), Glu-Trp-Ile EWI motif-containing protein F (EWLF), Prostaglandin F2-alpha receptor regulatory protein, Prostaglandin F2-alpha receptor-associated protein, or CD315.
• CD9P-1 CD9 partner 1
• EWLF Glu-Trp-Ile EWI motif-containing protein F
• Prostaglandin F2-alpha receptor regulatory protein Prostaglandin F2-alpha receptor-associated protein
• the full length amino acid sequence of the human PTGFRN protein (Uniprot Accession No. Q9P2B2) is shown at Table 3 as SEQ ID NO: 1.
• the PTGFRN polypeptide contains a signal peptide (amino acids 1 to 25 of SEQ ID NO: 1), the extracellular domain (amino acids 26 to 832 of SEQ ID NO: 1), a transmembrane domain (amino acids 833 to 853 of SEQ ID NO: 1), and a cytoplasmic domain (amino acids 854 to 879 of SEQ ID NO: 1).
• the mature PTGFRN polypeptide consists of SEQ ID NO: 1 without the signal peptide, i.e., amino acids 26 to 879 of SEQ ID NO: 1.
• a PTGFRN polypeptide fragment useful for the present disclosure comprises a transmembrane domain of the PTGFRN polypeptide.
• a PTGFRN polypeptide fragment useful for the present disclosure comprises the transmembrane domain of the PTGFRN polypeptide and (i) at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 amino acids at the N terminus of the transmembrane domain, (ii) at least five, at least 10, at least 15, at least 20, or at least 25 amino acids at the C terminus of the transmembrane domain, or both (i) and (ii).
• the fragments of PTGFRN polypeptide lack one or more functional or structural domains, such as IgV.
• the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 26 to 879 of SEQ ID NO: 1.
• the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1.
• the Scaffold X comprises an amino acid sequence at least about 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 33.
• the Scaffold X comprises the amino acid sequence of SEQ ID NO: 33, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
• the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
• the Scaffold X comprises the amino acid sequence of SEQ ID NO: 33 and 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or longer at the N terminus and/or C terminus of SEQ ID NO: 33.
• the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from both the N-terminus and C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking one or more functional or structural domains of the native protein.
• the scaffold moi eties are linked to one or more heterologous proteins.
• the one or more heterologous proteins can be linked to the N-terminus of the scaffold moieties.
• the one or more heterologous proteins can be linked to the C-terminus of the scaffold moieties.
• the one or more heterologous proteins are linked to both the N-terminus and the C-terminus of the scaffold moieties.
• the heterologous protein is a mammalian protein.
• the heterologous protein is a human protein.
• Scaffold X can be used to link any moiety, e.g., an IL- 12 moiety, to the luminal surface and on the exterior surface of the EV, e.g., exosome, at the same time.
• the PTGFRN polypeptide can be used to link an IL-12 moiety inside the lumen (e.g., on the luminal surface) in addition to the exterior surface of the EV, e.g., exosome.
• Scaffold X is a scaffold protein that is capable of anchoring the IL- 12 on the luminal surface of the EV and/or on the exterior surface of the EV.
• EVs e.g., exosomes
• EVs comprise an internal space (i.e., lumen) that is different from that of the naturally occurring EVs.
• the EV can be changed such that the composition in the luminal surface of the EV, e.g., exosome has the protein, lipid, or glycan content different from that of the naturally-occurring exosomes.
• engineered EVs e.g., exosomes
• a scaffold moiety e.g., exosome proteins, e.g., Scaffold Y
• modification or a fragment of the scaffold moiety that changes the composition or content of the luminal surface of the EV, e.g., exosome.
• modifications or fragments of the exosome protein that can be expressed on the luminal surface of the EV, e.g., exosome can be used for the aspects of the present disclosure.
• the exosome proteins that can change the luminal surface of the EVs include, but are not limited to, the myristoylated alanine rich Protein Kinase C substrate (MARCKS) protein, the myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1) protein, the brain acid soluble protein 1 (BASP1) protein, or any combination thereof.
• MARCKS myristoylated alanine rich Protein Kinase C substrate
• MARCKSL1 myristoylated alanine rich Protein Kinase C substrate like 1
• BASP1 brain acid soluble protein 1
• Scaffold Y comprises the MARCKS protein (Uniprot accession no. P29966).
• the MARCKS protein is also known as protein kinase C substrate, 80 kDa protein, light chain.
• the full-length human MARCKS protein is 332 amino acids in length and comprises a calmodulin-binding domain at amino acid residues 152-176.
• Scaffold Y comprises the MARCKSL1 protein (Uniprot accession no. P49006).
• the MARCKSL1 protein is also known as MARCKS-like protein 1, and macrophage myristoylated alanine-rich C kinase substrate.
• the full-length human MARCKSL1 protein is 195 amino acids in length.
• the MARCKSL1 protein has an effector domain involved in lipid-binding and calmodulin-binding at amino acid residues 87-110.
• the Scaffold Y comprises the BASP1 protein (Uniprot accession number P80723).
• the BASP1 protein is also known as 22 kDa neuronal tissue- enriched acidic protein or neuronal axonal membrane protein NAP-22.
• the full-length human BASP1 protein sequence (isomer 1) is 227 amino acids in length. An isomer produced by an alternative splicing is missing amino acids 88 to 141 from SEQ ID NO: 49 (isomer 1).
• Table 4 provides the full-length sequences for the exemplary Scaffold Y disclosed herein (i.e., the MARCKS, MARCKSL1, and BASPl proteins).
• the mature BASP1 protein sequence is missing the first Met from SEQ ID NO: 49 and thus contains amino acids 2 to 227 of SEQ ID NO: 49.
• Scaffold Y useful for the present disclosure comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 2 to 227 of SEQ ID NO: 49.
• a Scaffold Y useful for the present disclosure comprises the amino acid sequence of SEQ ID NO: 49, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
• the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
• the protein sequence of SEQ ID NO: 49 without Met at amino acid residue 1 of the SEQ ID NO: 49 is sufficient to be a Scaffold Y for the present disclosure (e.g., scaffold moiety linked to an IL-12 moiety.
• the Scaffold Y comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 49 without Met at amino acid residue 1 of the SEQ ID NO: 49.
• Scaffold Y-engineered EVs e.g., exosomes described herein can be produced from a cell transformed with a sequence set forth in SEQ ID NO: 49 without Met at amino acid residue 1 of the SEQ ID NO: 49.
• the Scaffold Y protein useful for the present disclosure comprises an "N-terminus domain” (ND) and an "effector domain” (ED), wherein the ND and/or the ED are associated with the luminal surface of the EV, e.g., an exosome.
• the Scaffold Y protein useful for the present disclosure comprises an intracellular domain, a transmembrane domain, and an extracellular domain; wherein the intracellular domain comprises an "N-terminus domain” (ND) and an "effector domain” (ED), wherein the ND and/or the ED are associated with the luminal surface of the EV, e.g., an exosome.
• the term "associated with” refers to the interaction between a scaffold protein with the luminal surface of the EV, e.g., and exosome, that does not involve covalent linking to a membrane component.
• the scaffolds useful for the present disclosure can be associated with the luminal surface of the EV, e.g., via a lipid anchor (e.g., myristic acid), and/or a polybasic domain that interacts electrostatically with the negatively charged head of membrane phospholipids.
• the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic compounds e.g., sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be added to the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the EVs, e.g., exosomes, in an effective amount and in an appropriate solvent with one or more ingredients enumerated herein or known in the art, as desired.
• dispersions are prepared by incorporating the EVs, e.g., exosomes, into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients.
• methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• the EVs e.g., exosomes
• compositions comprising exosomes can also be by transmucosal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of, e.g., nasal sprays.
• the pharmaceutical composition comprising EVs, e.g., exosomes is administered intravenously into a subject that would benefit from the pharmaceutical composition.
• the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti el al., PNAS 105(46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or into the liver.
• the pharmaceutical composition comprising exosomes is administered as a liquid suspension.
• the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration.
• the depot slowly releases the EVs, e.g., exosomes, into circulation, or remains in depot form.
• compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
• the pharmaceutically-acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto.
• the pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
• compositions described herein comprise the EVs, e.g., exosomes, described herein and optionally a pharmaceutically active or therapeutic agent.
• the therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.
• Dosage forms are provided that comprise a pharmaceutical composition comprising the EVs, e.g., exosomes, described herein.
• the dosage form is formulated as a liquid suspension for intravenous injection.
• the dosage form is formulated as a liquid suspension for intratumoral injection.
• the preparation of exosomes is subjected to radiation, e.g., X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.
• radiation e.g., X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.
• the preparation of exosomes is subjected to gamma irradiation using an irradiation dose of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 kGy.
• the preparation of exosomes is subjected to X-ray irradiation using an irradiation dose of more than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.
• the present disclosure also provides methods of treating a disease or disorder in a subject in need thereof comprising administering the extracellular vesicle described herein to the subject.
• statins to increase exosome production is likely incompatible with a manufacturing approach that requires continuous upstream cell culture platform that operates over the course of several weeks.
• data generated to confirm the observed increase in EVs induced by statins was cholesterol dependent pointed to a potential solution to sustain cells exposed to statin by supplementing the media with supplemental cholesterol.
• a HEK293 cell line engineered to overexpress a PTGFRN-HiBit luciferase reporter on the engineered EVs showed reduced proliferation in the presence of 10 nM simvastatin due to toxicity during treatment (FIG. 2A).
• proliferation returns to levels comparable to the untreated control.
• the PTGFRN-HiBit reporter cell line used in previous Example was also employed here. Cells were seeded at 2e6vc/ml in 10 mL of a proprietary seed train media. Cells were cultured in a humidified incubator set at 37 °C and 8% CO2 and agitated at 200 rpms.
• the combination treatment also produced an increase in EV associated reporter activity beginning on day 8, peaking on day 11 and remaining steady for the remaining 3 days until termination of the experiment (FIG. 4B). Over those last four days, the mean daily increase in EV associated reporter activity measured in the clarified harvest was 7.5-fold.
• the statin/cholesterol combo treatment showed a 2.5-fold increase in secreted reporter activity relative to the untreated controls, the mean 7.5-fold increase seen in this study appears unexpected.
• six days of treatment in this new study corresponds to day 10, at which point the mean induction of secreted reporter activity had only reached 4.7-fold.
• producer cells will be cultured according to the methods above, and extracellular vesicles will be isolated. The extracellular vesicles will be purified and subjected to biochemical, biophysical and functional analysis.

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