Nanocarriers for the Treatment of Pneumonia

Abstract

To provide lung-targeted extracellular vesicles (EVs) loaded with an anti-inflammatory cargo, as well as compositions, systems, and methods for making the same.SOLUTION: Provided is a therapeutic extracellular vesicle for use in a method for treating acute respiratory distress syndrome (ARDS), the therapeutic extracellular vesicle being produced by nanotransfecting skin cells with a non-viral vector encoding a fusion protein comprising CD200 and a heterologous exosome or lysosomal transmembrane protein under conditions suitable for vesicle secretion, and collecting extracellular vesicles produced in the skin cells, the therapeutic extracellular vesicle loaded with a therapeutic cargo, the therapeutic cargo comprising miR146a or Y-27632, and the method administering the therapeutic extracellular vesicle to a patient suffering from ARDS.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 747,987, filed on October 19, 2018, and incorporates by reference in its entirety the content of U.S. Provisional Application No. 62 / 747,987 as part of this application specification.

[0002] Sequence Listing This application includes a sequence listing submitted in electronic form as an ASCII.txt file titled "321501 - 2340 Sequence Listing_ST25" created on October 18, 2019. The entire content of the sequence listing is incorporated by reference as part of this application specification.

Background Art

[0003] Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and bilateral pulmonary infiltrates, and is characterized by reduced lung compliance. Currently, there is no approved treatment for ARDS.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Disclosed herein is a nanocarrier system for efficiently targeting lung cells loaded with anti - inflammatory cargo and modulating inflammation in the lungs affected by ARDS. These nanocarriers are obtained by in - vitro cell or in - vivo tissue transfection using various transfection techniques (such as bulk electroporation, nanoelectroporation, tissue nanotransfection, viral transfection, etc.). ​​​​​​​​​​​The disclosed nanocarrier carries anti-inflammatory cargo such as microRNA-146a. These are custom-made extracellular vesicles (EVs) that target specific receptors in the lung microenvironment. Functionalization is achieved through ligand modification, and the target gene is introduced. [Means for solving the problem]

[0005] As shown in Figure 1, for example, plasmid DNs encoding a specific cargo or ligand By transfecting A, the EV can be fitted with anti-inflammatory cargo and / or a thinner. Modification with ligands targeting cells becomes possible, depending on the inflammatory pathway that needs to be controlled. Improving the cargo and varying the degree of surface decoration depending on the type of target cell. It is possible.

[0006] For example, type II alveolar epithelial cells and lung macrophages produce lung surfactants, respectively. Plasmids encoding protein A (SPA) and membrane glycoprotein CD200 It can be targeted using genes. EV functionalized with SPA is a receptor for type II cells. While interacting with P63 / CKAP4, EV functionalized with CD200 interacts with the alveolar macromolecules. It preferentially interacts with CD200R receptors in lophages.

[0007] EV is functionalized by SDF1, which interacts with the pulmonary vascular receptor CXCR7. For example, it can be useful in introducing endovascular access (EV).

[0008] As shown in Figure 2, after surface modification, film permeation occurs, with diffusion into the EV due to the concentration gradient. Sexual pharmacological compounds (e.g., Y-27632 Rho inhibitor, manufactured by Calbiochem) It can carry anti-inflammatory cargo.

[0009] Details of one or more embodiments of the present invention will be described below with reference to the accompanying drawings. Other features, purposes, and advantages are described and drawn below, as well as the claims. reveal. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic diagram illustrating the production of custom-made nanocarriers loaded with anti-inflammatory cargo and / or decorated with cell-targeting ligands after transfection of cells or tissues. [Figure 2] Figure 2 is a schematic diagram showing EV after surface modification with other anti-inflammatory cargoes, such as membrane-permeable pharmacological compounds. [Figure 3A] Figures 3A to 3E show the characterization results of designer EVs derived from test tubes. Figures 3A and 3B show designer EVs loaded with miR-146a, isolated 24 hours after nanotransfection of primary mouse embryonic fibroblasts (PMEF). The particle size was in the range of 200 nm, and the EV aggregation was in the range of several billion particles per mL. Figure 3C shows the qRT-PCR characteristics of miR-146A-loaded designer EVs obtained from mouse dendritic cells 48 hours after nanotransfection using a plasmid encoding miR-146A and a sham / control plasmid (*p value = 0.041). Figure 3D shows the relative expression of miR-146A in A549 lung epithelial cells and differentiated THP-1 monocytes treated with these miR-146A or sham designer EVs (*p value < 0.001). Figure 3E shows primary alveolar epithelial cells from C57BL / 6 mice after 48 hours of treatment with fluorescently labeled miR-146a designer EV derived from PMEF, with white arrows highlighting EV uptake. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] The same as above. [Figure 3G] The same as above. [Figure 3H] The same as above. [Figure 3I] The same as above. [Figure 4A] In FIGS. 4A to 4F, the characteristics of designer EVs derived from a living body were shown. FIG. 4A is a schematic diagram of in vivo tissue nanotransfection (TNT) of the skin and subsequent isolation of designer EVs. FIG. 4B shows a particle concentration on the order of 10 trillion EVs per 1 cm2 of skin. FIG. 4C shows an average particle size in the range of 109 - 135 nm. FIG. 4D shows that both pseudo EVs and miR-146a designer EVs expressed positivity for the membrane EV marker tetraspanin CD9. FIG. 4E shows the qRT-PCR characteristics of these bio-derived miR-146A designer EVs, indicating successful loading of the molecular cargo. FIGS. 4F and 4G show the in vivo production state of labeled designer EVs, where GFP-labeled designer EVs were produced by transfected skin cells (white arrows) 24 hours after TNT of the skin containing the plasmid encoding CD63-GFP (EV tracer), as compared to control tissues from non-transfected animals (FIG. 4G). [Figure 4B] The same as above. [Figure 4C] The same as above. [Figure 4D] The same as above. [Figure 4E] The same as above. [Figure 4F] The same as above. [Figure 4G] The same as above. [Figure 5A] In FIGS. 5A to 5F, it is shown that lung inflammation is regulated by bio-derived designer EVs. The effects of miR-146A designer EVs 4 hours after ventilation treatment improved physiological parameters (FIGS. 5A to 5D), a significant decrease in BAL protein content (FIG. 5E), and a tendency for a decrease in the secretion amount of inflammatory cytokines (FIG. 5F, *p-value = 0.022). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 6] Figures 6A and 6B show functionalized designer EVs targeting inflamed lung tissue. Designer EVs can be functionalized to target various intracellular compartments in the alveolar space. In vivo images of organs collected from animals treated for 24 hours with designer EVs functionalized with CD200 ligand targeting alveolar macrophages are shown in Figure 6A. Figure 6B shows IF images of lung tissue treated with fluorescently labeled functionalized designer EVs 24 hours later, showing examples of successful uptake (arrows). [Modes for carrying out the invention]

[0011] The disclosures of this application include lung target extracellular vesicles (EVs) carrying anti-inflammatory cargo, and their The disclosed EV includes a fusion comprising a lung targeting portion. It contains ligands that target the lungs, such as proteins. It also contains the disclosed fusion protein. Compositions containing EVs are also disclosed. In some embodiments, the EVs carry anti-inflammatory cargo. Furthermore, EV-producing cells designed to produce disclosed EVs are also disclosed. A method for producing disclosed EVs, including culturing disclosed EV-producing cells under conditions suitable for production, is also being developed. This method further includes a method for purifying extracellular viable cells (EVs) from cells.

[0012] Before disclosing this application in more detail, the disclosures herein are not limited to any particular embodiment, and also include It should be noted that improvements are permitted. The terms used in this disclosure describe specific embodiments. It is used solely for the purpose of, and the scope of this disclosure is not limited to the attached claims. It is also understood that this is not intended to limit the scope of the term in question.

[0013] As for the range of numerical values ​​described herein, the numerical values ​​within that range have an upper limit and Between the lower limits, or any other value within the stated range, or between them, unless otherwise specified in the context. Unless otherwise indicated, this disclosure includes up to one-tenth of the lower limit unit. The upper and lower limits of the narrow range may be independently included in the narrower range within this disclosure, and also, Within the stated range, special exclusion limits may be set. Where one or both of the limits are included, the scope of excluding one or both is also included in this disclosure. It can be done.

[0014] Unless otherwise defined, all technical and scientific terms used in this disclosure are defined in this disclosure. This specification has the same meaning as commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in the implementation of this disclosure. While other methods and materials can be used for testing, preferred methods and materials are described below.

[0015] All publications and patents cited in this disclosure are subject to change as individual publications or patents may be specific to each other. The methods and related publications are incorporated herein by reference as if they were individually shown and are used in connection with the cited publications. Any cited publications are incorporated herein by reference to disclose and describe the materials and / or other materials. Although the purpose is to disclose this information before the filing date of this application, this disclosure may result in such disclosure This should not be interpreted as acknowledging that there are no prior rights to the publication. Furthermore, the provision The stated issue date may differ from the actual issue date, which may need to be confirmed individually.

[0016] As will be obvious to those skilled in the art, reading this disclosure will reveal that each of the individual items described and illustrated in this disclosure Embodiments may be described in a manner that does not deviate from the scope or spirit of this disclosure, or from any other feature. Having easily separable or combinable distinct components and features. This can be understood. Any of the listed methods are valid in the order they are listed, or logically so. If so, they can be executed in any order.

[0017] Embodiments of this disclosure, unless otherwise shown, are within the scope of the art of chemistry, biology, etc. It uses technologies such as physics.

[0018] The following examples will be useful to those skilled in the art for understanding the methods disclosed and claimed herein and the methods described herein. The use is presented in its entirety for complete disclosure and explanation. Numerical values ​​(e.g., quantity, temperature, etc.) are included. We have strived to ensure accuracy in this regard, but in some cases, explanations may be necessary for errors or deviations. Unless otherwise specified, the unit for components is parts by weight, temperature is in Celsius, and pressure is in atmospheric pressure. Pressure or pressure near pressure. Standard temperature and pressure are defined as 20°C and 1 atmosphere. It can be done.

[0019] Before describing the embodiments of this disclosure in detail, unless otherwise indicated, this disclosure does not specify certain materials. The text mentions that there is potential for improvement, not limited to materials, reagents, reaction materials, and manufacturing processes. The terms used in this disclosure are intended solely to describe specific embodiments. This disclosure is not intended to be limited to that. In addition, it is possible to carry out each process in a different order.

[0020] As used in this disclosure and attached claims, if a subject is singular "a", When written with "an" and the phrase "the", unless it is clear from the context, And, the subject can include plural forms.

[0021] As for the EV of the disclosure, in some embodiments, if it can be produced by cells Any of them will do. Cells secrete extracellular vesicles (EVs) with a wide range of diameters and functions. These include apoptotic vesicles (1-5 μm) and microvesicles (100-1000 nm in size). , and endosomal vesicles known as exosomes (50-150 nm) Born.

[0022] The extracellular vesicles disclosed can be prepared by methods known in the art. For example, the extracellular vesicles of the disclosed molecule encode a ligand that targets cells in eukaryotic cells. It can be prepared by expressing NA. In some embodiments, the cells also exhibit anti-inflammatory properties. It expresses mRNA encoding symptomatic cargo. It has cell-targeting ligands and anti-inflammatory properties. The mRNA for the cargo is transfected into production cells suitable for the production of disclosed EVs. It can be expressed from the chemist. For cell-targeting ligands and anti-inflammatory cargoes. mRNA is the same vector (for example, the ligand and anti-inflammatory that the vector targets cells). (When sex cargo mRNA is expressed from separate promoters), or when targeting cells The ligand and mRNA for the anti-inflammatory cargo can be expressed from separate vectors. The mRNA for ligands and anti-inflammatory cargo targeting the vector or cells is generated. The vector can be packaged into a kit designed for the preparation of extracellular vesicles of the disclosed vector. ru.

[0023] Compositions comprising EVs having the disclosed targeted ligand are also disclosed. In some embodiments, The EV is carrying an anti-inflammatory cargo of disclosure. The EV is designed to secrete disclosure. The cells that produce the cells have also been disclosed.

[0024] EVs such as exosomes are B lymphocytes, T lymphocytes, dendritic cells (DCs), and most EVs are produced by many different types of cells, including immune cells. For example, glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells They are also produced. The EVs used in the compositions and methods of disclosure include the specific cells described above. They can be induced from any suitable cell. Not limited to EV-producing cells suitable for mass production. Typical examples include dendritic cells (e.g., immature dendritic cells) and human fetal kidney 293 (HEK) cells. , 293T cells, Chinese hamster ovary (CHO) cells, and human ESC-derived cells It contains leaf stem cells. EV reduces the occurrence of immune responses in patients who have received exosome transfusions. To avoid this, use autologous patient-derived, allogeneic haplotype-compatible stem cells or allogeneic stem cells. It can also be obtained from there. For this purpose, any EV-producing cells can be used. ru.

[0025] Furthermore, a method for loading an anti-inflammatory cargo onto the disclosed EV, which secretes the disclosed EV This involves manipulating and culturing the EV-producing cells of disclosure. This method further This also includes purifying extracellular viable cells (EVs) from cells.

[0026] EVs produced from cells can be collected from the culture medium by any appropriate method. Typically, EVs This involves centrifugation, filtration, or a combination of these methods to extract the cell culture or tissue supernatant. It can be prepared. For example, to obtain larger spherical particles, use a low speed (<20 Centrifugation is performed at 0,000g, followed by high-speed spheroidization of EVs (>100,000g) ) Centrifugation, size filtration using a suitable filter, gradient ultracentrifugation (for example, EV can be prepared using a sucrose gradient or a combination of these methods.

[0027] The disclosed EV is expressed on the surface of lung cells in the lungs, and the targeting portion that binds to the cell surface It is targeted by expression. A suitable example of the targeted part is the exoprotein. Assuming expression on the surface, short peptides, scFv, and complete proteins are used. For example, the part of a peptide that targets is usually less than 100 amino acids in length, for example. It has less than 50 amino acids, less than 30 amino acids, and a minimum length of 10, 5, or 3 amino acids. That's fine.

[0028] In some embodiments, type II alveolar epithelial cells are subjected to pulmonary surfactant protein A(S) EVs functionalized with SPA can be targeted by type II cells. It interacts with the receptor P63 / CKAP4. In some embodiments, the targeting moiety is SPA1 has the following amino acid sequence: MWLCLPLALNLILMAASGAV CEVKDVCVGTPGIPGECGEKGEPGERGPPGLPAHLDEELQ ATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSNGQSITF DAIQEACARAGGRIAVPRNPEENEEAIASFVKKYNTYAYVG LTEGPSPGDFRYSDGTPVNYTNWYRGEPAGRGKEQCVEMY TDGQWNDRNCLYSRLTICEF (Sequence ID 1), or receptors on type II cells The identity of P63 / CKAP4 with Sequence ID No. 1, which can interact with it, is at least 65%. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , is a manifold and / or fragment thereof.

[0029] In some embodiments, the targeting ligand is a fragment of SPA1, and less However, 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, 156, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, The sequence of amino acids 195, 196, 197, 198, or 199 of sequence number 1 It is either or a variety thereof.

[0030] In some embodiments, the targeted portion is an SPA2 portion having the following amino acid sequence. A: MWLCLPLALTLILMAASGAACEVKDVCVGSPGIPGTPG SHGLPGRDGRDGVKGDPGPPGMGPPGETPCPPGNNGLPG APGVPGERGEKGEAGERGPPGLPAHLDEELQATLHDFRHQ ILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACAR AGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGPSPGD FRYSDGTPVNYTNWYRGEPAGRGKEQCVEMYTDGQWNDRN CLYSRLTICEF (SEQ ID NO: 2) or the P63 / CKA receptor on type II cells The identity of P4 with Sequence ID No. 2, which can interact with P4, is at least 65%, 70%, 71%, and 72%. %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% Manifolds and / or a fragment thereof.

[0031] In some embodiments, the targeting ligand is a fragment of SPA2, and less However, 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, 156, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, A sequence of amino acids containing sequence numbers 245, 246, 247, or 248 of sequence number 2, or It is a variety of that variety.

[0032] In some embodiments, lung macrophages use the membrane glycoprotein CD200 It can be targeted. CD200-activated EVs are CD200 in alveolar macrophages. It preferentially interacts with the R receptor. In some embodiments, the targeting moiety is as follows: CD200 having noacid sequence 3: MERLVIRMPFCHLSTYSLVWVM AAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIV TWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQ NSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQ PIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENS TVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTV TDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRH RNQDREP (SEQ ID NO: 3), or SEQ ID NO: 3 which can interact with lung macrophages. The identity with at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, and 7% 6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 In its manifold and / or fragments, the percentages are 6%, 97%, 98%, and 99%. be.

[0033] In some embodiments, the targeted ligand is a fragment of CD200, and less Kutomo 100, 110, 120, 130, 140, 141, 142, 143, 144, 1 45, 156, 147, 148, 149, 150, 151, 152, 153, 154, 1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 1 65, 166, 167, 168, 169, 170, 171, 172, 173, 174, 1 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 1 85, 186, 187, 188, 189, 190, 191, 192, 193, 194, 1 95, 196, 197, 198, 199, 200, 201, 202, 203, 204, 2 05, 206, 207, 208, 209, 210, 211, 212, 213, 214, 2 15, 216, 217, 218, 219, 220, 221, 222, 223, 224, 2 25, 226, 227, 228, 229, 230, 231, 232, 233, 234, 2 35, 236, 237, 238, 239, 240, 241, 242, 243, 244, 2 45, 246, 247, 248, 249, 250, 251, 252, 253, 254, 2 55, 256, 257, 258, 259, 260, 261, 262, 263, 264, 2 Contains the sequence of amino acids 65, 266, 267, 268, or 269 of sequence number 3. or a variety thereof.

[0034] In some cases, ligands that target cells are transmitted across exosomes or lysosomal membranes. It can be expressed on the surface of EVs as a fusion protein with other proteins. Proteins are known to be associated with exosomes. In other words, they are morphologically exosomes. They are incorporated into sosomes. Examples include Lamp-1, Lamp-2, CD13, CD86, flotillin, Syntaxin-3, CD2, CD36, CD40, Cd40l , CD41a, CD44, CD45, ICAM-1, Integrin alpha4, LiCAM, LFA-1, Mac-1 Alpha and Beta, Vti-IA and B, C D3 Epsilon and Zeta, CD9, CD18, CD37, CD53, CD63, CD 81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II) immunoglobulins, MHC-I or MHC-II components, TCR beta and tetrasperm Nin is one example, but it is not limited to these.

[0035] The extracellular vesicles of the disclosed cells can carry additional anti-inflammatory agents, but the extracellular vesicles are anti-inflammatory. The drug is delivered to the target cells. Suitable anti-inflammatory agents include therapeutic agents (e.g., small molecule drugs). Examples include therapeutic proteins and therapeutic nucleic acids (e.g., therapeutic RNA), but this The disclosure is not limited to these. In some embodiments, the extracellular vesicles of the disclosure are used for therapeutic RNA (this opening It includes (also called "cargo RNA" in the diagram). For example, in some embodiments, Furthermore, fusion proteins containing motifs that target cells trigger the secretion of extracellular vesicles from cells. The purpose is to pack cargo RNA into extracellular vesicles before it is released, and the substances present in the cargo RNA RNA domains that bind to one or more RNA motifs (e.g., the cytoplasm of a fusion protein) This includes the C-terminus of the sol. Thus, the fusion protein "targets cells." It can function as both a "protein" and a "packaging protein." Some implementations In terms of morphology, packaging proteins are extracellular vesicle-loading proteins or "EVs". It is called a "carrying protein."

[0036] In some mechanisms, cargo RNA is miRNA, shRNA, mRNA, n cRNA, sgRNA, or any combination thereof. For example, some implementations In this context, the anti-inflammatory agent is microRNA 146a.

[0037] Additional anti-inflammatory microRNAs that can be used include miR-155, miR-9, and m Examples include iR-210, miR-146b, and miR-181. Anti-inflammatory miR -155, miR-9, miR-210, miR-146b are used for high tidal volume ventilation and Acute lung injury (ALI), induced lung injury (VILI), and osteoarthritis are among the conditions that can be treated. It has been shown that upward control occurs during ALI / ARDS using Uss's model. On the other hand, MiR-181 modulates the inflammatory response of macrophages via IL-1α. 9. miR-511, miR-146a, miR-23b, and miR-181a are, It has already been reported as an anti-inflammatory miRNA that is upregulated in the lungs, particularly during sepsis and ARDS. It is being done.

[0038] Designer EV also contains key growth factors, regulatory proteins, and anti-inflammatory cytokines. It efficiently transports nutrients, reduces lung inflammation and damage, and further enhances the function of the lungs during ARDS / VILI. It can be used to enhance tissue repair. For example, anti-inflammatory cytokines Interleukin-10 and Interleukin-4, Regulatory Protein-Secreting Leukocyte Protea It can carry enzyme inhibitors (SLPIs) and keratinocyte growth factors (KGFs). These have been shown to reduce mortality from ARDS and to regulate inflammation. ru.

[0039] The cargo RNA of the disclosed extracellular vesicle may be of any suitable length. For example, However, in some embodiments, the cargo RNA is at least about 10 nt, 20 nt, 30 nt , 40nt, 50nt, 100nt, 200nt, 500nt, 1000nt, 2000 It may have a nucleotide length of nt, 5000nt or more. In other embodiments, Cargo RNA consists of approximately 5000 nt, 2000 nt, 1000 nt, 500 nt, and 200 nt. t, 100nt, 50nt, 40nt, 30nt, 20nt, or 10nt or less They may have a rheotide length. In a further embodiment, cargo RNA may have these design genes. Nucleotide lengths within the range of the cleotide length, for example, nucleotides in the range of approximately 10 nt to 5000 nt. The creotide length, or other ranges, may be disclosed. The cargo RNA of the extracellular vesicle is, for example, If the cargo RNA contains mRNA or another relatively long RNA, then the relatively long one That's good too.

[0040] In some embodiments, anti-inflammatory cargo is a substance secreted by cells and then carried into the EV. This refers to a pharmacological compound that has membrane permeability. For example, in some embodiments, The anti-inflammatory cargo contains Y-27632 Rho inhibitor, which can diffuse into EVs via a concentration gradient. It contains carbiochem (see Figure 2).

[0041] According to the method of disclosure, clinically relevant lipophilic compounds and other anti-inflammatory membrane-permeable compounds This can be installed in EVs. An example of this type of compound is statins, more specifically, One example is simvastatin, which is used in clinical settings to lower blood cholesterol levels. Simvastatin is effective locally in inflamed lungs via designer EVs. It has been introduced to suppress inflammation and accelerate recovery in patients with conditions such as ALI / ARDS or VILI. It is useful for that.

[0042] In some embodiments, the anti-inflammatory cargo is loaded onto the EV by diffusion due to a concentration gradient. It is possible.

[0043] This disclosure also describes how to use EVs. For example, using the extracellular vesicles of the disclosure The disclosed anti-inflammatory cargo may be introduced into the target lung cells, but this method does not involve introducing the disclosed E into the target cells. This also includes contact with V. Therefore, this disclosure includes a method for treating the target lung disease. The composition, including an EV that targets the lungs and carries the cargo of the present disclosure, is therapeutically effective. This also includes methods of treatment involving dose administration. In some embodiments, the subject develops ARDS.

[0044] The disclosed designer EV essentially designs ligands for EV modification, which is used in the lungs. It can also be used to regulate a wide variety of inflammatory conditions in different organ systems. Lung disease Examples of cases include ventilator-induced lung injury (VILI), pulmonary fibrosis, and sepsis, influenza Infectious diseases such as the flu and pneumonia are examples.

[0045] The EV of the disclosure may be prescribed as part of a pharmaceutical composition for the treatment of lung diseases or disorders. , administer the pharmaceutical composition to patients who require it when treating lung diseases or disorders, and the cargo It can be introduced into target lung cells.

[0046] The disclosed EV can be administered to a subject by any appropriate means, whether human or animal. The methods of administration to these targets include parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal administration. You can choose from the following. Typically, the delivery method is by injection. Alternatively, the injection can be intramuscular or intravascular (e.g., intravenous). The doctor will determine the appropriate dosage for each patient. The necessary route of administration can be determined.

[0047] EV is preferably introduced as a composition. The composition may be administered parenterally, intramuscularly, intracerebrally, or intravascularly. It can be prescribed for administration (including intravenously), subcutaneously, or transdermally. Parenteral administration set Examples of such products include sterile aqueous solutions containing buffers, diluents, and other suitable additives. In addition to this, EV contains pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and It may be formulated as a pharmaceutical composition containing other pharmaceutically acceptable carriers or excipients. stomach.

[0048] Parenteral administration is generally characterized by injection, such as subcutaneous, intramuscular, or intravenous injection. For preparations used for parenteral administration, a sterile injectable solution is used in combination with a solvent immediately before use. Lyophilized powders including solutions and subcutaneous tablets, sterile suspensions for injection, and vehicles immediately before use. Examples include sterile, dry, insoluble products for injection used in combination with sterile emulsions. The solution is water It can be either aqueous or non-aqueous.

[0049] Suitable carriers for intravenous administration include physiological saline or phosphate-buffered saline (P BS), as well as glucose, polyethylene glycol, and polypropylene glycol Examples include solutions containing thickeners and solubilizers such as rubbing acid, and mixtures thereof. Pharmaceutically acceptable carriers for use as oral formulations include aqueous vehicles, non-aqueous vehicles, Antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspensions and dispersants, emulsifiers, chelating agents Or chelating agents and other pharmaceutically acceptable substances. Aqueous vehicles and For example, sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water Examples include injections, dextrose, and Ringer's lactate injection. Non-aqueous parenteral vehicles and These include plant-derived fatty oils such as cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride The parenteral preparation, which contains benzethonium chloride and is packaged in a multi-dose container, has bacteriostatic properties. It is necessary to add it at a concentration that exhibits fungiostatic activity. As an isotonic agent, sodium chloride and Dextrose is one example. Phosphates and citrates are examples of buffer solutions. Sodium bisulfate is an example of an anti-caking agent. Procaine hydrochloride is an example of a local anesthetic. Examples include sodium carboxymethylcellulose, as a suspending and dispersing agent. Examples include hydroxypropyl methylcellulose and polyvinylpyrrolidone.

[0050] Polysorbate 80 (TWEEN® 80) is an example of an emulsifier. EDTA is an example of a chelating agent or sequestering agent for metal ions. This includes ethyl alcohol, polyethylene glycol, and propylene for water-miscible vehicles. Examples include glycol, sodium hydroxide for pH adjustment, hydrochloric acid, citric acid, or lactic acid. The concentration of the pharmaceutically active compound must be an effective amount to produce the desired pharmacological effect by injection. It is adjusted to be such. The exact dosage is determined as is known in the industry for patients or animals. It depends on the person's age, weight, and condition.

[0051] A unit dose of parenteral formulation can be filled into an ampoule, vial, or syringe with needle. Yes. All preparations used for parenteral administration are, as is already practiced in this art, It must be kept sterile.

[0052] The composition is administered in a therapeutically effective amount. The dosage is determined according to various factors, especially when receiving treatment. The severity of the patient's condition, age, and weight; the route of administration; and the necessary care plan should be determined accordingly. Good. The doctor will determine the necessary route of administration and dosage for a specific patient. The optimal dosage depends on the individual patient. Although it may be changed depending on the relative efficacy of the adult, generally, in vitro and in vivo animal models The dosage can be estimated based on the EC50 found to be effective by Dell. The dosage ranges from 0.01 mg to 100 mg per kg of body weight. A typical daily dose depends on the specific composition. The efficacy in adults, the age, weight, and condition of those receiving treatment, the severity of the disease, and the frequency of administration. Depending on the route, the dosage is approximately 0.1 to 50 mg per kg of body weight, preferably approximately 0.1 mg / kg. The dosage is 10 mg / kg. Administration should be done by intramuscular injection or systemic (intravenous or subcutaneous) injection. Depending on the cause, the dosage of the constituent components will be adjusted.

[0053] The dose for a single intramuscular injection is preferably in the range of about 5 to 20 μg. Single or multiple doses. The dose for a single systemic injection is preferably in the range of 10 to 100 mg / kg relative to body weight. ru.

[0054] The clearance of the constituents (and the degradation of any target molecule) is causing the patient to be unable to receive treatment. If you have to take it repeatedly, for example, once or more times a day, week, month, or year There is. A person skilled in the art can determine the repetition of drug administration based on the residence time and concentration of the constituents in body fluids or tissues. The number of treatments can be easily estimated. Even after successful treatment, patients can receive maintenance therapy. In some cases, it may be desirable to use 0.01 mg / kg of the constituent material per kg of body weight. A maintenance dose ranging from 100 mg per kg is administered at least once a day, or once every 20 years.

[0055] Numerous embodiments of the present invention have been described. Nevertheless, the spirit of the present invention and Various improvements can be made within the limits that do not deviate from the specified range. However, The embodiments are included in the following claims. [Examples]

[0056] Example 1 Figure 3 shows the characteristics evaluation and implementation results of bio-derived designer EVs. Figure 3A shows qRT- This bar graph shows the characteristics of EVs carrying miR-146a and SH via PCR. An EV equipped with SH-CT or miR is introduced via an intubator inserted into the trachea before ventilation. Figure 3B shows an EV (MV protocol) with SH-CT or miR-146A installed. Exhalation under 0 cmH2O conditions for 4 hours, with a tidal volume of 12 cc / kg and a respiratory rate of 150 breaths / min. Figure 3C shows a bar graph of physiological measurements of animals treated with terminal positive pressure (MV) for 4 hours. Changes in BAL protein content in animals treated with SH and miR-146a after the procedure. This is a bar graph showing the results. Figure 3D shows mock plasmids (fake control-SH) or This bar graph shows the average size distribution of extracellular vesicles (EVs) containing the anti-inflammatory miR-146a.

[0057] Bio-derived designer EVs carrying the anti-inflammatory miR-146a are called miR-146a(O A plasmid encoding riGene, which is found in the back of C57BL6 mice (8-10 weeks old). It was obtained by nanotransfection of skin. 24 hours later, the EV after installation was collected from a skin biopsy and tested using the ExoQuick kit (Sys It was isolated using temBio. After isolation, the EV was kept in granular form and stored in 0.9 for subsequent use. The EV was suspended in a % sodium chloride solution. The EV loading efficiency was measured via qRT-PCR at miR The copy number of -146a was quantified and measured.

[0058] Designer EVs were evaluated in vivo. Simply put, they have the potential to induce significant lung damage. Mechanical ventilation (MV) was performed according to a previously reported ventilation protocol. The conditions were: tidal volume of 12 cc / kg, respiratory rate of 120 breaths / min, and 4 hours under 0 cmH2O conditions. Positive end-expiratory pressure was observed. In these experiments, intratracheal injection (2 μL / g mouse weight) was administered before MV. Designer EVs were introduced into the lungs of these mice via (using a certain amount). Lung physiological function and acid Prime saturation data is collected during the ventilation process. After 4 hours, the animals are euthanized and bronchoalveolar lavage is performed. BAL (Beauty-Aligned Laser) test data and tissue samples were collected.

[0059] Example 2: In-vitro approach to manufacturing designer EVs Using nanochannel electroporation, the target cargo is extracted from donor cells. Overexpression is allowed within the control range, and the EV content and degree of surface modification are set to the desired range. This was achieved by using nanochannel electroporation, which improved transfection efficiency and precision. The cytoplasmic viability rate could be increased to 100%, and there were no limitations due to capsid size. Using this approach, primary cultures of mouse dendritic cells and embryonic fibroblasts, as well as Designer containing the anti-inflammatory miR-146A obtained from multiple cell sources such as st cell lines. EVs were obtained (Figures 3A-3E). Based on the characteristics evaluation results of the EV content, compared with the falsely loaded EVs... This demonstrated that the target molecular cargo had been successfully incorporated (Figure 3C). Next, this Effective use of such EVs when culturing human lung epithelial cells and human macrophages Gene expression was regulated (Figure 3D). Preneuronal factor genes ASCL1, BRN2, and MY Designer EVs carrying T1l(ABM) were also obtained from early primary cultures of mouse fibroblasts. As a result of examining the dynamic release and uptake of designer EVs equipped with ABM, donor cells EV emissions from the substance peaked 24 hours after transfection, at which point the concentration was It was revealed that the number was on the order of billions of particles / ml. The genes packed inside the EV The copy number was approximately three orders of magnitude higher than the original copy number delivered to the "donor" cells, before EV release. It was suggested that the therapeutic cargo is amplified within the cell.

[0060] Example 3: In vivo approaches to manufacturing designer EVs Nanochannel-based tissue transfection to induce designer extracellular proteins (EVs) from in vivo tissues. Doing so would likely allow the patient's own tissue to be used as a mass-produced EV bioreactor. This is thought to be the case, and it has important clinical and bridging implications for production. According to the method, approximately 1 cm 2 From the skin of mice, approximately 10 samples were taken with the anti-inflammatory miR-146A. Trillions of EVs can be obtained (Figure 4). Effective use of such EVs can improve mechanical ventilation. This can suppress inflammation (Figure 5).

[0061] Example 3: Considering the need for life-saving emergency care, prolonged hospitalization, and delayed recovery in cases of ARDS onset, The burden on the healthcare system related to ARDS is enormous. According to the g Association (2013), there are approximately 190,000 cases of ARDS annually in the United States. This is occurring. The mortality rate for ARDS patients is high, reaching 38-45%. ARDS is usually It is caused by blunt trauma to the lungs or underlying conditions such as sepsis. When damage occurs to the lungs, The patient develops sepsis, a severe systemic inflammatory response caused by infection, which puts their life at risk. As a result, many ICU patients are dying. A characteristic of ARDS is severe hypoxia, Impaired alveolar capillary barrier function, small airway flooding due to decreased production of pulmonary surfactant, and One example is the activation of pro-inflammatory pathways. Mechanical ventilation supports oxygen therapy while Mechanical stress exacerbates initial injury, leading to ventilator-induced lung injury with a higher mortality rate. This can cause VILI. All of these factors contribute to the underlying lung damage. Positive feedback loops can worsen the condition, exacerbate inflammation, and potentially lead to multiple organ failure and death. It exacerbates the condition. Currently, there is no cure for ARDS, but anti-inflammatory cargo targeting the lungs can be used. There are high expectations for this approach. Cell-based (e.g., endothelial progenitor cells, mesenchymal stem cells) Using this treatment method, for example, it can reduce inflammation and improve nutritional mechanisms and anti-inflammatory / infection mechanisms. Lung repair is promoted through this process. However, this is partly due to excessive external processing. Cell therapy based on progenitor cells that may cause tumor formation or highly immunogenic reactions. Serious concerns remain regarding the safety of the treatment. The identity of the specific drug has not yet been revealed. While this has become easier, there are still questions about how to efficiently introduce medication to inflamed lungs. This remains a major challenge for severely ill patients. Overcoming these limitations will be key. The anti-inflammatory and antibacterial molecular cargo is efficiently and selectively processed by the functionalized designer EV. It has been introduced to reduce lung inflammation and damage and enhance the function of tissue repair during sepsis-induced ARDS. Table 1 shows the design details of the designer EV equipped with molecular cargo. [Table 1]

[0062] We optimized the experimental protocol and obtained designer EVs derived from biological tissue. In the field of stenography, as shown in Table 1, 8-10 week old C57BL6 mice By nanotransfecting the skin with plasmids encoding each factor in vivo... This resulted in the acquisition of bio-derived designer EVs loaded with anti-inflammatory or antibacterial cargo. As previously reported, the Lansfection platform is a projection / contact photolithography platform. Manufactured in a cleanroom by combining with deep reactive ion etching. EVs were collected from skin biopsies at various time points after transfection. To separate the skin samples, the gentleMACS dissociative agent was used to dissociate and equalize them. The EVs were selectively precipitated using a total exosome isolation kit and used later. It was stored at -80°C for use.

[0063] Functionalizing designer EVs and lung epithelium and macrophages, which are critical sites of ARDS inflammation The functionalization designer EV delivers targeted delivery to the cell compartment of the lung surface. Using plasmids of ductate protein A (SPA) and membrane glycoprotein CD200 Obtained by nanotransfection (Figure 6). Functionalized EV by SPA. It interacts with the p63 / CKAP4 receptor on alveolar lung cells and the Toll-like receptors on these cells. It is also expected to bind to -2(TLR2) and weaken the activation of the NFκβ inflammatory pathway. EVs functionalized with 200 preferentially interact with the CD200R receptor on alveolar macrophages. It tends to act in a way that modulates the pro-inflammatory state. Additional targeting of the endothelium The ligand is used in combination with SPA and CD200, and the design is introduced via systemic circulation. This made it easier for Iner EV to interact with the pulmonary microvascular system. Enabling EVs preferentially interact with CXCR7 receptors in the pulmonary vascular system. This allows them to, Under damaged conditions, it is controlled upward, and then, as the designer EV moves into the alveolar space, it enters the pulmonary vascular system. It becomes easier to adhere. Identifying the optimal administration route for designer EVs is also important. This is an important factor for optimizing for therapeutic use. Therefore, three non-invasive administration routes By comparing (Table 2), we determined the optimal introduction method for therapeutic use. [Table 2]

[0064] Depending on the method of introduction, functional and non-functional anti-inflammatory, antibacterial, or pseudo-designer EVs may be used. Dose-sensitivity analysis was performed on healthy and diseased animals treated with the drug (Table 3). ). In this analysis, bronchoalveolar lavage specimens (BAL), blood, and lung tissue were collected and introduced. The relative expression levels of genes encoding the molecular cargo, as well as key pro-inflammatory factors. And anti-inflammatory factors (Table 4) are quantified via qRT-PCR. This analysis reveals that The optimal dosage and concentration for significantly regulating lung inflammation in the body can be determined. The result is the transfected skin surface required to obtain the optimal concentration of designer EV. It correlates with the product. Regarding the accumulation and clearance of off-target parts, quantitative in vivo analysis is required. The tests are conducted based on the fabric's characteristics (Figure 6). [Table 3] [Table 4]

[0065] In addition to in vivo studies, the results of extensive in vitro characterization of EVs have revealed the true nature of EVs. This is well understood. This evaluation includes, for example, Nanosight and zeta potential analysis. Using the Izer system, size distribution, charge, and as a function of cargo and surface modification. This includes analysis of particle concentration. The EV loading efficiency is calculated for each plasmid DNA. Using the absolute quantitative qRT-PCR protocol derived from the standard curve, the EVs were packed together. This is determined by quantifying the copy number of each gene. These results are obtained by transdermal tranquilizers. Correlates with the sfected copy number. Using the whole RNA sequence, anti-inflammatory, antibacterial, or Characterizing the contents of a designer EV loaded with a cargo made of pseudomolecules, thereby the designer - Obtain detailed information on the morphology of coding and non-coding RNAs packed within EVs. Furthermore, functional and non-functional designer EVs (at the membrane and cytosol levels) Comparative proteomics analysis was conducted to determine the anti-inflammatory efficiency and the packing efficiency of antimicrobial molecular cargo. Furthermore, it determines the expression efficiency of membrane ligands.

[0066] To better understand the mechanism of action of designer EVs, a mouse model of lung injury was developed. The in vivo performance evaluation results in Table 3) were compared with a well-established test tube model (Table 3) regarding damage. 5) was adopted as a benchmark. In the test tube model, for a specific cell population This enables effective and systematic research on designer EVs, while in in vivo models, multiple To better understand responses under pathophysiological conditions where interactions between organ systems are important. It is possible. In vivo, designer EVs are effective in reducing inflammation and bacterial infections. The sex was evaluated using a mouse model of sepsis-induced ARDS and a two-hit model. These models observe the level of inflammation attenuation from specific concentrations of designer EVs under infection. This helps determine the potential impact. Using test tube models of barotrauma and volumetric trauma, Designer EVs were probed on cellular components isolated from lung tissue and bacterial cultures. [Table 5]

[0067] In in vivo injury models, designer EVs are used at various doses (e.g., single vs. repeated) and time. The drug was administered at a single point (e.g., before vs. after injury) (Table 2). This modeled sepsis-induced ARDS. To do this, 8-10 week old C57BL6 mice are subjected to cecal ligation and puncture (CLP) or pseudo-laparotomy. The subjects were selected. Pulmonary edema was diagnosed after CLP based on ultrasound imaging using the Vevo2100 system. The evaluation was conducted by sacrificing animals and obtaining BAL, blood, and major organs (i.e., lungs, heart, The spleen, liver, and kidneys were collected after being perfused with sterile saline for downstream analysis. Using the two-hit model, for example, when an infected patient requires mechanical ventilation, This will allow for the reproduction of conditions that are more clinically relevant. In this model, C is 8-10 weeks old. 57BL6 mice were first subjected to CLP or pseudomotomy, and 24 hours after CLP, harmful Protocol (tidal volume 12cc / kg, 120 breaths / min, and FlexiVent Using FX and SCIREQ, under conditions of 4 hours (positive end-tidal pressure under 0 cmH2O), the machine... The subjects were subjected to mechanical ventilation. After collecting data on lung physiological function and oxygen saturation during the MV process, At the end of the music video, an animal is sacrificed by an overdose of anesthetic, and its BAL, blood, and major organs are taken. The samples are collected after perfusing with sterile saline for downstream analysis. Cell content of BAL samples. This refers to the inflammatory cell count (i.e., alveolar macrophages, lymphocytes, eosinophils, neutrophils). Determined by (and mast cells). Increased protein content affects alveolar capillary barrier function. BAL protein content was also measured because it correlates with loss of integrity. After protein quantification... Using custom-made cytokine arrays, pro-inflammatory and anti-inflammatory sites To express the caynes and factors, all BAL and plasma samples were screened. (Table 4). Relative expression of molecular cargo introduced into the body (as described in Table 1). (For therapies) and key anti-inflammatory and pro-inflammatory molecular markers for these therapies The effect on the expression of (listed in Table 4) was evaluated via qRT-PCR.

[0068] In the case of an in vivo injury model, a co-culture of primary lung epithelial cells and mouse-derived macrophages. They are exposed to barotrauma or volumetric trauma. Barotrauma is a type of periodic pressure trauma previously published. This is induced by using a model. Therefore, cells are co-cultured on a Transwell insert. Then, pressurize the top compartment for 24 hours at a frequency of 0.2 Hz and a height of 0-20 cmH. A periodic oscillating pressure of 20°C is applied to simulate the same conditions as with a ventilator and a normal respiratory rate. The treatment was rated. Volumetric trauma was treated using the Flexcell FX-5000 tension system. This was simulated. In this model, cells are placed in an improved 6-well lattice with an elastomer base. Co-culture on a rate and incubate in a cell culture incubator at 1.25 Hz for 24 hours, 10-2 The stretching was repeated continuously and periodically with a 0% elongation rate. Major pro-inflammatory molecules (Table 4) Secretion into the culture medium was monitored. To evaluate the formation and retention of tight junctions, ZO-1 was used. As an indicator of epithelial-endothelial barrier function after trauma, staining and imaging were performed via IF. Table 5 shows the experimental groups selected for evaluation.

[0069] Unless otherwise specified, all technical and scientific terms used in this disclosure are as defined in the disclosure. It has the same meaning as commonly understood by those skilled in the art in the field. (Referenced herein) The publications and referenced materials mentioned herein are incorporated herein by reference.

[0070] Those skilled in the art will recognize numerous embodiments of the present invention described in this disclosure that are equivalent to specific embodiments, and This can be confirmed by conventional experiments, but is included in the following claims. It is intended to be.

Claims

1. A therapeutic extracellular vesicle for use in the treatment of acute respiratory distress syndrome (ARDS), The therapeutic extracellular vesicles express a lung-targeting fusion protein that is derived from skin cells and contains CD200, which can bind to CD200R, and an allogeneic exosome or lysosomal transmembrane protein. The aforementioned therapeutic extracellular vesicle is equipped with therapeutic cargo, The aforementioned therapeutic cargo contains miR146a or Y-27632, and The therapeutic extracellular vesicle, wherein the treatment method comprises administering the therapeutic extracellular vesicle to a patient with ARDS.

2. A therapeutic extracellular vesicle derived from skin cells expressing a lung-targeting fusion protein containing CD200 and an allogeneic exosome or lysosomal transmembrane protein that can bind to CD200R, and carrying a therapeutic cargo containing miR146a or Y-27632.

3. The therapeutic extracellular vesicle according to claim 2, wherein the therapeutic cargo comprises miR146a.

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