Nanocarriers for lung inflammation therapy

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 is a continuation of U.S. Provisional Application No. 62 / 747,987, filed October 19, 2018. The entire contents of U.S. Provisional Application No. 62 / 747,987, which claims priority, are incorporated herein by reference in its entirety. This document is incorporated by reference as constituting part thereof.

[0002] Sequence Listing This application is filed on October 18, 2019 under the title "321501-2340 Sequential Application No. ASCII.txt file titled "License Listing_ST25" and The entire contents of the sequence listing may be incorporated herein by reference in its entirety as part of the present application. This document is incorporated by reference as constitutive thereof. [Background technology]

[0003] Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and diffuse bilateral pulmonary infiltrates. It is characterized by causing pulmonary embolism and reducing lung compliance. Currently, There is nothing that has been done. Summary of the Invention [Problem to be solved by the invention]

[0004] Disclosed herein are methods for efficiently targeting lung cells with anti-inflammatory cargo and inhibiting AR These nanocarrier systems are used to regulate lung inflammation in patients with DS. The carriers are compatible with various transfection techniques (bulk electroporation, nano Electroporation, tissue nanotransfection, viral transfection Transfection of cells in vitro or tissues in vivo using The disclosed nanocarriers are loaded with anti-inflammatory cargo such as microRNA-146a. Custom-made extracellular vesicles (EVs) that target specific receptors in the lung microenvironment. Functionalization is achieved via ligand modification, and targeted genes are introduced. [Means for solving the problem]

[0005] As shown in FIG. 1, for example, a plasmid DNA encoding a particular cargo or ligand Loading EVs with anti-inflammatory cargo by transfecting A and / or Depending on the inflammatory pathway that needs to be controlled, the cells can be modified with ligands that target them. Modifying the cargo and varying the degree of surface decoration depending on the target cell type can be done.

[0006] For example, type II alveolar epithelial cells and pulmonary macrophages each express pulmonary surfactant. Plasmid genes encoding glycoprotein A (SPA) and the membrane glycoprotein CD200 SPA-functionalized EVs can target type II cells using the receptors While interacting with p63 / CKAP4, EVs functionalized with CD200 were associated with alveolar macrophages. It preferentially interacts with the CD200R receptor in phages.

[0007] EVs are functionalized with SDF1, which interacts with the receptor CXCR7 in the pulmonary vasculature. , for example, to be useful for the delivery of EVs via intravascular methods.

[0008] As shown in Figure 2, after surface modification, EVs also showed a membrane permeability that diffused into the EVs due to the concentration gradient. pharmacological compounds (e.g., Y-27632 Rho inhibitor, Calbiochem) ) can be loaded with anti-inflammatory cargo.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will become apparent from the following description and drawings, as well as from the claims. reveal. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing the production of custom-made nanocarriers loaded with anti-inflammatory cargo and / or decorated with cell-targeting ligands following transfection of cells or tissues. [Diagram 2] FIG. 2 is a schematic showing EVs after surface modification with other anti-inflammatory cargo, e.g., membrane-permeable pharmacological compounds. [Figure 3A] Figures 3A to 3E show the characterization results of test tube derived designer EVs. Figures 3A and 3B show miR-146a loaded designer EVs isolated 24 hours after nanotransfection of primary mouse embryonic fibroblasts (PMEFs). The particle size was in the range of 200 nm and the EV concentration was in the range of billions of particles per mL. Figure 3C shows qRT-PCR characterization of miR-146A loaded designer EVs obtained from mouse dendritic cells 48 hours after nanotransfection with miR-146A encoding plasmid and mock / 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 mock designer EVs (*p value<0.001). In Figure 3E, we show C57BL / 6 mouse primary alveolar epithelial cells after treatment with fluorescently labeled miR-146a designer EVs derived from PMEFs for 48 h, 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] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 4A] Figures 4A to 4F show the characterization of biogenic designer EVs. Figure 4A is a schematic of in vivo tissue nanotransfection (TNT) of skin and subsequent isolation of designer EVs. Figure 4B shows particle concentrations on the order of 10 trillion EVs per cm2 of skin. Figure 4C shows average particle sizes ranging from 109 to 135 nm. Figure 4D shows that both mock EVs and miR-146a designer EVs expressed positivity for the membrane EV marker tetraspanin CD9. Figure 4E shows qRT-PCR characterization of these biogenic miR-146a designer EVs, demonstrating their successful loading of molecular cargo. In vivo production of labeled designer EVs is shown in Figures 4F and 4G, where 24 hours after TNT of skin containing a plasmid encoding CD63-GFP (an EV tracer), GFP-labeled designer EVs are clearly produced by transfected skin cells (white arrows) compared to control tissue from non-transfected animals (Figure 4G). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 5A] Figures 5A to 5F show that biogenic designer EVs modulate lung inflammation. After 4 hours of ventilation, miR-146A designer EVs improved physiological parameters (Figures 5A to 5D), significantly reduced BAL protein content (Figure 5E), and showed a trend toward reduced secretion of inflammatory cytokines (Figure 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] In Figures 6A and 6B, we show functionalized designer EVs targeting inflamed lungs. Designer EVs can be functionalized to target various cellular compartments in the alveolar space. In vivo images of organs collected from animals treated for 24 hours with designer EVs functionalized with CD200 ligand to target alveolar macrophages are shown in Figure 6A. In Figure 6B, IF images of lung tissue treated with fluorescently labeled functionalized designer EVs after 24 hours are shown, showing an example of successful uptake (arrow). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure relates to lung-targeted extracellular vesicles (EVs) loaded with anti-inflammatory cargo, as well as their Compositions, systems, and methods of making. The disclosed EVs include fusion moieties that include a lung targeting moiety. Also included are lung targeting ligands, such as proteins. Also included are the disclosed fusion proteins. Also disclosed are compositions comprising EVs carrying an anti-inflammatory cargo. In some embodiments, the EVs carry an anti-inflammatory cargo. Also disclosed are EV-producing cells engineered to produce the disclosed EVs. Also disclosed is a method for producing the disclosed EVs, comprising culturing the disclosed EV-producing cells under conditions suitable for production. The method further includes a method for purifying EVs from cells.

[0012] Before disclosing the present application in more detail, it should be noted that the disclosure is not limited to particular embodiments, It should be noted that modifications may be made. The terminology used in this disclosure describes specific embodiments. and the scope of the present disclosure is limited only by the appended claims. It is also understood that no limitation of such term is intended.

[0013] For numerical ranges described herein, the numerical values ​​within the range include the upper and lower limits of the range. Between the lower limit, or any other value or interval within the stated range, unless the context clearly indicates otherwise. Unless otherwise indicated, every tenth of the lower limit is included in the disclosure. The upper and lower limits of the narrower ranges may independently be included in the narrower ranges within this disclosure and may also be included in the Specific excluded limits may be made within the ranges listed. Where one or both limits are inclusive, ranges excluding either or both are also included in this disclosure. can be.

[0014] Unless otherwise defined, all technical and scientific terms used in this disclosure are defined by the The terms have the same meaning as commonly understood by a person skilled in the art in the specified technical field. Any methods and materials similar or equivalent to those described in the above may also be used in the practice of this disclosure. Any method and material may be used for the assay, but the preferred methods and materials are described below.

[0015] All publications and patents cited in this disclosure are without limitation, unless specifically indicated otherwise by each individual publication or patent. The methods and methods related to the cited publications are incorporated herein by reference as if each was individually indicated. Any references herein are incorporated by reference in their entirety to disclose and describe the materials and / or information contained therein. It is the intent of the present disclosure to be made prior to the filing date of this application, however, that the disclosure may be misleading in any way as to why such disclosure is necessary. Nothing in this document shall be construed as an admission that there are no prior rights to the work. The publication dates stated may be different from the actual publication dates which must be independently confirmed.

[0016] As will be apparent to one of ordinary skill in the art upon reading this disclosure, the individual The embodiments may be modified from any other feature without departing from the scope or spirit of the present disclosure. Having distinct components and features that can be easily separated or combined Any listed methods are understood to be true in the order or logical order listed. can be executed in any order.

[0017] The embodiments of the present disclosure are directed to, unless otherwise indicated, chemical, biological, and / or other methods within the skill of the art. Use techniques such as physics.

[0018] The following examples are provided to those of skill in the art to illustrate, by way of example, the methods and methods disclosed and claimed herein. Numerical values ​​(e.g. amounts, temperatures, etc.) are presented to fully disclose and explain the use. Every effort has been made to ensure accuracy, but explanations for errors and deviations may be necessary. Unless otherwise stated, materials are in parts by weight, temperatures are in degrees Celsius, and pressures are in atmospheres. Standard temperature and pressure are defined as 20°C and 1 atmosphere. can be.

[0019] Before describing the embodiments of the present disclosure in detail, it is to be understood that the present disclosure is not limited to the specific materials unless otherwise indicated. The present invention is not limited to the materials, reagents, reaction materials, and manufacturing processes, and may be subject to improvement. The terminology used in this disclosure is for the purpose of describing particular embodiments only. This disclosure is based on, and is not intended to be limited to, any particular case where it is logical and consistent. In other cases, the steps may be performed in a different order.

[0020] As used in this disclosure and the appended claims, an object may be referred to in the singular form "a," " When used with the words "an" and "the," the and the plural form is also included.

[0021] In some embodiments, the disclosed EVs include those that can be produced by cells. Cells secrete extracellular vesicles (EVs) with a wide range of diameters and functions. , which include apoptotic vesicles (1-5 μm in size), microvesicles (100-1000 nm in size) and small vesicles of endosomal origin known as exosomes (50-150 nm). Can be enjoyed.

[0022] The disclosed extracellular vesicles can be prepared by methods known in the art. For example, the disclosed extracellular vesicles can include mRNAs encoding cell-targeting ligands in eukaryotic cells. In some embodiments, the cells can also be prepared by expressing anti-inflammatory NA. The cells express mRNA encoding the anti-inflammatory cargo. The mRNA for the cargo of the vector is transfected into a producer cell suitable for the production of the disclosed EV. For cell-targeting ligands and anti-inflammatory cargo, The mRNA can be expressed by the same vector (e.g., a ligand and an anti-inflammatory vector that targets the cells). (when the mRNA for the functional cargo is expressed from a separate promoter) or to target cells The mRNA for the ligand and the anti-inflammatory cargo can be expressed from separate vectors. The vector or cells express mRNA for a ligand and an anti-inflammatory cargo that targets the cells. The vectors expressing the disclosed vectors can be packaged in kits designed for the preparation of the disclosed extracellular vesicles. do.

[0023] Also disclosed are compositions comprising EVs having the disclosed targeting ligands. In some embodiments, The EVs are loaded with the disclosed anti-inflammatory cargo. EVs engineered to secrete the disclosed EVs. Production cells are also disclosed.

[0024] EVs, such as exosomes, are secreted by B lymphocytes, T lymphocytes, dendritic cells (DCs), and most EVs are produced by many different types of cells, including immune cells of all types. For example, glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells The EVs used in the disclosed compositions and methods include those containing the specific cells described above. EV-producing cells suitable for large-scale production include, but are not limited to, EV-producing cells suitable for large-scale production. Specific examples include dendritic cells (e.g., immature dendritic cells), human embryonic kidney 293 (HEK) cells, and the like. , 293T cells, Chinese hamster ovary (CHO) cells, and human ESC-derived EVs are also known to reduce the development of immune responses in patients who receive exosomes. To avoid this, autologous patient-derived, allogeneic haplotype-matched stem cells or allogeneic stem cells can be used. For this purpose, any EV-producing cell can be used. do.

[0025] Also disclosed are methods of loading anti-inflammatory cargo onto the disclosed EVs, including secreting the disclosed EVs. and culturing the disclosed EV-producing cells. This also includes purifying EVs from cells.

[0026] EVs produced by cells can be collected from the culture medium by any suitable method. from cell culture or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, to obtain larger particle spherical EVs, a slower speed (<20 ,000g) followed by centrifugation at high speed (>100,000g) for spheronization of EVs. ) and then size filtration using an appropriate filter, gradient ultracentrifugation (e.g. EVs can be prepared using either the sucrose gradient method or a combination of these methods.

[0027] The disclosed EVs are expressed on the surface of lung cells in the lung and contain targeting moieties that bind to the cell surface. Suitable examples of targeting moieties include those in which the targeting moiety is an exosome. Short peptides, scFvs and full proteins are available for expression on the surface of the Peptide targeting moieties are typically less than 100 amino acids in length, e.g. , less than 50 amino acids, less than 30 amino acids, with a minimum length of 10, 5 or 3 amino acids. That's fine.

[0028] In some embodiments, type II alveolar epithelial cells are treated with pulmonary surfactant protein A (S EVs functionalized with SPA can be targeted to type II cells. In some embodiments, the targeting moiety interacts with the receptor P63 / CKAP4. SPA1 has the following amino acid sequence: MWLCPLALNLILMAASGAV CEVKDVCVGTPGIPGECGEKGEPGERGPPGLPAHLDEELQ ATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSNGQSITF DAIQEACARAGGRIAVPRNPEENEEAIASFVKKYNTYAYVG LTEGPSPGDFRYSDGTPVNYTNWYRGEPAGRGKEQCVEMY TDGQWNDRNCLYSRLTICEF (SEQ ID NO: 1) or receptor on type II cells A compound that can interact with P63 / CKAP4 has at least 65% identity with SEQ ID NO:1 , 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% , and / or fragments thereof.

[0029] In some embodiments, the targeting ligand is a fragment of SPA1, At least 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, or 199 consecutive amino acids of SEQ ID NO:1. or a variant thereof.

[0030] In some embodiments, the targeting moiety is an SPA2 moiety having the amino acid sequence There is:MWLCPLALTLILMAASGAACEVKDVCVGSPGIPGTPG SHGLPGRDGRDGVKGDPGPPGMGPPGETPCPPGNNGLPG APGVPGERGEKGEAGERGPPGLPAHLDEELQATLHDFRHQ ILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACAR AGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGPSPGD FRYSDGTPVNYTNWYRGEPAGRGKEQCVEMYTDGQWNDRN CLYSRLTICEF (SEQ ID NO:2) or the receptor P63 / CKA on type II cells P4 interacting with SEQ ID NO:2. %, 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%, and / or a fragment thereof.

[0031] In some embodiments, the targeting ligand is a fragment of SPA2, At least 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, or 245, 246, 247, or 248 consecutive amino acids of SEQ ID NO:2; is its manifold.

[0032] In some embodiments, the lung macrophages are characterized using the membrane glycoprotein CD200. CD200-functionalized EVs can target CD200 in alveolar macrophages. In some embodiments, the targeting moiety preferentially interacts with the R receptor. CD200 has the sequence: MERLVIRMPFCHLSTYSLVWVM AAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIV TWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQ NSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQ PIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENS TVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTV TDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRH RNQDREP (SEQ ID NO: 3) or SEQ ID NO: 3 capable of interacting with pulmonary macrophages Identities of 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 6%, 97%, 98%, 99%, of the variants and / or their fragments be.

[0033] In some embodiments, the targeting ligand is a fragment of CD200, At least 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 65, 266, 267, 268, or 269 consecutive amino acids of SEQ ID NO:3 Or a variant thereof.

[0034] In some cases, cell-targeting ligands are delivered via exosomal or lysosomal transmembrane delivery. They can be expressed on the surface of EVs as fusion proteins with other proteins. Proteins are known to be associated with exosomes, i.e., they form exosomes. 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 tetraspars Examples of such compounds include, but are not limited to, nin.

[0035] The disclosed extracellular vesicles can further be loaded with anti-inflammatory agents. Suitable anti-inflammatory agents include therapeutic agents (e.g., small molecule drugs) , therapeutic proteins, and therapeutic nucleic acids (e.g., therapeutic RNA), which In some embodiments, the disclosed extracellular vesicles are not limited to, but include, but are not limited to, therapeutic RNA (such as those described herein). For example, in some embodiments, the nucleic acid further comprises a nucleic acid sequence similar to that described above, but also referred to as "cargo RNA" in the present specification. In addition, fusion proteins containing cell-targeting motifs have been shown to induce secretion of extracellular vesicles from cells. The cargo RNA is packaged into extracellular vesicles before it is delivered to the endonuclease. An RNA domain that binds to one or more RNA motifs (e.g., the cytoplasmic domain of a fusion protein) In this way, the fusion protein is a "cell-targeting" In some embodiments, the protein can function as both a "packaging protein" and a "packaging protein." In morphology, the packaging protein is a type of extracellular vesicle-loaded protein or “EV These are called "carrying proteins."

[0036] In some embodiments, the cargo RNA is selected from the group consisting of miRNA, shRNA, mRNA, cRNA, sgRNA, or any combination thereof. For example, in some embodiments In this instance, the anti-inflammatory agent is microRNA 146a.

[0037] Additional anti-inflammatory microRNAs that can be used include miR-155, miR-9, and These include miR-210, miR-146b, and miR-181. Anti-inflammatory miRs -155, miR-9, miR-210, and miR-146b mediate high tidal volume ventilation and Mammals with acute lung injury (ALI), induced lung injury (VILI), and osteoarthritis It has been shown to be upregulated during ALI / ARDS in mouse models. MiR-181 regulates the inflammatory response of macrophages via IL-1α. 9, miR-511, miR-146a, miR-23b, and miR-181a Previously reported as an anti-inflammatory miRNA that is upregulated in the lungs, especially during sepsis and ARDS. It is being done.

[0038] Designer EVs also secrete key growth factors, regulatory proteins, and anti-inflammatory cytokines. It efficiently transports inflammatory cytokines to reduce lung inflammation and injury, and also promotes tissue remodeling during ARDS / VILI. It can be used to enhance tissue repair. For example, it can be used to promote the production of anti-inflammatory cytokines. Interleukin-10 and interleukin-4, regulatory proteins secretory leukocyte proteases It can carry keratinocyte growth factor (KGF), a phospholipase inhibitor (SLPI), and These have been shown to reduce mortality from ARDS and modulate inflammation. do.

[0039] The cargo RNA of the disclosed extracellular vesicles may be of any suitable length. For example, in some embodiments, the cargo RNA is at least about 10 nt, 20 nt, 30 nt, , 40nt, 50nt, 100nt, 200nt, 500nt, 1000nt, 2000 In other embodiments, the nucleic acid sequence may have a length of 5000 nt, 5000 nt or more. The cargo RNA is approximately 5000nt, 2000nt, 1000nt, 500nt, 200nt t, 100nt, 50nt, 40nt, 30nt, 20nt, or 10nt or less In a further embodiment, the cargo RNA may have a length of 100 bp. Nucleotide lengths within the range of nucleotide length, for example, nucleotides in the range of about 10 nt to 5000 nt. The cargo RNA of the disclosed extracellular vesicles may have a length of, for example, 10 nucleotides or other ranges. For example, if the cargo RNA contains mRNA or another relatively long RNA, Good too.

[0040] In some embodiments, the anti-inflammatory cargo is a cargo that is loaded onto an EV after being secreted by a cell. For example, in some embodiments, The anti-inflammatory cargo includes the Y-27632 Rho inhibitor, which can diffuse into EVs via a concentration gradient ( Calbiochem) (see Figure 2).

[0041] According to the disclosed methods, clinically relevant lipophilic compounds and other anti-inflammatory membrane-permeable compounds One example of this type of compound is statins, more specifically: Simvastatin, which is used in clinical practice to lower blood cholesterol levels, is one example. Simvastatin is effectively delivered locally to inflamed lungs via designer EVs. It has been introduced to reduce inflammation and speed up recovery in patients with, for example, ALI / ARDS or VILI. It helps to.

[0042] In some embodiments, the anti-inflammatory cargo is loaded onto the EVs by diffusion along a concentration gradient. can be done.

[0043] The present disclosure also describes methods of using EVs, for example, using the disclosed extracellular vesicles. The disclosed anti-inflammatory cargo may be introduced into target lung cells, but the method includes administering the target cells to the subject E. The present disclosure also includes contacting a subject with V. Accordingly, the present disclosure includes methods of treating a pulmonary disease in a subject. The composition comprising the cargo-loaded lung-targeted EVs of the present disclosure is therapeutically effective. Also included are methods of administering a therapeutically effective amount to treat a subject. In some embodiments, the subject develops ARDS.

[0044] The disclosed designer EVs essentially aim to improve lung function by designing ligands for EV modification. It can be used to regulate a wide variety of inflammatory conditions in different organ systems, as well as in lung disease. Examples of these include ventilator-induced lung injury (VILI), pulmonary fibrosis, and sepsis and influenza. Examples of infectious diseases include influenza and pneumonia.

[0045] The disclosed EVs may be formulated as part of a pharmaceutical composition for the treatment of a pulmonary disease or disorder. The pharmaceutical composition is administered to a patient in need thereof to treat a pulmonary disease or disorder, and the cargo is It can be introduced into target lung cells.

[0046] The disclosed EVs can be administered to a subject by any suitable means. The method of administration to the subject may be parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal. The delivery method can be selected from the following. Usually, the delivery method is by injection. Or, injections may be intramuscular or intravascular (e.g., intravenous). Physicians should determine the dosage for each particular patient. The required route of administration can be determined.

[0047] The EVs are preferably introduced as a composition, which may be administered parenterally, intramuscularly, intracerebrally, or intravascularly. The composition may be formulated for parenteral (including intravenous), subcutaneous, or transdermal administration. The compositions include sterile aqueous solutions that also contain buffers, diluents, and other suitable additives. In addition, EV may contain pharma- ceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and and other pharma- ceutical compositions containing a pharma- ceutical agent or excipient. stomach.

[0048] Parenteral administration is generally characterized by injection, such as subcutaneous, intramuscular, or intravenous. Preparations for parenteral administration include sterile injection preparations that are used in combination with a solvent immediately before use. Freeze-dried powders, including solutions and subcutaneous tablets, sterile suspensions for injection, and vehicles ready for use and sterile dry insoluble products for injection in combination with sterile emulsions. The aqueous solution may be either aqueous or non-aqueous.

[0049] For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS). BS), as well as glucose, polyethylene glycol, and polypropylene glycol Examples of suitable thickening and solubilizing agents include solutions containing thickening agents and solubilizing agents such as ethanol, and mixtures thereof. Pharmaceutically acceptable carriers for use in oral formulations include aqueous vehicles, non-aqueous vehicles, Antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering agents or chelating agents, and other pharma- ceutically acceptable substances. Examples include sodium chloride injection, Ringer's injection, isotonic dextrose injection, and sterile water. Injection, dextrose and lactated Ringer injection. Non-aqueous parenteral vehicle group and Examples of oils that are derived from plants include cottonseed oil, corn oil, sesame oil, and peanut oil. , phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, and benzalkonium chloride. In parenteral preparations packaged in multiple-dose containers containing benzethonium chloride, It is necessary to add it at a concentration that exhibits fungistatic or fungistatic properties. Dextrose is one of the buffers. Buffers include phosphate and citrate. Antioxidants include sodium hydrogen sulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, Hydroxypropyl methylcellulose and polyvinylpyrrolidone.

[0050] Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles. glycol, sodium hydroxide for pH adjustment, hydrochloric acid, citric acid, or lactic acid. The concentration of the pharmacologic active compound is an amount effective to produce the desired pharmacological effect upon injection. The exact dose will be adjusted for each patient or animal, as is known in the art. The amount of blood flow depends on the age, weight, and condition of the patient.

[0051] Ampoules, vials, or syringes with needles can be filled with unit doses of parenteral preparations. All preparations for parenteral administration may be prepared by the methods described herein, as previously practiced in the art. It must be kept sterile.

[0052] The compositions are administered in a therapeutically effective amount, the dosage of which will vary according to a variety of factors, particularly the type of treatment being administered. The severity, age, and weight of the patient; the route of administration; and the required regimen should determine the severity of the disease. The route of administration and dosage required for a particular patient will be determined by the physician. The optimal dosage depends on the individual Although this may vary depending on the relative efficacy of the adult, it is generally used in in vitro and in vivo animal models. The dose can be estimated based on the EC50 found to be effective by the Typical daily doses range from 0.01 mg to 100 mg per kg of body weight. Adult efficacy, age, weight, and condition of the subject to be treated, severity of illness, frequency of administration and Depending on the route, about 0.1 to 50 mg / kg body weight, preferably about 0.1 mg / kg The dose ranges from 1 to 10 mg / kg. The dose can be administered intramuscularly or systemically (intravenously or subcutaneously). Varying doses of the construct are administered depending on whether the compound is to be administered or not.

[0053] The dosage for a single intramuscular injection is preferably in the range of about 5 to 20 μg. The dosage for a single systemic injection is preferably in the range of 10 to 100 mg / kg of body weight. do.

[0054] Clearance of the construct (and degradation of any target molecule) may limit treatment to patients. If it must be taken repeatedly, for example, daily, weekly, monthly, or more than once a year Those skilled in the art will recognize that repetition of dosing may be necessary based on residence time and concentration of the constituents in bodily fluids or tissues. The number of treatments can be easily estimated. Even after successful treatment, patients should be placed on maintenance therapy. In this case, it may be desirable to limit the composition to 0.01 mg / kg body weight. Maintenance doses ranging from 1 kg to 100 mg are given at least once daily to once every 20 years.

[0055] A number of embodiments of the present invention have been described. Nonetheless, the spirit and scope of the present invention will be understood to be within the scope of the present invention. Various modifications may be made without departing from the scope of the present invention. Embodiments thereof are encompassed within the scope of the following claims. EXAMPLES

[0056] Example 1 Figure 3 shows the characteristics and transfection results of designer EVs derived from living organisms. 1 is a bar graph showing the characteristics of miR-146a and SH-loaded EVs via PCR. SH-CT or miR-loaded EVs were delivered via an intubation tube inserted into the trachea before ventilation. Figure 3B shows the results of EVs carrying SH-CT or miR-146A (MV protocol: Tidal volume 12cc / kg, respiratory rate 150 breaths / min, 4 hours of expiration under 0cm H2O Figure 3C shows bar graphs of physiological measurements from animals treated with MV for 4 hours. Changes in BAL protein content in animals treated with SH and miR-146a after FIG. 3D shows a bar graph showing the results of the experiment using mock plasmid (mock control-SH) or A bar graph of the average size distribution of EVs loaded with anti-inflammatory miR-146a is shown.

[0057] Designer EVs derived from living organisms carrying anti-inflammatory miR-146a were synthesized using miR-146a (O The plasmid encoding the riGene was transfected into the dorsal part of C57BL6 mice (8-10 weeks old). The skin was nanotransfected. After 24 hours from the start of the experiment, loaded EVs were collected from skin biopsies and purified using the ExoQuick kit (Systolic After isolation, EVs were kept in a granular state and diluted with 0.9% ethanol for subsequent use. EV loading efficiency was assessed by qRT-PCR using miR-associated ELISA. The copy number of -146a was quantified and measured.

[0058] Designer EVs were evaluated in vivo. Briefly, they showed the potential to induce severe lung injury. Mechanical ventilation (MV) was performed according to a previously published protocol. The conditions were tidal volume 12cc / kg, respiratory rate 120 breaths / min, and 0 cmH2O for 4 hours. In these experiments, an intratracheal injection (2 μL / g of mouse weight) was administered before MV. Designer EVs were then delivered to the lungs of these mice via a 100-μl volume of 100 μg / ml ethanol. Bronchoalveolar saturation data are collected during the ventilation process. After 4 hours, animals are euthanized and bronchoalveolar lavage is performed. BAL laboratory data and tissues were collected.

[0059] Example 2: In vitro approach to producing designer EVs Nanochannel-based electroporation is used to deliver the cargo of interest within donor cells. Overexpression within a controlled range allows the EV content and degree of surface modification to be set within the desired range Nanochannel-based electroporation improved transfection efficiency and cell viability. The cell viability could be increased to up to 100% and was not limited by capsid size. Using this approach, we have developed primary cultures of mouse dendritic cells and embryonic fibroblasts, as well as human Designer anti-inflammatory miR-146A-loaded cells derived from multiple cell sources, including mouse cell lines EVs were obtained (Figures 3A-3E). The characterization results of the EV content showed that compared with mock-loaded EVs, It was shown that the target molecular cargo was successfully incorporated into the endothelial cells (Fig. 3C). These EVs can be effectively used to culture human lung epithelial cells and human macrophages. Gene expression was modulated (Figure 3D). The proneural factor genes ASCL1, BRN2, and MY Designer EVs loaded with T1l(ABM) were also derived from early primary cultures of mouse fibroblasts. The dynamic release and uptake of designer EVs loaded with ABM was examined, and the donor cells EV release from the cells peaked 24 hours after transfection, at concentrations of It was revealed that the number of particles packed inside EVs was on the order of several billion particles / ml. The copy number exceeded the original copy number delivered to the “donor” cells by approximately three orders of magnitude prior to EV release. These results suggest that the therapeutic cargo is amplified within the cells.

[0060] Example 3: In vivo approach to producing designer EVs Nanochannel-based tissue transfection to induce designer EVs from in vivo tissues This could potentially allow patients to use their own tissues as bioreactors for mass production of EVs. This is thought to be important for clinical and production purposes. According to the method, about 1 cm 2 Approximately 10 cells carrying anti-inflammatory miR-146A were isolated from mouse skin. A trillion EVs can be obtained (Figure 4). By effectively utilizing these EVs, It can suppress inflammation caused by the above-mentioned condition (Figure 5).

[0061] Example 3: Given the need for emergency medical care, prolonged hospital stays, and delayed recovery in the event of ARDS, ARDS places a significant burden on the healthcare system. According to the g Association (2013), there are approximately 190,000 cases of ARDS per year in the United States. Mortality in ARDS patients is high, reaching 38-45%. ARDS is usually It can be caused by blunt trauma to the lungs or an underlying condition such as sepsis. When lung damage occurs, They may develop sepsis, a severe systemic inflammatory response caused by infection, putting their lives at risk. As a result, many ICU patients die. ARDS is characterized by severe hypoxia, Alveolar-capillary barrier dysfunction, small airway flooding due to reduced production of pulmonary surfactant, and Activation of proinflammatory pathways. Mechanical ventilation supports oxygen therapy, whereas , mechanical stress exacerbates the initial injury and leads to ventilator-induced lung injury with a higher mortality rate ( All of these factors can contribute to the underlying lung injury. This positive feedback loop can further exacerbate the condition, promoting inflammation and leading to multiple organ failure and death. Currently, there is no cure for ARDS, but there are treatments available that use anti-inflammatory cargo targeted to the lungs. There is great promise for cell-based approaches (e.g., endothelial progenitor cells, mesenchymal stem cells) With this treatment, for example, you can reduce inflammation and stimulate trophic and anti-inflammatory / infective mechanisms. This may be due in part to excessive extracorporeal processing. , and precursor cell-based cellular therapy that may lead to tumor formation and highly immunogenic responses. Serious concerns remain about the safety of certain therapeutic agents. While the details are clear, the efficient delivery of therapeutic drugs to inflamed lungs remains unclear. This remains a major challenge for critically ill patients. Anti-inflammatory and antibacterial molecular cargoes can be efficiently and selectively delivered by functionalized designer EVs. was introduced to reduce lung inflammation and injury and enhance tissue repair during sepsis-induced ARDS. Table 1 shows the design details of the designer EVs carrying molecular cargo. [Table 1]

[0062] The experimental protocol was optimized to obtain designer EVs derived from living tissue. In the dot field, 8-10 week old C57BL6 mice were cultured as described in Table 1. Plasmids encoding each factor were nanotransfected into the skin in vivo. By using these techniques, we obtained designer EVs of biological origin that were loaded with anti-inflammatory or antibacterial cargo. The transfection platform was fabricated using projection / contact photolithography as previously reported. Manufactured in a clean room using a combination of lithography and deep reactive ion etching EVs were collected from skin biopsies at various time points after transfection. To isolate the fibroblasts, the skin samples were dissociated and homogenized using gentleMACS dissociation agent. A total exosome isolation kit was used to selectively precipitate EVs for later use. The mixture was stored at -80°C for further use.

[0063] We functionalized designer EVs to target lung epithelium and macrophages, which are key sites of ARDS inflammation. Functionalized designer EVs target the delivery of pulmonary surface antigens to cellular compartments. Plasmids encoding septant protein A (SPA) and the membrane glycoprotein CD200 were used. Functionalized EVs by SPA interacts with the receptor p63 / CKAP4 on alveolar pneumocytes and the Toll-like receptor on these cells It is predicted that CD4+ binds to TLR2 and attenuates activation of the NF-κβ inflammatory pathway. EVs functionalized with CD200 preferentially interact with the CD200R receptor on alveolar macrophages. These tend to act in a manner that modulates the proinflammatory state. The ligands are used in combination with SPA and CD200 to detect designs delivered via the systemic circulation. This allows the inner EVs to easily interact with the pulmonary microvasculature. Activated EVs preferentially interact with the CXCR7 receptor in the pulmonary vasculature, which Upregulated under injury conditions and subsequently transported into the pulmonary vasculature as designer EVs migrated to the alveolar space Identifying the optimal administration route for designer EVs is also a step forward in identifying the best ways to administer designer EVs. This is an important step to optimize for therapeutic use. (Table 2) were compared to determine the optimal delivery method for therapeutic use. [Table 2]

[0064] For each delivery method, functional and non-functional anti-inflammatory, antibacterial, or fake designer EVs Dose sensitivity analyses were performed for healthy and diseased animals treated with (Table 3 In this analysis, bronchoalveolar lavage specimens (BAL), blood, and lungs were collected and The relative expression of genes encoding targeted molecular cargoes, as well as key proinflammatory properties and anti-inflammatory factors (Table 4) are quantified via qRT-PCR. The optimal dose concentration for significantly modulating pulmonary inflammation in vivo will be determined. The results show that the number of transfected skin surfaces required to obtain optimal concentrations of designer EVs is Accumulation and clearance of off-target moieties are correlated with the amount of off-target moieties in the target compound. The test is based on fabric investigation (Figure 6). [Table 3] [Table 4]

[0065] In addition to in vivo studies, extensive in vitro characterization of EVs has provided a better understanding of their nature. This evaluation is carried out using, for example, Nanosight and Zeta Potential Analyzers. Size distribution, charge, and surface modification as a function of cargo and surface modification using a fluoroscopy system. The loading efficiency of EVs was determined for each plasmid DNA produced. Using an absolute quantitative qRT-PCR protocol determined from a standard curve, the amount of DNA packed into each EV was These results are determined by quantifying the copy number of each gene that is transfected into the skin. Using total RNA sequencing, we have identified anti-inflammatory, antibacterial, or Characterize the contents of designer EVs carrying pseudomolecular cargo, thereby - Gain detailed information on the morphology of coding and non-coding RNA packaged within EVs Furthermore, the expression of functional and non-functional designer EVs (at the membrane and cytosolic levels) Comparative proteomic analysis was performed to assess the anti-inflammatory efficiency and the packing efficiency of antimicrobial molecular cargo, As well as determining the efficiency of membrane ligand expression.

[0066] To better understand the mechanism of action of designer EVs, we investigated a mouse model of lung injury ( Table 3. Results of in vivo performance evaluations in well-established in vitro models of injury (Table 5) was used as a benchmark for the test tube model. This will enable effective and systematic research into designer EVs, while in vivo models have multiple To better understand responses under pathophysiological conditions where interactions between multiple organ systems are important In vivo, the effectiveness of designer EVs in reducing inflammation and bacterial infections was The efficacy of this compound was evaluated in a mouse model of sepsis-induced ARDS and in a two-hit model. These models demonstrate that the level of inflammation attenuation observed from specific concentrations of designer EVs under infection Use test tube models of barotrauma and volutrauma to help determine the potential effects of Designer EVs were probed on cellular components isolated from lung tissue and bacterial cultures. [Table 5]

[0067] In vivo injury models involve the administration of designer EVs at various doses (e.g., single vs. repeated) and times. The 10-mg dose was administered at two different time points (e.g., pre-injury vs. post-injury) (Table 2). To obtain the cecal ligation and puncture (CLP)-induced pulmonary bypass, 8–10 week-old C57BL6 mice were cultured underwent either cecal ligation and puncture (CLP) or sham laparotomy. Pulmonary edema was diagnosed after CLP using ultrasound imaging with the Vevo2100 system. The animals were sacrificed and BAL, blood, and major organs (i.e., lungs, heart, Spleen, liver, and kidneys) were collected after perfusion with sterile saline for downstream analysis. Using the two-hit model, it is possible to prevent infection in infected patients who require mechanical ventilation, such as in cases where This model allows the reproduction of a more clinically relevant condition. 57BL6 mice were first subjected to CLP or sham laparotomy, and then 24 hours after CLP, Protocol (tidal volume 12cc / kg, 120 breaths / min, and FlexiVent Using FX and SCIREQ, the device was operated under the condition of 0 cm H2O positive end-expiratory pressure for 4 hours. After mechanical ventilation, pulmonary physiology and oxygen saturation data were collected during the MV procedure. At the end of MV, animals were sacrificed by anesthetic overdose and BAL, blood, and major organs were removed. Collect after perfusion with sterile saline for downstream analysis. Cellular content of BAL samples is the inflammatory cell count (i.e., alveolar macrophages, lymphocytes, eosinophils, neutrophils, etc.) The increase in protein content was associated with impaired alveolar-capillary barrier function. BAL protein content was also measured to correlate with loss of integrity. , Pro- and anti-inflammatory sites using custom-made cytokine arrays All BAL and plasma samples were screened for expression of kinases and factors. The relative expression of the delivered molecular cargo in vivo (treatments listed in Table 1) was and key anti-inflammatory and pro-inflammatory molecular markers for these therapies. (listed in Table 4) were assessed via qRT-PCR for their effect on expression.

[0068] For in vivo injury models, co-cultures of primary lung epithelial cells with mouse-derived macrophages Subjects are subjected to barotrauma or volumetric trauma. Barotrauma is a type of traumatic injury that has been previously published This can be induced using the model by co-culturing cells on transwell inserts. The apical compartment was then pressurized for 24 hours at a frequency of 0.2 Hz from 0 to 20 cm H A periodic oscillatory pressure of 200 is applied to simulate the conditions of a ventilator and normal breathing rate. Volume trauma was performed using the Flexcell FX-5000 tension system. In this model, cells were simulated in a modified 6-well plate with an elastomeric bottom. Co-culture on a 10-2000 cycle at 1.25 Hz for 24 h in a cell culture incubator. The cells were stretched continuously and cyclically at a stretch rate of 0.05%. To assess the formation and maintenance of tight junctions, ZO-1 was secreted into the culture medium. As an indicator of epithelial-endothelial barrier function after trauma, these were stained and imaged via IF. The experimental groups employed in the evaluation are shown in Table 5.

[0069] Unless otherwise stated, all technical and scientific terms used in this disclosure are within the meaning of the disclosure. The terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The publications and cited materials are incorporated herein by reference.

[0070] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. These features can be confirmed through routine experimentation, but are believed to be within the scope of the following claims. It is intended to.

Claims

1. Pulmonary surfactant protein A (SPA) or CD200, and exosomes or or a lysosomal transmembrane protein.

2. An extracellular vesicle comprising the fusion protein of claim 1.

3. The extracellular vesicle of claim 2, further comprising a therapeutic cargo.

4. The extracellular vesicle of claim 3, wherein the therapeutic cargo comprises miR146a.

5. The extracellular vesicle of claim 3 or 4, wherein the therapeutic cargo comprises Y-27632.

6. A cell comprising a nucleic acid encoding the fusion protein of claim 1.

7. The cell of claim 6 , further comprising a nucleic acid encoding a therapeutic RNA.

8. A method for producing extracellular vesicles, comprising the steps of: Culturing the cells and isolating extracellular vesicles secreted by said cells. A method comprising:

9. 10. The method of claim 8, further comprising loading the extracellular vesicles with a therapeutic agent.

10. A method for treating a lung disorder in a subject, comprising administering to said subject an extracellular microorganism according to any one of claims 3 to 5. The method comprises administering to the subject a therapeutically effective amount of a compound comprising:

11. 11. The method of claim 10, wherein the pulmonary disorder comprises acute respiratory distress syndrome (ARDS).

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