Method for producing extracellular vesicles

Abstract

The present disclosure provides methods of generating extracellular vesicles as well as methods of increasing extracellular vesicle production from producer cells that exhibit reduced gene and / or protein function in the cholesterol biosynthetic pathway wherein the producer cells are contacted with cholesterol.

Description

Technical Field

[0001] The present disclosure relates to the production of extracellular vesicles (eg, exosomes) by producer cells, wherein the producer cells are contacted with (i) a cholesterol biosynthesis pathway inhibitor and (ii) cholesterol. Background Art

[0002] Extracellular vesicles, particularly exosomes, have attracted considerable attention as a novel means of efficiently delivering a variety of payloads to all cell types within living organisms. As efforts to translate exosome biology into novel drugs accelerate, a technological gap has emerged between the current state-of-the-art technologies for producing exosomes and the capabilities required to support large-scale clinical and commercial manufacturing. To this end, significant efforts have focused on maintaining the growth and productivity of producer cell lines in vitro; however, maximizing exosome yield remains a challenge. Therefore, novel methods for efficiently, cost-effectively, and reliably producing extracellular vesicles at high titers are needed. Summary of the Invention

[0003] Some aspects of the present disclosure relate to a method of increasing the number of extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthesis pathway inhibitor and (ii) cholesterol.

[0004] Some aspects of the present disclosure relate to a method of producing extracellular vesicles (EVs) from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthesis pathway inhibitor and (ii) cholesterol.

[0005] In some aspects, the producer cells are contacted with (i) the cholesterol biosynthesis pathway inhibitor and (ii) cholesterol for greater than 8 days.

[0006] In some aspects, the EVs produced by the producer cells have increased yield compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthesis pathway inhibitor. In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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.

[0007] In some aspects, the cholesterol biosynthesis pathway inhibitor comprises a statin, cariprazine, PROTAC, AY9944, or BM15766. In some aspects, the statin comprises atorvastatin, lovastatin, pitavastatin, pravastatin, fluvastatin, cerivastatin, rosuvastatin, simvastatin, or a combination thereof.

[0008] In some aspects, the statins are administered at 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 160nM, about 170nM, about 180nM, about 190nM, about 200nM, about 210nM, about 220nM, about 230nM, about 240nM, about 250nM, about 260nM, about 270nM, about 280nM, about 290nM, about 300nM, about 310nM, about 320nM, about 330nM, about 340nM, about 350nM, about 360nM, about 370nM, about 380nM, about 390nM, about 400nM, about 410nM, about 420nM, about 430nM, about 440nM M, about 450nM, about 460nM, about 470nM, about 480nM, about 490nM, about 500nM, about 510nM, about 520nM, about 530nM, about 540nM, about 550nM, about 560nM, about 570nM, about 580nM, about 590nM, about 600nM, about 610nM, about 620nM, about 630nM, about 640nM, about 650nM, about 660nM, about 670nM, about 680nM, about 690nM, about 700nM, about 710nM, about 720nM, about 7 The present invention relates to a method for contacting an infected human fetus with a concentration of about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about 1,000 nM, about

[0009] In some aspects, the statin is administered 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 0.1 nM to about 10 nM. In some aspects, the statin is contacted at a concentration of about 10 nM. In some aspects, the statin is contacted at a concentration of about 15 nM. In some aspects, the statin is contacted at a concentration of about 20 nM.

[0010] In some aspects, the cholesterol biosynthetic pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 10 nM. In some aspects, the cholesterol biosynthetic pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 15 nM. In some aspects, the cholesterol biosynthetic pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 20 nM.

[0011] In some aspects, the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 10 nM. In some aspects, the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 25 nM. In some aspects, the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 50 nM.

[0012] In some aspects, the producer cells are at about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM. , about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM , about 38 μM, about 39 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 150 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM or about 1,000 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 0.1 μM to about 100 μM, about 0.1 μM to about 75 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 40 μM, about 0.1 μM to about 30 μM, about 0.1 μM to about 25 μM, about 0.1 μM to about 20 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, 1 μM to about 30 μM, or about 1 μM to about 25 μM, about 1 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 2 μM to about 3 μM, about 20 μM to about 30 μM, about 25 μM to about 30 μM, or about 20 μM to about 25 μM.

[0013] In some aspects, the producer cells are contacted with cholesterol at a concentration of about 2.5 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 20 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 25 μM.

[0014] In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 2.5 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 25 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 2.5 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 25 μM.

[0015] In some respects, compared to the producer cell that (i) contacts with cholesterol biosynthetic pathway inhibitor and (ii) is not contacted with cholesterol, producer cell has the vigor of increase.In some respects, compared to the producer cell that (i) contacts with cholesterol biosynthetic pathway inhibitor and (ii) is not contacted with cholesterol, producer cell has the vigor of increase.In some respects, vigor increases 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%.

[0016] In some aspects, the EVs produced by the producer cells have reduced cholesterol content per EV compared to EVs produced by producer cells that have not been contacted with the cholesterol biosynthesis pathway inhibitor. In some aspects, 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%. In some aspects, 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%.

[0017] Some aspects of the present disclosure relate to a method of increasing the number of extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with dimethyl sulfoxide (DMSO). Some aspects of the present disclosure relate to a method of producing extracellular vesicles (EVs) from producer cells, the method comprising contacting the producer cells with DMSO. In some aspects, the producer cells produce EVs at 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 0.40%, about 0.50%, about 0.60%, about 0.70%, about 0.80%, about 0.90%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about 1.00%, about About 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 9%, about 10%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about In some aspects, 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 0.5%, 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%. In some aspects, the producer cells are contacted with DMSO at a concentration of about 0.1%.

[0018] In some aspects, the EVs produced by the producer cells do not differ in average size distribution compared to EVs produced by producer cells that have not been contacted with the cholesterol biosynthesis pathway inhibitor.

[0019] In some aspects, the producer cells are mammalian cells. In some aspects, the producer cells are HEK293 cells, HEK293S cells, HEK293SF cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, AGE. Neuronal precursor cells, Amniotic fluid cells, adipose-derived 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. In some aspects, the producer cells are stem cells.

[0020] In some aspects, the EV further comprises a scaffold portion. In some aspects, the scaffold portion comprises a scaffold X. In some aspects, scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basic immunoglobulin (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin beta-1 (ITGB1 protein); integrin alpha-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins) and any combination thereof. In some aspects, the scaffold portion is a PTGFRN protein. In some aspects, the scaffold portion 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.

[0021] In some aspects, the scaffold portion comprises a scaffold Y. In some aspects, the scaffold Y is selected from the group consisting of: a myristoylated alanine-rich protein kinase C substrate (MARCKS protein); a myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein); a brain acid-soluble protein 1 (BASP1 protein); and any combination thereof.

[0022] In some aspects, the EV further comprises at least one therapeutic agent attached to the scaffold portion. In some aspects, the EV further comprises at least one therapeutic agent. In some aspects, the therapeutic agent comprises a cytokine, a small molecule, a growth factor, an antigen, an antisense oligonucleotide, siRNA, shRNA, miRNA, dsDNA, lncRNA, PROTAC, an adjuvant, an immunomodulator, or any combination thereof. In some aspects, the therapeutic agent is IL-12. In some aspects, the therapeutic agent comprises an IL-2 polypeptide. In some aspects, the therapeutic agent is a STING agonist. In some aspects, the therapeutic agent is an antisense oligonucleotide. In some aspects, the antisense oligonucleotide targets Kras, STAT3, Nras, STAT6, CEBP / b, NLRP3, or any combination thereof.

[0023] Some aspects of the present disclosure relate to producer cells for use in the methods disclosed herein. Some aspects of the present disclosure relate to producer cells made by the methods disclosed herein.

[0024] Some aspects of the present disclosure relate to extracellular vesicles produced by the methods disclosed herein or the producer cells disclosed herein. Some aspects of the present disclosure relate to a bioreactor comprising the producer cells disclosed herein or the extracellular vesicles disclosed herein.

[0025] Some aspects of the present disclosure relate to a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering an extracellular vesicle disclosed herein. Some aspects of the present disclosure relate to the use of an extracellular vesicle disclosed herein for treating or preventing a disease or condition in a subject in need thereof. Some aspects of the present disclosure relate to an extracellular vesicle disclosed herein for use in treating or preventing a disease or condition in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a line graph illustrating producer cell viability (%) after culture in control medium or medium supplemented with increasing concentrations of rosuvastatin for up to 6 days.

[0027] Figures 2A to 2B Figure 2 illustrates producer cell proliferation after culturing for up to 6 days in medium containing cholesterol ("CH"), simvastatin ("SIM"), or both. Figure 2A ) and EV production as measured by secreted luciferase activity ( Figure 2B ) line graph.

[0028] Figures 3A to 3B Figure 2 illustrates producer cell proliferation after 15 days of culture in medium containing DMSO, cholesterol ("CH"), simvastatin ("SIM"), or both cholesterol and simvastatin. Figure 2A ) and vitality ( Figure 2B ) line graph.

[0029] Figures 4A to 4B is exemplified by the effects of DMSO or cholesterol ("CHOL") ( Figure 4A ) or a combination of cholesterol ("CHOL") and simvastatin ("SIM") Figure 4B Line graph of EV production as measured by secreted luciferase activity after culture in ) medium for up to 14 days. DETAILED DESCRIPTION

[0030] The present disclosure relates to methods for increasing the number of extracellular vesicles (EVs) produced from producer cells, the methods comprising: (i) inhibiting a biosynthetic pathway in producer cells; and (ii) contacting producer cells with cholesterol. In some aspects, the biosynthetic pathway is inhibited by contacting producer cells with a cholesterol biosynthetic pathway inhibitor (e.g., statins). Therefore, some aspects of the present disclosure relate to methods for increasing the number of extracellular vesicles (EVs) produced from producer cells, the methods comprising contacting producer cells with (i) a cholesterol biosynthetic pathway inhibitor (e.g., statins) and (ii) cholesterol.

[0031] The present disclosure relates to a method for producing extracellular vesicles (EVs) produced from producer cells, the method comprising: (i) inhibiting a biosynthetic pathway in producer cells; and (ii) contacting the producer cells with cholesterol. In some aspects, the biosynthetic pathway is inhibited by contacting the producer cells with a cholesterol biosynthetic pathway inhibitor (e.g., statins). Therefore, some aspects of the present disclosure relate to a method for producing extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthetic pathway inhibitor (e.g., statins) and (ii) cholesterol.

[0032] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or process steps described, as these specific compositions or process steps may of course vary. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated or combined with features of any other several aspects without departing from the scope or spirit of the present disclosure. Any described method can be carried out in the order of events described or in any other order that is logically possible.

[0033] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0034] I. Definition

[0035] To facilitate understanding of this description, certain terms are first defined. Additional definitions are provided throughout the detailed description.

[0036] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "nucleotide sequence" is understood to mean one or more nucleotide sequences. Thus, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. It should be further noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for the use of exclusive terms such as "solely," "only," or the use of negative limitations in conjunction with the recitation of claim elements.

[0037] Furthermore, the term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] It should be understood that wherever herein aspects are described using the language "comprising," otherwise similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary for those skilled in the art of many of the terms used in this disclosure.

[0040] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numerals defining the range. In the case of enumerated value ranges, it will be understood that each intermediate integer value and each fraction thereof, as well as each subrange between such values, is also specifically disclosed. The upper and lower limits of any range may be independently included within the range or excluded from the range, and each range including any, not including any or including two limits is also encompassed within the present disclosure. Therefore, the scope described herein should be understood as a shorthand for all values ​​within the range, including the endpoints described. For example, a range of 1 to 10 is understood to include any number, combination of numerals, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0041] Where values ​​are explicitly enumerated, it will be understood that values ​​of approximately the same quantity or amount as the enumerated values ​​are also within the scope of the present disclosure. Where combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed separately, their combinations are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded individually or in any combination with other alternatives are also hereby disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are hereby disclosed.

[0042] Nucleotides are represented by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0043] Amino acid sequences are written left to right in amino to carboxyl orientation.Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0044] The headings provided herein are not limitations of the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0045] The terms "about" or "approximately" are used herein to mean approximately, roughly, around a certain area, or in a certain area. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the listed values. In general, the term "about" can modify the numerical value above or below the specified value up or down (higher or lower), for example, with a variance of 10%. In some aspects, the terms used herein mean within 5% of the reference amount, for example, about 50% is understood to cover a range of values ​​from 47.5% to 52.5%.

[0046] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that wraps an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) with a diameter smaller than the diameter of the cell from which they are derived. Typically, the diameter of an extracellular vesicle is in the range of 20 nm to 1000 nm, and may include various macromolecular payloads that are in the internal space (i.e., lumen), displayed on the outer surface of the extracellular vesicle, and / or span the membrane. In some aspects, the payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular vesicle comprises a scaffold portion. For example, but not limited to, extracellular vesicles include apoptotic bodies, cell debris, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells and / or cultured cells. In some aspects, extracellular vesicles are produced by cells expressing one or more transgene products.

[0047] As used herein, the term "exosome" refers to a cell-derived small (diameter between 20 nm and 300 nm, more preferably between 40 nm and 200 nm) vesicle that contains a membrane that encapsulates an internal space (i.e., lumen) and is produced from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes are extracellular vesicles of a species. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a monosaccharide, a polysaccharide or a glycan) or other molecules. In some aspects, exosomes contain a scaffold portion. Exosomes can be derived from producer cells and can be isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. In some aspects, the exosomes of the present disclosure are produced by cells expressing one or more transgenic products.

[0048] As used herein, the term "nanovesicle" refers to a cell-derived small (diameter between 20nm and 250nm, more preferably between 30nm and 150nm) vesicle, which comprises a membrane that wraps the inner space, and is produced from the cell by direct or indirect manipulation so that the producer cell does not produce the nanovesicle in the absence of the manipulation. Suitable manipulation of producer cells includes but is not limited to continuous extrusion, alkaline solution treatment, ultrasonic treatment, or a combination thereof. In some cases, the generation of nanovesicles can cause the destruction of producer cells. Preferably, the nanovesicle colony is substantially free of a vesicle derived from producer cells by direct budding from the plasma membrane or late endosome fusion with the plasma membrane. The nanovesicle comprises lipids or fatty acids and polypeptides, and optionally comprises a payload (e.g., therapeutic agent), an acceptor (e.g., targeting moiety), polynucleotides (e.g., nucleic acid, RNA, or DNA), sugar (e.g., monosaccharide, polysaccharide, or polysaccharide) or other molecules. In some aspects, the nanovesicle comprises a scaffold portion. Nanovesicles, when derived from producer cells according to the manipulations described, can be isolated from the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0049] As used herein, the term "surface engineered EVs (e.g., exosomes)" (e.g., Scaffold X engineered EVs (e.g., exosomes)) refers to EVs (e.g., exosomes) having a membrane or surface of an EV (e.g., exosome) modified in its composition such that the surface of the engineered EV (e.g., exosome) is different from the surface of the EV (e.g., exosome) before modification or the surface of a naturally occurring EV (e.g., exosome). The engineering can be performed on the surface of the EV (e.g., exosome) or in the membrane of the EV (e.g., exosome) such that the surface of the EV (e.g., exosome) is altered. For example, the membrane is modified in its composition of proteins, lipids, small molecules, carbohydrates, etc. The composition can be altered by chemical, physical, or biological methods, or by being produced from cells that have been previously or simultaneously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by being produced from cells that have been previously modified by genetic engineering. In some aspects, the surface-engineered EVs (e.g., exosomes) comprise an exogenous protein (i.e., a protein not naturally expressed by the EVs (e.g., exosomes)) or a fragment or variant thereof, which can be exposed on the surface of the EVs (e.g., exosomes) or can be an anchor point (attachment) for a portion exposed on the surface of the EVs (e.g., exosomes). In other aspects, the surface-engineered EVs (e.g., exosomes) comprise a higher expression (e.g., higher amount) of a native exosomal protein (e.g., Scaffold X) or a fragment or variant thereof, which can be exposed on the surface of the EVs (e.g., exosomes) or can be an anchor point (attachment) for a portion exposed on the surface of the EVs (e.g., exosomes).

[0050] As used herein, the term "lumen-engineered exosomes" (e.g., Scaffold Y engineered exosomes) refers to EVs (e.g., exosomes) having a membrane or lumen modified in its composition such that the lumen of the engineered EV (e.g., exosome) is different from the lumen of the EV (e.g., exosome) before modification or the lumen of a naturally occurring EV (e.g., exosome). The engineering can be performed directly in the lumen of the EV (e.g., exosome) or in the membrane such that the lumen of the EV (e.g., exosome) is altered. For example, the membrane is modified in its composition of proteins, lipids, small molecules, carbohydrates, etc. such that the lumen of the EV (e.g., exosome) is modified. The composition can be altered by chemical, physical, or biological methods, or by production from cells previously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by production from cells previously modified by genetic engineering. In some aspects, the lumen-engineered exosomes comprise an exogenous protein (i.e., a protein not naturally expressed by EVs (e.g., exosomes)) or a fragment or variant thereof that can be exposed in the lumen of the EV (e.g., exosome) or can be an anchor point (attachment) for a portion exposed on the inner layer of the EV (e.g., exosome). In other aspects, the lumen-engineered EVs (e.g., exosomes) comprise a more highly expressed native exosomal protein (e.g., Scaffold X or Scaffold Y) or a fragment or variant thereof that can be exposed in the lumen of the exosome or can be an anchor point (attachment) for a portion exposed in the lumen of the exosome.

[0051] The term "modified" when used in the context of EVs (e.g., exosomes described herein) refers to changes or engineering of EVs (e.g., exosomes and / or their producer cells) such that the modified EVs (e.g., exosomes) differ from naturally occurring EVs (e.g., exosomes). In some aspects, the modified EVs (e.g., exosomes) described herein comprise a membrane that differs from the membrane of naturally occurring EVs (e.g., exosomes) in terms of the composition of proteins, lipids, small molecules, carbohydrates, etc. (e.g., the membrane comprises a higher density or amount of native exosomal proteins and / or the membrane comprises proteins that do not naturally occur in exosomes (e.g., IL-12 moieties)). In certain aspects, such modifications to the membrane alter the outer surface of the EVs (e.g., exosomes) (e.g., surface-engineered EVs (e.g., exosomes) described herein). In certain aspects, such modifications to the membrane alter the lumen of the EVs (e.g., exosomes) (e.g., lumen-engineered EVs (e.g., exosomes) described herein).

[0052] As used herein, the term "scaffold moiety" refers to a molecule that can be used to anchor an IL-12 moiety or any other compound of interest to an EV (e.g., an exosome), the luminal surface, or the external surface of an EV (e.g., an exosome). In certain aspects, the scaffold moiety comprises a synthetic molecule. In certain aspects, the scaffold moiety comprises a non-polypeptide moiety. In other aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that naturally occurs in an EV (e.g., an exosome). In certain aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that does not naturally occur in an EV (e.g., an exosome). In certain aspects, the scaffold moiety is Scaffold X. In certain aspects, the scaffold moiety is Scaffold Y. In other aspects, the scaffold moiety comprises both Scaffold X and Scaffold Y. In certain aspects, the scaffold portion comprises Lamp-1, Lamp-2, CD13, CD86, Flotillin, Syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, integrin α4, L1CAM, LFA-1, Mac-1 α and β, Vti-1A and B, CD3ε and ζ, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II), immunoglobulins, MHC-I or MHC-II components, TCRβ, a tetraspanin, or a combination thereof.

[0053] As used herein, the term "scaffold X" refers to an exosomal protein recently identified on the surface of exosomes. See, for example, U.S. Patent No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold X proteins include: prostaglandin F2 receptor negative regulator ("PTGFRN protein"); basic immunoglobulin ("BSG protein"); immunoglobulin superfamily member 2 ("IGSF2 protein"); immunoglobulin superfamily member 3 ("IGSF3 protein"); immunoglobulin superfamily member 8 ("IGSF8 protein"); integrin beta-1 ("ITGB1 protein"); integrin alpha-4 ("ITGA4 protein"); 4F2 cell surface antigen heavy chain ("SLC3A2 protein"); and a class of ATP transporters ("ATP1A1 protein," "ATP1A2 protein," "ATP1A3 protein," "ATP1A4 protein," "ATP1B3 protein," "ATP2B1 protein," "ATP2B2 protein," "ATP2B3 protein," "ATP2B protein"). In some aspects, the scaffold X protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring another moiety to the exterior or lumenal surface of an EV (e.g., an exosome). In some aspects, scaffold X can anchor a moiety (e.g., a therapeutic protein, such as an IL-12 moiety) to the exterior or lumenal surface of an exosome.

[0054] As used herein, the term "scaffold Y" refers to an exosomal protein recently identified within the lumenal surface of an exosome. See, for example, International Publication No. WO / 2019 / 099942, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: myristoylated alanine-rich protein kinase C substrate ("MARCKS protein"); myristoylated alanine-rich protein kinase C substrate-like 1 ("MARCKSL1 protein"); and brain acid-soluble protein 1 ("BASP1 protein"). In some aspects, the scaffold Y protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring a portion to the lumenal surface of an EV (e.g., an exosome). In some aspects, scaffold Y can anchor the IL-2 portion to the lumen of an EV (e.g., an exosome).

[0055] As used herein, the term "fragment" of a protein (e.g., a therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence of the protein, the portion missing from the N-terminus and / or C-terminus, or any portion thereof, as compared to the naturally occurring protein. As used herein, the term "functional fragment" refers to a protein fragment that retains protein function. Thus, in some aspects, a functional fragment of a scaffold X protein retains the ability to anchor a portion to the luminal or external surface of an EV (e.g., an exosome). Similarly, in some aspects, a functional fragment of a scaffold Y protein retains the ability to anchor a portion to the luminal surface of an EV (e.g., an exosome). Whether a fragment is a functional fragment can be assessed by any known method for determining the protein content of an EV (e.g., an exosome), including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein, such as GFP. In certain aspects, functional fragments of Scaffold X protein retain the ability of naturally occurring Scaffold X protein, e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor a moiety. In some aspects, functional fragments of Scaffold Y protein retain the ability of naturally occurring Scaffold Y protein, e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor another molecule.

[0056] As used herein, the term "variant" of a molecule (e.g., a functional molecule, antigen, scaffold X and / or scaffold Y) refers to a molecule that shares certain structural and functional identities with another molecule after comparison by methods known in the art. For example, a variant of a protein may include a substitution, insertion, deletion, frameshift, or rearrangement in another protein.

[0057] In some aspects, variants of Scaffold X comprise variants that are at least about 70% identical to full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter, or a fragment (e.g., a functional fragment) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter. In some aspects, variants of PTGFRN or variants of fragments share at least about 70%, 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%, or at least about 99% sequence identity with PTGFRN according to SEQ ID NO: 1 or a functional fragment thereof.

[0058] In some aspects, variants of Scaffold Y include variants having at least 70% identity with MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1. In some aspects, variants of MARCKS or variants of a fragment share at least about 70%, 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%, or at least about 99% sequence identity with MARCKS according to SEQ ID NO: 47 or a functional fragment thereof. In some aspects, variants of MARCKSL1 or variants of a fragment share at least about 70%, 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%, or at least about 99% sequence identity with MARCKSL1 according to SEQ ID NO: 48 or a functional fragment thereof. In some aspects, the variants of BASP1 or variants of the fragments share at least about 70%, 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%, or at least about 99% sequence identity with BASP1 according to SEQ ID NO: 49 or a functional fragment thereof. In some aspects, the variants of the Scaffold Y protein or variants of the fragments retain the ability to specifically target the lumen of EVs (e.g., exosomes). In some aspects, Scaffold Y comprises one or more mutations, such as conservative amino acid substitutions.

[0059] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, an amino acid string can be conservatively replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0060] The term "percentage of sequence identity" or "percentage of identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because the target sequence nucleotides or amino acids are counted, not the nucleotides or amino acids from the reference sequence.

[0061] The percentage ratio of sequence identity is by determining the quantity of the position that identical amino acid residue or nucleic acid base occur in two sequences to obtain the quantity of matching positions, the quantity of matching positions is divided by the sum of the position in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage ratio calculation. The determination of the sequence identity percentage ratio between the comparison of sequence and two sequences can be completed using the easily available software that can be used for online use and downloading. Suitable software programs can be obtained from various sources, and are used for the comparison of protein and nucleotide sequences. A kind of program suitably for determining sequence identity percentage ratio is bl2seq, which is a part of the BLAST program suite that can be obtained from the National Center for Biotechnology Information (National Center for Biotechnology Information) BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithm to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are for example Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0062] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also note that length values ​​will always be integers.

[0063] Those skilled in the art will appreciate that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-sequence comparisons driven only by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, which can be obtained from www.clustal.org. Another suitable program is MUSCLE, which can be obtained from www.drive5.com / muscle / . ClustalW2 and MUSCLE can alternatively be obtained from, for example, EBI.

[0064] It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystal protein structures), functional data (e.g., the location of mutations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available at www.tcoffee.org and alternatively available from, for example, EBI. It will also be appreciated that the final alignment for calculating percent sequence identity can be planned automatically or manually.

[0065] Polynucleotide variants can contain changes in coding region, noncoding region or both.On the one hand, polynucleotide variants contain and produce silent substitution, addition or deletion but do not change the characteristic or active change of the coded polypeptide.On the other hand, due to the degeneracy of genetic code, nucleotide variants are produced by silent substitution.In other aspects, wherein 5-10, 1-5 or 1-2 amino acids are substituted, lack or added variants in any combination.Polynucleotide variants can produce for a variety of reasons, for example, for optimizing the codon expression of a specific host (codons in human mRNA are changed to other codons, for example bacterial hosts, such as Escherichia coli (E.coli)).

[0066] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0067] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without significant loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), which is incorporated herein by reference in its entirety, reported variant KGF proteins that had heparin binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, after deletion of 8 to 10 amino acid residues from the carboxyl terminus of this protein, interferon gamma exhibited up to ten times higher activity. (Dobeli et al., J. Biotechnology 7:199-216 (1988), which is incorporated herein by reference in its entirety.)

[0068] In addition, there is ample evidence that variants generally retain biological activities similar to naturally occurring proteins. For example, Gayle and colleagues (J.Biol.Chem 268:22105-22111 (1993), which is incorporated herein by reference in its entirety) conducted extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate more than 3,500 individual IL-1a mutants, with an average of 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that "most molecules can be changed with minimal effect on [binding or biological activity]. " (See abstract) In fact, of the more than 3,500 nucleotide sequences tested, only 23 unique amino acid sequences produced proteins whose activity was significantly different from that of the wild type.

[0069] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. In some aspects, Scaffold X and / or Scaffold Y are modified at any convenient location.

[0070] As used herein, the terms "linked to" or "conjugated to" are used interchangeably and refer to a covalent or non-covalent bond formed between a first moiety and a second moiety, e.g., a Scaffold X and an IL-12 moiety, e.g., a Scaffold moiety expressed in or on an extracellular vesicle and an IL-12 moiety, e.g., a Scaffold X (e.g., a PTGFRN protein) located in the luminal surface or on the outer surface of an extracellular vesicle, respectively.

[0071] The term "encapsulated" or grammatically different forms of the term (e.g., encapsulation or encapsulating) refers to the state or process of having a first part (e.g., an antigen) located inside a second part (e.g., an EV, e.g., an exosome) without chemically or physically connecting the two parts. In some aspects, the term "encapsulated" can be used interchangeably with "in the lumen of." Non-limiting examples of encapsulating a first part (e.g., an antigen) into a second part (e.g., an EV, e.g., an exosome) are disclosed elsewhere herein.

[0072] As used herein, the term "producer cell" refers to a cell used to produce EVs (e.g., exosomes). Producer cells can be cells cultured in vitro or in vivo. Producer cells include, but are not limited to, cells known to be effective in producing EVs (e.g., exosomes), such as HEK293 cells, C2C12 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, s9f cells, fHDF fibroblasts, AGE. Neuronal precursor cells, In some aspects, the producer cells are antigen presenting cells. In some aspects, the producer cells are bacterial cells. In some aspects, the producer cells are dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells or cells derived from any of these cells or any combination thereof. In some aspects, the producer cells are not bacterial cells. In other aspects, the producer cells are not antigen presenting cells.

[0073] As used herein, the term "association" refers to a covalent bond or non-covalent bond formed between a first portion (e.g., a protein, such as an IL-12 portion or an IL-2 portion) and a second portion (e.g., an extracellular vesicle) respectively; or the first portion (e.g., a protein, such as an IL-12 portion or an IL-2 portion) is encapsulated into a second portion (e.g., an extracellular vesicle). For example, in some aspects, a scaffold portion, e.g., scaffold X (e.g., PTGFRN protein) is expressed in or on an extracellular vesicle, and a protein (e.g., IL-12 portion) is loaded on the outer surface of the extracellular vesicle. On the one hand, the term "association with ... " means a covalent bond, a non-peptide bond, or a non-covalent bond. For example, the amino acid cysteine ​​comprises a thiol group that can form a disulfide bond or a bridge with the sulfhydryl group on the second cysteine ​​residue. Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, π hydrogen bonds, bent bonds, dipole bonds, π reverse bonds, double bonds, triple bonds, quadruple bonds, pentapole bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, Haptor numbers, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-energy barrier hydrogen bonds, or symmetric hydrogen bonds), van der Walls forces, London dispersion forces, mechanical bonds, halogen bonds, gold affinity, intercalation, stacking, entropic forces, or chemical polarity. In other aspects, the term "associated with" means that a first portion (e.g., an extracellular vesicle) encapsulates a second portion (e.g., a protein, such as an IL-12 portion). In some aspects, the first portion and the second portion can be connected to each other. In other aspects, the first portion and the second portion are not physically and / or chemically connected to each other.

[0074] As used herein, the term "loaded" or grammatical variations of the term (e.g., loaded or loaded) refers to the state or process of associating a first portion (e.g., an IL-12 portion or an IL-2 portion, a STING agonist) with a second portion (e.g., an EV, e.g., and an exosome). In some aspects, the first portion is chemically or physically linked to the second portion. In some aspects, the first portion is not chemically or physically linked to the second portion. In some aspects, the first portion is present within the second portion, e.g., within the lumen of an EV (e.g., an exosome), e.g., "encapsulated." In some aspects, the first portion is associated with the outer surface of the second portion, e.g., linked or conjugated to the surface of an EV (e.g., an exosome), e.g., "surface display" of the second portion.

[0075] As used herein, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," and "extracted," and "extracting," are used interchangeably and refer to the state (e.g., multiple known or unknown amounts and / or concentrations) of a desired preparation of EVs that has been subjected to one or more purification processes, e.g., selection or enrichment of a desired EV preparation. In some aspects, as used herein, isolation or purification is the process of removing, partially removing (e.g., fractionating) EVs from a sample containing producer cells. In some aspects, the isolated EV composition has no detectable undesirable activity, or alternatively, the level or amount of the undesirable activity is at or below an acceptable level or amount. In other aspects, the isolated EV composition has an amount and / or concentration of the desired EVs that is at or above an acceptable amount and / or concentration. In other aspects, the isolated EV composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999% or greater than 99.9999%. In some aspects, the isolated EV preparation is substantially free of residual biological products. In some aspects, the isolated EV preparation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free or 90% free of any contaminating biological substances. Residual biological products can include non-biological materials (including chemicals) or undesirable nucleic acids, proteins, lipids or metabolites. Substantially free of residual biological products can also mean that the EV composition does not contain detectable producer cells and only EVs are detectable.

[0076] As used herein, the term "statin" refers to a class of compounds that inhibit hydroxymethylglutaryl-CoA reductase (HMGCR) and reduce total cholesterol, low-density lipoprotein (LDL) and triglyceride concentrations while increasing high-density lipoprotein (HDL) concentrations. HMGCR is an enzyme responsible for converting HMG-CoA into mevalonate in the cholesterol synthesis pathway. Non-limiting examples of statins include atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0077] As used herein, "3-hydroxy-3-methylglutaryl-CoA reductase" or "HMG Co-A reductase" or "HMGCR" refers to an enzyme that catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting enzyme for cholesterol synthesis. A negative feedback mechanism mediated by sterol and non-sterol metabolites derived from mevalonate regulates HMGCR enzyme activity. In mammalian cells, HMGCR is normally inhibited by cholesterol produced by low-density lipoprotein (LDL) internalization and degradation via the LDL receptor. HMGCR is encoded by the human HMGCR gene, which is located on chromosome 5 (bases 75,336,334 to 75,362,116; NCBI reference sequence: NC_000005.10). The HMGCR protein has three isoforms produced by alternative splicing. The sequences are shown in Table 1 below.

[0078] Table 1. HMGCR protein isoforms.

[0079]

[0080]

[0081] As used herein, the term "HMGCR gene" refers to any transcript, genomic DNA, pre-mRNA or mRNA. As used herein, "HMGCR protein" refers to HMGCR isoform 1, HMGCR isoform 2 or HMGCR isoform 3 disclosed above, as well as variants and mutants thereof. As used herein, the term "HMGCR protein" also encompasses any fragment or variant of any isoform disclosed herein that has at least one function of a wild-type HMGCR protein.

[0082] As used herein, term " gene and / or protein function of reduction " refers to physical level (for example, owing to the gene sequence caused by editing from genome reduces, or owing to the protein caused by protein expression reduction reduces) and function reduction.For example, the reduction of HMGCR gene level can refer to the reduction of gene function, for example, owing to introducing stop codon or frameshift mutation, changing the epigenetic modification of transcription, or promoter gene or the sudden change or other changes of other genes of regulation HMGCR expression.In some aspects, the reduction of HMGCR gene level in modified cell refers to compared with reference cell (for example, untreated), the amount (for example, concentration) of genomic DNA, pre-mRNA and / or mRNA that can encode functional HMGCR albumen (for example, wild-type HMGCR albumen).Similarly, the reduction of HMGCR albumen can refer to the change causing functional HMGCR albumen (for example, wild-type HMGCR albumen) to express, includes but is not limited to the change (for example, sudden change or post-translational modification) causing loss of function (partially or completely), or causes the activity change (for example, its enzymatic activity) of the molecule combined with HMGCR function site.

[0083] As used herein, the term "sterol regulatory element binding factor 2" or "SREBF2" refers to a transcription factor that is a master regulator of cholesterol synthesis that activates the expression of genes such as HMG-CoA reductase (HMGCR), HMG-CoA synthase (HMGCS), and mevalonate kinase (MVK). 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, lop13, SREBP2, bHLHd2, SREBP-2, AI608257, and SREBP2gc.

[0084] Table 2. HMGCR protein isoforms.

[0085]

[0086]

[0087] As used herein, the term "SREBF2 gene" refers to any transcript, genomic DNA, pre-mRNA, or mRNA. As used herein, "SREBF2 protein" refers to SREBF2 isoform 1 or SREBF2 isoform 2 disclosed above, as well as variants and mutants thereof. As used herein, the term "SREBF2 protein" also encompasses any fragment or variant of any isoform disclosed herein that has at least one function of the wild-type SREBF2 protein.

[0088] As used herein, the term "reduced gene and / or protein function" refers to both physical levels (e.g., reduction in gene sequence due to editing from the genome, or reduction in protein due to reduced protein expression) and functional reduction. For example, a reduction in SREBF2 gene levels can refer to a reduction in gene function, such as due to the introduction of a stop codon or frameshift mutation, epigenetic modifications that may alter transcription, mutations in promoter genes or other genes that regulate SREBF2 expression, or other changes. In some aspects, a reduction in SREBF2 gene levels in a modified cell refers to a reduction in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA that can encode a functional SREBF2 protein (e.g., a wild-type SREBF2 protein) as compared to a reference cell (e.g., an untreated cell). Similarly, reduction of SREBF2 protein can refer to changes that result in the expression of functional SREBF2 protein (e.g., wild-type SREBF2 protein), including but not limited to changes (e.g., mutations or post-translational modifications) that result in loss of function (partial or complete), or that result in altered activity of a molecule that binds to a functional site of SREBF2 (e.g., its transcriptional activation activity).

[0089] As used herein, the term "free IL-12 portion" means the portion of IL-12 that is not associated with extracellular vesicles, but is otherwise identical to the portion of IL-12 associated with extracellular vesicles. In particular, the free IL-12 portion is the same portion of IL-12 associated with extracellular vesicles when compared to the portion of IL-12 associated with the IL-12 portion. In some aspects, when the free IL-12 portion is compared to an extracellular vesicle comprising the IL-12 portion in terms of its efficacy, toxicity, and / or any other properties, the amount of the free IL-12 portion is the same as the amount of the IL-12 portion associated with EVs when compared to the portion of IL-12 associated with extracellular vesicles.

[0090] As used herein, the term "exoIL-12" refers to an exosome loaded with an IL-12 portion (e.g., an IL-12 protein or a fragment thereof). In some aspects, the IL-12 portion is associated with the outer surface of the exosome (e.g., surface display of the IL-12 portion). In some aspects, the IL-12 portion is connected or conjugated to the outer surface of the exosome. In some aspects, the IL-12 portion is connected or conjugated to a surface-exposed scaffold protein, e.g., Scaffold X protein (e.g., PTGFRN protein). In some aspects, the IL-12 portion is connected or conjugated to the lipid bilayer of the exosome. In some aspects, the exosome comprises an IL-12 portion located in the lumen of the exosome. In some aspects, the IL-12 portion is associated with the lumen surface of the exosome, e.g., with a scaffold protein, e.g., Scaffold X (e.g., PTGFRN). In some aspects, the IL-12 portion is encapsulated in the lumen of the exosome and is not associated with the scaffold protein.

[0091] As used herein, 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, inactivation, blocking, or retardation of a receptor-mediated biological response. A receptor can be modulated by an endogenous ligand or an exogenous ligand. Non-limiting examples of endogenous ligands include antibodies and peptides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. A ligand can be a complete ligand, a partial ligand, or a counter ligand.

[0092] As used herein, the term "pharmaceutical composition" refers to one or more compounds, e.g., EVs, of the compounds described herein, mixed or doped with, or suspended in, one or more other chemical components, such as, for example, pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate the preparation of EVs for administration to a subject. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" and grammatical variations thereof encompass any agent approved by a U.S. federal government regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans), as well as any carrier or diluent that does not cause undesirable physiological effects to an extent that would prohibit administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that can be used to prepare pharmaceutical compositions and that are generally safe, non-toxic, and desirable are encompassed.

[0093] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that comes into contact with an EV. Payloads that can be introduced into EVs and / or producer cells include therapeutic agents such as nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0094] The terms "administration," "administering," and variations thereof refer to the introduction of a composition (such as an EV) or an agent into a subject and include simultaneous and sequential introduction of compositions or agents. The composition or agent is introduced into a subject by any suitable route, including intratumorally, orally, pulmonary, intranasally, parenterally (intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0095] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. The compositions and methods described herein are suitable for both human therapy and veterinary applications. In some aspects, the subject is a mammal, and in other aspects, the subject is a human. As used herein, a "mammalian subject" includes all mammals, including but not limited to humans, livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0096] As used herein, the term "substantially free" means that a sample containing EVs (e.g., exosomes) contains less than 10% macromolecules on a mass / volume (m / v) percentage concentration basis. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m / v) macromolecules.

[0097] As used herein, " perfusion culture " refers to cell culture method or cell culture, wherein cells are continuously fed fresh culture medium, and remove waste culture medium continuously while cells are maintained in culture vessel. In some respects, the culture vessel for perfusion culture comprises a cell retention device, such as a capillary fiber or a membrane. The example of the perfusion bioreactor with a cell retention device comprises a bioreactor connected to a cell retention device (for example, a hollow fiber filter or an acoustic cell separator), such as an N-terminal production container (such as a stirred tank bioreactor). In some respects, perfusion culture is a single cell perfusion culture. In some respects, perfusion culture comprises using a single cell suspension perfusion bioreactor, wherein independent cells are separated to add fresh culture medium and remove waste culture medium. In some respects, perfusion culture comprises separating cells from waste culture medium by centrifugation.

[0098] As used herein, "fed-batch" refers to a cell culture method or cell culture in which cells are retained in a culture vessel until the cells are harvested, wherein an initial culture medium is added to the initial cell culture and additional feed medium is added to prevent nutrient depletion. In some aspects, feed medium is added only once during the culture. In some aspects, feed medium is added multiple times during the culture. In some aspects, fed-batch cultures can include constant fed-batch cultures and exponential fed-batch cultures.

[0099] As used herein, "treatment" or "treating" refers to, for example, reducing the severity of a disease or condition; shortening the course of a disease; ameliorating or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject suffering from a disease or condition, but not necessarily curing the disease or condition. The term also includes the prevention or prevention of a disease or condition or its symptoms. In one aspect, the term "treating" or "treatment" means inducing an immune response to an antigen in a subject. In some aspects, the disease or condition is cancer.

[0100] As used herein, "prevent" or "preventing" refers to reducing or lowering the incidence or severity of a particular outcome. In some aspects, prevention of an outcome is achieved by prophylactic treatment. In some aspects, EVs (e.g., exosomes) comprising a cytokine (e.g., an IL-12 portion) as described herein are administered prophylactically to a subject.

[0101] As used herein, the term "immunomodulator" refers to an agent (i.e., therapeutic agent) that acts on a target (e.g., target cell) in contact with an extracellular vesicle and regulates the immune system. Non-limiting examples of immunomodulators that can be introduced into EVs (e.g., exosomes) and / or producer cells include regulators such as checkpoint inhibitors, ligands of checkpoint inhibitors, cytokines, derivatives thereof, or any combination thereof. Immunomodulators can also include agonists, antagonists, antibodies, antigen-binding fragments, polynucleotides (such as siRNA), antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), peptide-coupled phosphorodiamidate morpholino oligomers (PPMOs), miRNAs, lncRNAs, mRNA DNAs, or small molecules.

[0102] II. Methods for Producing Extracellular Vesicles

[0103] The present disclosure relates to methods for increasing the number of extracellular vesicles (EVs) produced from producer cells, the methods comprising: (i) inhibiting a biosynthetic pathway in producer cells; and (ii) contacting producer cells with cholesterol. In some aspects, the biosynthetic pathway is inhibited by contacting producer cells with a cholesterol biosynthetic pathway inhibitor (e.g., statins). Therefore, some aspects of the present disclosure relate to methods for increasing the number of extracellular vesicles (EVs) produced from producer cells, the methods comprising contacting producer cells with (i) a cholesterol biosynthetic pathway inhibitor (e.g., statins) and (ii) cholesterol.

[0104] The present disclosure relates to a method for producing extracellular vesicles (EVs) produced from producer cells, the method comprising: (i) inhibiting a biosynthetic pathway in producer cells; and (ii) contacting the producer cells with cholesterol. In some aspects, the biosynthetic pathway is inhibited by contacting the producer cells with a cholesterol biosynthetic pathway inhibitor (e.g., statins). Therefore, some aspects of the present disclosure relate to a method for producing extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthetic pathway inhibitor (e.g., statins) and (ii) cholesterol.

[0105] Without wishing to be bound by theory, genetic and / or pharmacological inhibition of the cholesterol synthesis pathway in producer cells increases the yield of harvested EVs compared to producer cells that have not undergone genetic and / or pharmacological inhibition of the cholesterol synthesis pathway. This increase in EV production comes at the expense of producer cell viability. However, the applicants surprisingly discovered that, relative to control cells, inhibiting cholesterol biosynthesis in producer cells while also exposing the producer cells to cholesterol restored cell viability and maintained increased EV production.

[0106] In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 8 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 9 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 10 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 11 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 12 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 13 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 14 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 15 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 16 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 17 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 18 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 19 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 20 days. In some aspects, producer cells are contacted with (i) cholesterol biosynthetic pathway inhibitor and (ii) cholesterol for more than 21 days.

[0107] In some aspects, the EVs produced by the producer cells have increased yield compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthesis pathway inhibitor. In some aspects, the yield is increased by 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.

[0108] In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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. In some aspects, EV production is increased by at least about 1.5-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 2-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 2.5-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 3-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 3.5-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 4-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 4.5-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor. In some aspects, EV production is increased by at least about 5-fold compared to EVs produced by producer cells that have not been contacted with a cholesterol biosynthetic pathway inhibitor.

[0109] In some aspects, the producer cells have increased viability compared to EVs produced by producer cells that (i) are contacted with a cholesterol biosynthesis pathway inhibitor and (ii) are not contacted with cholesterol. In some aspects, the producer cells have increased viability compared to EVs produced by producer cells that (i) are not contacted with a cholesterol biosynthesis pathway inhibitor and (ii) are not contacted with cholesterol. In some aspects, the viability is increased by 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. In some aspects, the yield is increased by 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 by 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.

[0110] In some aspects, the viability of the producer cells is comparable to the viability of producer cells that have not been exposed to the cholesterol biosynthesis pathway inhibitor and / or cholesterol.

[0111] II.A. Cholesterol Biosynthesis Inhibition

[0112] Any method can be used to inhibit cholesterol biosynthesis in producer cells. In some aspects, producer cells are contacted with cholesterol biosynthesis pathway inhibitors. In some aspects, cholesterol biosynthesis pathway inhibitors include statins, cariprazine, PROTAC, AY9944 or BM15766. In some aspects, cholesterol biosynthesis pathway inhibitors include meglutol, clinofibrate, hesperetin 7-O-glucoside, compound 81 (also known as HMG499, and related structures thereof, described in Jiang et al., Nature Communications 9, 5138 (2018), which is incorporated herein by reference in its entirety) or Monacolin J.

[0113] In some aspects, the expression of one or more genes or their proteins in the cholesterol synthesis pathway can be inhibited or reduced. Non-limiting examples of genes include HMG CoA (3-hydroxy-3-methylglutaryl-CoA), SQLE (squalene monooxygenase), ACAT1 (acetyl-CoA acetyltransferase 1), ACAT2 (acetyl-CoA acetyltransferase 2), HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1), HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2), HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), PMVK (phosphomevalonate kinase), MVD (mevalonate decarboxylase), IDI1 (isopentenyl diphosphate delta isomerase 1), IDI2 (isopentenyl diphosphate delta isomerase 2), FD PS (farnesyl diphosphate synthase), GGPS1 (geranylgeranyl diphosphate synthase 1), FDFT1 (farnesyl diphosphate farnesyltransferase 1), SQLE (squalene epoxidase), LSS (lanosterol synthase), DHCR24 (24-dehydrocholesterol reductase), CYP51A1 (cytochrome P450 family 51 subfamily A polypeptide 1), TM7SF2 (transmembrane 7 superfamily member 2), FAXDC2 (C5orf4 fatty acid hydroxylase domain-containing 2), MSMO1 (SC4MOL methylsterol monooxygenase), NSDHL (NAD(P)-dependent steroid dehydrogenase-like), HSD17B7 (hydroxysteroid (1 7-β) dehydrogenase 7), EBP (emopam-binding protein (sterol isomerase)), SC5D (SC5DL sterol-C5-desaturase synthesis), DHCR7 (7-dehydrocholesterol reductase), CEL (carboxy ester lipase), LIPA (lipase A, lysosomal acid), SOAT1 (sterol O-acetyltransferase 1), SOAT2 (sterol O-acetyltransferase 2), ABCA1 (ATP-binding cassette, subfamily A), ABCG1 (ATP-binding cassette, subfamily G), SLCO2B1 (solute carrier organic anion transporter family member 2B1), SLCO1B3 (solute carrier organic anion transporter family member 1B3) ), LDLR (low-density lipoprotein receptor), APOE (apolipoprotein E), SREBF1 (sterol-binding element transcription factor 1), SREBF2 (sterol-binding element transcription factor 2), SCAP (SREBF molecular chaperone), MBTPS1 (S1P membrane-bound transcription factor peptidase site 1), MBTPS2 (S2P, membrane-bound transcription factor peptidase site 2), INSIG1 (insulin-induced gene 1), INSIG2 (insulin-induced gene 2), AMFR (GP78, autocrine motility factor receptor E3 ubiquitin-protein ligase), NR1H3 (LXRA, nuclear receptor subfamily 1H group member 3), NR1H2 (LXRB,Nuclear receptor subfamily 1H group member 2), RXRA (retinoic acid X receptor alpha), RXRB (retinoic acid X receptor beta) or MYLIP (IDOL, myosin regulatory light chain interacting protein). In some aspects, the gene is HMGCR. In other aspects, the gene is SREBF2.

[0114] In some aspects, the expression of SM (squalene monooxygenase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of ACAT1 (acetyl-CoA acetyltransferase 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of ACAT2 (acetyl-CoA acetyltransferase 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of MVK (mevalonate kinase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of PMVK (phosphomevalonate kinase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of IDI1 (isopentenyl diphosphate delta isomerase 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of IDI2 (isopentenyl diphosphate delta isomerase 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of FDPS (farnesyl diphosphate synthase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of GGPS1 (geranylgeranyl diphosphate synthase 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of FDFT1 (farnesyl diphosphate farnesyltransferase 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SQLE (squalene epoxidase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of LSS (lanosterol synthase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of DHCR24 (24-dehydrocholesterol reductase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of CYP51A1 (cytochrome P450 family 51 subfamily A polypeptide 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of TM7SF2 (transmembrane protein 7 superfamily member 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of FAXDC2 (C5orf4 fatty acid hydroxylase domain containing 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of MSMO1 (SC4MOL methylsterol monooxygenase) or its protein can be inhibited or reduced. In some aspects, the expression of NSDHL (NAD (P) dependent steroid dehydrogenase-like) or its protein can be inhibited or reduced. In some aspects, the expression of EBP (emopam binding protein (sterol isomerase)) or its protein can be inhibited or reduced. In some aspects, the expression of HSD17B7 (hydroxysteroid (17-β) dehydrogenase 7) or its protein can be inhibited or reduced. In some aspects, the expression of DHCR7 (7-dehydrocholesterol reductase) or its protein can be inhibited or reduced. In some aspects, the expression of CEL (carboxy ester lipase) or its protein can be inhibited or reduced. In some aspects, the expression of LIPA (lipase A, lysosomal acid) or its protein can be inhibited or reduced. In some aspects, the expression of SOAT1 (sterol O-acetyltransferase 1) or its protein can be inhibited or reduced.In some aspects, the expression of SOAT2 (sterol O-acetyltransferase 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of ABCA1 (ATP-binding cassette, subfamily A) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SLCO2B1 (solute carrier organic anion transporter family member 2B1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SLCO1B3 (solute carrier organic anion transporter family member 1B3) or a protein thereof can be inhibited or reduced. In some aspects, the expression of APOE (apolipoprotein E) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SREBF1 (sterol binding element transcription factor 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SREBF2 (sterol binding element transcription factor 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of SCAP (SREBF molecular chaperone) can be inhibited or reduced. In some aspects, the expression of MBTPS1 (S1P membrane-bound transcription factor peptidase site 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of MBTPS2 (S2P, membrane-bound transcription factor peptidase site 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of INSIG1 (insulin-induced gene 1) or a protein thereof can be inhibited or reduced. In some aspects, the expression of INSIG2 (insulin-induced gene 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of AMFR (GP78, autotaxin receptor E3 ubiquitin-protein ligase) or a protein thereof can be inhibited or reduced. In some aspects, the expression of NR1H3 (LXRA, nuclear receptor subfamily 1H group member 3) or a protein thereof can be inhibited or reduced. In some aspects, the expression of NR1H2 (LXRB, nuclear receptor subfamily 1H group member 2) or a protein thereof can be inhibited or reduced. In some aspects, the expression of RXRA (retinoid X receptor alpha) or a protein thereof can be inhibited or reduced. In some aspects, the expression of RXRB (retinoid X receptor beta) or a protein thereof can be inhibited or reduced. In some aspects, the expression of MYLIP (IDOL, Myosin Regulatory Light Chain Interacting Protein) or a protein thereof can be inhibited or reduced.

[0115] In some respects, method of the present disclosure comprises modifying producer cell so that it shows the reduction of gene and / or protein function in the cholesterol biosynthetic pathway of producer cell. In some respects, method of the present disclosure comprises cultivating the producer cell that shows the reduction of gene and / or protein function in the cholesterol biosynthetic pathway. In some respects, gene and / or protein reduced in the cholesterol biosynthetic pathway comprise HMGCR gene and / or HMGCR albumen.

[0116] HMGCR gene levels (e.g., the presence / disappearance of the entire gene or its portion, or gene function) can be measured by various methods known in the art. HMGCR protein levels (e.g., the presence / disappearance of HMGCR protein or its fragment, or quantitative or protein function) can be measured by various methods known in the art.

[0117] In some aspects, HMGCR gene expression reduces by about 2 times to about 20 times. In some aspects, HMGCR gene expression reduces by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times or about 20 times.

[0118] In some aspects, the cholesterol biosynthetic pathway gene and / or protein function reduction 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 cholesterol biosynthetic pathway gene and / or protein function reduction 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%.

[0119] In some aspects, the medicament that can reduce the gene and / or protein function in cholesterol biosynthetic pathway comprises gene editing technology.In some aspects, gene editing technology comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, large range nuclease, restriction endonuclease or their any combination.In some aspects, gene editing technology comprises siRNA.

[0120] In some aspects, the methods of the present disclosure include modifying producer cells to exhibit a reduction in gene and / or protein function in the cholesterol biosynthetic pathway of the producer cells. In some aspects, the methods of the present disclosure include culturing producer cells that exhibit a reduction in gene and / or protein function in the cholesterol biosynthetic pathway. In some aspects, the genes and / or proteins reduced in the cholesterol biosynthetic pathway include the SREBF2 gene and / or SREBF2 protein.

[0121] SREBF2 gene levels (e.g., presence / absence of the entire gene or a portion thereof, or gene function) can be measured by various methods known in the art. SREBF2 protein levels (e.g., presence / absence of SREBF2 protein or a fragment thereof, or quantification or protein function) can be measured by various methods known in the art.

[0122] In some aspects, reduction in SREBF2 gene expression levels and / or SREBF2 protein expression or function levels in producer cells can increase the yield of EVs produced by producer cells compared to EVs produced by producer cells that do not have reduced SREBF2 gene expression levels and / or SREBF2 protein expression levels. In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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.

[0123] In some aspects, SREBF2 gene expression is reduced by about 2-fold to about 30-fold. In some aspects, SREBF2 gene expression is reduced by 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, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, or about 30-fold.

[0124] In some aspects, the cholesterol biosynthetic pathway gene and / or protein function is reduced by 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 cholesterol biosynthetic pathway gene and / or protein function is reduced by 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%.

[0125] In some aspects, reduced HMGCR gene expression levels and / or expression or functional levels of HMGCR protein and / or contact with cholesterol in producer cells can increase the yield of EVs produced by producer cells compared to EVs produced by producer cells that do not have reduced HMGCR gene expression levels and / or HMGCR protein expression levels or are not contacted with cholesterol. In some aspects, the yield is increased by 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. In some aspects, the yield is increased by 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 by 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.

[0126] In some aspects, modifications of the methods described herein include contacting the producer cells with an agent capable of reducing the function of the SREBF2 gene and / or SREBF2 protein. In some aspects, the agent includes betulin (see Tang et al., Cell Metab. 2011 Jan 5; 13(1):44-56, which is incorporated herein by reference in its entirety). In some aspects, the agent includes fatostatin. In some aspects, the agent includes pseudoprotodioscin. In some aspects, the agent includes oligomeric amyloid beta 42. In some aspects, the agent includes luteolin. In some aspects, the agent includes clofibrate.

[0127] In some aspects, the modification comprises contacting the producer cell with an agent capable of reducing gene and / or protein function in the cholesterol biosynthetic pathway. In some aspects, prior to cultivation, the producer cell is modified by contacting the producer cell with a substance capable of reducing gene and / or protein function in the cholesterol biosynthetic pathway.

[0128] In some aspects, the medicament that can reduce the gene and / or protein function in cholesterol biosynthetic pathway comprises gene editing technology.In some aspects, gene editing technology comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, large range nuclease, restriction endonuclease or their any combination.In some aspects, gene editing technology comprises siRNA.

[0129] In some aspects, one or more protein inhibitors (e.g., small molecule HMGCR and / or SREBF2 inhibitors) can be used in methods of increasing the number of extracellular vesicles produced from producer cells and the methods of producing extracellular vesicles of the present disclosure.

[0130] In some aspects, protein inhibitor tools that can be used in the present disclosure include proteolysis targeting chimeras (PROTACs). PROTAC is a heterobifunctional small molecule with the following three chemical elements: a ligand that binds to the target protein, a ligand that binds to the 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. Unlike the competitive and occupancy-driven processes of traditional inhibitors, PROTAC's mode of action is catalytic and can promote target protein degradation at low exposure levels. In addition, traditional small molecule inhibitors generally inhibit the enzymatic activity of the target, while PROTAC not only affects the enzymatic activity of the protein, but also affects non-enzymatic activity by degrading the entire protein.

[0131] In some aspects, PROTAC comprises a statin (e.g., HMGCR ligand) linked to an 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 incorporated herein by reference in its entirety. In some aspects, PROTAC degrades HMCGR, as described in Li et al., J. Med. Chem. 2020, 63, 9, 4908-4928, which is incorporated herein by reference in its entirety.

[0132] In some aspects, the protein inhibitor comprises cariprazine.

[0133] II.A.1. Statins

[0134] In some aspects, by contacting the producer cells with an agent that can reduce the function of the HMGCR gene and / or HMGCR protein, cholesterol biosynthesis in the producer cells can be inhibited. In some aspects, the agent includes statins. Without wishing to be bound by theory, statins play a role by competitively blocking the active site of the first and key rate-limiting enzyme in the mevalonate pathway (e.g., the cholesterol biosynthetic pathway), HMG-CoA reductase (HMGCR). Inhibiting this site prevents substrate entry, thereby blocking the conversion of HMG-CoA into mevalonate. The active ingredient of statins is a modified 3,5-dihydroxyglutaric acid moiety, which is structurally similar to the endogenous substrate HMG-CoA and mevalonate coenzyme A transition state intermediate. This active site combines and inhibits HMG-CoA reductase activity during the stereoselective process that requires statins to have a 3R, 5R configuration. The molecular and clinical differences of statins are derived from the ring attached to the active moiety, which can be partially reduced naphthalene (lovastatin, simvastatin, pravastatin), pyrrole (atorvastatin), indole (fluvastatin), pyrimidine (rosuvastatin), pyridine (cerivastatin) or quinoline (pitavastatin). The substituents on the ring limit the solubility and pharmacological properties of statins. Hydrophilicity (pravastatin and rosuvastatin) is derived from the common active site plus other polar substituents, while lipophilicity (atorvastatin, lovastatin, fluvastatin, pitavastatin, simvastatin and cerivastatin) is derived from the addition of non-polar substituents. In some aspects, statins can be selected from atorvastatin, lovastatin, pitavastatin, pravastatin, fluvastatin, cerivastatin, rosuvastatin, mevastatin, simvastatin or a combination thereof. In some aspects, statins include simvastatin. In some aspects, statins include lovastatin, simvastatin, and / or pravastatin. In some aspects, statins include atorvastatin. In some aspects, statins include fluvastatin. In some aspects, statins include rosuvastatin. In some aspects, statins include cerivastatin. In some aspects, the statins include pitavastatin. In some aspects, the statins include pravastatin and / or rosuvastatin. In some aspects, the statins include atorvastatin, lovastatin, fluvastatin, pitavastatin, simvastatin, and / or cerivastatin.

[0135] In some aspects, statins (e.g., simvastatin) are administered at 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 15 0nM, about 160nM, about 170nM, about 180nM, about 190nM, about 200nM, about 210nM, about 220nM, about 230nM, about 240nM, about 250nM, about 260nM, about 270nM, about 280nM, about 290nM, about 300nM, about 310nM, about 320nM, about 330nM, about 340nM, about 350nM, about 360nM, about 370nM, about 380nM, about 390nM, about 400nM, about 410nM, about 420nM, about 430nM, about 440nM nM, about 450nM, about 460nM, about 470nM, about 480nM, about 490nM, about 500nM, about 510nM, about 520nM, about 530nM, about 540nM, about 550nM, about 560nM, about 570nM, about 580nM, about 590nM, about 600nM, about 610nM, about 620nM, about 630nM, about 640nM, about 650nM, about 660nM, about 670nM, about 680nM, about 690nM, about 700nM, about 710nM, about 720nM, about 730nM The present invention further comprises contacting the producer cells with a concentration of about 740 nM, about 750 nM, about 760 nM, about 770 nM, about 780 nM, about 790 nM, about 800 nM, about 810 nM, about 820 nM, about 830 nM, about 840 nM, about 850 nM, about 860 nM, about 870 nM, about 880 nM, about 890 nM, about 900 nM, about 910 nM, about 920 nM, about 930 nM, about 940 nM, about 950 nM, about 960 nM, about 970 nM, about 980 nM, about 990 nM or about 1,000 nM.In some aspects, the statin is administered 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 0.1 nM to about 10 nM. The method comprises contacting the patient with a concentration of about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM 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 about 20 nM, about 1 nM to about 10 nM, or about 10 nM to about 20 nM.

[0136] In some aspects, statins (e.g., simvastatin) contact producer cells with a concentration of about 10nM to about 10nM. In some aspects, statins (e.g., simvastatin) contact producer cells with a concentration of about 10nM to about 20nM. In some aspects, statins (e.g., simvastatin) contact producer cells with a concentration of about 10nM to about 15nM. In some aspects, statins (e.g., simvastatin) contact producer cells with a concentration of about 15nM to about 20nM.

[0137] In some aspects, the producer cells are contacted with about 10nM simvastatin. In some aspects, the producer cells are contacted with about 15nM simvastatin. In some aspects, the producer cells are contacted with about 20nM simvastatin. In some aspects, the producer cells are contacted with about 25nM simvastatin. In some aspects, the producer cells are contacted with about 30nM simvastatin. In some aspects, the producer cells are contacted with about 35nM simvastatin. In some aspects, the producer cells are contacted with about 40nM simvastatin. In some aspects, the producer cells are contacted with about 45nM simvastatin. In some aspects, the producer cells are contacted with about 50nM simvastatin.

[0138] In some aspects, the producer cells are contacted with about 10nM rosuvastatin. In some aspects, the producer cells are contacted with about 15nM rosuvastatin. In some aspects, the producer cells are contacted with about 20nM rosuvastatin. In some aspects, the producer cells are contacted with about 25nM rosuvastatin. In some aspects, the producer cells are contacted with about 30nM rosuvastatin. In some aspects, the producer cells are contacted with about 35nM rosuvastatin. In some aspects, the producer cells are contacted with about 40nM rosuvastatin. In some aspects, the producer cells are contacted with about 45nM rosuvastatin. In some aspects, the producer cells are contacted with about 50nM rosuvastatin.

[0139] In some aspects, the statin is contacted with the producer cells prior to harvesting the EV. 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 EV. In some aspects, statins are contacted with producer cells for 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. In some aspects, statins are contacted with producer cells before harvesting EV 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. In some aspects, statins are contacted with producer cells when harvesting EV. In some aspects, the statin is contacted with the producer cells for the duration of a continuous (eg, perfusion) cell culture process spanning about 30 days, about 35 days, or about 40 days.

[0140] In some aspects, the producer cells are modified prior to culturing by contacting the producer cells with a substance that reduces the function of genes and / or proteins in the cholesterol biosynthetic pathway.

[0141] In some aspects, the agent is at 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 16 nM, about 0nM, about 170nM, about 180nM, about 190nM, about 200nM, about 210nM, about 220nM, about 230nM, about 240nM, about 250nM, about 260nM, about 270nM, about 280nM, about 290nM, about 300nM, about 310nM, about 320nM, about 330nM, about 340nM, about 350nM, about 360nM, about 370nM, about 380nM, about 390nM, about 400nM, about 410nM, about 420nM, about 430nM, about 440nM, about 450nM, about 460nM, about 470nM, about 480nM, about 490nM, about 500nM, about 510nM, about 520nM, about 530nM, about 540nM, about 550nM, about 560nM, about 570nM, about 580nM, about 590nM, about 50nM, about 460nM, about 470nM, about 480nM, about 490nM, about 500nM, about 510nM, about 520nM, about 530nM, about 540nM, about 550nM, about 560nM, about 570nM, about 580nM, about 590nM, about 600nM, about 610nM, about 620nM, about 630nM, about 640nM, about 650nM, about 660nM, about 670nM, about 680nM, about 690nM, about 700nM, about 710nM, about 720nM, about 730nM, A concentration of about 740 nM, about 750 nM, about 760 nM, about 770 nM, about 780 nM, about 790 nM, about 800 nM, about 810 nM, about 820 nM, about 830 nM, about 840 nM, about 850 nM, about 860 nM, about 870 nM, about 880 nM, about 890 nM, about 900 nM, about 910 nM, about 920 nM, about 930 nM, about 940 nM, about 950 nM, about 960 nM, about 970 nM, about 980 nM, about 990 nM, or about 1,000 nM is contacted with the producer cells.In some aspects, the statin is administered 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 0.1 nM to about 10 nM. The method comprises contacting the patient with a concentration of about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM 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 about 20 nM, about 1 nM to about 10 nM, or about 10 nM to about 20 nM.

[0142] II.A.2. Gene Editing Tools

[0143] In some aspects, cholesterol biosynthesis is inhibited by genetically modifying producer cells. One or more gene editing tools can be used in methods for increasing the number of extracellular vesicles from producer cells and in the production of extracellular vesicles and methods of producing extracellular vesicles of the present disclosure.

[0144] II.A.2.a. CRISPR / Cas systems

[0145] In some aspects, the gene editing tools that can be used in the present disclosure include CRISPR / Cas systems. Such systems can use, for example, Cas9 nucleases, which, in some cases, are codon-optimized for the desired cell type to be expressed (e.g., CHO cells, e.g., MSC cells). The CRISPR / Cas system uses Cas nucleases, such as Cas9 nucleases, which target genomic sites by compounding with a synthetic guide RNA (gRNA), which hybridizes with the target DNA sequence just in front of the NGG motif identified by the gRNA and Cas nuclease (e.g., Cas9). This can result in double-strand breaks at three nucleotides upstream of the NGG motif. In some aspects, a modified version of the Cas nuclease (e.g., Cas9) can be used, thereby resulting in single-strand gaps rather than double-strand breaks. Additional fusion with other enzymes can achieve site-specific base editing in the absence of double-strand breaks. The uniqueness of the CRISPR / Cas9 system is that it is possible to target multiple different genomic sites simultaneously 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 use guide RNAs (gRNAs) comprising two independent molecules. In some aspects, the bi-molecule gRNA comprises a crRNA-like molecule ("CRISPR RNA" or "targeting RNA" or "crRNA" or "crRNA repeats") and a corresponding tracrRNA-like molecule ("trans-CRISPR RNA" or "activating RNA" or "tracrRNA" or "scaffold") molecule.

[0146] crRNA comprises the DNA targeting fragment (single strand) of gRNA and a nucleotide sequence that constitutes half of the double-stranded RNA (dsRNA) duplex of the gRNA protein binding fragment. The corresponding tracrRNA (activating RNA) comprises a nucleotide that forms the other half of the dsRNA duplex of the protein binding fragment of the gRNA. Therefore, a nucleotide of crRNA is complementary to and hybridizes with a nucleotide of tracrRNA to form a dsRNA duplex of the gRNA protein binding domain. Therefore, it can be said that each crRNA has a corresponding tracrRNA. crRNA additionally provides a single-stranded DNA targeting fragment. Therefore, gRNA comprises a sequence that hybridizes with the target sequence and tracrRNA. Therefore, crRNA and tracrRNA (as a corresponding pair) hybridize to form gRNA. If used for intracellular modification, 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 molecule will be used (e.g., humans).

[0147] The natural genes encoding three elements (Cas9, tracrRNA and crRNA) are typically organized in an operon. Naturally occurring CRISPR RNA is different because of the Cas9 system and organism, but generally contains a targeting fragment with a length between 21 and 72 nucleotides, flanked by two directional repeats (DR) with a length between 21 and 46 nucleotides (see, for example, WO2014 / 131833). In the case of Streptococcus pyogenes (S.pyogenes), the DR is 36 nucleotides in length, and the targeting fragment is 30 nucleotides in length. The DR at the 3' end is complementary to and hybridizes with the corresponding tracrRNA, which then binds to the Cas9 protein.

[0148] Alternatively, the CRISPR system used herein can further adopt a fused crRNA-tracrRNA construct (i.e., a single transcript), which works together with codon-optimized Cas9. This single RNA is commonly referred to as guide RNA or gRNA or single guide RNA or sgRNA. In gRNA, the crRNA portion is identified as a "target sequence" for a specific recognition site, and tracrRNA is commonly referred to as a "scaffold." In short, a short DNA fragment containing a target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid comprises a target sequence (in some aspects, about 20 nucleotides), a form (scaffold) of the tracrRNA sequence, and a suitable promoter active in the cell and elements necessary for proper processing in eukaryotic cells. Many of these systems rely on customized complementary oligonucleotides, which anneal to form double-stranded DNA and are then cloned into the gRNA expression plasmid.

[0149] The gRNA expression cassette and the Cas9 expression cassette are then introduced into the cells. See, e.g., 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 WY et al., Nat Biotechnol 2013 March; 31(3): 227-9; Jiang W et al., Nat Biotechnol 2013 March; 31(3): 233-9; Cronican et al., ACS Chem. Biol. 5(8): 747-52 (2010); and Cong L et al., Science 2013 Feb. 15; 339(6121): 819-23, each of which is incorporated herein by reference in its entirety. See also, for example, WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, and WO / 2013142578A1, each of which is incorporated herein by reference in its entirety.

[0150] In some aspects, the Cas9 nuclease can be provided in the form of a protein. In some aspects, the Cas9 protein can be provided in the form of a complex with a gRNA. In other aspects, the Cas9 nuclease can be provided in the form of a nucleic acid encoding a protein. The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA. In some aspects, the gRNA can be provided in the form of RNA. In other aspects, the gRNA can be provided in the form of a DNA encoding RNA. In some aspects, the gRNA can be provided in the form of a separate crRNA and tracrRNA molecule, or provided in the form of a separate DNA molecule encoding crRNA and tracrRNA respectively.

[0151] In some aspects, two separate Cas proteins (e.g., nickases) specific for target sites on each dsDNA chain can produce an overhang sequence that is complementary to an overhang sequence on another nucleic acid, or a separate region on the same nucleic acid. The overhang end produced by contacting the nucleic acid with two nickases specific for target sites on two dsDNA chains can be a 5' or 3' overhang end. For example, a first nickase can produce a single-strand break on the first dsDNA chain, while a second nickase can produce a single-strand break on the second dsDNA, so as to produce an overhang sequence. The target site of each nickase that produces a single-strand break can be selected so that the overhang end sequence produced is complementary to the overhang end sequence on different nucleic acid molecules. The complementary overhang ends of two different nucleic acid molecules can be annealed by the methods disclosed herein. In some aspects, the target site of the nickase on the first chain is different from the target site of the nickase on the second chain.

[0152] In some aspects, expression of the HMGCR and / or SREBF2 genes and their encoded HMGCR and / or SREBF2 proteins can be reduced by contacting the cell with, for example, a CRISPR (e.g., a CRISPR-Cas9 system) that is specific for the HMGCR and / or SREBF2 genes.

[0153] In some aspects, gene editing using CRISPR reduces (e.g., HMGCR and / or SREBF2) gene levels by 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% relative to the HMGCR and / or SREBF2 gene levels observed in a reference cell (e.g., a corresponding cell that has not undergone gene editing using CRISPR). In some aspects, HMGCR and / or SREBF2 gene levels can be measured using any technique known in the art, such as by digital droplet PCR.

[0154] In some aspects, the nucleic acid encoding gRNA or Cas9 disclosed herein is RNA or DNA. In other aspects, the RNA or DNA encoding gRNA or Cas9 disclosed herein is synthetic RNA or synthetic DNA respectively. In some aspects, synthetic RNA or DNA include at least one non-natural core base. In some aspects, all core bases of a certain class have been replaced with non-natural core bases (for example, all uridines in the polynucleotides disclosed herein can be replaced with non-natural core bases, such as 5-methoxyuridine or pseudouridine). In some aspects, polynucleotides (for example, synthetic RNA or synthetic DNA) only include natural core bases, that is, A, C, T and U in the case of synthetic DNA, or A, C, T and U in the case of synthetic RNA or synthetic DNA.

[0155] II.A.2.b. Meganucleases

[0156] In some aspects, gene editing tools for regulating VHL expression in cells include nucleases, such as meganucleases. Based on conserved sequence motifs, meganucleases have been classified into four families: the "LAGLIDADG," "GIY-YIG," "HNH," and "His-Cys box" families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds.

[0157] HEase is known for its long recognition site and tolerance to some sequence polymorphisms in its DNA substrate. Meganuclease domains, structure and function are known, see, for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764.

[0158] In some instances, naturally occurring variants and / or engineered derivatives of meganucleases are used. Methods for modifying kinetics, cofactor interactions, expression, optimal conditions, and / or recognition site specificity, and screening for activity are known, see, for example, Epinat et al., (2003) Nucleic Acids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853; WO2006097784; and WO2004031346; each of which is herein incorporated by reference in its entirety.

[0159] This document can use any meganuclease, including but not limited to I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP, PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII or any active variant or fragment thereof.

[0160] In some aspects, the meganuclease recognizes double-stranded DNA sequences of 12 to 40 base pairs. In some aspects, the meganuclease recognizes a target sequence that is a perfect match in a genome. In some aspects, the meganuclease is a homing nuclease. In some aspects, the homing nuclease is a homing nuclease of the "LAGLIDADG" family. In some aspects, the homing nuclease of the "LAGLIDADG" family is selected from I-SceI, I-CreI, I-Dmol, or a combination thereof.

[0161] II.A.2.c. TALEN

[0162] In some aspects, gene editing tools that can be used in the present disclosure include nucleases, such as transcription activator-like effector nucleases (TALENs). TAL effector nucleases are a class of sequence-specific nucleases that can be used to produce double-strand breaks at specific target sequences in the genomes of prokaryotes or eukaryotes. TAL effector nucleases are produced by fusing natural or engineered transcription activator-like (TAL) effects or their functional parts with the catalytic domain of a nuclease (e.g., FokI).

[0163] The unique modular TAL effector DNA binding domain allows the design of proteins with any specific DNA recognition specificity. Therefore, the DNA binding domain of the TAL effector nuclease can be engineered to recognize a specific DNA target site, thereby being used to produce a double-strand break at the desired target sequence. See WO 2010 / 079430; Morbitzer et al., (2010) PNAS10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res. (2010) doi:10.1093 / nar / gkq704; and Miller et al., (2011) Nature Biotechnology 29:143-148. All documents in these documents are incorporated herein by reference in their entirety.

[0164] Non-limiting examples of suitable TAL nucleases and methods for making suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Nos. 2011 / 0239315A1, 2011 / 0269234A1, 2011 / 0145940A1, 2003 / 0232410A1, 2005 / 0208489A1, 2005 / 0026157A1, 2005 / 0064474A1, 2006 / 0188987A1, and 2006 / 0063231A1, each of which is incorporated herein by reference in its entirety.

[0165] In various aspects, TAL effector nucleases are designed to cleave at or near a target nucleic acid sequence, for example, in a genomic locus of interest, wherein the target nucleic acid sequence is located at or near a sequence to be modified by a targeting vector. TAL nucleases suitable for use in the various methods and compositions provided herein include those specifically designed to bind to or near a target nucleic acid sequence to be modified by a targeting vector as described herein.

[0166] II.A.2.d. Zinc Finger Nucleases (ZFNs)

[0167] In some aspects, gene editing tools that can be used in the present disclosure include nuclease reagents, such as zinc finger nuclease (ZFN) systems. Zinc finger-based systems include fusion proteins that include two protein domains: a zinc finger DNA binding domain and an enzymatic domain. "Zinc finger DNA binding domain," "zinc finger protein" or "ZFP" is a domain within a protein or larger protein that binds to DNA in a sequence-specific manner through one or more zinc fingers, and the zinc finger is a region of amino acid sequence within the binding domain, and its structure is stabilized by the coordination of zinc ions. The zinc finger domain guides the activity of the enzymatic domain to the vicinity of the sequence by binding to the target DNA sequence, and thus induces modification of endogenous target genes near the target sequence. The zinc finger domain can be engineered to bind to almost any desired sequence. Therefore, after identifying a target genetic locus containing a target DNA sequence desired to be cut or recombined, one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell affects the modification of the target genetic locus.

[0168] In some aspects, the zinc finger binding domain comprises one or more zinc fingers. See, for example, Miller et al., (1985) EMBO J. 4: 1609-1614; Rhodes (1993) Scientific American February: 56-65; U.S. Patent No. 6,453,242. Each of these documents is incorporated herein by reference in its entirety. Typically, the length of a single zinc finger domain is about 30 amino acids. A single zinc finger binds to a trinucleotide (i.e., triplet) sequence (or a tetranucleotide sequence that can overlap one nucleotide with the tetranucleotide binding site of an adjacent zinc finger). Therefore, the length of the sequence (e.g., target sequence) to which the zinc finger binding domain is engineered to bind will determine the number of zinc fingers in the engineered zinc finger binding domain. For example, for a ZFP whose finger motif does 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, and so on. The binding sites for individual zinc fingers in a target site (i.e., subsites) need not be contiguous, but may be separated by one or several nucleotides, depending on the length and nature of the amino acid sequence between the zinc fingers (i.e., interfinger linkers) in the multi-finger binding domain. In some aspects, the DNA binding domain of an individual ZFN comprises between three and six individual zinc finger repeats, and each can recognize between 9 and 18 base pairs.

[0169] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, e.g., Beerli et al., (2002) Nature Biotechnol. 20:135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nature Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416; 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388; each of which is incorporated herein by reference in its entirety. Compared to naturally occurring zinc finger proteins, engineered zinc finger binding domains can have novel binding specificities. Engineering methods include, but are not limited to, rational design and various types of selection.

[0170] The selection of a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the method disclosed in U.S. Patent No. 6,453,242. It will be clear to those skilled in the art that a simple visual inspection of the nucleotide sequence can also be used for the selection of the target DNA sequence. Therefore, any method for selecting a target DNA sequence can be used for the methods described herein. The target site typically has a length of at least 9 nucleotides and is therefore bound by a zinc finger binding domain comprising at least three zinc fingers. However, for example, it is also possible that a 4-finger binding domain binds to a target site of 12 nucleotides, a 5-finger binding domain binds to a target site of 15 nucleotides, or a 6-finger binding domain binds to a target site of 18 nucleotides. It will be apparent that it is also possible that a larger binding domain (e.g., 7, 8, 9 and above) binds to a longer target site.

[0171] The enzymatic domain portion of the zinc finger fusion protein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al., (1997) Nucleic Acids Res. 25: 3379-3388. Additional enzymes that cut DNA are known (e.g., 51 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al., (ed.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of the cleavage domain.

[0172] Exemplary restriction endonucleases (restriction enzymes) suitable for use as enzymatic domains of the ZFPs described herein are found in many species and are capable of sequence-specific binding to DNA (at a recognition site) and cleaving the DNA at or near the binding site. Certain restriction enzymes (e.g., Type IIS) cleave DNA at a site removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al., (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al., (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al., (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al., (1994b) J. Biol. Chem. 269:31,978-31,982, each of which is incorporated herein by reference in its entirety.

[0173] II.A.2.e. Interfering RNA (RNAi)

[0174] In some aspects, gene editing tools that can be used to reduce VHL expression in cells include RNA interference molecules ("RNAi"). As used herein, RNAi is an RNA polynucleotide that mediates the reduction of endogenous target gene product expression by degrading target mRNA through endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complex (RISC)). Non-limiting examples of RNAi agents include microRNA (also referred to herein as "miRNA"), short hairpin RNA (shRNA), small interfering RNA (siRNA), RNA aptamers, or combinations thereof.

[0175] In some aspects, the gene editing tools that can be used in the present disclosure include one or more miRNAs. "miRNA" refers to a naturally occurring small non-coding RNA molecule with a length of about 21 to 25 nucleotides. In some aspects, the miRNA that can be used in the present disclosure is at least partially complementary to genes in the cholesterol synthesis pathway, such as HMGCR and / or SREBF2 mRNA molecules. MiRNA can downregulate (e.g., reduce) the expression of endogenous target gene products (i.e., HMGCR and / or SREBF2 proteins) by translational inhibition, cutting and / or deadenylation of mRNA.

[0176] In some aspects, the gene editing tools that can be used together with the present disclosure include one or more shRNAs." shRNA" (or "short hairpin RNA" molecule) refers to an RNA sequence comprising a double-stranded region and a loop region forming a hairpin loop at one end, which can be used to reduce and / or silence gene expression. The length of the double-stranded region on each side of the stem is typically about 19 nucleotides to about 29 nucleotides, and the length of the loop region is typically about three to about ten nucleotides (and 3' or 5' end single-stranded overhanging nucleotides are optional). shRNA can be cloned into a plasmid or into a non-replicating recombinant viral vector to introduce into the cell and cause the shRNA encoding sequence to be integrated into the genome. Therefore, shRNA can stably and continuously inhibit the translation and expression of endogenous target genes.

[0177] In some aspects, the gene editing tools that can be used for the present disclosure include one or more siRNAs. "siRNA" refers to a double-stranded RNA molecule with a length typically of about 21 to 23 nucleotides. siRNA associates with a multiprotein complex called RNA-induced silencing complex (RISC), during which the "follower" sense chain is enzymatically cut. Then, due to sequence homology, the antisense "guide" chain contained in the activated RISC guides RISC to the corresponding mRNA, and the same nuclease cuts the target mRNA, resulting in specific gene silencing. In some aspects, the length of siRNA is about 18, about 19, about 20, about 21, about 22, about 23 or about 24 nucleotides, and has 2 bases hanging at its 3' end. siRNA and shRNA are further described in Fire et al., Nature 391:19,1998 and U.S. Patent Nos. 7,732,417; 8202846; and 8,383,599. Each of these documents is incorporated herein by reference in its entirety.

[0178] II.A.2.f. Antisense Oligonucleotides (ASOs)

[0179] In some aspects, gene editing tools that can be used to reduce HMGCR and / or SREBF2 gene and / or HMGCR and / or SREBF2 protein expression in cells include antisense oligonucleotides. As used herein, "antisense oligonucleotides" or "ASOs" refer to oligonucleotides that can regulate target gene (i.e., HMGCR and / or SREBF2) expression by hybridizing with target nucleic acids, particularly hybridizing with continuous sequences on target nucleic acids. Antisense oligonucleotides are not double-stranded in nature, and therefore are not siRNA or shRNA.

[0180] In some aspects, the ASOs that can be used for the present disclosure are single-stranded. It should be understood that the single-stranded oligonucleotides of the present disclosure can form a hairpin or intermolecular duplex structure (a duplex between two molecules of the same oligonucleotide), as long as the degree of self-complementarity within or between the total length of the oligonucleotide is less than about 50%. In some aspects, the ASOs that can be used for the present disclosure can include one or more modified nucleosides or nucleotides, such as 2' sugar-modified nucleosides. Other modifications that can be performed on ASO (for example, such as those that can be used to suppress or reduce HMGCR and / or SREBF2 gene expression) are provided in, for example, U.S. Publication No. 2019 / 0275148A1.

[0181] In some aspects, ASOs can reduce the expression of HMGCR and / or SREBF2 proteins via nuclease-mediated degradation of HMGCR and / or SREBF2 transcripts (e.g., mRNA), wherein the ASOs are capable of recruiting nucleases, e.g., RNase H, such as RNaseH1. RNase H is a ubiquitous enzyme that hydrolyzes the RNA strand of RNA / DNA duplexes. Thus, in certain aspects, upon binding to a target sequence (e.g., HMGCR and / or SREBF2 mRNA), the ASOs can induce HMGCR and / or SREBF2 mRNA, thereby reducing the expression of HMGCR and / or SREBF2 proteins.

[0182] As disclosed herein, the above examples of gene editing tools are not intended to be limiting, and any gene editing tool available in the art can be used to reduce or inhibit expression of HMGCR and / or SREBF2 genes and / or HMGCR and / or SREBF2 proteins.

[0183] II.B. Cholesterol

[0184] Some aspects of the present disclosure relate to methods for producing EVs from producer cells, the methods comprising inhibiting the cholesterol biosynthetic pathway (e.g., by contacting the producer cells with statins) and contacting the producer cells with cholesterol. In some aspects, the producer cells are contacted with (i) statins and (ii) cholesterol. In some aspects, the producer cells are contacted with statins before contacting cholesterol. In some aspects, the producer cells are contacted with statins after contacting cholesterol. In some aspects, the producer cells are contacted with statins and cholesterol simultaneously.

[0185] In some aspects, the producer cells are cultured in a culture medium comprising statins and cholesterol. In some aspects, the producer cells are cultured in a culture 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 a culture medium for more than 6 days. In some aspects, the producer cells are cultured in a culture medium for more than 7 days. In some aspects, the producer cells are cultured in a culture medium for more than 8 days. In some aspects, the producer cells are cultured in a culture medium for more than 9 days. In some aspects, the producer cells are cultured in a culture medium for more than 10 days. In some aspects, the producer cells are cultured in a culture medium for more than 10 days. In some aspects, the producer cells are cultured in the culture medium for more than 11 days. In some aspects, the producer cells are cultured in the culture medium for more than 12 days. In some aspects, the producer cells are cultured in the culture medium for more than 13 days. In some aspects, the producer cells are cultured in the culture medium for more than 14 days. In some aspects, the producer cells are cultured in the culture medium for more than 15 days. In some aspects, the producer cells are cultured in the culture medium for more than 16 days. In some aspects, the producer cells are cultured in the culture medium for more than 17 days. In some aspects, the producer cells are cultured in the culture medium for more than 18 days. In some aspects, the producer cells are cultured in the culture medium for more than 19 days. In some aspects, the producer cells are cultured in the culture medium for more than 20 days. In some aspects, the producer cells are cultured in the culture medium for more than 21 days.

[0186] In some aspects, the producer cells are at about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM. , about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM , about 38 μM, about 39 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 150 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM or about 1,000 μM.

[0187] In some aspects, the producer cells are contacted with cholesterol at a concentration of about 0.1 μM to about 100 μM, about 0.1 μM to about 75 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 40 μM, about 0.1 μM to about 30 μM, about 0.1 μM to about 25 μM, about 0.1 μM to about 20 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, 1 μM to about 30 μM, or about 1 μM to about 25 μM, about 1 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 2 μM to about 3 μM, about 20 μM to about 30 μM, about 25 μM to about 30 μM, or about 20 μM to about 25 μM.

[0188] In some aspects, the producer cells are contacted with cholesterol at a concentration of about 2.5 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 5 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 6 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 7 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 8 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 9 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 10 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 15 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 20 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 25 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 30 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 35 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 40 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 45 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 50 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 60 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 70 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 80 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 90 μM. In some aspects, the producer cells are contacted with cholesterol at a concentration of about 100 μM.

[0189] In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 2.5 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 5 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 10 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 15 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 20 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 25 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 30 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 35 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 40 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 45 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 50 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 60 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 70 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 80 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 90 μM. In some aspects, producer cells are contacted with (i) simvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 100 μM.

[0190] In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 2.5 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 5 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 10 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 15 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 20 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 25 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 30 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 35 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 40 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 45 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 50 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 60 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 70 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 80 μM. In some aspects, producer cells are contacted with (i) rosuvastatin at a concentration of about 10mM, and contacted with (ii) cholesterol at a concentration of about 90 μM. In some aspects, the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and with (ii) cholesterol at a concentration of about 100 μM.

[0191] In some aspects, cholesterol is added to the culture medium. In some aspects, cholesterol is produced by cells in culture. In some aspects, cholesterol is produced by producer cells. In some aspects, cholesterol is synthetic cholesterol.

[0192] II.C.DMSO

[0193] Some aspects of the present disclosure relate to a method of increasing the number of extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with dimethyl sulfoxide (DMSO). Some aspects of the present disclosure relate to a method of producing extracellular vesicles (EVs) from producer cells, the method comprising contacting the producer cells with DMSO.

[0194] In some aspects, producer cells are cultivated in a culture medium comprising DMSO. In some aspects, producer cells are cultivated in a culture medium comprising DMSO and cholesterol. In some aspects, producer cells are cultivated in a culture medium comprising DMSO and statins. In some aspects, producer cells are cultivated in a culture medium comprising (i) DMSO, (ii) statins and (iii) cholesterol. In some aspects, producer cells are cultivated in a culture 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, producer cells are cultivated in a culture medium for more than 6 days. In some aspects, producer cells are cultivated in a culture medium for more than 7 days. In some aspects, the producer cells are cultured in the culture medium for more than 8 days. In some aspects, the producer cells are cultured in the culture medium for more than 9 days. In some aspects, the producer cells are cultured in the culture medium for more than 10 days. In some aspects, the producer cells are cultured in the culture medium for more than 10 days. In some aspects, the producer cells are cultured in the culture medium for more than 11 days. In some aspects, the producer cells are cultured in the culture medium for more than 12 days. In some aspects, the producer cells are cultured in the culture medium for more than 13 days. In some aspects, the producer cells are cultured in the culture medium for more than 14 days. In some aspects, the producer cells are cultured in the culture medium for more than 15 days. In some aspects, the producer cells are cultured in the culture medium for more than 16 days. In some aspects, the producer cells are cultured in the culture medium for more than 17 days. In some aspects, the producer cells are cultured in the culture medium for more than 18 days. In some aspects, the producer cells are cultured in the culture medium for more than 19 days. In some aspects, the producer cells are cultured in the culture medium for more than 20 days. In some aspects, the producer cells are cultured in the culture medium for more than 21 days.

[0195] In some aspects, the producer cells are at 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 0.40%, about 0.50%, about 0.60%, about 0.70%, about 0.80%, about 0.90%, about 10.10%, about 11.10%, about 12.10%, about 13.10%, about 14.10%, about 15.10%, about 16.10%, about 17.10%, about 18.10%, about 19.10%, about 1 About 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 9%, about 10%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about In some aspects, 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 0.5%, 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%. In some aspects, the producer cells are contacted with DMSO at a concentration of about 0.1%.

[0196] Book

[0197] III. Extracellular vesicles, such as exosomes

[0198] Disclosed herein are methods for producing EVs (e.g., exosomes). These methods improve EV (e.g., exosome) production yields.

[0199] In some aspects, EVs produced by producer cells (e.g., methods of increasing EV production and methods of producing EVs described herein) have reduced cholesterol content per EV compared to EVs produced by producer cells in which gene and / or protein function in the cholesterol biosynthetic pathway is not reduced (e.g., producer cells that have not been contacted with a statin as disclosed herein). In some aspects, 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%. In some aspects, 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%.

[0200] In some aspects, EVs produced by producer cells (e.g., methods of increasing EV production and methods of producing EVs described herein) have a similar or identical cholesterol content per EV as EVs produced by producer cells in which the function of genes and / or proteins in the cholesterol biosynthetic pathway is not reduced, such as EVs produced by producer cells that have not been contacted with a statin disclosed herein.

[0201] In some aspects, the EVs described herein (e.g., exosomes) are extracellular vesicles with a diameter between about 20 nm and 300 nm. In some aspects, the size of the EVs obtained from the producer cells described herein (e.g., having reduced gene and / or protein function in the cholesterol biosynthetic pathway) is not different in average size compared to EVs produced by producer cells that do not have reduced gene and / or protein function in the cholesterol biosynthetic pathway. In certain aspects, the EVs (e.g., exosomes) of the present disclosure have a diameter between about 20 nm to 290 nm, 20 nm to 280 nm, 20 nm to 270 nm, 20 nm to 260 nm, 20 nm to 250 nm, 20 nm to 240 nm, 20 nm to 230 nm, 20 nm to 220 nm, 20 nm to 210 nm, 20 nm to 200 nm, 20 nm to 190 nm, 20 nm to 180 nm, 20 nm to 170 nm, 20 nm to 160 nm, 20 nm to 150 nm, 20 nm to 140 nm, 20 nm to 130 nm, nm, 20nm to 120nm, 20nm to 110nm, 20nm to 100nm, 20nm to 90nm, 20nm to 80nm, 20nm to 70nm, 20nm to 60nm, 20nm to 50nm, 20nm to 40nm, 20nm to 30nm, 30nm to 300nm, 30nm to 290nm, 30nm to 280nm, 30nm to 270nm, 30nm to 260nm, 30nm to 250nm, 30nm to 240nm, 30nm to 230nm, 30nm to 220nm, 30nm to 210nm, 30nm to 200nm, 30nm to 190nm, 30nm to 180nm, 30nm to 170nm, 30nm to 160nm, 30nm to 150nm, 30nm to 140nm, 30nm to 130nm, 30nm to 120nm, 30nm to 110nm, 30nm to 100nm, 30nm to 90nm, 30nm to 80nm, 30nm to 70nm, 30nm to 60nm, 30nm to 50nm, 30nm to 40nm, 40nm to 300nm, 40nm to 290nm , 40nm to 280nm, 40nm to 270nm, 40nm to 260nm, 40nm to 250nm, 40nm to 240nm, 40nm to 230nm, 40nm to 220nm, 40nm to 210nm, 40nm to 200nm, 40nm to 190nm, 40nm to 180nm, 40nm to 170nm, 40nm to 160nm, 40nm to 150nm, 40nm to 140nm, 40nm to 130nm, 40nm to 120nm, 40nm to 110nm, 40nm to 100nm,40nm to 90nm, 40nm to 80nm, 40nm to 70nm, 40nm to 60nm, 40nm to 50nm, 50nm to 300nm, 50nm to 290nm, 50nm to 280nm, 50nm to 270nm, 50nm to 260nm, 50nm to 250nm, 50nm to 240nm, 50nm to 230nm, 50nm to 220nm, 50nm to 210nm, 50nm to 200nm, 50nm to 190nm, 50nm to 180nm, 50nm to 170nm, 50nm to 160nm, 50nm to 150nm, 50nm to 140nm, 50nm to 130nm, 50nm to 140nm 0nm to 120nm, 50nm to 110nm, 50nm to 100nm, 50nm to 90nm, 50nm to 80nm, 50nm to 70nm, 50nm to 60nm, 60nm to 300nm, 60nm to 290nm, 60nm to 280nm, 60nm to 270nm, 60nm to 260nm, 60nm to 250nm, 60nm to 240nm, 60nm to 230nm, 60nm to 220nm, 60nm to 210nm, 60nm to 200nm, 60nm to 190nm, 60nm to 180nm, 60nm to 170nm, 60nm to 160nm, 60nm to 150nm, 60 nm to 140nm, 60nm to 130nm, 60nm to 120nm, 60nm to 110nm, 60nm to 100nm, 60nm to 90nm, 60nm to 80nm, 60nm to 70nm, 70nm to 300nm, 70nm to 290nm, 70nm to 280nm, 70nm to 270nm, 70nm to 260nm, 70nm to 250nm, 70nm to 240nm, 70nm to 230nm, 70nm to 220nm, 70nm to 210nm, 70nm to 200nm, 70nm to 190nm, 70nm to 180nm, 70nm to 170nm, 70nm to 160nm, 70nm to 280nm 0nm to 150nm, 70nm to 140nm, 70nm to 130nm, 70nm to 120nm, 70nm to 110nm, 70nm to 100nm, 70nm to 90nm, 70nm to 80nm, 80nm to 300nm, 80nm to 290nm, 80nm to 280nm, 80nm to 270nm, 80nm to 260nm, 80nm to 250nm, 80nm to 240nm, 80nm to 230nm, 80nm to 220nm, 80nm to 210nm, 80nm to 200nm, 80nm to 190nm, 80nm to 180nm, 80nm to 170nm, 80nm to 160nm,80nm to 150nm, 80nm to 140nm, 80nm to 130nm, 80nm to 120nm, 80nm to 110nm, 80nm to 100nm, 80nm to 90nm, 90nm to 300nm, 90nm to 290nm, 90nm to 280nm, 90nm to 270nm, 90nm to 260nm, 90nm to 250nm, 90nm to 240nm, 90nm to 230nm, 90nm to 220nm, 90nm to 210nm, 90nm to 200nm, 90nm to 190nm, 90nm to The size of EVs (e.g., exosomes) described herein can be measured according to methods known in the art.

[0202] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise a bilipid membrane ("EV, e.g., exosome membrane") comprising an inner (luminal) surface and an outer surface. In some aspects, the inner (luminal) surface faces the inner core (i.e., lumen) of the EV (e.g., exosome). In some aspects, the outer surface can be in contact with an endosome, multivesicular body, or membrane / cytoplasm of a producer cell or a target cell.

[0203] In some aspects, the EV (e.g., exosome) membrane comprises lipids and fatty acids. In some aspects, the EV (e.g., exosome) membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine.

[0204] In some aspects, the EV (e.g., exosome) membrane comprises an inner leaflet and an outer leaflet. The composition of the inner leaflet and the outer leaflet can be determined by a cross-bilayer distribution assay known in the art, see, for example, Kuypers et al., Biohim Biophys Acta 1985 819: 170. In some aspects, the composition of the outer leaflet is a choline phospholipid between about 70%-90%, an acidic phospholipid between about 0%-15%, and a phosphatidylethanolamine between about 5%-30%. In some aspects, the composition of the inner leaflet is a choline phospholipid between about 15%-40%, an acidic phospholipid between about 10%-50%, and a phosphatidylethanolamine between about 30%-60%. In some aspects, EV (e.g., exosome) comprises about 40% to about 60% cholesterol. In some aspects, the EV produced by the producer cells has a reduced cholesterol content per EV compared to EVs produced by producer cells in which gene and / or protein function in the cholesterol biosynthesis pathway is not reduced.

[0205] In some aspects, the EV (eg, exosome) membrane comprises one or more polysaccharides, such as glycans.

[0206] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise an IL-12 moiety, wherein the IL-12 moiety is on the outer surface of the EV or is attached to the EV via a scaffold moiety on the luminal surface of the EV.

[0207] In some aspects, EVs (e.g., exosomes of the present disclosure) comprise an IL-12 moiety within the lumen of the EV. In other aspects, the EV comprises an IL-12 moiety on its outer surface, optionally connected via a first scaffold moiety (e.g., Scaffold X). In other aspects, the EV comprises an IL-12 moiety on its luminal surface, optionally connected via a scaffold moiety (e.g., Scaffold X or Scaffold Y).

[0208] III.A. Bracket

[0209] One or more scaffold moieties can be used to anchor the IL-12 moiety to the EV of the present disclosure. In some aspects, the IL-12 moiety is connected to a scaffold moiety. In some aspects, the EV comprises more than one scaffold moiety. In some aspects, the IL-12 moiety is connected to a first scaffold moiety, and the second moiety (e.g., a second polypeptide or polynucleotide) is connected to a second scaffold moiety. In some aspects, the first scaffold moiety and the second scaffold moiety are the same type of scaffold moieties, for example, both the first scaffold moiety and the second scaffold moiety are Scaffold moieties of different types, for example, the first scaffold moiety is Scaffold Y protein, and the second scaffold moiety is Scaffold X protein. In some aspects, the first scaffold moiety is Scaffold Y disclosed herein. In some aspects, the first scaffold moiety is Scaffold X disclosed herein. In some aspects, the second scaffold moiety is Scaffold Y disclosed herein. In some aspects, the second scaffold moiety is Scaffold X disclosed herein.

[0210] In some aspects, EV comprises one or more scaffold portions that are capable of anchoring, for example, a nIL-12 portion to an EV (e.g., an exosome) (e.g., on the lumen surface or on the outer surface). In some aspects, the scaffold portion is a polypeptide ("scaffold protein"). In some aspects, the scaffold protein comprises an exosomal protein or a fragment thereof. In other aspects, the scaffold portion is a non-polypeptide portion. In some aspects, the scaffold protein includes various membrane proteins enriched on the exosomal membrane, such as transmembrane proteins, integrins, and peripheral proteins. It may include various CD proteins, transporters, integrins, lectins, and cadherins. In some aspects, the scaffold portion (e.g., scaffold protein) comprises scaffold X. In other aspects, the scaffold portion (e.g., exosomal protein) comprises scaffold Y. In other aspects, the scaffold portion (e.g., exosomal protein) comprises both scaffold X and scaffold Y.

[0211] In some aspects, 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. III.A.1. Scaffold X Engineered EVs (e.g., Exosomes)

[0212] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise membranes that are modified in their composition. For example, their membrane composition can be modified by altering the protein, lipid, or glycan content of the membrane.

[0213] In some aspects, surface engineered EVs (e.g., exosomes) are produced by chemical and / or physical methods, such as PEG-induced fusion and / or ultrasonic fusion. In other aspects, surface engineered EVs (e.g., exosomes) are produced by genetic engineering. EVs (e.g., exosomes) produced by genetically modified producer cells or the progeny of genetically modified cells may contain modified membrane compositions. In some aspects, surface engineered EVs (e.g., exosomes) have a higher or lower density (e.g., a higher number) of scaffold portions (e.g., exosomal proteins, e.g., scaffold X), or include variants or fragments of scaffold portions.

[0214] For example, EVs engineered with a surface (e.g., Scaffold X) can be produced by cells (e.g., HEK293 cells) transformed with an exogenous sequence encoding a scaffold portion (e.g., an exosomal protein, e.g., Scaffold X) or a variant or fragment thereof. EVs comprising a scaffold portion expressed by an exogenous sequence can include a modified membrane composition.

[0215] Various modifications or fragments of the scaffold moiety can be used in various aspects of the present disclosure. For example, a scaffold moiety modified to have enhanced affinity for a binding agent can be used to generate surface-engineered EVs that can be purified using a binding agent. Scaffold moieties modified to more effectively target EVs and / or membranes can be used. Scaffold moieties modified to contain the minimum fragment required for specific and effective targeting to exosome membranes can also be used.

[0216] The scaffold portion can be engineered to be expressed as a fusion molecule, e.g., a fusion molecule of Scaffold X and an IL- 12 portion. For example, a fusion molecule can comprise a scaffold portion disclosed herein (e.g., Scaffold X, e.g., PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment or variant thereof) linked to an IL-12 portion.

[0217] In some aspects, the surface (e.g., Scaffold X) engineered EVs described herein exhibit superior characteristics compared to EVs known in the art. For example, compared to naturally occurring EVs or EVs generated using conventional exosomal proteins, surface (e.g., Scaffold X) engineered EVs contain a higher enrichment of modified proteins on their surfaces. Furthermore, compared to naturally occurring EVs or EVs generated using conventional exosomal proteins, the surface (e.g., Scaffold X) engineered EVs disclosed herein may have greater, more specific, or more controllable biological activity.

[0218] In some aspects, Scaffold X comprises a prostaglandin F2 receptor negative regulator (PTGFRN polypeptide). PTGFRN protein can also be referred to as CD9 partner 1 (CD9P-1), protein F (EWI-F) containing Glu-Trp-Ile EWI motif, prostaglandin F2-alpha receptor regulatory protein, prostaglandin F2-alpha receptor-associated protein or CD315. The full-length amino acid sequence of human PTGFRN protein (Uniprot accession number Q9P2B2) is shown as SEQ ID NO: 1 in Table 3. PTGFRN polypeptide contains a signal peptide (SEQ ID NO: 1 amino acid 1 to 25), an extracellular domain (SEQ ID NO: 1 amino acid 26 to 832), a transmembrane domain (SEQ ID NO: 1 amino acid 833 to 853) and a cytoplasmic domain (SEQ ID NO: 1 amino acid 854 to 879). Mature PTGFRN polypeptide consists of SEQ ID NO: 1 without a signal peptide, i.e., SEQ ID NO: 1 amino acids 26 to 879. In some aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide. In other aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide and (i) comprise 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) comprise 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).

[0219] In some aspects, fragments of a PTGFRN polypeptide lack one or more functional or structural domains, such as IgV.

[0220] In other aspects, Scaffold X comprises an amino acid sequence that is 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. In other aspects, Scaffold X comprises an amino acid sequence that is 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. In other aspects, Scaffold X comprises an amino acid sequence that is 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. In other aspects, Scaffold X comprises the amino acid sequence of SEQ ID NO: 33, except for 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 substitutions, insertions, deletions, or any combination thereof. In some aspects, Scaffold X comprises the amino acid sequence of SEQ ID NO: 33 and comprises 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 more amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 33.

[0221] Table 3. Exemplary Scaffold X protein sequences.

[0222]

[0223] Non-limiting examples of other Scaffold X proteins can be found in U.S. Patent No. US10195290B1, issued on February 5, 2019, which is incorporated by reference in its entirety.

[0224] In some aspects, the sequence encodes a fragment of the scaffold portion that lacks 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 portion that lacks 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 portion that lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from both the N-terminus and the C-terminus of the native protein. In some aspects, the sequence encodes a fragment of a scaffold portion that lacks one or more functional or structural domains of the native protein.

[0225] In some aspects, a scaffold portion, such as Scaffold X, such as a PTGFRN protein, is connected to one or more heterologous proteins. The one or more heterologous proteins can be connected to the N-terminus of the scaffold portion. The one or more heterologous proteins can be connected to the C-terminus of the scaffold portion. In some aspects, the one or more heterologous proteins are connected to the N-terminus and C-terminus of the scaffold portion. In some aspects, the heterologous protein is a mammalian protein. In some aspects, the heterologous protein is a human protein.

[0226] In some aspects, Scaffold X can be used to attach any moiety (e.g., an IL-12 moiety) to both the luminal and external surfaces of an EV (e.g., an exosome). For example, the PTGFRN polypeptide can be used to attach to the external surface of an EV (e.g., an exosome) as well as to the IL-12 moiety within the lumen (e.g., on the luminal surface). In some aspects, Scaffold X is a scaffold protein capable of anchoring IL-12 to the luminal surface of an EV and / or to the external surface of an EV.

[0227] III.A.2. Scaffold Y-engineered EVs (e.g., exosomes)

[0228] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise an interior space (i.e., a lumen) that is different from the interior space of naturally occurring EVs. For example, EVs can be modified such that the composition in the luminal surface of the EVs (e.g., exosomes) has a protein, lipid, or glycan content that is different from the protein, lipid, or glycan content of naturally occurring exosomes.

[0229] In some aspects, engineered EVs (e.g., exosomes) can be generated from cells transformed with exogenous sequences encoding a scaffold portion (e.g., an exosomal protein, e.g., Scaffold Y) or a modification or fragment of a scaffold portion that alters the composition or content of the luminal surface of an EV (e.g., an exosome). Various modifications or fragments of exosomal proteins that can be expressed on the luminal surface of an EV (e.g., an exosome) can be used in aspects of the present disclosure.

[0230] In some aspects, exosomal proteins that can alter the luminal surface of EVs (e.g., exosomes) include, but are not limited to, myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1) protein, brain acid-soluble protein 1 (BASP1) protein, or any combination thereof.

[0231] In some aspects, scaffold Y comprises MARCKS protein (Uniprot accession number P29966). MARCKS protein is also known as protein kinase C substrate, 80kDa protein, light chain. The full-length human MARCKS protein is 332 amino acids long and contains a calmodulin binding domain at amino acid residues 152-176. In some aspects, scaffold Y comprises MARCKSL1 protein (Uniprot accession number P49006). 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 long. MARCKSL1 protein has an effector domain at amino acid residues 87-110 that is involved in lipid binding and calmodulin binding. In some aspects, scaffold Y comprises BASP1 protein (Uniprot accession number P80723). BASP1 protein is also known as the 22kDa neuronal tissue-enriched acidic protein or neuronal axonal membrane protein NAP-22. The full-length human BASP1 protein sequence (isoform 1) is 227 amino acids in length. The isoform generated by alternative splicing lacks amino acids 88 to 141 (isoform 1) from SEQ ID NO: 49. Table 4 provides the full-length sequences of exemplary Scaffold Y disclosed herein (i.e., MARCKS, MARCKSL1, and BASP1 proteins).

[0232] Table 4. Exemplary Scaffold Y Protein Sequences.

[0233]

[0234] The mature BASP1 protein sequence lacks the first Met in SEQ ID NO:49 and therefore contains amino acids 2 to 227 of SEQ ID NO:49.

[0235] In other aspects, Scaffold Y useful in the present disclosure comprises an amino acid sequence that is 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. In other aspects, Scaffold Y useful in the present disclosure comprises the amino acid sequence of SEQ ID NO: 49, except for 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 substitutions, insertions, deletions, or any combination thereof.

[0236] In some aspects, the protein sequence of SEQ ID NO: 49 without a Met at amino acid residue 1 of SEQ ID NO: 49 is sufficient as Scaffold Y for the present disclosure (e.g., a scaffold portion attached to an IL-12 portion). In some aspects, Scaffold Y comprises an amino acid sequence that is 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 a Met at amino acid residue 1 of SEQ ID NO: 49.

[0237] Scaffold Y engineered EVs (eg, exosomes described herein) can be produced from cells transformed with the sequence shown in SEQ ID NO: 49 without a Met at amino acid residue 1 of SEQ ID NO: 49.

[0238] In some aspects, the scaffold Y protein useful in the present disclosure comprises an "N-terminal domain" (ND) and an "effector domain" (ED), wherein the ND and / or ED associate with the luminal surface of EVs (e.g., exosomes). In some aspects, the scaffold Y protein useful in the present disclosure comprises an intracellular domain, a transmembrane domain, and an extracellular domain; wherein the intracellular domain comprises an "N-terminal domain" (ND) and an "effector domain" (ED), wherein the ND and / or ED associate with the luminal surface of EVs (e.g., exosomes). As used herein, the term "associated with" refers to an interaction between a scaffold protein and the luminal surface of an EV (e.g., an exosome), the interaction not involving covalent attachment to a membrane component. For example, a scaffold useful in the present disclosure may be associated with the luminal surface of an EV, for example, via a lipid anchor (e.g., myristic acid) and / or a multi-domain that electrostatically interacts with the negatively charged head of a membrane phospholipid. In other aspects, the scaffold Y protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND associates with the luminal surface of the EV and the ED associates with the luminal surface of the EV through ionic interactions, wherein the ED comprises at least two, at least three, at least four, at least five, at least six or at least seven contiguous basic amino acids, such as lysine (Lys), in sequence.

[0239] In other aspects, the Scaffold Y protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND is associated with the luminal surface of EVs (e.g., exosomes) and the ED is associated with the luminal surface of EVs through ionic interactions, wherein the ED comprises at least two, at least three, at least four, at least five, at least six, or at least seven contiguous basic amino acids, e.g., lysine (Lys), in sequence.

[0240] In some aspects, NDs are associated with the luminal surface of EVs (e.g., exosomes) via lipidation, such as myristoylation. In some aspects, NDs have a Gly at the N-terminus. In some aspects, the N-terminal Gly is myristoylated.

[0241] In some aspects, the ED is associated with the luminal surface of the EV (e.g., exosome) through ionic interactions. In some aspects, the ED is associated with the luminal surface of the EV (e.g., exosome) through electrostatic interactions, particularly attractive electrostatic interactions.

[0242] In some aspects, the ED comprises (i) a basic amino acid (e.g., lysine), or (ii) two or more basic amino acids (e.g., lysine) adjacent to each other in the polypeptide sequence. In some aspects, the basic amino acid is lysine (Lys; K), arginine (Arg, R), or histidine (His, H). In some aspects, the basic amino acid is (Lys)n, where n is an integer between 1 and 10.

[0243] In other aspects, if the N-terminus of the ED is directly linked to a lysine at the C-terminus of the ND, i.e., the lysine is in the N-terminus of the ED and fused to a lysine in the C-terminus of the ND, then the ED comprises at least one lysine and the ND comprises a lysine at the C-terminus. In other aspects, when the N-terminus of the ED is linked to the C-terminus of the ND via a linker (e.g., one or more amino acids), the ED comprises at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines.

[0244] Non-limiting examples of scaffold Y proteins that can be used in the present disclosure are disclosed in International Publication No. WO / 2019 / 099942, which is incorporated herein by reference in its entirety.

[0245] In some aspects, the Scaffold Y proteins useful in the present disclosure do not contain an N-terminal Met. In some aspects, the Scaffold Y proteins contain a lipidated amino acid, such as a myristoylated amino acid, at the N-terminus of the scaffold protein, which serves as a lipid anchor. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is Gly. The presence of N-terminal Gly is an absolute requirement for N-myristoylation. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is synthetic. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is a glycine analog, such as allylglycine, butylglycine, or propargylglycine.

[0246] In other aspects, the lipid anchor can be any lipid anchor known in the art, such as palmitic acid or glycosylphosphatidylinositol. In unusual cases, such as by using a culture medium in which myristic acid is limiting, some other fatty acids (including shorter chain fatty acids and unsaturated fatty acids) can be attached to the N-terminal glycine. For example, in the BK channel, myristate has been reported to be attached post-translationally to internal serine / threonine or tyrosine residues via a hydroxyester bond. Membrane anchors known in the art are shown in the table below:

[0247]

[0248] III.B. Connectors

[0249] As described above, the extracellular vesicles (EVs) (e.g., exosomes) of the present disclosure can comprise one or more linkers that connect a molecule of interest (e.g., a payload, such as an IL-12 portion) to the EV (e.g., to the outer surface or lumen surface). In some aspects, the payload (e.g., a bioactive molecule) is connected to the EV directly or via a scaffold portion (e.g., scaffold X or scaffold Y). In some aspects, the payload (e.g., a bioactive molecule) is connected to the scaffold portion via a linker. In some aspects, the payload (e.g., a bioactive molecule) is connected to the second scaffold portion via a linker.

[0250] In some aspects, the payload (e.g., a bioactive molecule) is linked to the outer surface of the exosome via Scaffold X. In other aspects, the payload (e.g., a bioactive molecule) is linked to the luminal surface of the exosome via Scaffold X or Scaffold Y. The linker can be any chemical moiety known in the art.

[0251] In some aspects, two or more joints can be connected in series. When there are multiple joints, each joint in the joint can be the same or different. Usually, the joint provides flexibility or prevents / improves steric hindrance. The joint is typically not cut; However, in some aspects, this cutting may be desirable. Therefore, in some aspects, the joint can include one or more protease cleavage sites, and the one or more protease cleavage sites can be located in the sequence of the joint or at the joint side of either end of the joint sequence.

[0252] In some aspects, the linker is a peptide linker. In some aspects, the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, 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 about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 or at least about 100 amino acids.

[0253] In some aspects, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally occurring or non-naturally occurring peptide) comprising an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising mutations such as additions, substitutions, or deletions).

[0254] In some aspects, the linker is a non-polypeptide moiety.

[0255] The linker may be susceptible to cleavage (a "cleavable linker"), thereby facilitating release of the biologically active molecule (eg, the IL-12 moiety).

[0256] In some aspects, the linker is a "reduction-sensitive linker". In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an "acid-labile linker". In some aspects, the acid-labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, cis-aconitine linkers, hydrazide linkers, thiocarbamoyl linkers, or any combination thereof.

[0257] In some aspects, the linker comprises a non-cleavable linker.

[0258] III.C. Therapeutic Agents

[0259] In some aspects, the EVs described herein may further comprise at least one therapeutic agent or bioactive molecule. In some aspects, the therapeutic agent comprises a cytokine (e.g., IL-12 or IL-2), a small molecule, a growth factor, an antigen, an antisense oligonucleotide, siRNA, shRNA, miRNA, dsDNA, lncRNA, PROTAC, an adjuvant, an immunomodulator, or any combination thereof.

[0260] In some aspects, antisense oligonucleotides regulate the function of target gene.In some aspects, ASO regulates the function of the nucleic acid molecule encoding mammal STAT6 (for example, described in WO2021030776, the document is incorporated herein by reference), such as STAT6 nucleic acid, such as STAT6 transcript, including STAT6 pre-mRNA and STAT6 mRNA, or the naturally occurring variants of such nucleic acid molecules encoding mammal STAT6.In some aspects, ASO regulates the function of the nucleic acid molecule encoding mammal STAT3 (for example, described in WO2021030768, the document is incorporated herein by reference in its entirety), such as STAT3 nucleic acid, such as STAT3 transcript, including STAT3 pre-mRNA and STAT3 mRNA, or the naturally occurring variants of such nucleic acid molecules encoding mammal STAT3. In some aspects, the ASO modulates the function of a nucleic acid molecule encoding mammalian CEBP / b (e.g., described in WO2021030780, which is incorporated herein by reference in its entirety), such as a CEBP / b nucleic acid, e.g., a CEBP / b transcript, including CEBP / b pre-mRNA and CEBP / b mRNA, or a naturally occurring variant of such a nucleic acid molecule encoding mammalian CEBP / b. In some aspects, the ASO modulates the function of a nucleic acid molecule encoding mammalian NRAS (e.g., described in WO2021030769, which is incorporated herein by reference in its entirety), such as a NRAS nucleic acid, e.g., a NRAS transcript, including NRAS pre-mRNA and NRAS mRNA, or a naturally occurring variant of such a nucleic acid molecule encoding mammalian NRAS. In some aspects, ASOs modulate the function of nucleic acid molecules encoding mammalian KRAS (e.g., described in WO2021030781, which is incorporated herein by reference in its entirety), such as KRAS nucleic acids, e.g., KRAS transcripts, including KRAS pre-mRNA and KRAS mRNA, or naturally occurring variants of such nucleic acid molecules encoding mammalian KRAS. In the context of the present disclosure, the term "ASO" refers to a molecule formed by covalent bonds of two or more nucleotides (i.e., oligonucleotides).

[0261] In some aspects, the therapeutic agent comprises a STING agonist, as described in WO 2019 / 183578.

[0262] In some aspects, the therapeutic agent comprises an IL-12 polypeptide. In some aspects, the IL-12 polypeptide is any IL-12 polypeptide disclosed in U.S. Patent No. 10,723,782 or U.S. Publication No. 2022 / 0218811A1, each of which is incorporated herein by reference in its entirety. In some aspects, the therapeutic agent comprises an IL-2 polypeptide.

[0263] In some aspects, the present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering an extracellular vesicle as described herein. The present disclosure also provides uses of the extracellular vesicles as described herein for treating or preventing a disease or condition in a subject in need thereof. In some aspects, the present disclosure provides an extracellular vesicle as described herein for treating or preventing a disease or condition in a subject in need thereof.

[0264] IV. Producer Cells

[0265] The EVs (e.g., exosomes) disclosed herein can be produced from cells grown in vitro or from body fluids of a subject. When exosomes are produced by in vitro cell culture, various producer cells can be used, such as HEK293 cells, CHO cells, C2C12 and MSCs. In some aspects, the producer cells can be selected from HEK293 cells, HEK293S cells, HEK293SF cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, AGE. Neuronal precursor cells, Amniotic fluid cells, adipose-derived 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. In some aspects, the producer cells are stem cells.

[0266] Producer cells can be genetically and / or pharmacologically modified to reduce genes and / or protein functions in the cholesterol biosynthetic pathway, as described herein. In some aspects, modified producer cells can be further modified (e.g., genetically) to include exogenous sequences encoding proteins to produce EVs as described herein. Genetically modified producer cells can contain the exogenous sequence by transient or stable transformation. The exogenous sequence can be converted into a plasmid. In some aspects, the exogenous sequence is a vector. The exogenous sequence can be stably integrated into the genomic sequence of the producer cell at a targeted site or a random site. In some aspects, a stable cell line is produced for the production of cavity-engineered exosomes.

[0267] The exogenous sequence can be inserted into the genomic sequence of the producer cell, located within, upstream (5' end) or downstream (3' end) of the endogenous sequence encoding the exosomal protein. Various methods known in the art can be used to introduce exogenous sequences into producer cells. For example, cells modified using various gene editing methods (e.g., methods using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9 or TALEN) are within the scope of the present disclosure.

[0268] The exogenous sequence may comprise a sequence encoding a scaffold portion disclosed herein, or a fragment or variant thereof. Additional copies of a sequence encoding a scaffold portion may be introduced to generate exosomes as described herein (e.g., with a higher density of scaffold portions on the surface or lumen of an EV (e.g., exosome)). Exogenous sequences encoding modifications or fragments of a scaffold portion may be introduced to generate lumen-engineered and / or surface-engineered exosomes containing modifications or fragments of a scaffold portion.

[0269] In some aspects, producer cells can be modified, eg, transfected, with one or more vectors encoding a scaffold moiety linked to a protein.

[0270] In some aspects, the producer cells disclosed herein are further modified to include additional exogenous sequences. For example, additional exogenous sequences can be introduced to regulate endogenous gene expression, or to produce exosomes comprising a specific polypeptide as a payload (e.g., an IL-12 portion or an IL-2 portion). In some aspects, the producer cells are modified to include two exogenous sequences, one encoding a scaffold portion (e.g., scaffold X and / or scaffold Y) or a variant or fragment thereof, and the other encoding a payload. In some aspects, the producer cells are modified to include two exogenous sequences, one encoding a scaffold portion disclosed herein or a variant or fragment thereof, and the other encoding a protein that confers additional functionality to the exosomes. In some aspects, the producer cells are further modified to include one, two, three, four, five, six, seven, eight, nine or ten or more additional exogenous sequences.

[0271] In some aspects, the EVs (e.g., exosomes) of the present disclosure (e.g., surface engineered and / or cavity engineered exosomes) can be produced by cells transformed with sequences encoding the full-length mature scaffold moieties disclosed herein or the scaffold moieties linked to proteins (e.g., therapeutic proteins). Any of the scaffold moieties described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acids such as synthetic messenger RNA (mRNA).

[0272] In some aspects, the present disclosure provides a bioreactor comprising extracellular vesicle (EV) producer cells that exhibit a reduction in gene and / or protein function in the cholesterol biosynthetic pathway. In some aspects, the methods described herein are performed using a perfusion bioreactor. In some aspects, the bioreactor is connected to a cell retention device.

[0273] In some aspects, the producer cells can be cultivated in a bioreactor (e.g., a WAVE bioreactor, a stirred tank bioreactor, an oscillating bioreactor). The various configurations of bioreactors are known in the art, and suitable configurations can be selected as needed. In some aspects, cultivation is carried out in, for example, an N-1 or N-terminal container or a bioreactor. In some aspects, the culture vessel is a fed-batch or perfusion bioreactor. In some aspects, the bioreactor is connected to a cell separator. For example, an N-terminal production container, particularly a stirred tank bioreactor, can be used that is connected to a cell retention device (such as a hollow fiber membrane, for example, alternating tangential flow filtration (ATF), tangential flow filtration (TFF)) or an acoustic cell separator.

[0274] V. Pharmaceutical Compositions and Methods of Treatment

[0275] Provided herein are pharmaceutical compositions comprising EVs (e.g., exosomes) of the present disclosure having a desired purity and a pharmaceutically acceptable carrier or excipient in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier may be determined in part by the specific composition being administered and by the specific method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions comprising multiple extracellular vesicles. (See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st edition (2005)). Pharmaceutical compositions are typically formulated as sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0276] In some aspects, the pharmaceutical composition comprises one or more therapeutic agents and exosomes as described herein. In certain aspects, EVs (e.g., exosomes) are co-administered with one or more additional therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered before administering the additional therapeutic agent. In other aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered after administering the additional therapeutic agent. In other aspects, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered simultaneously with the additional therapeutic agent.

[0277] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenolic, butyl, or benzyl alcohols; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( less than about 10 residues); polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0278] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such culture media and compounds for pharmaceutically active substances is well known in the art. Except for the case where any conventional medium or compound is incompatible with the extracellular vesicles described herein, the use of the medium or compound in the composition is envisioned. Supplementary therapeutic agents may also be incorporated into the composition. Typically, the pharmaceutical composition is formulated to be compatible with its intended route of administration. EV (e.g., exosomes) can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intratumorally, intramuscularly, or in the form of an inhalant. In certain aspects, the pharmaceutical composition comprising exosomes is administered intravenously, for example, by injection. EV (e.g., exosomes) may optionally be administered in combination with other therapeutic agents that are at least partially effective in treating the disease, disorder, or condition for which EV (e.g., exosomes) is intended.

[0279] The solution or suspension may contain the following ingredients: a sterile diluent such as water, saline, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antimicrobial compound such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate or phosphate, and a compound for adjusting osmotic pressure such as sodium chloride or glucose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The formulation can be enclosed in an ampoule, disposable syringe or multiple-dose vial made of glass or plastic.

[0280] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Said composition is normally sterile and is fluid to the extent that it is easy to inject. Carrier can be a solvent or dispersion medium, and it contains for example water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol etc.) and suitable mixture thereof. Can for example by using coatings such as lecithin, by maintaining required particle size and by using surfactant to maintain suitable fluidity in the case of dispersion. The effect of preventing microorganisms can be achieved by various antibacterial agents and antifungal compounds (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal etc.). If desired, isotonic compounds can be added to the composition, for example sugar, as polyols such as mannitol, sorbitol and sodium chloride. Can be achieved by including a compound that delays absorption in the composition, for example, aluminum monostearate and gelatin to extend the absorption of injectable compositions.

[0281] Sterile injectable solutions can be prepared as desired by incorporating an effective amount of EVs (e.g., exosomes) in an appropriate solvent together with one or more of the ingredients listed herein or known in the art. Typically, dispersions are prepared by incorporating EVs (e.g., exosomes) into a sterile vehicle containing a basic dispersion medium and any desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. EVs (e.g., exosomes) can be administered in the form of depot injections or implant formulations, which can be formulated in a manner that allows for sustained or pulsatile release of EVs (e.g., exosomes).

[0282] Systemic administration of compositions containing exosomes can also be performed transmucosally. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved using, for example, nasal sprays.

[0283] In certain aspects, a pharmaceutical composition comprising EVs (e.g., exosomes) is administered intravenously to a subject who would benefit from the pharmaceutical composition. In certain other aspects, the composition is administered to the lymphatic system, for example, by intralymphatic injection or by intranodal injection (see, e.g., Senti et al., PNAS 105(46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or by injection into the liver.

[0284] In some aspects, the pharmaceutical composition comprising exosomes is administered in the form of a liquid suspension. In some aspects, the pharmaceutical composition is administered as a formulation capable of forming a reservoir after administration. In certain preferred aspects, the reservoir slowly releases EVs (e.g., exosomes) into the circulation or remains in a reservoir form.

[0285] Typically, pharmaceutically acceptable compositions are highly purified, free of contaminants, biocompatible and non-toxic, and suitable for administration to a subject. If water is a component of the vehicle, the water is highly purified and treated to be free of contaminants, such as endotoxins.

[0286] Pharmaceutically acceptable carriers can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and / or mineral oil, but are not limited thereto. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifier, a suspending agent and / or a preservative.

[0287] The pharmaceutical compositions described herein comprise EVs (eg, exosomes) described herein and optionally a pharmaceutically active agent or therapeutic agent. The therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.

[0288] Dosage forms are provided comprising pharmaceutical compositions comprising EVs (e.g., exosomes) as described herein. In some aspects, the dosage form is formulated as a liquid suspension for intravenous injection. In some aspects, the dosage form is formulated as a liquid suspension for intratumoral injection.

[0289] In certain aspects, the preparation of exosomes is subjected to radiation, e.g., X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays, to destroy residual replication-competent nucleic acids.

[0290] In certain aspects, the preparation of exosomes is subjected to gamma irradiation using an irradiation dose of more than 1 kGy, 5 kGy, 10 kGy, 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, or more than 100 kGy.

[0291] In certain aspects, the preparation of exosomes is subjected to X-ray irradiation using more than 0.1 mSv, 0.5 mSv, 1 mSv, 5 mSv, 10 mSv, 15 mSv, 20 mSv, 25 mSv, 30 mSv, 35 mSv, 40 mSv, 50 mSv, 60 mSv, 70 mSv, 80 mSv, 90 mSv, 100 mSv, 200 mSv, 30 0mSv, 400mSv, 500mSv, 600mSv, 700mSv, 800mSv, 900mSv, 1000mSv, 2000mSv, 3000mSv, 4000mSv, 5000mSv, 6000mSv, 7000mSv, 8000mSv, 9000mSv, 10000mSv or an exposure dose exceeding 10000mSv.

[0292] The present disclosure also provides methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject an extracellular vesicle described herein.

[0293] Examples

[0294] Example 1

[0295] The effect of rosuvastatin (ROS) treatment on producer cell viability was evaluated in a HEK293 cell line grown in a fed-batch mode, where cells were centrifuged on days 2 and 4 for complete medium exchange. Figure 1 The percentage of cell viability is shown for control cells and cells exposed to 10 nM, 25 nM, 50 nM, 75 nM, and 100 nM ROS. Cultivation in the presence of ROS resulted in a dose-dependent decrease in HEK293 cell viability over 6 days. Therefore, this method may have a time limit, as treated cells become stressed and eventually begin to die after 4 to 6 days of continuous treatment.

[0296] Reduced cell viability suggests that using statins to increase exosome production may be incompatible with manufacturing methods that require continuous upstream cell culture platforms to operate for weeks. However, data generated to confirm that the statin-induced increase in extracellular vesicles (EVs) is cholesterol-dependent suggest that maintaining statin-exposed cells by supplementing the culture medium with cholesterol is a potential solution. HEK293 cell lines engineered to overexpress the PTGFRN-HiBit luciferase reporter gene on engineered EVs showed reduced proliferation in the presence of 10 nM simvastatin due to toxicity during treatment ( Figure 2A However, when only 2.5 mM cholesterol and statins were added, proliferation was restored to levels comparable to the untreated control group.

[0297] To assess the effect on EV production, given that HiBit luciferase was genetically fused to the EV-associated protein PTGFRN, luciferase activity in the culture medium was measured as a surrogate for EV levels. Supplementation with additional cholesterol alone had no effect on secreted luc activity ( Figure 2B ). In contrast, treatment with 10 nM simvastatin had the expected spike in secreted luciferase in the cell culture medium at both the 4-day and 6-day time points tested. This induction was completely abolished when the culture medium was also spiked with 25 mM cholesterol. However, when supplemented with 10-fold less cholesterol, secreted luc activity still increased significantly by 146%, without any obvious adverse effects on cell proliferation. While its effect is not as dramatic as the 419% increase in secreted luciferase activity in cultures supplemented with statins alone, it still represents a significant improvement over continuous upstream manufacturing processes if it can be sustained for more than 6 days.

[0298] Example 2

[0299] To investigate whether the combined treatment of statins and cholesterol could be sustained for more than 6 days and to simulate continuous upstream manufacturing processing in a small-scale model, a semi-continuous batch fed rotary tube model was employed with daily culture medium changes. The model employed 50 mL conical tubes as culture vessels with ventilated caps for gas exchange. The tube format allowed for low culture volumes (10 mL) and the ability to process a large number of tubes in parallel because it was easy to directly centrifuge the tubes and aspirate the old culture medium. To avoid overgrowth of cells to the point where gas exchange was insufficient to meet the culture's demand for dissolved oxygen, daily cell bleeds were incorporated to remove biomass before daily culture medium changes, when necessary, to maintain cell density close to the desired target.

[0300] This article also uses the PTGFRN-HiBit reporter cell line used in previous examples. The cells were seeded at 2e6vc / ml in 10mL of proprietary seed culture medium. The cells were cultured in a humidified incubator at 37°C and 8% CO2 and stirred at 200rpm. On day 3, daily medium changes were started by spinning the test tubes at 300×g for 3 minutes, then aspirating the medium and resuspending the cell pellet in fresh 10mL of medium. On day 4, daily medium changes began to incorporate the following treatments and continued until the termination of the experiment on day 14. The following conditions were evaluated in duplicate:

[0301] 1. Unprocessed

[0302] 2. 0.1% DMSO vehicle control

[0303] 3. 10 μM cholesterol

[0304] 4. 10 nM simvastatin

[0305] 5. 15nM simvastatin

[0306] 6. 15nM simvastatin + 10μM cholesterol

[0307] On day 5, the culture approached the target cell density of 25 million viable cells / mL, and daily cell bleeds were initiated. These were necessary daily throughout the remainder of the experiment to maintain the culture at the desired target and typically represented 25% to 35% of the culture volume.

[0308] After 6 days of treatment (total day 10), both the 10 nM and 15 nM conditions were at the expected decline ( Figure 3A and Figure 3B ). At this point, due to the high cell mortality rate and the expectation of further decline, these concentration conditions were removed from the study. On day 11, the viability and cell number of the combined treatment began to decline slightly. In response, the culture medium supplement was changed to reduce the amount of simvastatin delivered daily (13nM) and increase the supplementation of cholesterol (12.5μM). This stabilized the culture for the remainder of the study, which was terminated on day 14.

[0309] To assess how these conditions affect EV levels in the cell culture medium, samples were removed from the cultures starting on day 5 and centrifuged at 300 × g for 3 minutes to remove cells. The cell culture supernatant was removed and centrifuged again, this time at 21,000 × g for 12 minutes to remove cell debris. The clarified harvest was then stored at -20°C until the culture was terminated. After the cell culture portion of the experiment was completed, the samples were thawed, aliquots were removed and diluted 350-fold in PBS. The diluted samples were transferred (50 μL) to a 5% PBS container containing an equal volume of Nano- HiBiT lysis reagent (Promega) was added to white-walled 96-well plates. Luciferase activity was then read on a plate reader as a surrogate for secreted EV levels.

[0310] Cholesterol supplementation alone had no effect on EV-associated luciferase activity during the 10-day measurement ( Figure 4A In contrast, DMSO vehicle control cultures showed an unexpected increase in luc activity, which began on day 7 and peaked at day 11, with 4.3-fold higher luc activity, before beginning to wane. Its inclusion in this study reflects the fact that DMSO was used as a solvent for simvastatin. Additional experiments will be conducted to further evaluate the effects of DMSO on producer cells and / or exosome production. Further experiments will be conducted to test the effects of other excipients and / or solutes present in the cholesterol formulation.

[0311] The combined treatment also produced an increase in EV-associated reporter gene activity, which began on day 8, peaked on day 11, and remained stable over the remaining 3 days until the end of the experiment ( Figure 4B ). Over the four days, the average daily increase in EV-associated reporter activity measured in clarified harvests was 7.5-fold. The average 7.5-fold increase observed in the present study seems unexpected, given that previous studies have shown that statin / cholesterol combination treatment exhibited a 2.5-fold increase in secreted reporter activity relative to untreated controls at the end of a six-day study. However, in this new study, six days of treatment corresponded to day 10, at which point the average induction of secreted reporter activity reached only 4.7-fold. One possible explanation for the elevated EV secretion after six days of combination treatment in this new study, relative to previous data, is that this new study incorporated daily media changes, whereas the previously used media was changed every other day. If this hypothesis proves to be accurate, it would bode well for the observation of such greater induction in fully continuous upstream cell culture operations used in manufacturing.

[0312] Example 6

[0313] To determine the effects of culturing producer cells in the presence of a combination of statins and cholesterol on extracellular vesicles, we will culture producer cells as described above and isolate extracellular vesicles. The extracellular vesicles will be purified and subjected to biochemical, biophysical, and functional analyses.

[0314] Example 7

[0315] To scale up the above approaches, process development work will be conducted at benchtop bioreactor scale to translate these findings from the reporter cell lines grown in a benchtop rotating tube semi-continuous model to clinically relevant cell lines grown in a scaled-down model for intensified continuous manufacturing.

[0316] Incorporated by reference

[0317] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference herein for all purposes.

[0318] Equivalent

[0319] Although various specific aspects have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. For those skilled in the art, many changes will become apparent after reading this description.

Claims

1. A method for increasing the number of extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthesis pathway inhibitor and (ii) cholesterol.

2. A method for producing extracellular vesicles (EVs) from producer cells, the method comprising contacting the producer cells with (i) a cholesterol biosynthesis pathway inhibitor and (ii) cholesterol.

3. The method of claim 1 or 2, wherein the producer cells are contacted with (i) the cholesterol biosynthesis pathway inhibitor and (ii) cholesterol for more than 8 days.

4. The method of any one of claims 1 to 3, wherein the EVs produced by the producer cells have an increased yield compared to EVs produced by producer cells that have not been contacted with the cholesterol biosynthesis pathway inhibitor.

5. The method of claim 4, wherein the yield is increased by 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.

6. The method of claim 4, wherein the yield is increased by 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.

7. The method of any one of claims 4 to 6, wherein the yield is increased by 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.

8. The method of any one of claims 1 to 7, wherein the cholesterol biosynthesis pathway inhibitor comprises a statin, cariprazine, a PROTAC, AY9944, or BM15766.

9. The method of claim 8, wherein the statin comprises atorvastatin, lovastatin, pitavastatin, pravastatin, fluvastatin, cerivastatin, rosuvastatin, simvastatin, or a combination thereof.

10. The method of claim 8 or 9, wherein the statin is at 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 14 0nM, about 150nM, about 160nM, about 170nM, about 180nM, about 190nM, about 200nM, about 210nM, about 220nM, about 230nM, about 240nM, about 250nM, about 260nM, about 270nM, about 280nM, about 290nM, about 300nM, about 310nM, about 320nM, about 330nM, about 340nM, about 350nM, about 360nM, about 370nM, about 380nM, about 390nM, about 400nM, about 410nM, about 420nM, about 430nM nM, about 440nM, about 450nM, about 460nM, about 470nM, about 480nM, about 490nM, about 500nM, about 510nM, about 520nM, about 530nM, about 540nM, about 550nM, about 560nM, about 570nM, about 580nM, about 590nM, about 600nM, about 610nM, about 620nM, about 630nM, about 640nM, about 650nM, about 660nM, about 670nM, about 680nM, about 690nM, about 700nM, about 710nM, about 720nM The present invention may be contacted with a concentration of about 750 nM, about 760 nM, about 770 nM, about 780 nM, about 790 nM, about 800 nM, about 810 nM, about 820 nM, about 830 nM, about 840 nM, about 850 nM, about 860 nM, about 870 nM, about 880 nM, about 890 nM, about 900 nM, about 910 nM, about 920 nM, about 930 nM, about 940 nM, about 950 nM, about 960 nM, about 970 nM, about 980 nM, about 990 nM, or about 1,000 nM.

11. The method of claim 8 or 9, wherein the statin is administered 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, about 0.1 nM to about 30 nM, about 0.1 nM to about 40 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 60 nM, about 0.1 nM to about 70 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 90 nM, about 0.1 nM to about The invention also provides a method for contacting a patient with a concentration of about 10 nM to about 10 nM, or about 10 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM 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 about 20 nM, about 1 nM to about 10 nM, or about 10 nM to about 20 nM.

12. The method of any one of claims 8 to 11, wherein the statin is contacted at a concentration of about 10 nM.

13. The method of any one of claims 8 to 11, wherein the statin is contacted at a concentration of about 15 nM.

14. The method of any one of claims 8 to 11, wherein the statin is contacted at a concentration of about 20 nM.

15. The method of any one of claims 1 to 11, wherein the cholesterol biosynthesis pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 10 nM.

16. The method of any one of claims 1 to 11, wherein the cholesterol biosynthetic pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 15 nM.

17. The method of any one of claims 1 to 11, wherein the cholesterol biosynthetic pathway inhibitor comprises simvastatin, which is contacted at a concentration of about 20 nM.

18. The method of any one of claims 1 to 11, wherein the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 10 nM.

19. The method of any one of claims 1 to 11, wherein the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 25 nM.

20. The method of any one of claims 1 to 11, wherein the cholesterol biosynthetic pathway inhibitor comprises rosuvastatin, which is contacted at a concentration of about 50 nM.

21. The method of any one of claims 1 to 20, wherein the producer cells are expressed at about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 2μM, about 13μM, about 14μM, about 15μM, about 16μM, about 17μM, about 18μM, about 19μM, about 20μM, about 21μM, about 22μM, about 23μM, about 24μM, about 25μM, about 26μM, about 27μM, about 28μM, about 29μM, about 30μM, about 31μM, about 32μM, about 33μM, about 34μM, about 35μM, about 36μM, A concentration of about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 150 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, or about 1,000 μM is contacted with cholesterol.

22. The method of any one of claims 1 to 20, wherein the producer cells are contacted with cholesterol at a concentration of about 0.1 μM to about 100 μM, about 0.1 μM to about 75 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 40 μM, about 0.1 μM to about 30 μM, about 0.1 μM to about 25 μM, about 0.1 μM to about 20 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, 1 μM to about 30 μM, or about 1 μM to about 25 μM, about 1 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 2 μM to about 3 μM, about 20 μM to about 30 μM, about 25 μM to about 30 μM, or about 20 μM to about 25 μM.

23. The method of any one of claims 1 to 22, wherein the producer cells are contacted with cholesterol at a concentration of about 2.5 μM.

24. The method of any one of claims 1 to 22, wherein the producer cells are contacted with cholesterol at a concentration of about 20 μΜ.

25. The method of any one of claims 1 to 22, wherein the producer cells are contacted with cholesterol at a concentration of about 25 μΜ.

26. The method of any one of claims 1 to 22, wherein the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and with (ii) cholesterol at a concentration of about 2.5 μΜ.

27. The method of any one of claims 1 to 22, wherein the producer cells are contacted with (i) simvastatin at a concentration of about 10 mM and with (ii) cholesterol at a concentration of about 25 μM.

28. The method of any one of claims 1 to 22, wherein the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and with (ii) cholesterol at a concentration of about 2.5 μΜ.

29. The method of any one of claims 1 to 22, wherein the producer cells are contacted with (i) rosuvastatin at a concentration of about 10 mM and with (ii) cholesterol at a concentration of about 25 μM.

30. The method of any one of claims 1 to 29, wherein the producer cell has increased viability compared to a producer cell that is (i) contacted with the cholesterol biosynthesis pathway inhibitor and (ii) not contacted with cholesterol.

31. The method of any one of claims 1 to 29, wherein the producer cell has increased viability compared to a producer cell that (i) has not been contacted with the cholesterol biosynthetic pathway inhibitor and (ii) has not been contacted with cholesterol.

32. The method of claim 30 or 31, wherein the activity 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%.

33. The method of any one of claims 1 to 32, wherein the EVs produced by the producer cells have a reduced cholesterol content per EV compared to EVs produced by producer cells that have not been contacted with the cholesterol biosynthesis pathway inhibitor.

34. The method of claim 33, wherein 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%.

35. The method of claim 34, wherein 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%.

36. A method of increasing the number of extracellular vesicles (EVs) produced from producer cells, the method comprising contacting the producer cells with dimethyl sulfoxide (DMSO).

37. A method of producing extracellular vesicles (EVs) from producer cells, the method comprising contacting the producer cells with DMSO.

38. The method of claim 36 or 37, wherein the producer cells are at 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. The present invention relates to a method for contacting an intravenous catheter with DMSO at a concentration of about 0.05%, about 0.1%, about 0.2%, 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%.

39. The method of any one of claims 36 or 37, wherein 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 0.5%, 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%.

40. The method of any one of claims 36 to 38, wherein the producer cells are contacted with DMSO at a concentration of about 0.1%.

41. The method of any one of claims 1 to 40, wherein the EVs produced by the producer cells have no difference in average particle size distribution compared to EVs produced by producer cells that have not been contacted with the cholesterol biosynthesis pathway inhibitor.

42. The method of any one of claims 1 to 41, wherein the producer cell is a mammalian cell.

43. The method of any one of claims 1 to 42, wherein the producer cells are HEK293 cells, HEK293S cells, HEK293SF cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, Neuronal precursor cells, Amniotic fluid cells, adipose-derived 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.

44. The method of any one of claims 1 to 43, wherein the producer cells are stem cells.

45. The method of any one of claims 1 to 44, wherein the EV further comprises a scaffold portion.

46. ​​The method of claim 45, wherein the scaffold portion comprises Scaffold X.

47. The method of claim 46, wherein scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basic immunoglobulin (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin beta-1 (ITGB1 protein); integrin alpha-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins) and any combination thereof.

48. The method of claim 47, wherein the scaffold moiety is a PTGFRN protein.

49. The method of claim 48, wherein the scaffold portion 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.

50. The method of claim 45, wherein the scaffold portion comprises scaffold Y.

51. The method of claim 50, wherein the scaffold Y is selected from the group consisting of: myristoylated alanine-rich protein kinase C substrate (MARCKS protein); myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein); brain acid-soluble protein 1 (BASP1 protein) and any combination thereof.

52. The method of any one of claims 45 to 51, wherein the EV further comprises at least one therapeutic agent attached to the scaffold portion.

53. The method of any one of claims 1 to 51, wherein the EV further comprises at least one therapeutic agent.

54. The method of claim 52 or 53, wherein the therapeutic agent comprises a cytokine, a small molecule, a growth factor, an antigen, an antisense oligonucleotide, siRNA, shRNA, miRNA, dsDNA, lncRNA, PROTAC, an adjuvant, an immunomodulator, or any combination thereof.

55. The method of claim 54, wherein the therapeutic agent is IL-12.

56. The method of claim 54, wherein the therapeutic agent comprises an IL-2 polypeptide.

57. The method of claim 54, wherein the therapeutic agent is a STING agonist.

58. The method of claim 54, wherein the therapeutic agent is an antisense oligonucleotide.

59. The method of claim 58, wherein the antisense oligonucleotide targets Kras, STAT3, Nras, STAT6, CEBP / b, NLRP3, or any combination thereof.

60. Producer cell for use in the method according to any one of claims 1 to 59.

61. A producer cell prepared by the method of any one of claims 1 and 3 to 59.

62. Extracellular vesicles produced by the method of any one of claims 1 to 59 or the producer cell of claim 60 or 61.

63. A bioreactor comprising the producer cells of claim 60 or 61 or the extracellular vesicles of claim 62.

64. A method of treating or preventing a disease or condition in a subject in need thereof, the method comprising administering the extracellular vesicles of claim 62.

65. Use of the extracellular vesicles of claim 62 for treating or preventing a disease or disorder in a subject in need thereof.

66. The extracellular vesicle of claim 62, for use in treating or preventing a disease or disorder in a subject in need thereof.

Images