Exosome-Based Therapies for Liver Fibrosis and Other Fibrosis-Related Diseases

Abstract

Lipid-based nanoparticle compositions, such as exosome compositions, containing therapeutic STAT3-targeting inhibitory RNA are provided herein. Methods of using such compositions to treat patients with fibrosis or fibrosis-related diseases are also provided. TIFF2023505273000011.tif103153

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 943,943, filed December 5, 2019. U.S. Provisional Patent Application No. 62 / 943,943 is incorporated herein by reference in its entirety.

[0002] 1. Field The present invention relates generally to the field of medicine. More specifically, the present invention relates to compositions and methods for treating fibrosis and fibrosis-related diseases. [Background technology]

[0003] 2.Background Liver fibrosis is characterized by excessive extracellular matrix (ECM) deposition in the liver, which replaces functional parenchyma and has serious health consequences worldwide (Hernandez-Gea et al., 2001). Currently, there is no effective antifibrotic therapy except for the reduction of ongoing liver damage or liver transplantation (Bataller et al., 2005). Effective treatment of liver fibrosis urgently requires innovative new approaches. Among the key regulators of liver fibrosis, the signal transducer and activator of transcription 3 (STAT3) signaling pathway is centrally implicated, leading to the activation of fibroblasts and hepatic stellate cells (HSCs) and the transformation of these cells into a myofibroblast-like phenotype (Chakraborty et al., 2017; Xiang et al., 2018; Pechkovsky et al., 2012). STAT3 is a transcription factor that is phosphorylated by Janus tyrosine kinases (JAKs) in response to cytokine activation. Upon activation, phosphorylated STAT3 dimerizes and translocates to the nucleus to activate transcription of cytokine-responsive downstream genes (Chakraborty et al., 2017). Cytokines that activate STAT3 include TGFβ1 (Meng et al., 2016), the IL-6 family of cytokines, and growth hormone (GH). STAT3 activation has been reported in fibrotic livers observed in patients and mouse models (Xiang et al., 2018; Choi et al., 2019), and STAT3 inhibition with sorafenib or other inhibitors partially ameliorates CCl4-induced liver fibrosis in mice (Choi et al., 2019; Su et al., 2015). STAT3 has emerged as a key weakness in liver fibrosis, but therapeutic targeting of STAT3 remains a challenge due to the lack of STAT3-specific inhibitors (Bartneck et al., 2014). Therefore, new means of inhibiting STAT3 signaling are needed to develop specific anti-fibrotic therapies. Summary of the Invention

[0004] overview The embodiments of the present disclosure include nanoparticles, compositions, pharmaceutical compositions, nucleic acids, inhibitory RNA molecules, methods for preparing therapeutic compositions, methods for isolating exosomes, methods for preparing lipid-based nanoparticles, and methods for treating subjects.The compositions of the present disclosure may include at least one, two, three, four, five, or more of the following components: liposomes, exosomes, inhibitory RNA, siRNA, shRNA, miRNA, growth factors, unmodified antisense oligonucleotides, modified antisense oligonucleotides, and antimicrobial agents.In some embodiments, any one or more of these components may be excluded from the compositions of the present disclosure. The method of the present disclosure may include at least one, two, three, four, or more of the following steps: administering a pharmaceutical composition, administering an exosome, administering a liposome, administering an inhibitory RNA, making a liposome, obtaining an exosome from a subject, purifying an exosome from a mesenchymal cell, making an inhibitory RNA, synthesizing an siRNA, preparing lipid nanoparticles, introducing an inhibitory RNA into a lipid-based nanoparticle, encapsulating an inhibitory RNA into a nanoparticle, diagnosing a subject as having fibrosis, and treating fibrosis in a subject. It is contemplated that in some embodiments, any one or more of these steps may be excluded from the method of the present disclosure.

[0005] In some embodiments, compositions are provided herein that include lipid-based nanoparticles that contain an inhibitory RNA that hybridizes to a STAT3 polynucleotide. In some aspects, the lipid-based nanoparticles include CD47 on their surface. In some aspects, the lipid-based nanoparticles include a growth factor on their surface. In some aspects, the lipid-based nanoparticles are liposomes or exosomes. In some aspects, the inhibitory RNA is an siRNA, shRNA, antisense oligonucleotide, miRNA, or pre-miRNA. In certain aspects, the antisense oligonucleotide is modified. In some aspects, the inhibitory RNA knocks down the expression of STAT3 protein. In some aspects, the inhibitory RNA is 18-30 nucleotides in size. In some embodiments, the inhibitory RNA is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, with any one of NO:1 to 5, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, or any range derivable therein.In some embodiments, an inhibitory RNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:1, or any range derivable therein, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein. In some embodiments, the inhibitory RNA comprises SEQ ID NO:1. In some embodiments, the inhibitory RNA comprises SEQ ID NO:2. having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity with NO:2, or any range derivable therein, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity with NO:2, or any range derivable therein; 5%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein. In some embodiments, the inhibitory RNA comprises SEQ ID NO:2.In some embodiments, an inhibitory RNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:3, or any range derivable therein, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:3, or any range derivable therein. 5%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein. In some embodiments, the inhibitory RNA comprises SEQ ID NO:3. In some embodiments, the inhibitory RNA comprises SEQ ID NO:4. having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, or any range derivable therein, with NO:4, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein. In some embodiments, the inhibitory RNA comprises SEQ ID NO:4.In some embodiments, an inhibitory RNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:5, or any range derivable therein, or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity to SEQ ID NO:5, or any range derivable therein. 5%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity, or any range derivable therein. In some embodiments, the inhibitory RNA comprises SEQ ID NO:5. In some embodiments, it is contemplated that any one or more of these components may be excluded from the compositions of the present disclosure. Also disclosed herein, in some aspects, is a method of preparing a therapeutic composition, the method comprising introducing an inhibitory RNA of the present disclosure (e.g., an inhibitory RNA that hybridizes to a STAT3 polynucleotide) into a lipid-based nanoparticle (e.g., a liposome, an exosome, etc.).

[0006] In some embodiments, provided herein is a pharmaceutical composition comprising any one of the lipid-based nanoparticles of this embodiment and an excipient. In some aspects, the composition is formulated for parenteral administration. In certain aspects, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In certain aspects, the composition further comprises an antibacterial agent. In certain aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0007] In some embodiments, provided herein is a method for treating fibrosis or a condition associated with fibrosis in a patient in need thereof, comprising administering to the patient a composition according to any one of the embodiments. In some aspects, administering the pharmaceutical composition results in delivery of inhibitory RNA to cells in the patient. In some aspects, the fibrosis is liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury. In some embodiments, the fibrosis is liver fibrosis. In some aspects, the pharmaceutical composition is administered by systemic administration. In certain aspects, the systemic administration is intravenous administration. In certain aspects, the method further comprises administering at least a second therapy to the patient. In some aspects, the patient is a human. In certain aspects, the lipid-based nanoparticles are exosomes, and the exosomes are autologous to the patient. In certain aspects, the exosomes are obtained from a body fluid sample obtained from the patient. In certain aspects, the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. In certain aspects, the exosome is obtained from mesenchymal cells. In certain aspects, the composition is administered more than once. In some embodiments, the composition is administered at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times, or any range derivable therein, or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times, or any range derivable therein. In certain aspects, administering a composition of the present disclosure (e.g., a composition comprising lipid-based nanoparticles containing inhibitory RNA that hybridizes to a STAT3 polynucleotide) reduces expression of one or more STAT3-related genes in cells (e.g., hepatocytes) of the patient. In some embodiments, administering the composition reduces expression of Col1a1 in hepatocytes of the patient. In some embodiments, administering the composition reduces expression of Acta2 in hepatocytes of the patient. In some embodiments, administering the composition reduces expression of Col1a2 in hepatocytes of the patient.In some aspects, administering the composition reduces expression of Vim in liver cells of the patient.

[0008] As used herein, "essentially free" is used herein to mean that, in terms of the named component, none of the named components are intentionally formulated into the composition and / or are present merely as contaminants or in trace amounts.Thus, the total amount of the named components resulting from unintentional contamination of the composition is significantly less than 0.05%, for example, less than 0.01%.In some embodiments, a composition that is "essentially free" of a named component contains 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.001%, 0.0001%, or less of the named component, or contains at most 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.001%, 0.0001%, or less of the named component. In some embodiments, a composition that is "essentially free" of a specified component is one in which the specified component is not detectable in any amount by standard analytical methods.

[0009] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0010] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to mutually exclusive alternatives, but this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0011] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for any measuring or quantitating method.

[0012] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that embodiments described herein in the context of the term "comprising" may also be practiced in the context of the term "consisting of" or "consisting essentially of."

[0013] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may be described in "use" claim language, e.g., the "use" of any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0014] The use of one or more sequences or compositions can be used based on any method described herein. Other aspects are discussed throughout the application. Any aspect discussed in one aspect of the disclosure applies to other aspects of the disclosure, and vice versa. For example, any step in the method described herein applies to any other method. Furthermore, any method described herein may have the exclusion of any step or combination of steps. It is understood that the embodiments in the Examples section are applicable to all aspects of the technology described herein.

[0015] [The present invention 1001] A composition comprising lipid-based nanoparticles containing an inhibitory RNA that hybridizes to a STAT3 polynucleotide. [The present invention 1002] The composition of claim 1001, wherein the lipid-based nanoparticles comprise CD47 on their surface. [The present invention 1003] The composition of any one of claims 1001 to 1002, wherein the lipid-based nanoparticles comprise a growth factor on their surface. [The present invention 1004] The composition of any one of claims 1001 to 1003, wherein the lipid-based nanoparticle is a liposome or an exosome. [The present invention 1005] The composition of any one of claims 1001 to 1004, wherein the inhibitory RNA is an siRNA, an shRNA, an antisense oligonucleotide, an miRNA, or a pre-miRNA. [The present invention 1006] The composition of the present invention 1005, wherein said inhibitory RNA is an antisense oligonucleotide, and said antisense oligonucleotide is modified. [The present invention 1007] The composition of any of claims 1001 to 1006, wherein said inhibitory RNA knocks down the expression of a STAT3 protein. [The present invention 1008] The composition of any of claims 1001 to 1007, wherein the inhibitory RNA has a size of 18 to 30 nucleotides. [The present invention 1009] The composition of any of claims 1001 to 1008, wherein the inhibitory RNA comprises SEQ ID NO:1. [The present invention 1010] The composition of any of claims 1001 to 1008, wherein the inhibitory RNA comprises SEQ ID NO:2. [The present invention 1011] The composition of any of claims 1001 to 1008, wherein the inhibitory RNA comprises SEQ ID NO:3. [The present invention 1012] The composition of any of claims 1001 to 1008, wherein the inhibitory RNA comprises SEQ ID NO:4. [The present invention 1013] The composition of any of claims 1001 to 1008, wherein the inhibitory RNA comprises SEQ ID NO:5. [The present invention 1014] A pharmaceutical composition comprising any one of the compositions of the present inventions 1001 to 1013 and an excipient. [The present invention 1015] A pharmaceutical composition of the present invention 1014, which is formulated for parenteral administration. [The present invention 1016] A pharmaceutical composition of the present invention 1015 formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1017] The pharmaceutical composition of any one of claims 1014 to 1016, further comprising an antibacterial agent. [The present invention 1018] The pharmaceutical composition of the present invention 1017, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. [The present invention 1019] A method for treating fibrosis or a condition associated with fibrosis in a patient in need thereof, comprising administering to the patient a pharmaceutical composition according to any one of claims 1014 to 1018. [The present invention 1020] The method of claim 1019, wherein administering said pharmaceutical composition results in delivery of said inhibitory RNA to cells in said patient. [The present invention 1021] The method of claim 1019 or 1020, wherein said fibrosis is liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury. [The present invention 1022] The method of claim 1021, wherein the fibrosis is hepatic fibrosis. [The present invention 1023] The method of any of claims 1019 to 1021, wherein the pharmaceutical composition is administered by systemic administration. [The present invention 1024] The method of claim 1023, wherein said systemic administration is intravenous administration. [The present invention 1025] The method of any one of claims 1019 to 1024, further comprising administering at least a second therapy to the patient. [The present invention 1026] The method of any one of claims 1019 to 1025, wherein the patient is a human. [The present invention 1027] The method of claim 1026, wherein said lipid-based nanoparticles are exosomes, and said exosomes are autologous to said patient. [The present invention 1028] Any of the methods of claims 1019 to 1027, wherein the step of administering the pharmaceutical composition reduces Col1a1 expression in liver cells of the patient. [The present invention 1029] The method of any of claims 1019 to 1027, wherein the step of administering the pharmaceutical composition reduces Acta2 expression in liver cells of the patient. [The present invention 1030] Any of the methods of claims 1019 to 1027, wherein the step of administering the pharmaceutical composition reduces Col1a2 expression in liver cells of the patient. [The present invention 1031] The method of any of claims 1019 to 1027, wherein the step of administering the pharmaceutical composition reduces Vim expression in liver cells of the patient. [The present invention 1032] 1032. The method of any one of claims 1019 to 1031, wherein said hepatic cells are hepatic stellate cells. [The present invention 1033] 1032. The method of any of claims 1019 to 1031, wherein said hepatic stellate cells are activated hepatic stellate cells. [The present invention 1034] A composition comprising lipid-based nanoparticles containing an inhibitory RNA that hybridizes to a STAT3 polynucleotide. [The present invention 1035] The composition of claim 1034, wherein the lipid-based nanoparticles comprise CD47 on their surface. [The present invention 1036] The composition of claim 1034, wherein the lipid-based nanoparticles comprise a growth factor on their surface. [The present invention 1037] The composition of the present invention, wherein the lipid-based nanoparticle is a liposome or an exosome. [The present invention 1038] The composition of the present invention, wherein the inhibitory RNA is an siRNA, an shRNA, an antisense oligonucleotide, an miRNA, or a pre-miRNA. [The present invention 1039] The composition of claim 1038, wherein said inhibitory RNA is an antisense oligonucleotide, and said antisense oligonucleotide is modified. [The present invention 1040] The composition of claim 1034, wherein said inhibitory RNA knocks down the expression of STAT3 protein. [The present invention 1041] The composition of the present invention, wherein the inhibitory RNA has a size of 18 to 30 nucleotides. [The present invention 1042] The composition of claim 1034, wherein the inhibitory RNA comprises SEQ ID NO:1. [The present invention 1043] The composition of claim 1034, wherein the inhibitory RNA comprises SEQ ID NO:2. [The present invention 1044] The composition of claim 1034, wherein the inhibitory RNA comprises SEQ ID NO:3. [The present invention 1045] The composition of claim 1034, wherein the inhibitory RNA comprises SEQ ID NO:4. [The present invention 1046] The composition of claim 1034, wherein the inhibitory RNA comprises SEQ ID NO:5. [The present invention 1047] A pharmaceutical composition comprising any one of the compositions according to the present inventions 1034 to 1046 and an excipient. [The present invention 1048] A pharmaceutical composition of the present invention 1047, which is formulated for parenteral administration. [The present invention 1049] A pharmaceutical composition of the present invention 1048 formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1050] The pharmaceutical composition of the present invention further comprising an antibacterial agent. [The present invention 1051] The pharmaceutical composition of the present invention 1050, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. [The present invention 1052] A method for treating fibrosis or a condition associated with fibrosis in a patient in need thereof, comprising administering to said patient a pharmaceutical composition according to any one of claims 1047 to 1051. [The present invention 1053] The method of claim 1052, wherein administering said pharmaceutical composition results in delivery of said inhibitory RNA to cells in said patient. [The present invention 1054] The method of claim 1052, wherein said fibrosis is liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury. [The present invention 1055] The method of claim 1054, wherein said fibrosis is hepatic fibrosis. [The present invention 1056] The method of claim 1052, wherein said pharmaceutical composition is administered by systemic administration. [The present invention 1057] The method of claim 1056, wherein said systemic administration is intravenous administration. [The present invention 1058] The method of claim 1052, further comprising administering to said patient at least a second therapy. [The present invention 1059] The method of claim 1052, wherein the patient is a human. [The present invention 1060] The method of claim 1059, wherein said lipid-based nanoparticles are exosomes, and said exosomes are autologous to said patient. [The present invention 1061] The method of claim 1060, wherein said exosomes are obtained from a body fluid sample obtained from said patient. [The present invention 1062] The method of claim 1061, wherein said body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. [The present invention 1063] The method of claim 1060, wherein said exosomes are obtained from mesenchymal cells. [The present invention 1064] The method of claim 1052, wherein the composition is administered more than once. [The present invention 1065] The method of claim 1052, wherein administering said pharmaceutical composition reduces Col1a1 expression in liver cells of said patient. [The present invention 1066] The method of claim 1052, wherein administering said pharmaceutical composition reduces Acta2 expression in liver cells of said patient. [The present invention 1067] The method of claim 1052, wherein administering said pharmaceutical composition reduces Col1a2 expression in liver cells of said patient. [The present invention 1068] The method of claim 1052, wherein administering said pharmaceutical composition reduces Vim expression in liver cells of said patient. [The present invention 1069] The method of claim 1052, wherein said hepatic cells are hepatic stellate cells. [The present invention 1070] The method of claim 1052, wherein said hepatic stellate cells are activated hepatic stellate cells. [The present invention 1071] A method for preparing a therapeutic composition comprising the step of introducing an inhibitory RNA that hybridizes to a STAT3 polynucleotide into a lipid-based nanoparticle. [The present invention 1072] 1072. The method of claim 1071, wherein the lipid-based nanoparticles comprise CD47 on their surface. [The present invention 1073] 13. The method of claim 1071 or 1072, wherein the lipid-based nanoparticles comprise a growth factor on their surface. [The present invention 1074] The method of any one of claims 1071 to 1073, wherein the lipid-based nanoparticle is a liposome or an exosome. [The present invention 1075] The method of any one of claims 1071 to 1074, wherein the inhibitory RNA is an siRNA, an shRNA, an antisense oligonucleotide, an miRNA, or a pre-miRNA. [The present invention 1076] The method of claim 1075, wherein said inhibitory RNA is an antisense oligonucleotide, and said antisense oligonucleotide is modified. [The present invention 1077] The method of any one of claims 1071 to 1076, wherein said inhibitory RNA knocks down the expression of a STAT3 protein. [The present invention 1078] The method of any of claims 1071 to 1077, wherein the inhibitory RNA has a size of 18 to 30 nucleotides. [The present invention 1079] The method of any one of claims 1071 to 1078, wherein the inhibitory RNA comprises SEQ ID NO:1. [The present invention 1080] The method of any one of claims 1071 to 1078, wherein the inhibitory RNA comprises SEQ ID NO:2. [The present invention 1081] The method of any one of claims 1071 to 1078, wherein the inhibitory RNA comprises SEQ ID NO:3. [The present invention 1082] The method of any one of claims 1071 to 1078, wherein the inhibitory RNA comprises SEQ ID NO:4. [The present invention 1083] The method of any one of claims 1071 to 1078, wherein the inhibitory RNA comprises SEQ ID NO:5. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain particular aspects of the present invention, are given by way of example only, as various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Liver IVIS imaging. [Figure 2A]Liver parenchymal damage. Figure 2A shows H&E stained sections of livers from fibrotic mice treated with various exosome treatments. [Figure 2B] Liver parenchymal damage. Figure 2B shows quantification of the levels of fibrosis seen in Figure 2A. [Figure 3A] Lung parenchymal damage. Figure 3A shows H&E stained sections of lungs from fibrotis mice treated with various exosome treatments. [Figure 3B] Lung Parenchymal Damage. Figure 3B shows quantification of the levels of fibrosis seen in Figure 3A. [Figure 4] Figures 4A-4F. Knockdown efficiency of STAT3 in primary HSCs and biodistribution of exosomes. Figures 4A and 4B show relative Stat3 expression in HSCs treated with 5 μg / 2 billion iExosiRNA-STAT3 (Figure 4A) or iExomASO-STAT3 (Figure 4B). Figure 4C shows representative images of the listed organs analyzed for the presence of DiR-labeled exosomes in non-fibrotic (sham) (left panel) and fibrotic (right panel) mice (n=1). Figures 4D-4F show immunofluorescence imaging (Figure 4D) and quantification (Figures 4E and 4F) of AF647-labeled exosomes and DAPI in frozen liver tissues of the listed groups (three fields for each tissue analyzed). Scale bar: 100 μm. Data presented as mean ± SEM. For Figures 4A and 4D, an unpaired two-tailed t-test was used. For Figure 4B, one-way ANOVA with Sidak's post-hoc analysis was used: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 5]Figures 5A-5K. Figure 5A shows images of mouse hepatic stellate cells (HSCs) cultured for 7 days (left panel). Scale bar: 100 μm. Immunofluorescence staining of α-SMA and DAPI of primary mouse HSCs (middle and right panels). Scale bar: 100 μm. Figures 5B and 5C show qPCR analysis of STAT3. Figure 5D shows a diagram of CCl4 and iExosome / siRNA / mASO treatment schedules. The upper arrow indicates CCl4 injection (day 0) and the lower arrow indicates iExosome / siRNA / mASO injection (day 9). Figures 5E and 5F show immunohistochemical staining (Figure 5E) and quantification (Figure 5F) of collagen I in mice treated with 1 μg of 1 billion iExosiRNA-STAT3 or iExomASO-STAT3 (three fields of view for each tissue analyzed). n=3; Scale bar: 100 μm. Figures 5G and 5H show immunofluorescence staining (Figure 5G) and quantification (Figure 5H) of α-SMA in mice treated with 1 μg of 1 billion iExosiRNA-STAT3 or iExomASO-STAT3 (three fields for each tissue analyzed). n=3; scale bar: 100 μm. Figure 5I shows H&E staining of liver from mice treated with 1 μg of 1 billion iExosiRNA-STAT3 or iExomASO-STAT3 (five fields for each tissue analyzed). n=5; scale bar: 100 μm. Figures 5J and 5K show the percentage of necrotic (Figure 5J) and degenerated hepatocytes (Figure 5K). Data are presented as mean ± SEM. Each point in the graph represents a separate mouse. Figure 5B (left panel), unpaired two-tailed Student's t-test. Figure 5B (right panel) and Figures 5C-5K One-way ANOVA and Sidak's post-hoc analysis; p values ​​are indicated in all graphs. *p<0.05; **p<0.01; ****p<0.0001; ns: not significant. [Figure 6]Figures 6A-6J. iExosomes targeting STAT3 reduced liver fibrosis. Figures 6A and 6B show STAT3 mRNA relative expression in livers of mice treated with iExosiRNA-STAT3 or iExomASO-STAT3 at 1 μg / billion (Figure 6A) or 5 μg / 2 billion (Figure 6B) of the indicated treatments. n=4-5 separate mice in the 5 μg / 2 billion group; n=4-5 separate mice in the 1 μg / billion group, one-way ANOVA was used. Figures 6C and 6D show representative Sirius red staining (Figure 6C) and quantification (Figure 6D) of liver sections from the 1 μg / billion treatment group (3 fields for each tissue analyzed). n=5-6 separate mice, one-way ANOVA. Scale bar: 100 μm. Graph shows percent Sirius red positive area. Figures 6E and 6F show representative Sirius red staining (Figure 6E) and quantification (Figure 6F) of liver sections from the 5 μg / 2 billion treatment group (three fields for each tissue analyzed). n=3-5 separate mice. Scale bar: 100 μm. Graphs show percent Sirius red positive area. Figures 6G and 6H show representative images (three fields for each tissue analyzed) of collagen I immunohistochemical staining (Figure 6G) and quantification of the percent of collagen I+ area / field (100×) (Figure 6H). n=3. Figures 6I and 6J show α-SMA immunofluorescence staining (Figure 6I; three fields for each tissue analyzed) and quantification of the number of α-SMA+ cells / field (100×) (Figure 6J). n=3-4 separate mice; Scale bar: 100 μm. Data shown as mean ± SEM. Each point in the graph represents a separate mouse. One-way ANOVA or unpaired two-tailed t-test unless otherwise stated; p values ​​are shown on all graphs. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns: not significant. [Figure 7]Figures 7A-7K. STAT3-targeted iExosomes preserved functional parenchyma in liver. Figures 7A-7D show relative Col1a1 expression (Figures 7A and 7B) and Acta2 expression (Figures 7C and 7D) in livers receiving the indicated treatments. n=4-5 separate mice for 5 μg / 2 billion group; n=5 separate mice for 1 μg / 1 billion group. One-way ANOVA was used for statistical analysis in the 1 μg / 1 billion group. Figures 7E and 7F show serum levels of ALT in mice receiving the indicated treatments. n=4-5 separate mice for 5 μg / 2 billion group; n=4-5 separate mice for statistical analysis in the 1 μg / 1 billion group, one-way ANOVA was used. Figures 7G and 7H show serum levels of AST in mice receiving the indicated treatments. n=4-5 separate mice for the 5 μg / 2 billion group; n=4-5 separate mice for the 1 μg / 1 billion group. One-way ANOVA was used for statistical analysis in the 1 μg / 1 billion group. Figure 7I shows H&E staining of paraffin-embedded liver sections (3-5 fields for each tissue analyzed). n=4-5 separate mice; scale bar: 100 μm. Figures 7J and 7K show the percentage of necrotic and degenerated hepatocytes. Data are presented as mean ± SEM. Each point in the graph represents a separate mouse. One-way ANOVA or unpaired two-tailed t-test; p values ​​are shown in all graphs. *p<0.05; **p<0.01; ****p<0.0001; ns: not significant. [Figure 8A] Shown is H&E staining of the listed organs in mice treated with 5 μg / 2 billion iExosiRNASTAT3 or iExomASO-STAT3. [Figure 8B] H&E staining of the listed organs in mice treated with 1 μg / billion iExosiRNASTAT3 or iExomASO-STAT3 is shown. [Figure 9A]Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9A shows a heatmap illustrating the relative intensity of all probes across experimental groups (siCntrl iExo (n=3), siSTAT3 iExo (n=3), mASO Scrbl iExo (n=3), and mASO STAT3 iExo (n=3). Euclidean clustering of both rows and columns using log2-transformed mRNA-Seq expression data. [Figure 9B] Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9B shows a volcano plot illustrating the number of differentially regulated genes in the liver of the listed experimental groups. [Figure 9C] Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9C shows a volcano plot illustrating the number of differentially regulated genes in the liver of the listed experimental groups. [Figure 9D] Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9D shows a heatmap of STAT3 signaling. [Figure 9E] Reprogramming of the fibrotic liver transcriptome during EiExosome treatment. Figure 9E shows selected genes associated with ECM deposition and remodeling. [Figure 9F] Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9F shows a diagram of target gene differences using Gene Ontology (GO) analysis (WebGestalt) enrichment. [Figure 9G] Reprogramming of the fibrotic liver transcriptome during iExosome treatment. Figure 9G shows a diagram of the target gene differences using Gene Ontology (GO) analysis (WebGestalt) enrichment. [Figure 9H] Reprogramming of fibrotic liver transcriptome during iExosome treatment. Figure 9H shows the interaction network generated by NetworkAnalyst for STAT3 signaling and ECM-related genes. [Figure 10A] Col1a1 knockout in activated hepatic stellate cells. Figure 10A shows Sirius red staining aimed at assessing ECM and collagen I-associated fibrosis, demonstrating that genetic loss (Col1a1cKO) of collagen I from activated hepatic stellate cells or αSMA+ myofibroblasts in the context of fibrosis is significantly reduced. [Figure 10B] Col1a1 knockout in activated hepatic stellate cells. Figure 10B shows results demonstrating that collagen I is significantly reduced in Col1a1cKO with liver fibrosis. [Figure 10C] Col1a1 knockout in activated hepatic stellate cells. Figure 10C shows results demonstrating that liver histology is significantly improved in Col1a1cKO with liver fibrosis. [Figure 10D] Col1a1 knockout in activated hepatic stellate cells. Figure 10D shows quantification of the results from Figures 10A-10C. [Figure 10E] Col1a1 knockout in activated hepatic stellate cells. Figure 10E shows quantification of the results from Figures 10A-10C. [Figure 10F] Col1a1 knockout in activated hepatic stellate cells. Figure 10F shows quantification of the results from Figures 10A-10C. [Figure 10G] Col1a1 knockout in activated hepatic stellate cells. Figure 10G shows gene expression data demonstrating that many of the global expression patterns associated with liver fibrosis are significantly ameliorated in Col1a1cKO mice with liver fibrosis. [Figure 10H] Col1a1 knockout in activated hepatic stellate cells. Figure 10H shows gene expression data demonstrating that Col1a1cKO mice with liver fibrosis have significantly ameliorated many of the global expression patterns associated with liver fibrosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description Provided herein is an exosome engineered to carry inhibitory RNA molecules, including antisense oligonucleotides (ASO) and siRNA, that target STAT3, a mediator of organ fibrosis, including liver fibrosis and lung fibrosis.These engineered exosomes can be used to treat fibrosis, including liver fibrosis and lung fibrosis.The exosomes obtained from mesenchymal stem cells are highly distributed in lung and liver, making the delivery of ASO or siRNA efficient.

[0018] I. Lipid-Based Nanoparticles The lipid-based nanoparticle is a liposome, an exosome, a lipid preparation, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). The lipid-based nanoparticle may be positively charged, negatively charged, or neutral. The lipid-based nanoparticle may include the necessary components that enable transcription and translation, signal transduction, chemotaxis, or other cellular functions. In one embodiment, it is intended that one or more of these items may be excluded.

[0019] Lipid-based nanoparticles may include CD47 on their surface. CD47 (integrin-associated protein) is a transmembrane protein expressed on the surface of most tissues and cells. CD47 is a ligand for Signal Regulatory Protein Alpha (SIRP-α) expressed on the surface of phagocytes such as macrophages and dendritic cells. Activated CD47-SIRP-α initiates a signaling cascade that inhibits phagocytosis. Thus, without being bound by theory, the expression of CD47 on the surface of exosomes may block phagocytosis by macrophages (see WO2016 / 201323, which is incorporated herein by reference in its entirety).

[0020] A. Liposomes "Liposome" is a generic term that includes various unilamellar and multilamellar lipid vesicles formed by forming a closed lipid bilayer or aggregate. Liposomes may be characterized as having a vesicular structure with a bilayer membrane that generally comprises phospholipids, and an internal medium that generally comprises an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0021] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components self-reorganize before forming a closed structure that traps water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0022] In certain aspects, a polypeptide, nucleic acid, or small molecule drug may, for example, be placed in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that binds both the liposome and the polypeptide / nucleic acid, entrapped within the liposome, or complexed with the liposome.

[0023] The liposomes used according to this embodiment can be prepared by various methods as known to those skilled in the art. For example, phospholipids, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. The lipids are then mixed with polypeptides, nucleic acids, and / or other components. Tween 20 is added to the lipid mixture so that Tween 20 is about 5% of the composition's weight. Excess tert-butanol is added to this mixture so that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparations can be stored at -20°C and used for up to 3 months. When needed, the lyophilized liposomes are reconstituted by dissolving in 0.9% saline.

[0024] Alternatively, liposomes can be prepared by mixing lipids dissolved in a solvent in a container, e.g., a glass pear-shaped flask. The volume of the container should be 10 times the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator under negative pressure at about 40°C. Typically, the solvent is removed within about 5 minutes to 2 hours, depending on the desired liposome volume. The composition can be placed in a desiccator and further dried under reduced pressure. Dried lipids tend to deteriorate over time, so they are generally discarded after about one week.

[0025] The dried lipids can be dissolved and hydrated in sterile pyrogen-free water at approximately 25-50 mM phospholipid by shaking until all of the lipid film is resuspended. The aqueous liposome solution can then be divided into aliquots, each placed into a vial, lyophilized, and sealed under vacuum.

[0026] The dried lipid or lyophilized liposomes prepared as described above may be dehydrated, dissolved in a solution of protein or peptide, reconstituted, and diluted to an appropriate concentration with a suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. Any additional unencapsulated materials, such as agents including, but not limited to, hormones, drugs, nucleic acid constructs, etc., are removed by centrifugation at 29,000×g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of encapsulated additional materials or active agents can be determined according to standard methods. After determining the amount of additional materials or active agents encapsulated in the liposome preparation, the liposomes may be diluted to an appropriate concentration and stored at 4° C. until use. Pharmaceutical compositions containing liposomes usually contain a sterilized pharma- ceutical acceptable carrier or diluent, e.g., water or saline.

[0027] Additional liposomes that may be useful with this embodiment include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068, all of which are incorporated by reference in their entirety without disclaimer.

[0028] In preparing such liposomes, any protocol described herein or known to those skilled in the art can be used.More non-limiting examples of preparing liposomes are described in U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; International Application PCT / US85 / 01161 and PCT / US89 / 05040, each of which is incorporated herein by reference.

[0029] In certain embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., DOPC liposome). As used herein, "neutral liposome" or "uncharged liposome" is defined as a liposome having one or more lipid components that result in an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that within a particular population (e.g., a population of liposomes), some lipid components, if any, contain a charge that is not counterbalanced by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1% of the components contain an uncounterbalanced charge). In certain embodiments, neutral liposomes may contain a majority of lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., about pH 7).

[0030] The liposome and / or lipid-based nanoparticle of this embodiment may include phospholipid. In certain embodiments, one type of phospholipid may be used in making liposomes (e.g., neutral phospholipid, e.g., DOPC may be used to make neutral liposomes). In other embodiments, multiple types of phospholipids may be used to make liposomes. Phospholipids may be derived from neutral or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), so these compounds may be particularly useful for making neutral liposomes. In certain embodiments, phospholipid DOPC is used to generate uncharged liposomes. In certain embodiments, non-phospholipid lipids (e.g., cholesterol) may be used.

[0031] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myris ... 1-Palmitoyl-2-stearoylphosphatidylcholine ("PMPC"), 1-Palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-Stearoyl-2-Palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), "), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyris ... dipalmitoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristoyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,These include, but are not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyleoylphosphatidylcholine ("POPC"), palmitoyleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0032] B. Exosomes As used herein, the terms "microvesicle" and "exosome" refer to membranous particles having a diameter (or largest dimension if the particle is not a spheroid) of about 10 nm to about 5000 nm, more typically 30 nm to 1000 nm, and most typically about 50 nm to 750 nm, where at least a portion of the exosome's membrane is obtained directly from a cell. Exosomes of the present disclosure have a diameter of at least 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 2000nm, 3000nm, 4000nm, or 5000nm, or any range derivable therein, and at most 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 5000nm, or 5000nm, or any range derivable therein. The size of the exosome may be about 100 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, or 5000 nm, or any range derivable therein, or about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, or 5000 nm, or any range derivable therein. Most commonly, the size (average diameter) of the exosome is up to 5% of the size of the donor cell. Thus, exosomes specifically contemplated include exosomes shed from cells.

[0033] Exosomes may be detected in or isolated from any suitable sample type, such as, for example, bodily fluids. The term "isolated" as used herein refers to separation from the natural environment and is intended to include at least partial purification, and may include substantial purification. The term "sample" as used herein refers to any sample suitable for the methods provided by the present invention. The sample may be any sample that contains exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, joint fluid, breast milk, sweat, tears, synovial fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention can be extracted from any known source that contains blood cells or components thereof, such as, for example, venous, arterial, peripheral, tissue, cord, etc. For example, samples can be obtained and processed using well-known and routine clinical methods (e.g., procedures for collecting and processing whole blood). In one aspect, an exemplary sample can be peripheral blood taken from a subject with cancer.

[0034] Exosomes may also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from tissue sources, it may be necessary to obtain a single cell suspension and then homogenize the tissue to lyse the cells and release the exosomes. When isolating exosomes from tissue samples, it is important to select homogenization and lysis procedures that do not destroy the exosomes. The exosomes contemplated herein are preferably isolated from body fluids dissolved in a physiologically acceptable solution, such as buffered saline, growth medium, various aqueous media, etc.

[0035] Exosomes may be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. In some embodiments, exosomes may be isolated from cell culture media. Although not necessary, exosomes of higher purity may be obtained if the fluid sample is clarified before precipitation with a volume-excluding polymer to remove debris from the sample. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by numerous methods well known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatant. Exemplary methods of exosome isolation are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes may also be isolated by flow cytometry as described in (Combes et al., 1997).

[0036] One commonly accepted protocol for isolating exosomes involves ultracentrifugation, which is often combined with a sucrose density gradient or sucrose cushion to float the relatively low density exosomes. Isolation of exosomes by sequential fractional centrifugation is complicated by the potential for overlap in size distribution with other microvesicles or macromolecular complexes. In addition, centrifugation may provide an insufficient means of separating vesicles based on size. However, exosomes can be highly enriched when sequential centrifugation is combined with sucrose gradient ultracentrifugation.

[0037] Size-based exosome isolation using alternatives to the ultracentrifugation route is another option. Successful exosome purification has been reported using an ultrafiltration procedure that is less time-consuming than ultracentrifugation and does not require the use of special equipment. Similarly, a commercial kit (EXOMIR™, Bioo Scientific) can be used that uses positive pressure to move fluids to remove cells, platelets, and cellular debris in one microfilter and capture vesicles larger than 30 nm in a second microfilter. However, in this process, exosomes are not recovered and their RNA content is extracted directly from the material captured in the second microfilter and can then be used for PCR analysis. Using HPLC-based protocols, these processes require dedicated equipment and are difficult to scale up, but potentially high purity exosomes could be obtained. A significant problem is that both blood and cell culture media contain numerous nanoparticles (some of which are non-vesicles) in the same size range as exosomes. For example, some miRNA may be contained in extracellular protein complexes rather than in exosomes. However, treatment with a protease (eg, proteinase K) can be carried out to eliminate any contamination with "extraexosomal" proteins.

[0038] In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosomes in a sample, such as techniques involving immune-specific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, typically involves attaching antibodies directed against proteins found on the surface of certain cell types to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, the beads attach to and surround the specific cells. The sample is then placed in a strong magnetic field, causing the beads to pellet on one side. After removing the blood, the captured cells are retained together with the beads. Many variations of this general method are known in the art and are suitable for use to isolate exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then antibodies are added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins that have been found to be found on cancer cell-derived exosomes include ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLITRK4 (SLIT and NTRK-like protein 4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and glypican-1 (GPC1). Cancer cell-derived exosomes can be isolated, for example, using antibodies or aptamers against one or more of these proteins.

[0039] It should be noted that not all proteins expressed in a cell are found in the exosomes secreted by that cell. For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells, but are not found in the exosomes secreted by MCF-7 cells (Baietti et al., 2012). As another example, a study found that 190 / 190 pancreatic ductal adenocarcinoma patients have higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015, the entirety of which is incorporated herein by reference). Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes.

[0040] 1. Exemplary Protocol for Harvesting Exosomes from Cell Cultures On day 1, seed a sufficient number of cells (e.g., about 5 million cells) into a T225 flask containing medium containing 10% FBS so that the cells are about 70% confluent the next day. On day 2, aspirate the medium on the surface of the cells, wash the cells twice with PBS, and then add 25-30 mL of basal medium (i.e., no PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. Although a 48-hour incubation is preferred, some cell lines are sensitive to serum-free medium, and therefore the incubation time must be reduced to 24 hours. Note that FBS contains exosomes that strongly skew the NanoSight results.

[0041] On day 3 / 4, collect the medium and centrifuge at 800 x g for 5 min at room temperature to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and recentrifuge the medium at 2000 x g for 10 min to remove other large debris and large vesicles. Pass the medium through a 0.2 μm filter and then aliquot it into ultracentrifuge tubes (e.g., 25 x 89 mm Beckman Ultra-Clear) using 35 mL / tube. If the volume of medium per tube is less than 35 mL, fill the remainder of the tube with PBS to make it 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2-4 hours at 4 °C using a SW 32 Ti rotor (k-factor 266.7, RCF max 133,907). Carefully aspirate the supernatant until approximately 1 inch of liquid remains. Tilt the tube and slowly dispense the remaining medium into an aspirating pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifugation at 28,000 rpm for 1-2 h can be repeated to further purify the exosome population.

[0042] Finally, resuspend the exosome pellet in 210 µL of PBS. If there are multiple ultracentrifuge tubes for each sample, use the same 210 µL PBS to successively resuspend each exosome pellet. For each sample, take 10 µL and add to 990 µL of H2O for use in nanoparticle tracking analysis. Use the remaining 200 µL exosome-containing suspension for downstream processes or store immediately at -80 °C.

[0043] 2. Exemplary Protocol for Extracting Exosomes from Serum Samples First, thaw the serum sample on ice. Then, dilute 250 μL of cell-free serum sample with 11 mL of PBS. Filter through a 0.2 μm pore filter. Ultracentrifuge the diluted sample at 150,000 × g at 4 °C overnight. The next day, carefully discard the supernatant and wash the exosome pellet with 11 mL PBS. Ultracentrifuge a second time at 150,000 × g at 4 °C for 2 h. Finally, carefully discard the supernatant and resuspend the exosome pellet in 100 μL PBS for analysis.

[0044] C. Exemplary Protocols for Electroporating Exosomes and Liposomes 1×10 8 100 nm exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) are mixed with 1 μg siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Exosomes or liposomes are electroporated using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, exosomes or liposomes are treated with protease-free RNase followed by the addition of 10× concentrated RNAse inhibitor. Finally, exosomes or liposomes are washed with PBS under ultracentrifugation as described above.

[0045] II. Inhibitory RNA A. Antisense Oligonucleotides Antisense oligonucleotide (ASO) therapeutics are single-stranded nucleic acid therapeutics, typically about 16-30 nucleotides in length, that are complementary to a target nucleic acid sequence in target cells in culture or in an organism.

[0046] In some embodiments, the substance is a single-stranded antisense RNA molecule, a single-stranded antisense DNA molecule, or a single-stranded antisense polynucleotide that contains both DNA and RNA. In certain embodiments, the antisense molecule is an ASO that contains both DNA and RNA. The antisense molecule is complementary to a sequence in a target mRNA, for example, STAT3 mRNA. The antisense molecule can stoichiometrically inhibit translation by base pairing to the mRNA and physically interfering with the translation machinery. The antisense molecule may have at least 15-30 nucleotides or at most 15-30 nucleotides that are complementary to the target mRNA. For example, an antisense molecule may have a sequence of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides, or any range or value derivable therein, or at most 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides, or any range or value derivable therein, that are complementary to a target mRNA.

[0047] In some embodiments, the ASO is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or any range derivable therein. ASOs may contain between 8 and 50 nucleotides, or ... In some embodiments, the ASO is comprised of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or any range or value derivable therein. Any of these values ​​can be used to define a range for the number of nucleotides in the ASO. For example, the ASO may be comprised of 8-50, 15-30, or 20-25 nucleotides.

[0048] In one aspect of the disclosure, the agent is a single-stranded antisense nucleic acid molecule (ASO). Antisense oligonucleotides (ASOs) are synthetic molecules that, in some embodiments, comprise 18-21 nucleotides in length and are complementary to the mRNA sequence of a target gene. ASOs bind to the cognate mRNA sequence by sequence-specific hybridization, resulting in cleavage and inactivation of the mRNA, thereby inhibiting expression of the target gene.

[0049] 1. ASO Modification In certain embodiments, the ASO of the present disclosure may be modified. "Modified ASO" refers to a molecule in which one or more of the nucleic acid components, i.e., sugar, base, and phosphate moieties, are different from those that occur naturally, for example, those that occur in the human body. Some modifications to ASO are described in the art. These modifications are aimed at improving ASO properties, such as resistance to nucleases, permeability across biological membranes, solubility, stability, or adjustment of pharmacokinetic and pharmacodynamic properties, while maintaining specificity for target mRNA. For example, modifications on nucleotides may include, but are not limited to, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In one embodiment, it is contemplated that one or more of these modifications may be excluded.

[0050] The patents directed to antisense nucleic acid, chemical modification, and therapeutic use are provided in, for example, U.S. Patent No. 5,898,031, which is related to therapeutic compounds containing chemically modified RNA, and U.S. Patent No. 6,107,094, which is related to the method of using these compounds as therapeutic substances, U.S. Patent No. 7,432,250, which is related to the method of treating patients by administering single-stranded chemically modified RNA-like compounds; and U.S. Patent No. 7,432,249, which is related to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds.U.S. Patent No. 7,629,321, which is related to the method of cleaving target mRNA using a single-stranded oligonucleotide having multiple RNA nucleosides and at least one chemical modification, relates to each of the patents listed in this paragraph, which are incorporated herein by reference in their entirety.

[0051] A modified base Therapeutic nucleic acids may contain natural bases (i.e., A, G, U, C, or T) or modified bases (e.g., 7-deazaguanosine, inosine, etc.). Base modifications include the incorporation of modified bases (or modified nucleosides or nucleotides) that are variations of the standard base, sugar, and / or phosphate backbone chemical structures that occur in ribonucleic acids (i.e., A, C, G, and U) and deoxyribonucleic acids (i.e., A, C, G, and T). Included in this range are, for example, Gm (2'-methoxyguanylic acid), Am (2'-methoxyadenylic acid), Cf (2'-fluorocytidylic acid), Uf (2'-fluorouridylic acid), Ar (riboadenylic acid). Aptamers may also include cytosine or any cytosine-related base, including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, 5-hydroxymethylcytosine, 2-thiocytosine, 5-halocytosine (e.g., 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, and 5-iodocytosine), 5-propynylcytosine, 6-azocytosine, 5-trifluoromethylcytosine, N4,N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine, carbazole cytidine, or pyridoindole cytidine. The aptamer may further comprise guanine or any guanine-related base including 6-methylguanine, 1-methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 2-propylguanine, 6-propylguanine, 8-haloguanine (e.g., 8-fluoroguanine, 8-bromoguanine, 8-chloroguanine, and 8-iodoguanine), 8-aminoguanine, 8-sulfhydrylguanine, 8-thioalkylguanine, 8-hydroxyguanine, 7-methylguanine, 8-azaguanine, 7-deazaguanine, or 3-deazaguanine.The aptamer may further comprise adenine or any adenine related base including 6-methyladenine, N6-isopentenyladenine, N6-methyladenine, 1-methyladenine, 2-methyladenine, 2-methylthio-N6-isopentenyladenine, 8-haloadenine (e.g., 8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and 8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine, 8-thioalkyladenine, 8-hydroxyadenine, 7-methyladenine, 2-haloadenine (e.g., 2-fluoroadenine, 2-bromoadenine, 2-chloroadenine, and 2-iodoadenine), 2-aminoadenine, 8-azaadenine, 7-deazaadenine, or 3-deazaadenine. Uracil, or 5-halouracil (e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil), 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, 1-methylpseudouracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil , 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methylaminomethyluracil, 5-propynyluracil, 6-azouracil, or 4-thiouracil.

[0052] Other examples of modified base variants known in the art include, for example, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-methoxycytidine, 5-carboxymethylaminomethyl-2-thiolysine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, bD-galactosylqueosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, bD-mannosylqueosine, 5-methoxycarbonylmethyluridine, 5-methylaminomethyl-2-thiouridine, Toxuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-bD-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-bD-ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, urdine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid (v), wybutoxosine, pseudomycin These include, but are not limited to, douridine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-bD-ribofuranosylpurin-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, and wybutosine, 3-(3-amino-3-carboxypropyl)uridine.

[0053] U.S. Patent Nos. 3,687,808, 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, Also included are modified nucleobases described in Nos. 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, and 5,681,941, each of which is incorporated herein by reference in its entirety.

[0054] b.Modified sugar Modified sugar moieties for use in ASOs are well known in the art and are described, for example, in U.S. Patent No. 9,045,754, which is incorporated herein by reference in its entirety. Modified sugars can be used to alter, typically increase, the affinity of the ASO for the ASO target and / or increase nuclease resistance. For example, in some embodiments, the binding affinity of the ASO for the ASO target can be increased by incorporating a substituent into the nucleoside subunit of the ASO. In some embodiments, the substituent is a T substituent, which is a substituent located at the 2' position of the pentofuranosyl sugar moiety of the nucleoside subunit of the ASO. The substituents include, but are not limited to, fluoro, alkoxy, amino-alkoxy, allyloxy, imidazolylalkoxy, and polyethylene glycol. Alkoxy and aminoalkoxy groups generally include lower alkyl groups, particularly C1-C9 alkyl. In certain embodiments, the 2' substituent is 2'-O-methyl. Polyethylene glycol is of the structure (O-CH2-CH2)nO-alkyl. In certain embodiments, the substituent is a polyethylene glycol substituent of the formula (-O-CH2-CH2)nO-alkyl, where n=1 and alkyl=CH3. This modification has been shown to increase the affinity of the oligonucleotide to the oligonucleotide target and the nuclease resistance of the oligonucleotide. See U.S. Patent No. 7,629,321 cited above. Another particularly useful 2'-substituent for increasing binding affinity is the 2'-fluoro group.

[0055] Examples of modified nucleoside and nucleotide sugar backbone variants known in the art include, but are not limited to, those having a 2' ribosyl substituent, e.g., F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, ONO2, NO2, N3, NH2, OCH2CH2OCH3, O(CH2)2ON(CH3)2, OCHOCH2N(CH3)2, O(C1-10 alkyl), O(C2-10 alkenyl), O(C2-10 alkynyl), S(C1-10 alkyl), S(C2-10 alkenyl), S(C2-10 alkynyl), NH(C1-10 alkyl), NH(C2-10 alkenyl), NH(C2-10 alkynyl), and O-alkyl-O-alkyl. Desirable 2'-ribosyl substituents include 2'-methoxy (2'-OCH3), 2'-aminopropyloxy (2'OCH2CH2CH2NH2), 2'-O-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2), 2'-amino (2'-NH2), and 2'-fluoro (2'-F). The 2'-substituent may be in the arabino (up) or ribo (down) position. One or more of these variations may be excluded from embodiments of the present disclosure.

[0056] Another modified ASO class that is known in the art and can be used in the ASO of the present disclosure contains alkyl modifications at the 2' position of the ribose moiety. These ASOs have been developed to improve the binding affinity and hybridization stability with target mRNA and to increase the nuclease resistance of ASOs. In this category, the most commonly used ASOs are 2'-O-methyl (2'-OME) and 2'-O-methoxyethyl (2'-MOE) ASOs. ASOs with this type of modification cannot activate RNAseH. Therefore, to induce RNAseH activation, chimeric ASOs have been developed in which a central gap region consisting of a phosphorothioate deoxyribose core is flanked by nuclease-resistant arms, such as 2'-OME or 2'-MOE, which have large nuclease resistance. As a result, a "gapmer" is generated in which RNAseH is located in the central gap and can activate target-specific mRNA degradation while the arms prevent ASO degradation. This category of ASOs has a higher affinity for mRNA, exhibits better tissue uptake, is more resistant to nucleases, has a longer in vivo half-life, and is less toxic compared to the first class of modified ASOs.

[0057] Further ASO classes, known in the art and can be used in the ASOs of the present disclosure, contain furanose ring modifications together with modifications of phosphate linkages, ribose moieties, or nucleotides. These modifications were designed to improve the nuclease stability, target affinity, and pharmacokinetic profile of ASOs. Common examples of the third ASO category are locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino phosphoramidate (MF). This category of ASOs is highly stable in biological fluids due to its high resistance to degradation by nucleases and peptidases. This category of ASOs also shows strong hybridization affinity with mRNA. In addition, PNAs can recognize double-stranded DNA and regulate gene expression or induce mutations by strand invasion of chromosomal double-stranded DNA. This category of ASOs also does not activate RNAseH and relies on steric hindrance of the ribosomal machinery to stop translation. This category of ASOs is uncharged and therefore does not bind to serum proteins. Being uncharged reduces the likelihood of non-specific interactions but increases the rate of clearance from the body. Their electrostatically neutral backbones may reduce solubility and make uptake more difficult.

[0058] A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars (BNAs), including methyleneoxy (4'-CH2-O-2') BNAs and ethyleneoxy (4'-(CH2)2-O-2' bridged) BNAs; substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and 4'-thio modified sugars. Sugars can also be substituted, particularly with sugar mimetic groups. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications which disclose the preparation of such modified sugars include U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,5 91,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO2005 / 121371.

[0059] C modified internucleotide linkage The nucleic acid therapeutic agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be on any nucleotide at any position on the sense strand or antisense strand or both strands (in the nucleic acid therapeutic agent containing a sense strand). For example, the internucleotide linkage modification may be on any nucleotide on the sense strand or antisense strand. Each internucleotide linkage modification may be in a staggered pattern on the sense strand or antisense strand. Alternatively, the sense strand or antisense strand may contain internucleotide linkage modifications in a staggered pattern. The staggered pattern of the internucleotide linkage modification on the sense strand may be the same as or different from the antisense strand, and the staggered pattern of the internucleotide linkage modification on the sense strand may have a change compared to the staggered pattern of the internucleotide linkage modification on the antisense strand.

[0060] In certain embodiments, the ASOs of the disclosure comprise one or more nucleoside subunits linked by phosphorus linkages, including phosphodiester, phosphorothioate, 3' (or -5') deoxy-3'- (or -5') thio-phosphorothioate, phosphorodithioate, phosphoroselenate, 3'- (or -5') deoxyphosphinate, boranophosphate, 3'- (or 5'-) amino phosphoramidate, hydrogen phosphonate, boranophosphate ester, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester phosphorus linkages. In some embodiments, the ASOs of the disclosure comprise nucleoside subunits linked by carbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetyl, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino bonds.

[0061] For example, one of the modified ASO classes described in the art and that can be used in the ASO of the present disclosure is one in which one of the non-bridging oxygen atoms in the phosphate group of the ASO is replaced with a sulfur group (phosphorothioate), a methyl group (methylphosphonate), or an amine group (phosphoramidate). These ASOs are more resistant to nucleases and have a longer plasma half-life compared to phosphodiester oligonucleotides. These ASOs can activate RNAseH, have a negative charge that facilitates delivery to cells, and have suitable pharmacokinetics. Among these modifications, phosphorothioate modification is the most widely used. For example, the FDA-approved ASO drug Vitravene and the majority of other ASO drugs in clinical trials are phosphorothioate ASOs.

[0062] Furthermore, the bases in nucleotides may be linked by bonds other than phosphodiester bonds, so long as they do not interfere with hybridization. Thus, inhibitory nucleic acids may be peptide nucleic acids, in which the constituent bases are linked by peptide bonds rather than phosphodiester bonds. Inhibitory nucleic acids may be prepared by converting RNA to cDNA using known methods (see, for example, Ausubel et. al., Current Protocols in Molecular Biology Wiley 1999). Inhibitory nucleic acids may also be cRNA (see, for example, Park et. al., (2004) Biochem. Biophys. Res. Commun. 325(4):1346-52).

[0063] B. Small interfering RNA siRNA (e.g., siNA) is well known in the art.For example, siRNA and double-stranded RNA are described in U.S. Patent No. 6,506,559 and U.S. Patent No. 6,573,099, and U.S. Patent Application No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161, and No. 2004 / 0064842.All of these are incorporated herein by reference in their entirety.

[0064] The nucleic acid components in siRNA do not have to be the same type or uniform throughout (e.g., siRNA may comprise nucleotides and nucleic acids or nucleotide analogues). Typically, siRNA forms a double-stranded structure. The double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present disclosure, siRNA may comprise only one nucleic acid (polynucleotide) or nucleic acid analogue, and form a double-stranded structure by complementing itself (e.g., forming a hairpin loop). The double-stranded structure of the siRNA may comprise at least 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive nucleobases. The siRNA may comprise 250, 300, 350, 400, 450, 500 or more contiguous nucleobases, or at most 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous nucleobases, including all ranges and values ​​therein. The siRNA may comprise 17-35 contiguous nucleobases, 18-30 contiguous nucleobases, 19-25 nucleobases, 20-23 contiguous nucleobases, or 20-22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize to a complementary nucleic acid (which may be another portion of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[0065] The agent of the present disclosure useful for carrying out the method of the present disclosure includes, but is not limited to, siRNA.Typically, the introduction of double-stranded RNA (dsRNA), which may be alternatively referred to herein as small interfering RNA (siRNA), induces strong and specific gene silencing, a phenomenon called RNA interference or RNAi.RNA interference has been called "co-suppression", "post-transcriptional gene silencing", "sense suppression" and "quelling".RNAi is an attractive biotechnology tool because it provides a means to knock out the activity of specific genes.

[0066] There are several factors that need to be considered when designing RNAi, such as the nature of the siRNA, the permanence of the silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into the organism typically contains exon sequences. Furthermore, the RNAi process is dependent on homology. Therefore, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of mutual interference between homologous but non-gene specific sequences. Preferably, the siRNA shows 80%, 85%, 90%, 95%, 98% or even 100% identity between the siRNA sequence and the gene to be inhibited, or any range or value that can be derived therein, or shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity, or any range or value that can be derived therein. Sequences with less than about 80% identity with the target gene have a much smaller effect. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0067] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that contain 19-25 nucleotides or any range or value derivable therein, contain at least 19-25 nucleotides or any range or value derivable therein, or contain at most 19-25 nucleotides or any range or value derivable therein, and can regulate gene expression. In the context of the present invention, the siRNA is, in some embodiments, less than 500, 200, 100, 50, or 25 nucleotides in length. In some embodiments, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0068] Target gene generally refers to a polynucleotide that includes a region that codes for a polypeptide, or a region that regulates the replication, transcription, or translation, or other processes important for the expression of a polypeptide, or a polynucleotide that includes both a region that codes for a polypeptide and a region that is functionally linked to the region that regulates expression.Any gene that is expressed in a cell can be targeted.Preferably, the target gene is a gene that is involved in or associated with the progression of a cellular activity that is important for disease, or a gene that is of particular interest as a research subject.

[0069] siRNA can be obtained from commercial suppliers, natural sources, or can be synthesized by any of the many techniques known to those skilled in the art.For example, one of the commercial suppliers of pre-designed siRNA is Ambion®, Austin, Tex. Another commercial supplier is Qiagen® (Valencia, Calif.).The inhibitory nucleic acid applicable in the compositions and methods of the present disclosure can be any nucleic acid sequence that is found by any supplier to be a verified down-regulator of the protein of interest.It is understood that without undue experimentation and using the disclosure of the present disclosure, further siRNA can be designed and used to carry out the method of the present disclosure.

[0070] The siRNA may also contain one or more nucleotide changes. Such changes may include, for example, the addition of non-nucleotide material to the end of the 19-25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. The nucleotides in the RNA molecules of the present disclosure may also contain non-standard nucleotides, including non-natural nucleotides or deoxyribonucleotides. The double-stranded oligonucleotides may contain modified backbones, such as phosphorothioates, phosphorodithioates, or other modified backbones known in the art, and may contain non-natural internucleoside linkages. Further modifications of siRNA (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in US Patent Application Publication No. 2004 / 0019001 and US Patent No. 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all of the above-mentioned modified nucleic acids or RNAs are referred to as modified siRNAs.

[0071] Since exosomes are known to contain DICER and active RNA processing RISC complex (see PCT publication WO2014 / 152622, the entirety of which is incorporated herein by reference), the shRNA introduced into exosomes by transfection can be matured into RISC complex-bound siRNA in exosomes themselves. Alternatively, mature siRNA itself can be introduced into exosomes or liposomes by transfection. Any inhibitory nucleic acid can be applied in the compositions and methods of the present disclosure, as long as such inhibitory nucleic acid is found by any supplier to be a verified down-regulator of protein of interest.

[0072] III. Treatment of Disease Many serious diseases in mammals, including humans, are associated with fibrosis. As used herein, "fibrosis" includes any condition involving the formation of fibrous tissue, whether such formation is desired or undesired. Such conditions include, but are not limited to, connective tissue inflammation, fibroma formation (fibromatosis), fibrosis (including pulmonary fibrosis and hepatic fibrosis), fibroelastosis (endocardial fibrosis), fibromyopathy formation, uterine fibroid formation, fibroidoma formation, fibromyxoma formation, and fibrocystitis (including cystic fibrosis).

[0073] In some embodiments, fibrosis that can be treated in animals with STAT3 expression inhibitors include, but are not limited to, liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), radiation-induced lung injury, or radiation-induced lung injury resulting from cancer treatment.In some embodiments, fibrosis that can be treated include pulmonary fibrosis (e.g., pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), radiation-induced lung injury, or radiation-induced lung injury resulting from cancer treatment), skin fibrosis, kidney fibrosis, liver fibrosis (e.g., cirrhosis), gastrointestinal fibrosis (e.g., fibrosis of the gastrointestinal tract, fibrosis associated with gastrointestinal inflammation, fibrosis associated with inflammatory bowel disease, fibrosis associated with ulcerative colitis, fibrosis associated with Crohn's disease, intestinal fibrosis, small intestinal fibrosis, iliac fibrosis, cecal fibrosis, or colon fibrosis), cardiac fibrosis (e.g., atrial fibrosis, endomyocardial fibrosis, or myocardial infarction), brain fibrosis (e.g., glial scar), or other forms of fibrosis, including, but not limited to, arteriosclerosis, arthrofibrosis (e.g., of the knee, shoulder, or other joints), Crohn's disease (e.g., of the intestine), Dupuytren's contracture (e.g., of the hand or fingers), keloids (e.g., of the skin), mediastinal fibrosis (e.g., of the soft tissues of the mediastinum), myelofibrosis (e.g., of the bone marrow), Peyronie's disease (e.g., of the penis), nephrogenic systemic fibrosis (e.g., of the skin), progressive massive fibrosis (e.g., a complication of coal workers' pneumoconiosis), retroperitoneal fibrosis (e.g., of the soft tissues of the retroperitoneal space), scleroderma / systemic sclerosis (e.g., of the skin or lungs), adhesive capsulitis (e.g., of the shoulder), or other organ fibrosis.

[0074] Animals that can be treated include, but are not limited to, mammals, rodents, primates, monkeys (e.g., macaques, rhesus monkeys, pig-tailed macaques), humans, dogs, cats, pigs, birds (e.g., chickens), cows, mice, rabbits, and rats. As used herein, the terms "individual," "subject," and "patient" are used interchangeably and may refer to both human and non-human subjects. In some cases, the animal is in need of treatment (e.g., by exhibiting symptoms of disease or fibrosis).

[0075] The term "treat" (and its variants, e.g., "treatment") as used herein should be considered in its broadest context. In particular, the term "treat" does not necessarily mean that an animal is treated until complete recovery. Thus, "treat" includes amelioration of symptoms, relief from symptoms or effects associated with a condition, reducing the severity of a condition, or preventing, prophylactically ameliorating, or otherwise reducing the risk of developing a particular condition. As used herein, reference to "treating" an animal includes, but is not limited to, prophylactic and therapeutic treatments. Any of the compositions (e.g., pharmaceutical compositions) described herein can be used to treat an animal.

[0076] When related to the treatment of fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury), treatment may include, but is not limited to, prophylactic and therapeutic treatment. Thus, treatment may include, but is not limited to, preventing fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); reducing the risk of fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); reducing the risk of fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); ameliorate or reduce the symptoms of fibrosis (e.g., hepatic fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); induce a body response that resists fibrosis (e.g., hepatic fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); inhibit the development or progression of fibrosis (e.g., hepatic fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); inhibiting or preventing the onset of conditions associated with fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); reducing the severity of fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury) or conditions associated with fibrosis; The treatment may include, but is not limited to, causing regression of one or more of (e.g., a reduction in the amount of fibrosis); causing remission of fibrosis (e.g., liver fibrosis, lung fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury); or preventing the recurrence of fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury). In some embodiments, the treatment does not include prophylactic treatment of fibrosis (preventing or ameliorating future fibrosis).

[0077] Treatment of an animal (e.g., a human) can be performed using any suitable administration method (e.g., the administration methods disclosed herein) and with any suitable amount of a STAT3 expression inhibitor (e.g., siRNA or ASO). In some embodiments, the treatment method includes treating an animal for fibrosis (e.g., liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury). Some embodiments of the present disclosure include a method for treating a subject (e.g., an animal, e.g., a human or primate) with a composition (e.g., siRNA or ASO) (e.g., a pharmaceutical composition) comprising one or more STAT3 expression inhibitors, comprising one or more administrations of one or more such compositions, where the compositions may be the same or different.

[0078] Treatment with a STAT3 expression inhibitor can result in a decrease in expression of one or more genes associated with STAT3 activity (e.g., regulated by STAT3 activity). In some embodiments, the treatment of the present disclosure reduces expression of one or more genes associated with STAT3 activity, including, for example, Col1a1, Acta2, Col1a2, and / or Vim. In some embodiments, the treatment of the present disclosure reduces expression of Col1a1 in target cells (e.g., hepatocytes) of the patient. Exemplary target cells of the present disclosure include hepatocytes, e.g., activated hepatic stellate cells and αSMA. + Contains myofibroblasts.

[0079] In some embodiments, other fibrosis treatments are optionally included and can be used with the treatments of the invention described herein. Other fibrosis treatments can include any known fibrosis treatment suitable for treating fibrosis. Examples of known fibrosis treatments include antibiotics (e.g., penicillin, methicillin, oxacillin, nafcillin, cavenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin, ticarcillin clavulanic acid, piperacillin tazobactam, cephalosporins, cephalexin, cefdinir, cefprozil, cefaclor, cefepime, sulfa, sulfamethoxazole, trimethoprim, erythromycin / sulfisoxazole, macrolides, erythromycin, clarithromycin, azithromycin, tetracycline, tetrasalicylic acid, tetracycline ... These include, but are not limited to, administration of anti-inflammatory drugs (e.g., cyclosporine, doxycycline, minocycline, tigecycline, vancomycin, imipenem, meripenem, colistimethate / colistin, aminoglycosides, tobramycin, amikacin, gentamicin, quinolones, aztreonam, or linezolid), anti-inflammatory drugs (e.g., NSAIDs, aspirin, ibuprofen, naproxen, corticosteroids, cortisol, corticosterone, cortisone, or aldosterone), bronchodilators (e.g., albuterol or levalbuterol hydrochloride), mucus thinners (e.g., hypertonic saline or domase alfa), and anti-fibrotic drugs (e.g., pirfenidone, nintedanib, N-acetylcysteine, ivacaftor, or lumacaftor / ivacaftor). Other fibrosis treatments may also include performing non-pharmacological respiratory therapy, such as, but not limited to, airway clearance techniques (e.g., postural drainage and chest percussion, exercise, breathing exercises, or mechanical devices, such as high-frequency chest compression vests or positive expiratory pressure therapy). Other fibrosis treatments may also include organ transplants (e.g., lung, skin, kidney, liver, heart, small intestine, or colon). It is contemplated that one or more other fibrosis treatments may be excluded in embodiments of the present disclosure.

[0080] In some embodiments, administration of an opioid receptor inhibitor, naltrexone, pirfenidone, nintedanib, or a combination thereof, can be used as part of a treatment regime (e.g., as another fibrosis treatment). Administration of an opioid receptor inhibitor, naltrexone, pirfenidone, nintedanib, or a combination thereof can include separate administration (i.e., in a different composition than the STAT3 expression inhibitor) or can be added to a composition that includes the STAT3 expression inhibitor.

[0081] In some embodiments, the additional optional treatment (e.g., as another fibrosis treatment) may also include one or more of surgical intervention, hormonal therapy, immunotherapy, and adjuvant systematic therapy.It is intended that one or more additional optional treatments may be excluded in the embodiments of the present disclosure.

[0082] When treating a disease, the appropriate dosage of the therapeutic composition will depend on the type of disease being treated, as defined above, the severity and course of the disease, the patient's medical history and response to the agent, and the judgment of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

[0083] The methods and compositions for treatment and prevention can be provided in a combined amount effective to achieve the desired effect.Tissue, tumor, or cell can be contacted with one or more compositions or pharmacological preparations containing one or more of the above-mentioned agents, or by contacting tissue, tumor, and / or cell with two or more separate compositions or preparations.It is also contemplated that such combination therapy can be used with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

[0084] Coadministration may include coadministration of two or more agents in the same dosage form, coadministration in separate dosage forms, and separate administration. That is, the therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and coadministered. Or, the therapeutic composition and another therapeutic agent can be coadministered, where both agents are in separate formulations. In another option, a therapeutic agent can be administered and the other therapeutic agent can be administered immediately thereafter, or vice versa. In another administration protocol, the therapeutic composition and another therapeutic agent can be administered minutes apart, hours apart, or days apart.

[0085] IV. Pharmaceutical Compositions It is contemplated that exosomes expressing or containing therapeutic agents can be administered systemically or locally to enhance telomerase activity.Exosomes expressing or containing therapeutic agents can be administered intravenously, intrathecally, and / or intraperitoneally.Exosomes expressing or containing therapeutic agents can be administered alone or in combination with a second drug.

[0086] It is not intended that the present invention be limited by the specific nature of the therapeutic preparation.For example, such compositions can be provided in the form of a formulation together with physiologically acceptable liquid, gel, solid carrier, diluent or excipient.These therapeutic preparations can be administered to mammals for veterinary use, such as veterinary use with livestock, and clinical use in humans, as with other therapeutic substances.In general, the dosage required for therapeutic efficacy varies according to the type of use and method of administration, as well as the specific requirements of each individual subject.

[0087] When clinical application is intended, it may be necessary to prepare a pharmaceutical composition comprising exosomes in a form suitable for the intended use. In general, a pharmaceutical composition may comprise an effective amount of one or more exosomes and / or additional agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as, for example, a human, as appropriate. Preparation of pharmaceutical compositions comprising exosomes or additional active ingredients disclosed herein is known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. In addition, it is understood that for administration to animals (e.g., humans), preparations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards.

[0088] Furthermore, according to certain aspects of the present disclosure, compositions suitable for administration may be provided dissolved in a pharma- ceutically acceptable carrier with or without an inert diluent. As used herein, a "pharma- ceutically acceptable carrier" refers to any and all aqueous solvents known to those skilled in the art (e.g., water, alcoholic / aqueous solutions, ethanol, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), vegetable oils, and injectable organic esters such as ethyl oleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, and the like. , preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, noble gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof), isotonic agents (e.g., sugars and sodium chloride), absorption retarders (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, fillers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquid and nutritional supplements, such similar materials and combinations thereof. The carrier must be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffering agents. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0089] Although pharmaceutically acceptable carriers are specifically formulated for administration to humans, in certain embodiments, it may be desirable to use pharmaceutically acceptable carriers that are formulated for administration to non-human animals but are not acceptable for administration to humans (e.g., due to government regulations).Unless a conventional carrier is incompatible with the active ingredient (e.g., harmful to the recipient or harmful to the therapeutic effectiveness of the composition contained in the carrier), its use in therapeutic or pharmaceutical compositions is contemplated.According to certain aspects of the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc.Such procedures are routine for those skilled in the art.

[0090] Certain embodiments of the present disclosure may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, whether they need to be sterile for the route of administration, such as injection. The compositions may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing the target cells directly, via a catheter, via lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the foregoing known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[0091] Exosomes can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.Typically, such compositions can be prepared in either liquid solutions or suspensions.Solid dosage forms can also be prepared, suitable for use in preparing solutions or suspensions by adding liquid prior to injection.The preparation can also be emulsified.

[0092] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. The dosage form must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0093] Once formulated, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, for example, formulated for parenteral administration, such as injections or aerosols for delivery to the lungs, or formulated for edible administration, such as drug release capsules.

[0094] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response, i.e., the desired route and treatment regimen discussed above. The amount to be administered depends on the desired effect, depending on the number of treatments and the unit dose. The actual dosage of the composition of the present disclosure administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the extent of disease invasion, previous or concurrent therapeutic interventions, the patient's idiopathic disease, the route of administration, and the potency, stability, and toxicity of the particular therapeutic agent. For example, the dosage may also include a dosage of about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such ranges include doses therebetween) or more, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges of about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. As another example, the dose may include about 1 billion to about 500 billion exosomes per administration (such ranges include doses therebetween) or more exosomes, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges of about 1 million exosomes to about 500 billion exosomes, about 5 million exosomes to about 250 billion exosomes, etc. In one example, the dose may include about 150 billion exosomes in a volume of 5 mL, and such a dose may be administered to a human patient weighing 70 kg. The physician responsible for administration will determine the concentration of active ingredient in the composition and the dose appropriate for the individual subject in any given situation.

[0095] The actual dosage of the composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount may vary according to the subject's response. The physician responsible for administration will, in any given situation, determine the concentration of active ingredient in the composition and the dosage appropriate for the individual subject.

[0096] In certain embodiments, the pharmaceutical composition may, for example, contain at least about 0.1% or at most about 0.1% of the active compound. In other embodiments, the active compound may, for example, contain about 2% to about 75% or about 25% to about 60% of the weight of the unit, and any range derivable therein. Naturally, in each therapeutically useful composition, the amount of active compound may be prepared in such a way that a suitable dosage is obtained in a particular unit dose of the active compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimes may be desirable.

[0097] In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 milligrams / kg / body weight, or more, per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include ranges based on the above numbers, such as about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0098] V. Kits and Diagnostics In various aspects of the present disclosure, kits are contemplated that contain the necessary components for purifying exosomes from body fluids or tissue culture media. In other aspects, kits are contemplated that contain the necessary components for isolating exosomes and transfecting them with therapeutic nucleic acids, therapeutic proteins, or inhibitory RNA. The kit may contain one or more sealed vials that contain any of such components. In some embodiments, the kit may also contain suitable container means, such as eppendorf tubes, assay plates, syringes, bottles, or tubes, that are containers that do not react with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass. The kit may further contain instructions that outline the procedural steps of the methods described herein, following substantially the same procedures as those described herein or known to those skilled in the art. The information in the instructions may be in a computer readable medium that contains machine readable instructions that, when executed using a computer, display a real or virtual procedure of purifying exosomes from a sample and transfecting the exosomes with a therapeutic cargo. EXAMPLES

[0099] VI. Working Examples The following examples are included to demonstrate certain aspects of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples demonstrate techniques discovered by the inventors to function well in the practice of the invention, and are therefore believed to constitute preferred embodiments for practicing the invention. However, in light of this disclosure, it should be understood by those of skill in the art that many changes can be made in the specific embodiments disclosed and still obtain a similar or similar result without departing from the spirit and scope of the invention.

[0100] Example 1 – Exosome localization to the liver of CC14-induced liver fibrosis mouse model Liver fibrosis was induced in adult Balb / c mice by ip injection of CC14 every other week for 6 weeks. Mice with and without liver fibrosis were both administered 109 Dye-labeled exosomes or dye alone were administered intravenously. Mice were euthanized 3 hours after administration. DiR-labeled exosomes given to mice were assayed using IVIS imaging (Figure 1). PHK67-labeled exosomes given to mice were assayed using confocal microscopy. Exosomes localized to the liver of both healthy and fibrotic mice. However, localization levels were increased in fibrotic mice compared to healthy mice.

[0101] Example 2 – Reduction of liver fibrosis after administration of exosomes loaded with STAT3 inhibitor RNA Liver fibrosis was induced in C57Bl6 adult mice by ip injection of CC14 every other week for 6 weeks. MSC-derived exosomes (1.5 billion exosomes with 1.5 μg siRNA or ASO per dose per injection) were then administered every 48 hours. Exosomes contained scrambled siRNA, STAT3 siRNA, unmodified STAT3 antisense oligonucleotide, modified scrambled antisense oligonucleotide, or modified STAT3 antisense oligonucleotide. After treatment, mice were euthanized, their livers were sectioned, processed for hematoxylin and eosin (H&E) staining, and stained sections were imaged (Figure 2A). Fibrosis levels were quantified (Figure 2B). Results show that STAT3 siRNA, unmodified STAT3 antisense oligonucleotide, and modified STAT3 antisense oligonucleotide reduced fibrosis levels.

[0102] Example 3 – Reduction of pulmonary fibrosis following administration of exosomes loaded with STAT3 inhibitor RNA Mice were induced with bleomycin to induce pulmonary fibrosis. MSC-derived exosomes (2 billion exosomes containing 5 μg siRNA or ASO per dose per injection) were then administered every 48 hours. The exosomes contained STAT3 siRNA, unmodified STAT3 antisense oligonucleotide, modified scrambled antisense oligonucleotide, or modified STAT3 antisense oligonucleotide. After treatment, mice were euthanized, their lungs were sectioned, processed for hematoxylin and eosin (H&E) staining, and the stained sections were imaged (Figure 3A). Fibrosis levels were quantified (Figure 3B). Results show that STAT3 siRNA reduced fibrosis levels to the greatest extent.

[0103] Example 4 – Exosome-mediated STAT3 therapeutic targeting in liver fibrosis result To confirm the efficiency of iExosomes (exosomes engineered to deliver a nucleic acid payload) loaded with siRNA or ASO targeting STAT3, primary hepatic stellate cells (HSCs) isolated from wild-type (WT) mice were cultured for 7 days, which resulted in spontaneous activation of HSCs (Figure 4A) (Zhai et al., 2019; Lu et al., 2014; Mederacke et al., 2013). HSC activation is characterized by the expression of α-smooth muscle actin (α-SMA) (Figure 4B). iExosomes loaded with STAT3-targeting siRNA or ASO were cultured for 7 days, which resulted in spontaneous activation of HSCs (Figure 4A) (Zhai et al., 2019; Lu et al., 2014; Mederacke et al., 2013). siRNA-STAT3 or iExo mASO-STAT3 Treatment reduced Stat3 mRNA levels in HSCs (Figures 4C and 4D) with similar efficiency compared to lipid-based transfection reagents (Figures 5A and 5B).

[0104] Tropism of exogenously administered exosomes in mice was previously reported (Mendt et al., 2018; Kamerkar et al., 2017). This included several GI organs such as the pancreas and liver. We confirmed the liver tropism of human mesenchymal stromal cell (MSC)-derived exosomes toward the liver of healthy mice (Figure 4C). To further investigate the biodistribution of exosomes in fibrotic tissues, DiR-labeled exosomes were administered intraperitoneally (ip) to carbon tetrachloride (CCl4)-induced fibrotic liver mice and WT mice. Results showed that specific accumulation signals were associated with exosomes in normal liver and pancreas, and low amounts of signals were detected in kidney, intestine, and spleen (Figure 4C). However, fibrotic liver showed a greater enrichment of DiR-labeled exosomes compared to healthy liver. Furthermore, we demonstrated that Alexa-Fluor 647 (AF647)-tagged siSTAT3 or modified ASO (mASO) STAT3 was delivered to exosomes (iExosomes) by electroporation after ip injection (24 h) of tagged iExosome / siSTAT3 / mASO STAT3. siRNA647-STAT3 or iExo mASO647-STAT3 The results demonstrated that fluorescently labeled siRNA and ASO accumulated in the liver at a higher rate than naked siRNA or ASO (Figure 4D-F).

[0105] To verify the function of iExosomes in treating liver fibrosis in vivo, WT mice were subjected to twice weekly ip injections of CCl4 to induce chronic liver fibrosis (Figure 5D). After inducing fibrosis, on day 9, mice were treated with either naked siRNA or ASO, or with iExosomes. siRNA Or iExo mASOiExosomes containing siRNA or ASO targeting STAT3 were administered at two doses: 1 billion exosomes electroporated with 1 μg siRNA or ASO (1 μg / billion iExo) and 2 billion exosomes electroporated with 5 μg siRNA or ASO (5 μg / 2 billion iExo). siRNA targets both human and mouse STAT3, while ASO was designed to target human STAT3, exhibiting three nucleotide mismatches compared to the mouse sequence. ASO designs included unmodified (umASO) and modified ASO (mASO, see Methods). Controls included untreated mice and mice treated with non-targeting control siRNA (siCntrl), or mouse exosomes containing ASO (modified scramble [mASO Scrbl] or umASO STAT3). iExosomes targeting STAT3 with siRNA or mASO were administered at 1 μg / billion iExo. siRNA-STAT3 and 5μg / 2 billion iExo siRNA-STAT3 or iExo mASO-STAT3 significantly reduced Stat3 expression in fibrotic livers upon treatment with iExo at 5 μg / 2 billion compared to siRNA (siRNA-STAT3) or ASO (mASO-STAT3) alone (Figures 6A and 6B). siRNA-STAT3 or iExo mASO-STAT3 Superior efficacy was observed in umASOs (Figures 6A and 6B). umASOs did not significantly suppress Stat3 in vivo, likely as a result of reduced stability in this setting. In contrast, high stability of mASOs correlated with robust targeting of Stat3 (Figures 6A and 6B). Both siRNA and mASO-targeted iExosomes showed similar potency in suppressing Stat3 expression in fibrotic liver (Figures 6A and 6B).

[0106] Repeated exposure to the hepatotoxin CCl4 induces highly visible inflammation and liver injury, which promotes progressive fibrosis and the accumulation of activated HSCs or myofibroblasts (Mederacke et al., 2013). Sirius red and collagen I staining of liver sections were applied to evaluate extracellular matrix (ECM) deposition. Results showed that iExo at 5 μg / 2 billion siRNA-STAT3 or iExo mASO-STAT3 The ECM was significantly reduced in mice treated with 1 μg / billion iExo siRNA-STAT3 or iExo mASO-STAT3 It was found that a moderate reduction in fibrosis was observed in mice treated with 5 μg / 2 billion iExo (Figures 6C-6H, Figures 5D and E). siRNA-STAT3 or iExo mASO-STAT3 Treatment with 5 μg / 2 billion iExo also significantly inhibited type I collagen deposition compared with mice treated with siRNA-STAT3 or mASO-STAT3 alone (Figure 6E-H). siRNA-STAT3 or iExo mASO-STAT3 In mice treated with 1 μg / billion iExo, the expression pattern of α-SMA, a well-established marker of activated HSCs (aHSCs), in fibrotic liver (Figure 4B) was significantly inhibited (Figure 6J). siRNA-STAT3 or iExo mASO-STAT3 No significant reduction was observed in mice treated with (Figure 5H).

[0107] In addition to STAT3 downregulation at the transcriptional level, 5 μg / 2 billion iExo siRNA-STAT3 or iExo mASO-STAT3 Treatment with 5 μg / 2 billion iExo resulted in significant transcriptional suppression of the α1 chain of type I collagen (Col1a1) and smooth muscle actin (Acta2) compared with treatment with siRNA-STAT3 or mASO-STAT3 (Figures 7A-7D). Liver function, as measured by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, was improved by treatment with 5 μg / 2 billion iExo. siRNA-STAT3 and iExo mASO-STAT3Mice treated with 5 μg / 2 billion iExo showed significant improvement, and to some extent with siRNA-STAT3 or mASO-STAT3 (Figures 7E-7H). siRNA-STAT3 and iExo mASO-STAT3 In the treatment, both ALT and AST levels were reduced to levels close to those in healthy control mice (Figures 7E-7H). Histopathological evaluation of CCl4-induced liver fibrosis showed hepatocellular degeneration, focal bridging necrosis, and considerable structural destruction of the lobule structure (Figures 7I-7K, untreated group). Compared with control treatments, including treatment with siRNA-STAT3 or mASO-STAT3, mice were treated with 5 μg / 2 billion iExo siRNA-STAT3 and iExo mASO-STAT3 The percentage of hepatocellular necrosis and degeneration was significantly reduced when 1 μg / billion iExo was administered (Figures 7I-7K). siRNA-STAT3 or iExo mASO-STAT3 A slight improvement in liver histopathology was observed in mice treated with iExo (Figures 5I-5K). siRNA-STAT3 or iExo mASO-STAT3 The treatment did not result in observable cytotoxicity in other organs (FIGS. 8A and 8B).

[0108] To confirm the effect of iExosome treatment on gene expression in target cells, we administered 5 μg / 2 billion iExosomes. siRNA-STAT3 and iExo mASO-STAT3 We performed RNA sequencing of whole livers from mice treated with iExo. Differentially expressed genes (DEGs) from each experimental group were plotted in a heatmap (Figure 9A). Significant expression changes for a given gene were defined as a ratio increase or decrease of more than 2-fold, with a corrected p-value of <0.05. From the heatmap and volcano plot, we found that iExo siRNA-STAT3 and iExo mASO-STAT3 Gene expression was found to be altered by treatment compared to the respective controls (Figures 9A-9C). siRNA-STAT3Liver transcriptome analysis from the iExo group revealed increased expression of 1,918 genes and decreased expression of 2,460 genes. mASO-STAT3 Liver transcript analysis of the group revealed increased expression of 2,140 genes and decreased expression of 2,021 genes (Figures 9B and 9C). Gene clusters involved in STAT3 signaling, including SPP1 and Thbs1 (Arriazu et al., 2017; Breitkopf et al., 2005), which play important roles in the development of liver fibrosis, were suppressed after iExosome treatment (Figure 9D). Commonly differentially deregulated genes associated with STAT3 signaling in liver fibrosis were related to ECM deposition and remodeling (Figure 9E). iExosome treatment suppressed the expression of classical fibrosis-related genes, including Col1a1, Acta2, Col1a2, and Vim (Figure 9E). This suggests that STAT3 is a critical mediator of liver fibrosis. Overrepresentation analysis demonstrated that DEGs were mainly enriched in ECM receptor interaction pathways (Figures ​(Figures9F​and9G), and downregulated genes were also enriched for pathways involved in xenobiotic metabolism by cytochrome P450, protein digestion and absorption, primary bile acid biosynthesis, linoleic acid metabolism, and chemical carcinogenesis (Figures9F​and9G). A similar set of altered downstream pathways was found in iExo siRNA-STAT3 Treatment and iExo mASO-STAT3 STAT3 mRNA was significantly increased in both IL-1 and IL-2 treatments (Figures 9F and 9G), supporting that this dataset is a useful tool for further investigating the STAT3 regulatory pathway in liver fibrosis. To further explore the association between STAT3 signaling and target ECM genes in liver fibrosis, a STAT3 mRNA-related ECM regulatory network was constructed based on DEGs. As shown in Figure 9H, the constructed network showed associations in 24 ECM-related genes. This network analysis identified STAT3 as a key node in the regelation of ECM deposition for future clinical treatment.

[0109] Taken together, these studies support previous reports of a critical role for STAT3 in promoting liver fibrosis in hepatocytes and stellate cells (Wang, Lafdil, Kong et al., 2011). STAT3 deregulation in liver fibrosis is complex, with protective functions in hepatocytes and profibrotic functions in aHSCs / myofibroblasts (Chakraborty et al., 2017; Wang, Lafdil, Kong et al., 2011; Wang, Lafdil, Wang et al., 2011). The antifibrotic outcome of the STAT3-targeted iExosome approach may reflect preferential uptake by aHSCs / myofibroblasts (Figure 5C). This is consistent with previous reports using adipose-derived mesenchymal stem cell-derived exosomes that were shown to block liver fibrosis by exosomal miR-181-5p, which suppresses STAT3 expression (Qu et al., 2017). Although various inhibitors have shown efficacy in mice, their specificity in targeting STAT3 remains to be validated (Beebe et al., 2018). The disclosed approach provides gene targeting specificity and may be combined with additional siRNA targets. Furthermore, the role of exosomes in therapeutic approaches for liver cancer is also emerging (Lou et al., 2020). Transformed hepatocytes in liver cancer rely on STAT3 expression (Wang, Lafdil, Wang et al., 2011; Jung et al., 2017), and iExosomes targeting STAT3 may provide benefits in limiting liver cancer progression. Previous studies in pancreatic cancer have shown the development of clinical grade exosomes with siRNA targeting of oncogenic Kras (Mendt et al., 2018; Kamerkar et al., 2017). This supports the possibility of clinical application for exosome-based STAT3 inhibition in liver fibrosis.

[0110] method HSC isolation and α-SMA staining Primary mouse HSCs were isolated from 8-week-old female Balb / c mice as previously described (Vinas et al., 2003). Primary mouse HSCs were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 20% ​​FBS (Gemini). Culture-activated primary HSCs (day 7) were immunostained overnight with Cy3-α-SMA antibody (Sigma, C6198, 1:200). Representative images at 200× magnification were captured with an Axiovert 200 and Axiocam HRc camera (Zeiss).

[0111] Real-time PCR analysis Total RNA was isolated from liver tissue using TRIzol™ (Invitrogen, 15596018) and cDNA was generated by High-Capacity cDNA Reverse Transcriptase Kit (Life Technology) according to the manufacturer's instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master Mix. Total amount of target gene mRNA was normalized to GAPDH expression. The following primer sequences: TIFF0007674354000001.tif48159 was used. Statistical analysis of variance was performed using ΔCt. Fold changes are shown and normalized to the control comparator, which was set to 1.

[0112] Exosome purification and electroporation Bone marrow-derived MSCs were obtained from the Cell Therapy Laboratory at the University of Texas MD Anderson Cancer Center and cultured in αMEM (Corning) supplemented with 20% FBS, 1% penicillin-streptomycin (Corning), 1% L-glutamine (Corning), and 1% non-essential amino acids (NEAA, Gibco). MSCs at passages 4–6 were used for exosome collection. Exosomes were purified by a differential centrifugation process according to our established protocols (Mendt et al., 2018; Kamerkar et al., 2017). Briefly, cells were grown to 70–80% confluency, washed with 1× PBS (Corning), and cultured in serum-free medium (αMEM with 1% penicillin-streptomycin, 1% L-glutamine, and 1% NEAA) for 48 h. The supernatant was collected, centrifuged at 800×g for 5 min, then 2,000×g for 10 min, and filtered through a 0.2 μm filter (Thermo Fisher). The filtered supernatant was centrifuged at 100,000×g for 3 h at 4°C in a SW 32 Ti rotor (Beckman). The supernatant was aspirated and the pellet was resuspended in 100 μL of 1×PBS. The concentration and size of the exosomes were verified by nanoparticle tracking analysis (NTA, NanoSight LM10, Malvern). Aliquots of 10 billion exosomes were stored at −80°C before use. The low-dose mixture contained 1 billion total exosomes according to NTA and 1 μg of siRNA or antisense oligo (ASO) in 100 μl of PlasmaLyte (Medline, BHL2B2544XH). In contrast, the high dose mixture contained 2 billion total exosomes and 5 μg siRNA or ASO in 100 μl PlasmaLyte. 400 μl of the RNAi-exosome mixture was loaded into a cuvette and then electroporated at 400 V, 125 μF, and ∞ ohms. The cuvette was immediately transferred to ice. siSTAT3 sequence:sense strand TIFF0007674354000002.tif4128, antisense TIFF0007674354000003.tif4128 (Sigma-Aldrich). The mASO Scrbl sequence is The umASO STAT3 sequence was: TIFF0007674354000005.tif4128. The mASO STAT3 sequence was TIFF0007674354000006.tif4154. "m" indicates 2'O-methoxy-ethyl base. * indicates phosphorothioate linkage. siCntrl was obtained from Sigma-Aldrich (SIC001, Sigma-Aldrich). ASO was synthesized by Integrated DNA Technologies, Inc. siRNAs with equal potential potency were designed to target mouse STAT3 and human STAT3. ASOs were also designed to target mouse STAT3 and human STAT3, but were potentially less potent against mouse STAT3 due to three nucleotide mismatches with the mouse sequence.

[0113] Visualization of exosome biodistribution in vivo Mice were treated with CCl4 to induce liver fibrosis (as detailed below). For biodistribution of MSC-derived exosomes, 8 × 10 exosomes labeled with XenoLight DiR (1,1'-dioctadecyltetramethyliodotricarbocyanine iodide, Perkin Elmer, Cat. 125964) were used as previously described (Mendt et al., 2018). 9 Purified exosomes were injected ip (100 μl) into healthy (sham) and fibrotic Balb / c mice. Diluted DiR (100 μl) was injected as a control. Mice were euthanized 6 hours after injection and various tissues (kidney, spleen, liver, pancreas, and intestine) were collected and immediately imaged. Briefly, 5 × 10 9Each MSC-derived exosome was labeled with 1 μM DiR, then incubated at 37°C for 1 h and 4°C for 15 min, and then dissolved in 10 ml of 1× PBS and washed by ultracentrifugation at 40,000 g for 3 h at 4°C (Mendt et al., 2018). 9 100 μl of DiR was resuspended in 100 μl of 1× PBS. For the control group, 1 μl of DiR was diluted in 11 ml of 1× PBS by ultracentrifugation for 3 h following the same procedure as above. The control sample (DiR only) was resuspended in 100 μl of 1× PBS. The fluorescence intensity of the mutant organs was imaged and quantified using an IVIS 200 small animal imaging system (PerkinElmer) equipped with a 780 nm Em filter and a 710 nm Ex filter.

[0114] Visualization of labeled siSTAT3 and mASO STAT3 localization in liver tissue Mice were treated with CCl4 to induce liver fibrosis (as detailed below). Prior to injection, MSC-derived exosomes were electroporated with AF647-tagged siRNA and mASO. These AF647-labeled iExosomes and AF647-tagged naked siRNA or mASO were then injected ip into WT Balb / c mice and fibrotic mice. Sectioned liver specimens were stained with DAPI and then mounted. Images were obtained by confocal laser scanning microscope (Zeiss LSM800), and then quantified by counting the number of nuclei of AF647-positive cells and dividing the number by the total number of nuclei. Three random fields per organ were captured (200×).

[0115] mouse Liver fibrosis was induced in Balb / c mice (8-week-old females purchased from Jackson laboratory). Liver injury was induced by ip injection of CCl4 (Sigma, 56-23-5) in 100 μl olive oil at a dose of 10% twice weekly for 37 days. Control mice received olive oil lacking CCl4 (Figure 5D). After 9 days, mice were randomized into 13 groups. Mice were also ip injected with 1 billion engineered exosomes at 1 μg siRNA / ASO or 2 billion engineered exosomes at 5 μg siRNA / ASO or siRNA-STAT3 / mASO-STAT3 alone in a 100 μl volume of PlasmaLyte (Medline) every other day. Mice were euthanized within 24 hours of the last iExosome injection. All protocols and procedures were approved by the MDACC Institute for Animal Care and Use Committee.

[0116] Sirius Red staining and quantification Livers were fixed in 10% neutral buffered formalin and paraffin embedded. 5 μm-thick paraffin sections were used for Sirius red staining. After rinsing three times and staining with Weigert's hematoxylin for 8 min, slides were counterstained with picrosirius red for 1 h. To quantify liver fibrosis, three independent Sirius red-stained sections from each mouse were analyzed using a counting grid. The percent area of ​​liver fibrosis was calculated as previously described (Whittaker et al., 1994).

[0117] immunohistochemistry Tissues were fixed overnight in 10% formalin, dehydrated, and paraffin embedded. 5 μm sections were then processed for analysis. Heat-mediated antigen retrieval was performed in 1 mM EDTA-TE (pH 9.0) for 1 h. Sections were blocked with 4% CWFS gelatin (Aurion) in TBS for collagen I (Southern Biotech, 1310-01, 1:200) staining 1 h prior to overnight incubation with primary antibody. After incubation with biotinylated anti-goat (Vector Laboratories, BA9500, 1:400), sections were reacted with ABC for 30 min and then developed with DAB according to the manufacturer's protocol.

[0118] Liver function evaluation Mouse blood was collected from the retro-orbital plexus. Serum was then immediately isolated by centrifugation at 6,000 rpm at 4°C for 10 min and stored at -80°C before use. Serum ALT and AST content were measured by the Department of Veterinary Pathology at MDACC.

[0119] Hematoxylin and eosin staining Liver tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections with a thickness of 5 μm were stained with hematoxylin and eosin (H&E). Five separate 200× fields were randomly selected for each slide, and the number of necrotic and degenerated hepatocytes was manually counted using the counting tool in Adobe Photoshop 7.0. Hepatocytes were defined as necrotic by the presence of clumped, densely stained cytoplasm and the absence of nuclei (Krishna et al., 2017). Hepatocyte degeneration was confirmed by the finding of cell swelling and enlargement, among others, as previously reported (Lackner et al., 2008).

[0120] RNA sequencing Total RNA was extracted from liver using TRIzol™ (Invitrogen, 15596018) and purified according to the manufacturer's instructions. RNA integrity was confirmed using the RNA 6000 Nano assay by the MDACC Sequencing and ncRNA Program core. Liver RNA sequencing was performed using Illumina TrueSeq stranded mRNAseq MDACC Sequencing and ncRNA Program core. Genomic mapping was performed using TopHat software (v2.0.9; available on the World Wide Web at ccb.jhu.edu / software / tophat / index.shtml). We used the Cufflinks algorithm to identify transcripts from the RNA-Seq data, and for gene expression profiling, we used DESeq2 (available on the World Wide Web at bioconductor.org / packages / release / bioc / html / DESeq2.html) to determine differentially expressed genes. False discovery rate (FDR) was performed to determine the significance threshold for p-values ​​for multiple testing. Significantly expressed genes were determined by an FDR of less than 0.05. Gene Set Enrichment Analysis (GSEA) and gene annotation were performed using WebGestalt 2019 ( Available on the World Wide Web at webgestalt.org / ) (He et al., 2019). The STAT-ECM gene interaction regulatory network was generated using NetworkAnalyst 3.0 (available on the World Wide Web at networkanalyst.ca / ) (Zhou et al., 2019).

[0121] statistical analysis Statistical analyses used are detailed in the figure briefs. Data are expressed as mean ± standard error of the mean. p<0.05 was considered statistically significant. Statistical significance was established using one-way ANOVA or unpaired two-tailed Student's t-test with Welch's correction using GraphPad Prism (GraphPad Software). The significance of statistical tests was reported in the graphs as follows: **** (p<0.0001), *** (p<0.001), ** (p<0.01), * (p<0.05), n.s.(p>0.05).

[0122] Example 5 – Col1a1 knockout in hepatic stellate cells Activated hepatic stellate cells or αSMA + Myofibroblasts were treated with a knockout mutation of the Col1a1 gene (Col1a1 cKO We generated mice carrying Col1a1. Liver fibrosis was induced in one group of mice by ip injection of CCl4, whereas a control group did not receive CCl4. Both the fibrotic and control ("healthy") groups were sacrificed and analyzed. Compared to wild-type (WT), Col1a1 cKO A significant reduction in collagen I and liver fibrosis was observed in Col1a1 mice (Figure 10A-F). Compared to WT, Col1a1 mice had liver fibrosis. cKO Many of the global expression patterns associated with liver fibrosis were significantly ameliorated in mice (Figures 10G and 10H).

[0123] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with respect to certain specific embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or order of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. For example, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0124] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007674354000007.tif201160TIFF0007674354000008.tif238160TIFF0007674354000009.tif136160

Claims

1. A pharmaceutical composition comprising lipid-based nanoparticles containing a STAT3 inhibitory RNA that hybridizes to a STAT3 polynucleotide, for use in treating fibrosis or a fibrosis-related condition in a patient in need thereof, wherein the inhibitory RNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5.

2. The pharmaceutical composition of claim 1, wherein the lipid-based nanoparticles comprise CD47 and / or a growth factor on their surface.

3. The pharmaceutical composition of claim 1 or 2, wherein the lipid-based nanoparticle is an exosome or a liposome.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the inhibitory RNA knocks down expression of STAT3 protein.

5. A pharmaceutical composition according to any one of claims 1 to 4, further comprising an excipient.

6. A pharmaceutical composition according to any one of claims 1 to 5, formulated for parenteral administration.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, further comprising an antibacterial agent, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

8. A pharmaceutical composition described in any one of claims 1 to 7, wherein administration of the pharmaceutical composition results in delivery of the inhibitory RNA to cells in the patient.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the fibrosis is liver fibrosis, pulmonary fibrosis, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis (IPF), or radiation-induced lung injury.

10. A pharmaceutical composition according to any one of claims 1 to 8, wherein the fibrosis is liver fibrosis.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, which is administered by systemic administration and / or is administered more than once.

12. The pharmaceutical composition of any one of claims 1 to 11, further comprising administering at least a second therapy to said patient.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the patient is a human.

14. The pharmaceutical composition of claim 13, wherein the lipid-based nanoparticles are exosomes and the exosomes are autologous to the patient.

15. The pharmaceutical composition of claim 13 or 14, wherein the lipid-based nanoparticles are exosomes, and the exosomes are obtained from mesenchymal cells.

16. The pharmaceutical composition of any one of claims 13 to 15, wherein the lipid-based nanoparticles are exosomes, and the exosomes are from blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum from a patient.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein administration of the pharmaceutical composition reduces Col1a1, Acta2, Col1a2, or Vim expression in liver cells of the patient.

18. The pharmaceutical composition of claim 17, wherein the hepatic cells are hepatic stellate cells or activated hepatic stellate cells.

19. A method for preparing a therapeutic composition, comprising the step of introducing a STAT3 inhibitory RNA that hybridizes to a STAT3 polynucleotide into a lipid-based nanoparticle, wherein the inhibitory RNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5.

20. The method of claim 19, wherein the lipid-based nanoparticles comprise CD47 and / or a growth factor on their surface.

21. The method of claim 19 or 20, wherein the lipid-based nanoparticle is an exosome or a liposome.

22. A method described in any one of claims 19 to 21, wherein the inhibitory RNA knocks down expression of STAT3 protein.

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