Use of exosomes for targeted delivery of therapeutic agents

Abstract

Provided herein is a method for using exosomes, which function similarly to minicells, to deliver therapeutic substances to diseased or injured cells. In particular, the exosomes can be targeted to specific body regions using a growth factor gradient. The gradient also serves to induce protein expression within the exosomes from transfected nucleic acids in the desired target.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 649,057, filed March 28, 2018, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. This ASCII copy was created on March 21, 2019, is named UTFC.P1363WO_ST25.txt, and is 3 kilobytes in size.

[0003] 1. Field The present invention relates generally to the fields of biology, medicine, and oncology. More particularly, the present invention relates to the use of exosomes for targeted delivery of therapeutic agents to diseased or disordered cells. [Background technology]

[0004] 2. Description of Related Technology Exosomes are small extracellular vesicles (EVs) with a lipid bilayer that contain proteins and polynucleotides, including messenger RNA (mRNA), non-coding RNA, and double-stranded genomic DNA (Kalluri, 2016; Raposo and Stoorvogel, 2013). First discovered as a by-product of reticulocyte differentiation (Harding et al., 1984; Raposo and Stoorvogel, 2013), exosomes are now generally accepted to be secreted by virtually all mammalian cells and found in all body fluids (El-Andaloussi et al., 2013; Kalluri, 2016).

[0005] Exosomes are part of a larger group of extracellular vesicles that also includes microvesicles and apoptotic bodies (Colombo et al., 2014). Among extracellular vesicles, exosomes are distinguished by their unique biogenesis, typically via the endocytic pathway. Endocytic vesicles mature into late endosomes, also known as multivesicular bodies, which contain numerous intracellular vesicles (ILVs) generated by invagination of the endosomal membrane. Upon appropriate fusion of these multivesicular bodies with the plasma membrane, exosomes are released into the extracellular space and enter the circulation (Bastos et al., 2017; Colombo et al., 2014). As a result of their endocytic origin, the exosome membrane has a polarity similar to that of the plasma membrane and contains membrane proteins anchored with an intracellular domain facing the lumen and an extracellular domain facing the extracellular space. While the protein content of exosomes varies depending on their cellular origin, some proteins generally appear to be enriched. These include members of the tetraspanin family, as well as components of the endocytic and ILV maturation pathways, such as Rab proteins and members of the ESCRT complex. Interestingly, various proteomic studies using exosomes derived from many different cell types have identified many components related to the protein translation machinery, such as eukaryotic initiation factors, ADP-ribosylation factors, and ribosomal proteins (Pisitkun et al., 2004; Valadi et al., 2007). Furthermore, proteomic analysis suggests that some of the transcription and translation regulators identified in exosomes are delivered to recipient cells, altering gene and protein expression patterns (Ung et al., 2014).

[0006] Among the proteins commonly identified in exosomes are growth factor receptors such as epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB family of growth factor receptors, which also includes HER2, HER3, and HER4 (Seshacharyulu et al., 2012). Upon binding to a ligand such as epidermal growth factor (EGF), the receptor dimerizes to form homodimers or heterodimers with other members of the ErbB family (Seshacharyulu et al., 2012). This dimerization activates the receptor's intrinsic kinase activity, thereby autophosphorylating various critical tyrosine residues on the cytoplasmic domain. This autophosphorylation recruits various adaptor proteins containing SH2 and PTB (phosphotyrosine-binding) domains, such as Shc and GRB2, which mediate various downstream signaling activities, including the synthesis of associated proteins (Normanno et al., 2006; Tomas et al., 2014). Phosphorylated EGFR is eventually ubiquitinated and transported to the endosomal pathway, where it is recycled back to the membrane, or it remains in the late endosomal pathway leading to incorporation into multivesicular bodies or lysosomal degradation (Tomas et al., 2014). Because multivesicular bodies give rise to exosomes, post-phosphorylation recycling of EGFR (and other growth factors) likely contributes to membrane localization within these extracellular vesicles.

[0007] EGFR signaling has been shown to be important for the progression of various malignancies, including glioblastoma, lung cancer, and breast cancer (Lim et al., 2016; Liu et al., 2012; Masuda et al., 2012; Morgillo et al., 2016; Westphal et al., 2017; Zhang et al., 2013). Perhaps for this reason, most studies of EGFR in exosomes have been conducted in the context of cancer development. EGFR signaling has been specifically linked to the cellular uptake and secretion patterns of exosomes derived from various sources. In mantle cell carcinoma cells, incubation with gefitinib (an EGFR inhibitor) has been shown to dramatically decrease exosome uptake rates (Hazan-Halevy et al., 2015). Treatment of lung cancer cells with gefitinib increases exosome secretion, which mediates horizontal transfer of cisplatin resistance (Li et al., 2016). EGFR transmission via cancer cell-derived exosomes has also been shown to alter microenvironmental components such as endothelial cells and T cells (Al-Nedawi et al., 2009; Huang et al., 2013). More recently, exosomes derived from gastric cancer cells containing EGFR were shown to deliver EGFR to liver stromal cells and mediate metastasis (Zhang et al., 2017). Finally, exosomes derived from breast cancer cells were shown to contain functional phosphorylated EGFR and translocate it to monocytes, where it mediates monocyte survival by activating the ERK pathway (Song et al., 2016).

[0008] Delivery of EGFR and members of the protein translation machinery by exosomes appears to have clear biological importance in the context of cell-cell communication; moreover, these properties may be exploited to target therapeutic delivery to certain tissues and to induce therapeutic protein production at the desired delivery site. Summary of the Invention

[0009] overview Herein, growth factor stimulation induced protein synthesis in exosomes. Exosomes containing DNA, RNA, and proteins can respond to biological stimuli and initiate properties such as migration, proliferation, initiation of signaling networks / cascades, transcription, and protein translation. Thus, in one embodiment, exosomes capable of functioning similarly to minicells are provided herein. As further discussed below, these minicell-like exosomes can be used in numerous therapeutic approaches to treat various diseases and / or disorders.

[0010] In one aspect, provided herein is a method of treating a disease or disorder in a patient in need thereof, the method comprising the steps of: (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (c) administering the transfected exosomes to the patient; and (d) providing a growth factor gradient to a site of the disease or disorder to attract the exosomes to the site and stimulate production of the therapeutic protein at the site, thereby treating the disease in the patient.

[0011] In some aspects, the method is further defined as a method for administering therapeutic protein to diseased cells in patients.In some aspects, the exosome obtained in step (a) is obtained from the body fluid sample obtained from patients.In some aspects, the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, eye exudate / tear or serum.In some aspects, the nucleic acid is mRNA, plasmid or cDNA.

[0012] In some aspects, the disease or disorder is cancer, injury, autoimmune disorder, neurological disorder, gastrointestinal disorder, infectious disease, kidney disease, cardiovascular disorder, eye disorder, skin disease or disorder, urogenital disorder, or bone disease or disorder. In certain aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In some aspects, the site of the disease or disorder is a tumor. In some aspects, the cancer is metastatic. In certain aspects, the site of the disease or disorder is a metastatic nodule.

[0013] In some aspects, the therapeutic protein is a kinase, phosphatase, or transcription factor.In certain aspects, the therapeutic protein corresponds to the wild-type version of a protein that is mutated or inactivated in cells at the site of disease or disorder.In certain aspects, the therapeutic protein corresponds to the dominant-negative version of a protein that is overactive in cells at the site of disease or disorder.In certain aspects, the disease or disorder is cancer, and the therapeutic protein is a tumor suppressor.In some aspects, the exosome comprises CD47 on its surface.In some aspects, the transfection comprises electroporation.

[0014] In some aspects, the method further comprises administering at least a second therapy to the patient, hi some aspects, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

[0015] In one aspect, there is provided a method of treating a disease or disorder in a patient in need thereof, the method comprising the steps of: (a) obtaining exosomes having growth factor receptors on their surface; (b) transfecting the exosomes with a therapeutic agent; (c) administering the transfected exosomes to the patient; and (d) providing a growth factor gradient to a site of the disease or disorder to attract the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the disease in the patient.

[0016] In some aspects, the method is further defined as a method for administering a therapeutic substance to diseased cells in a patient.In some aspects, the exosome obtained in step (a) is obtained from a body fluid sample obtained from a patient.In certain aspects, the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum.

[0017] In some aspects, the therapeutic substance is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. In certain aspects, the antibody binds to an intracellular antigen. In certain aspects, the antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. In certain aspects, the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. In certain aspects, the gene editing system is a CRISPR / Cas system. In certain aspects, the therapeutic protein is a kinase, phosphatase, or transcription factor. In certain aspects, the therapeutic protein corresponds to a wild-type version of a protein that is mutated or inactivated in cells at the site of a disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant-negative version of a protein that is overactive in cells at the site of a disease or disorder. In some aspects, the small molecule drug is an imaging agent.

[0018] In some aspects, the disease or disorder is cancer, injury, autoimmune disorder, neurological disorder, gastrointestinal disorder, infectious disease, kidney disease, cardiovascular disorder, eye disorder, skin disease or disorder, genitourinary disorder, or bone disease or disorder. In certain aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In some aspects, the site of the disease or disorder is a tumor. In some aspects, the cancer is metastatic. In some aspects, the site of the disease or disorder is a metastatic nodule. In some aspects, the disease or disorder is cancer, and the therapeutic protein is a tumor suppressor. In some aspects, the disease or disorder is cancer, and the therapeutic substance is an inhibitory RNA that targets an oncogene.

[0019] In some aspects, the exosome comprises CD47 on its surface. In some aspects, the transfection comprises electroporation. In some aspects, the method further comprises administering at least a second therapy to the patient. In some aspects, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

[0020] In a further aspect, the exosomes for use according to the above embodiment are contained in a tissue scaffold matrix. For example, such a matrix may be a synthetic matrix, for example, a matrix that can be degraded or absorbed by tissue. In a further aspect, the matrix may be a living tissue matrix. In some aspects, the exosomes of the above embodiment are cultured within the matrix.

[0021] 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 component resulting from unintentional contamination of the composition is significantly less than 0.01%. Most preferred are compositions in which the amount of the named component is undetectable using standard analytical methods.

[0022] 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.

[0023] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to refer to mutually exclusive alternatives, but this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second, or more.

[0024] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0025] [The present invention 1001] 1. A method of treating a disease or disorder in a patient in need thereof, comprising: (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (c) administering the transfected exosomes to a patient; (d) providing a growth factor gradient to the site of the disease or disorder to attract the exosomes to the site and stimulate production of the therapeutic protein at the site, thereby treating the disease in the patient. The method comprises: [The present invention 1002] The method of claim 1001 further defined as a method of administering a therapeutic protein to diseased cells in a patient. [The present invention 1003] 1001. The method of claim 1001, wherein the exosomes obtained in step (a) are obtained from a body fluid sample obtained from said patient. [The present invention 1004] 1004. The method of claim 1003, wherein said body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. [The present invention 1005] 1001. The method of claim 1001, wherein said nucleic acid is mRNA. [The present invention 1006] 1001. The method of claim 10, wherein said nucleic acid is a plasmid. [The present invention 1007] 1001. The method of claim 1001, wherein said nucleic acid is cDNA. [The present invention 1008] 1001. The method of claim 1001, wherein said disease or disorder is cancer, an injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an eye disorder, a skin disease or disorder, a genitourinary disorder, or a bone disease or disorder. [The present invention 1009] 1008. The method of claim 1008, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1010] The method of claim 1008, wherein the site of the disease or disorder is a tumor. [The present invention 1011] The method of claim 1008, wherein the cancer is metastatic. [The present invention 1012] 10. The method of claim 10, wherein the site of the disease or disorder is a metastatic nodule. [The present invention 1013] 1001. The method of claim 1001, wherein said therapeutic protein is a kinase, phosphatase, or transcription factor. [The present invention 1014] 1001. The method of claim 1001, wherein said therapeutic protein corresponds to a wild-type version of a protein that is mutated or inactivated in cells at the site of said disease or disorder. [The present invention 1015] 1001. The method of claim 1001, wherein said therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in cells at the site of said disease or disorder. [The present invention 1016] 1001. The method of claim 1001, wherein said disease or disorder is cancer and said therapeutic protein is a tumor suppressor. [The present invention 1017] 1001. The method of claim 1001, wherein said exosomes comprise CD47 on their surface. [The present invention 1018] 1001. The method of claim 1001, wherein the transfection comprises electroporation. [The present invention 1019] 1002. The method of claim 1001, further comprising administering at least a second therapy to said patient. [The present invention 1020] 1020. The method of claim 1019, wherein said second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. [The present invention 1021] 1001. The method of claim 1001, wherein the exosomes are contained in a tissue scaffold matrix. [The present invention 1022] 1. A method of treating a disease or disorder in a patient in need thereof, comprising: (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with a therapeutic agent; (c) administering the transfected exosomes to a patient; (d) providing a growth factor gradient to the site of the disease or disorder to attract the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the disease in the patient. The method comprises: [The present invention 1023] The method of claim 1022 further defined as a method of administering a therapeutic agent to diseased cells in a patient. [The present invention 1024] The method of claim 1022, wherein the exosomes obtained in step (a) are obtained from a body fluid sample obtained from said patient. [The present invention 1025] 1025. The method of claim 1024, wherein said body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. [The present invention 1026] 1023. The method of claim 1022, wherein the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. [The present invention 1027] 1027. The method of claim 1026, wherein said antibody binds to an intracellular antigen. [The present invention 1028] 1027. The method of claim 1026, wherein said antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. [The present invention 1029] 1027. The method of claim 1026, wherein said inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. [The present invention 1030] 1026. The method of claim 1026, wherein the gene editing system is a CRISPR / Cas system. [The present invention 1031] 1027. The method of claim 1026, wherein said therapeutic protein is a kinase, phosphatase, or transcription factor. [The present invention 1032] 1027. The method of claim 1026, wherein said therapeutic protein corresponds to a wild-type version of a protein that is mutated or inactivated in cells at the site of said disease or disorder. [The present invention 1033] 1027. The method of claim 1026, wherein said therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in cells at the site of said disease or disorder. [The present invention 1034] 1027. The method of claim 1026, wherein said small molecule drug is an imaging agent. [This invention 1035] 1023. The method of claim 1022, wherein said disease or disorder is cancer, an injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an eye disorder, a skin disease or disorder, a genitourinary disorder, or a bone disease or disorder. [The present invention 1036] 1035. The method of claim 1035, wherein said cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [This invention 1037] The method of claim 1035, wherein the site of the disease or disorder is a tumor. [The present invention 1038] The method of claim 1035, wherein the cancer is metastatic. [This invention 1039] The method of claim 1038, wherein the site of the disease or disorder is a metastatic nodule. [The present invention 1040] 1023. The method of claim 1022, wherein said disease or disorder is cancer and said therapeutic protein is a tumor suppressor. [This invention 1041] 1023. The method of claim 1022, wherein said disease or disorder is cancer and said therapeutic agent is an inhibitory RNA that targets an oncogene. [The present invention 1042] 1023. The method of claim 1022, wherein the exosome comprises CD47 on its surface. [This invention 1043] The method of claim 1022, wherein the transfection comprises electroporation. [This invention 1044] 1023. The method of claim 1022, further comprising administering at least a second therapy to said patient. [This invention 1045] 104. The method of claim 1044, wherein said second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. [The present invention 1046] The method of claim 1022, wherein the exosomes are contained in a tissue scaffold matrix. 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 preferred embodiments of the present invention, are given by way of example only, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0026] 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]Figures 1A-E. EGFR phosphorylation in exosomes. Figure 1A - Immunoblot of EGFR expression on exosomes obtained from various human and mouse cell lines. The exosome marker CD81 was used as a loading control to confirm the exosomal origin of the protein extracts. Figure 1B - Immunoblot showing EGFR phosphorylation on exosomes derived from MDA-MB-231 cells but not MCF10A cells after incubation with 500 ng / ml rhEGF at 37°C for 15 minutes. Phosphorylation levels were detected using an antibody specific for the Tyr1068 residue of EGFR. EGFR levels were shown as a loading control to confirm differences in phosphorylation. Band densitometry quantification was performed using ImageJ software. Figure 1C - Immunoblot showing the presence of EGFR adaptor proteins Shc and GRB2, as well as high levels of phosphorylated ERK protein, in exosomes derived from MDA-MB-231 cells stimulated and unstimulated with rhEGF for 15 minutes at 37°C. The exosome marker CD81 was used as a loading control to confirm the exosomal origin of the protein extracts. Figure 1D - GRB immune complexes were obtained using a GRB2-specific antibody from protein extracts of MDA-MB-231 exosomes incubated and unincubated with 500 ng / ml rhEGF for 15 minutes at 37°C. Immunoblot analysis of the immune complexes demonstrates that GRB2 binds to EGFR only upon rhEGF stimulation. A nonspecific isotype control IgG was used as a negative control for GRB2 pulldown. Equal amounts of stimulated and unstimulated extracts were probed for β-actin as input controls. Figure 1E - A similar experiment repeated twice using Shc antibodies for pull-down experiments also demonstrated binding to EGFR only after stimulation with 500 ng / ml rhEGF for 15 minutes at 37°C. A nonspecific isotype control IgG was used as a negative control for Shc pull-down. Equal amounts of stimulated and unstimulated extracts were probed for b-actin as input controls. [Figure 2]Figures 2A-G. EGFR phosphorylation alters exosomal content. Figure 2A - Luciferase-based ATP quantification assay performed on protein extracts obtained from unstimulated exosomes or exosomes stimulated with rhEGF for 15 minutes at 37°C. Luminescence derived from luciferin was measured using a plate reader and expressed in arbitrary units. Significance was determined using the Mann-Whitney test (n=4). Figure 2B - Immunoblot analysis of exosomes derived from MDA-MB-231 cells incubated with 500 ng / ml rhEGF for 48 hours at 37°C. Increased pEGFR and GRB2 levels are shown compared to unstimulated exosomes. Figure 2C - Cellular component binding analysis of mass spectrometry data obtained for unstimulated MDA-MB-231 exosomes or MDA-MB-231 exosomes incubated with 500 ng / ml EGF for 48 hours at 37°C. A list of significant proteins identified for stimulated and unstimulated exosomes was obtained and used as input for the open-access FunRich functional enrichment analysis tool to identify the subcellular origin of the identified proteins. Figure 2D - Venn diagram illustrating the overlap of proteins identified in control MDA-MB-231 exosomes and exosomes incubated with 500 ng / ml rhEGF at 37°C for 48 hours. Figure 2E - Individual EGFR and GRB2 protein scores obtained from mass spectrometry analysis of MDA-MB-231 exosomes incubated with 500 ng / ml rhEGF for 48 hours or not. Figure 2F - BCA analysis of protein extracts obtained from control MDA-MB-231 exosomes and exosomes incubated with 500 ng / ml rhEGF at 37°C for 48 hours. Significance was determined using the Mann-Whitney test (n=3).Figure 2G - Immunoblot analysis of β-actin expression in protein extracts obtained from control MDA-MB-231 exosomes and exosomes incubated with 500ng / ml and 1000ng / ml rhEGF for 48 hours at 37°C (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). [Figure 3]Figure 3A-F. Exosomes contain functional components for transcription and translation. Figure 3A - Ultra-performance liquid chromatography-mass spectrometry (UPLCMS) was used to detect free amino acids in MCF10A-, MDA-MB-231-, HDF-, E10-, and NIH-3T3-derived exosomes. Data are displayed as a heatmap using normalized signal intensity (log10). As indicated in the color legend, hotter colors correspond to higher intensity levels. Figure 3B - Immunoblot of eIF4A1, eIF3A, and eIF1A in exosomal protein extracts obtained from E10-, NIH-3T3-, MCF10A-, HDF-, and MDA-MB-231-derived exosomes. CD9 was used as a loading control. Figure 3C - In vitro translation assay using protein lysates from MCF10A- and MDA-MB-231-derived exosomes incubated with pEMT7-GFP cDNA expression plasmid. Protein lysates obtained from cells were used as controls. Figure 3D - Immunoblot analysis of RNA polymerase II in exosomal protein extracts. CD9 was shown as a loading control. Figure 3E - Autoradiography of exosomes derived from MDA-MB-231 and E10 cells cultured in the presence of 35S-methionine. Exosomes alone and exosomes cultured in the presence of 35S-methionine and cycloheximide were used as controls. Figure 3F - BCA quantification of protein extracts obtained from exosomes immediately after isolation or after 24 and 48 hours of incubation in cell-free conditions. Significance was determined using one-way ANOVA followed by Tukey's multiple comparison test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, n=3). [Figure 4]Figures 4A-J. Exosomes synthesize new proteins through DNA transcription and cap-dependent mRNA translation. Figure 4A - qPCR analysis of GFP mRNA levels in exosomes isolated from MDA-MB-231 cells and isolated from non-electroporated, mock-electroporated, or electroporated with the pCMV-GFP plasmid with or without α-amanitin. Expression levels were normalized to GAPDH. Figure 4B - Transmission electron microscopy images of immunogold labeling with an anti-GFP antibody of exosomes electroporated with the GFP plasmid and incubated in cell-free conditions for 48 hours (bottom row). Secondary antibody alone was used as a negative control (top row). Gold particles are shown as black dots. Scale bar, 100 nm. Figure 4C - Immunoblot of GFP protein expression in exosomes electroporated with the pCMV-GFP plasmid and incubated at 37°C for 12 hours, 2 days, or 1 week. Exosomes alone and mock-electroporated exosomes were used as negative controls. The exosome marker TSG101 was used as a loading control for the presence of exosomes. Figure 4D - Immunoblot of GFP protein expression in exosomes electroporated with GFP plasmid and incubated for several time periods up to one month. Non-electroporated exosomes were used as a negative control. The exosome marker CD63 was used as a loading control for the presence of exosomes. Figure 4E - Immunoblot of GFP protein expression in exosomes electroporated with GFP plasmid immediately after isolation (0 h) or after incubation in cell-free conditions (24 h) and cultured as described above. Mock-electroporated exosomes were used as a negative control. The exosome marker TSG101 was used as a loading control for the presence of exosomes. Figure 4F - Immunoblot of GFP protein expression in exosomes electroporated with pCMV-GFP plasmid and cultured with the translation inhibitor cycloheximide. Exosomes only and mock-electroporated exosomes were used as negative controls.TSG101 was used as a loading control for the presence of exosomes.Band densitometry was performed using ImageJ software. Figure 4G - Immunoblot of GFP protein expression in exosomes electroporated with GFP plasmid and incubated with the transcription inhibitor α-amanitin. Exosomes alone and mock-electroporated exosomes were used as negative controls. TSG101 was used as a loading control for the presence of exosomes. Band densitometry was performed using ImageJ software. Figure 4H - Schematic diagram of the bicistronic plasmid (pCDNA3-rLuc-polIRESfLuc) used as a reporter for cap-dependent or cap-independent translation. Figure 4I - Renilla (r-Luc) and firefly luciferase (f-Luc) activities measured by bioluminescence after 48 hours of incubation of exosomes electroporated with the bicistronic plasmid. Non-electroporated exosomes were used as a negative control. FIG. 4J - Luminescence counts measured from exosomes electroporated with or without a plasmid carrying firefly luciferase expressed under the CMV promoter and then incubated for 48 hours. [Figure 5]Figures 5A-E. Protein translation in exosomes produces functional proteins and is increased by growth factor stimulation. Figure 5A - Confocal microscopy showing the presence of GFP in electroporated MCF10A cells and MCF10A cells incubated with MDA-MB-231-derived exosomes pretreated with cycloheximide, electroporated with pCMV-GFP plasmid, and preincubated for 48 hours. MCF10A cells treated with non-electroporated MDA-MB-231-derived exosomes were used as a negative control. Figure 5B - Immunoblot analysis of GFP expression in protein lysates derived from MDA-MB-231-derived exosomes electroporated with p53-GFP expression plasmid. Non-electroporated exosomes were used as a negative control. TSG101 was used as a loading control for the presence of exosomes. Figure 5C - p21 mRNA expression in MDA-MB-231 cells treated with mock-electroporated MDA-MB-231-derived exosomes or exosomes electroporated with p53-GFP plasmid with and without cycloheximide. Expression levels were normalized to the housekeeping gene GAPDH. Figure 5D - Immunoblot of exosomes isolated from MDA-MB-231 cells incubated with 100 μg / ml of the translation inhibitor cycloheximide. Exosome lysates were examined for β-actin and GAPDH. Band densitometry quantification was performed using ImageJ software. Figure 5E - Immunoblot of GFP protein expression in exosomes electroporated with pCMV-GFP plasmid and then incubated for 48 hours at 37°C in the presence of different concentrations of rhEGF. Mock-electroporated exosomes and exosomes without rhEGF incubation were shown as negative controls. The exosome marker CD81 was used as a loading control. Band densitometry quantification was performed using ImageJ software. [Figure 6]Figures 6A-C. Exosomes derived from MDA-MB-231 cells exhibit chemotaxis toward a growth factor gradient. Figure 6A - Schematic diagram showing the setup of the exosome retrograde migration assay. Briefly, 10 x 10 exosomes isolated from MDA-MB-231 cells were placed in the bottom well of a Corning Transwell® system. HTS Transwell® inserts with 400 nm pores were placed on top of exosome suspensions containing either PBS, 20% FBS, or 10,000 ng / ml rhEGF and incubated at 37°C. To assess exosome motility, the number of exosomes on the top insert was measured using a Nanosight NTA after various time points. Figures 6B and 6C - Quantification of MDA-MB-231 exosomes on the top insert of the retrograde migration assay by Nanosight NTA after 4 hours (Figure 6B) and 24 hours (Figure 6C) of incubation at 37°C. Significance was determined using one-way ANOVA followed by the Newman-Keuls multiple comparison test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, n=3). [Figure 7]Figures 7A-D. Tumor-bearing mice show increased protein synthesis in the presence of delivered exosomes. Figure 7A - Schematic illustrating the experimental design of the in vivo translation experiment. Briefly, female Balb / C mice were orthotopically injected with 4T1 tumors and allowed to grow to 500 mm3. Following this, the mice were injected with 30 billion MDA-MB-231 exosomes electroporated with the pCMV-mCherry plasmid. Twelve hours after exosome injection, the mice were euthanized and serum was collected for exosome extraction. Figure 7B - Tumor growth in mice injected with 4T1 tumors and electroporated exosomes or 4T1 tumors alone showed comparable growth rates. Figure 7C - Nanosight NTA analysis of exosomes extracted from the serum of healthy mice injected with electroporated exosomes, and 4T1 tumor-bearing mice injected with electroporated exosomes and without exosome injection. All exosomes show a similar size peak around 100 nm. Figure 7D - Nanosight NTA quantification of serum exosomes shown in Figure 7C. There was no significant difference in the amount of exosomes obtained from the serum of different animals, although there was a trend toward more exosomes in 4T1 tumor-bearing mice injected with MDA-MB-231 electroporated exosomes. [Figure 8]Figures 8A-G. Characterization of exosomes. Figure 8A - Nanoparticle tracking analysis of exosomes collected from MDA-MB-231 cells obtained using Nanosight NTA 2.1 analysis software. The left graph shows the size distribution of particles in solution. It shows an average size of 104 nm, with no peak at larger sizes. The right graph shows the size and concentration distribution of particles in solution. Figure 8B - Atomic force microscope image of exosomes (left image). The right graph shows the particle distribution in the analyzed area. Figure 8C - Transmission electron micrograph of MDA-MB-231 exosomes. Scale bar - 100 nm. Figure 8D - Transmission electron micrograph of immunogold-labeled MDA-MB-231 exosomes using anti-CD9 antibody. Gold particles are shown as black dots. Scale bar - 100 nm. Figure 8E - Immunoblot analysis of exosome markers CD9, CD63, and TSG101 in protein extracts of exosomes obtained from different cell lines. Figure 8F - Imaging flow cytometry analysis of exosomes derived from MDA-MB-231 cells bound to 0.4 μm beads using antibodies for the markers CD9, CD81, CD82, and CD63. Figure 8G - Representative images of LB culture plates incubated with E. coli and either MDA-MB-231 (top) or MCF10A (bottom) exosomes. The E. coli-inoculated side (left) showed colony formation, while the exosome-inoculated side (right) showed no colony formation. [Figure 9]Figures 9A-B. EGFR phosphorylation and downstream biological activity in exosomes derived from MDA-MB-231 cells. Figure 9A - Immunoblot of protein extracts obtained from MDA-MB-231 cells and probed for p-EGFR and GRB2. β-actin was used as a loading control. Figure 9B - Immunoblot of immune complexes obtained using anti-EGFR antibody pull-down of protein lysates derived from MDA-MB-231 exosomes incubated with 500 ng / ml EGFR for 15 minutes at 37°C or not. Immune complexes were probed for GRB2. A nonspecific isotype control IgG was used as a negative control for GRB2 pull-down. Equal amounts of stimulated and unstimulated extracts were probed for β-actin as input controls. [Figure 10] Proteomic analysis of exosomes derived from various cell types. Heatmap showing the binary identification of every protein in a protein translation pathway in the Reactome (Croft et al., 2014) database in mass spectrometry data from mouse liver cells (Valadi et al., 2007), mouse fibroblasts (Luga et al., 2012), human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et al., 2013), human thymus tissue (Skogberg et al., 2013), and human urine (Gonzales et al., 2009). Black indicates the protein's presence in each dataset, while white indicates its absence. The summary column indicates how ubiquitous each protein is across all datasets analyzed. Warm colors indicate widespread distribution among different exosome types. [Figure 11]Figure 11A-B. Proteomic analysis of exosomes from various sources. Figure 11A - Heatmap showing the number of proteins identified in mass spectrometry data from mouse liver cells (Valadi et al., 2007), mouse fibroblasts (Luga et al., 2012), human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et al., 2013), human thymus tissue (Skogberg et al., 2013), and human urine (Gonzales et al., 2009) associated with various pathways related to protein translation in the Reactome (Croft et al., 2014) database. Warmer colors represent a higher number of identified proteins per pathway. FIG. 11B - Heatmap depicting protein scores of proteins associated with protein translation identified in mass spectrometry from exosomes isolated from HDF, NIH3T3, MDA-MB231, MCF10A, and E10 cells. Warmer colors represent higher protein scores. [Figure 12]Figures 12A-C. Exosomes contain nucleic acids and proteins associated with the protein translation machinery. Figure 12A - 18S and 28S rRNA were quantified by qPCR using RNA extracted from exosomes of NIH-3T3, E10, 67NR, 4T1, HDF, MCF10A, MCF7, and MDA-MB-231 cell lines. rRNA expression levels were normalized to U6 snRNA expression. The bars in each group represent NIH 3T3, E10, 67NR, 4T1, HDF, MCF10A, MCF7, and MDA-MB-231 from left to right. Figure 12B - Using RNA extracted from 4T1 exosomes and cells, the presence of tRNAMet, tRNAGly, tRNALeu, tRNASer, and tRNAVal was identified by digital qPCR. The bars in each group represent Leu, Met, Val, Ser, and Gly from left to right. Figure 12C - Immunoprecipitation of eIF4A1 demonstrating the presence of eIF3A MCF10A and MDA-MB-231 derived exosomes. MB231 and MCF10A cell lysates were used as positive controls. The exosome marker CD82 was used as a loading control. [Figure 13]Figures 13A-E. DNA transcription and mRNA translation in exosomes derived from MCF10A and MDA-MB-231 cells. Figure 13A - Immunoblot of GFP protein expression in exosomes isolated from MCF10A cells electroporated with pCMV-GFP plasmid and incubated at 37°C for various periods. Exosomes only and mock-electroporated exosomes were used as negative controls. CD63 was used as a loading control to confirm the presence of exosomes. Figure 13B - Plot illustrating the amount of green exosomes detected by NanoSight in MCF10A-derived exosomes electroporated with GFP plasmid. MCF10A-derived exosomes, MCF10A-derived mock-electroporated exosomes, and exosomes electroporated with α-amanitin and cycloheximide were used as negative controls. Figure 13C - Flow cytometry analysis of beads attached to exosomes after electroporation with GFP plasmid using high voltage. The percentage of beads with green fluorescent signal is shown. Figure 13D - Immunoblot of ovalbumin protein levels in MDA-MB-231 exosomes electroporated with pCMV-Ova plasmid and incubated at 37°C for 48 hours. β-Actin was used as a loading control. Figure 13E - p21 mRNA expression in MDA-MB-231 cells treated with mock-electroporated MDA-MB-231-derived exosomes, or with exosomes electroporated with p53-GFP plasmid and immediately added to cells (0 hour), or incubated at 37°C for 48 hours in cell conditions before treatment (48 hours). Exosomes were added to cells for 30 minutes or 48 hours before RNA extraction. Expression levels were normalized to the housekeeping gene GAPDH. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description Extracellular vesicles (EVs), including exosomes, are nanosized intracellular communication vehicles with a lipid bilayer surrounding cytosol-like material. Exosomes are involved in several physiological processes and contain DNA, RNA, and proteins. It is generally assumed that all contents in exosomes originate from the cell and remain intact until they enter another cell and deposit their contents. Exosomes are released in large numbers from all cells and are considered to be garbage bags that carry cellular components as payloads into the extracellular space, with no biological significance in themselves.

[0028] Provided herein are exosomes that behave similarly to minicells, biologically respond to stimuli, and exhibit the ability to proliferate and migrate just like cells, but lack a defined nucleus. These exosomes exhibit chemotaxis toward serum factors, and when stimulated with growth factors such as EGF, they phosphorylate the EGFR receptor on their surface, initiating a signaling cascade that leads to the transcription and translation of new proteins. When injected into tumor-bearing mice, these exosomes preferentially accumulate in tumors. Collectively, the ability of these exosomes to translate proteins and respond to growth factors allows exosomes to play functional roles in regulating tissue homeostasis and disease.

[0029] I. Aspects of the Invention Extracellular vesicles, particularly exosomes, have attracted considerable attention over the past few years due to the identification of several components, including DNA, RNA, and proteins. Furthermore, exosomes have been implicated in influencing many diverse biological processes by transferring their contents to recipient cells in various tissues and facilitating a unique form of intercellular communication (Bastos et al., 2017). The ability of exosomes to serve as delivery vehicles for therapeutic agents, particularly in the context of cancer or neurological pathologies, has also been reported (El-Andaloussi et al., 2012; Kamerkar et al., 2017). However, the precise patterns of systemic distribution and organ tropism of exosomes remain poorly understood.

[0030] However, the nuclear and cytoplasmic components of exosomes are not only used for passive transfer into recipient cells, but also can respond to external stimuli by phosphorylating growth factor receptors such as EGFR and generate new proteins through active transcription and translation. External stimuli of exosomes can initiate novel biological activities, such as retrograde migration. It is thought that exosomes may function similarly to primitive, albeit minicells, in their finely tuned function in response to external stimuli. Indeed, it has recently been suggested that exosomes could potentially serve as extant representatives of protocellular ribosomes. This requires the inclusion of rRNA, a finding confirmed in this study (Sinkovicsm, 2015). Exosomes actively respond biologically to growth factor gradients and migrate. Actin remodeling may be involved. Therefore, actin polymerization patterns may be an interesting target for modulating exosome biodistribution.

[0031] Vesicles such as prostasomes from various species have been shown to contain various components of the glycolytic pathway, enabling ATP generation under cell-free conditions (Ronquist et al., 2013a; Ronquist et al., 2013b). Although direct transcription in exosomes has not previously been reported, one study found that exosomes from dairy cows infected with bovine leukemia virus exhibited reverse transcriptase activity (Yamada et al., 2013). Recently, it was demonstrated that exosomes isolated from cancer cells can independently produce mature miRNAs (Melo et al., 2014). This demonstrated that exosomes possess the endogenous ability to synthesize functional proteins de novo through DNA transcription coupled with mRNA translation. Platelets can translate proteins from mRNA molecules that remain in the platelet after megakaryocytic differentiation (Weyrich et al., 2004). However, DNA transcription to generate new mRNA molecules within platelets has not been reported. Furthermore, centers of mRNA translation activity associated with polyribosomes and mRNA-binding proteins have been observed in dendritic spines, even when they are disconnected from the larger cell body (Aakalu et al., 2001; Smith et al., 2001; Steward and Levy, 1982). Therefore, it is clear that some cellular structures have retained the capacity for protein biosynthesis, even in the absence of a nucleus, likely to rapidly support specific biological functions. In addition to protein translation, exosomes can also transcribe DNA via RNA polymerase II. It is well established that basal transcription of naked DNA in nucleosome-free regions is possible with minimal transcription machinery components: RNA Pol II and a cocktail of six general transcription factors (GTFs) (Lorch et al., 2014; Nagai et al., 2017). Exosomes have also been shown to contain numerous transcription factors that can be delivered to cells to alter protein expression patterns (Ung et al., 2014).Thus, exosomes are thought to contain naked DNA residues that are not bound to chromatin and can undergo transcription in the presence of these minimal transcriptional components.

[0032] This study also suggests that the components required for transcription / translation are likely exhausted within 24 hours, resulting in limited transcription and translation rates. Because it has been suggested that different subpopulations of exosomes may have distinct molecular characteristics (Willms et al., 2016), it is possible that only a small subset of exosomes is capable of de novo protein synthesis. Newly synthesized proteins in exosomes are functionally active, suggesting proper protein conformation. Exosomes have been shown to contain not only ribosomal components but also several molecular chaperones, such as Hsp60 and Hsp70. Ribosomes themselves play an important role in cotranslational protein folding. For example, ribosomes can promote secondary structure formation in newly formed proteins. Ribosomes also serve as a platform for the binding of chaperones, which can assist in the proper folding of nascent proteins (Kramer et al., 2009). It is thought that these exosomal components may contribute to the stabilization of newly formed proteins. Physical constraints, such as those in the exosome lumen, can also have a stabilizing effect on protein folding (Rao and Cruz, 2013). However, we cannot rule out the possibility that many proteins exhibit inappropriate conformations or misfolding. These may still have important biological implications, as evidenced by the recent uncovering of unexpected features of the "dark proteome," suggesting that proteins with unknown structures or intrinsically disordered regions may have important physiological functions (Perdigao et al., 2015).

[0033] A thorough and quantitative identification of proteins naturally synthesized in exosomes is necessary to fully understand the biological significance of this process. However, it is clear that this could have a profound impact on the reevaluation of our understanding of eukaryotic biology. Recent studies suggest that cells can selectively import mRNA into exosomes (Raposo and Stoorvogel, 2013). This raises the possibility that mRNA selectively loaded into exosomes can be translated into proteins that are silenced in the cell of origin, as demonstrated in this study as proof of concept. Newly synthesized proteins were identified in exosomes up to one month after translation, suggesting that exosome-mediated protein production may lead to significantly extended protein half-lives, likely due to low levels of proteolytic enzymes.

[0034] Collectively, these results demonstrate that exosomes possess previously unrecognized biological activities with potentially significant impacts on body homeostasis and tissue pathogenesis. It can be speculated that growth factor gradients may play a role in the systemic tropism of exosomes within the body. Disrupting the natural pattern of growth factor production could immediately affect exosome redistribution and delivery patterns. These response patterns may have potential implications, particularly in determining intercellular communication between distant body sites. The fact that exosomes change their protein expression patterns in response to these extracellular cues suggests that they may act as a primary response to tissue injury. In conclusion, these findings provide novel insights into the fundamental biology of exosomes and inform their biological functions in organismal homeostasis and their potential impact in disease states.

[0035] I. Lipid-Based Nanoparticles In some embodiments, the lipid-based nanoparticles are liposomes, exosomes, lipid preparations, or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP:cholesterol vesicles). The lipid-based nanoparticles may be positively charged, negatively charged, or neutral. The lipid-based nanoparticles may contain components necessary for transcription and translation, signal transduction, chemotaxis, or other cellular functions.

[0036] 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 generally comprising phospholipids and an internal medium generally comprising 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.

[0037] Multilamellar liposomes contain 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 to form a closed structure, trapping 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.

[0038] In certain aspects, the polypeptide, nucleic acid, or small molecule drug may be, for example, 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.

[0039] Liposomes used in accordance with this embodiment can be prepared by various methods 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 it accounts for approximately 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 preparation can be stored at -20°C and used for up to 3 months. When needed, the lyophilized liposomes are reconstituted by dissolving them in 0.9% saline.

[0040] Alternatively, liposomes can be prepared by dissolving lipids in a solvent and mixing them in a container, such as 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 approximately 40°C. The solvent is usually removed within approximately 5 minutes to 2 hours, depending on the desired liposome volume. The composition can be further dried under reduced pressure in a desiccator. Dried lipids tend to deteriorate over time and are generally discarded after approximately one week.

[0041] The dried lipids can be dissolved and hydrated in sterile, pyrogen-free water at approximately 25-50 mM phospholipid by shaking until the lipid film is fully resuspended. The aqueous liposome solution can then be divided into aliquots, each placed in a vial, lyophilized, and sealed under vacuum.

[0042] The dried lipid or lyophilized liposomes prepared as described above can be dehydrated, reconstituted by dissolving in a protein or peptide solution, and diluted to an appropriate concentration with an appropriate solvent, such as DPBS. The mixture is then vigorously shaken in a vortex mixer. Any additional unencapsulated materials, such as active substances including, but not limited to, hormones, drugs, nucleic acid constructs, etc., are removed by centrifugation at 29,000 x g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., approximately 50-200 mM. The amount of encapsulated additional materials or active substances can be determined according to standard methods. After determining the amount of additional materials or active substances encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically contain a pharmaceutically acceptable sterile carrier or diluent, such as water or saline.

[0043] 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.

[0044] When preparing such liposome, can use any protocol described herein or known to those skilled in the art.Other non-limiting examples of preparing liposome are described in U.S. Patent No. 4,728,578, U.S. Patent No. 4,728,575, U.S. Patent No. 4,737,323, U.S. Patent No. 4,533,254, U.S. Patent No. 4,162,282, U.S. Patent No. 4,310,505 and U.S. Patent No. 4,921,706; International Application No. PCT / US85 / 01161 and U.S. Patent No. PCT / US89 / 05040, each of which is incorporated herein by reference.

[0045] In certain embodiments, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). As used herein, "neutral liposomes" or "uncharged liposomes" are defined as liposomes 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, if any, lipid components 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).

[0046] The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In certain embodiments, one type of phospholipid may be used to prepare the liposomes (e.g., a neutral phospholipid, such as DOPC, may be used to prepare neutral liposomes). In other embodiments, multiple types of phospholipids may be used to prepare the liposomes. The phospholipids may be derived from neutral or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), these compounds may be particularly useful for preparing neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, a lipid other than a phospholipid (e.g., cholesterol) may be used.

[0047] 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-myristoylphosphatidylcholine ("MPPC"), 1-myris ... lysine monophosphate ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelitis ("DSPG"), and glycerol monophosphate ("PDPG"). Dioleoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), di Myristoyl 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"), palmitoyloeoylphosphatidylcholine ("POPC"), palmitoyloeoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0048] 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 5,000 nm, more typically 30 nm to 1,000 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. Most commonly, the size (average diameter) of an exosome is up to 5% of the size of the donor cell. Thus, exosomes specifically contemplated include exosomes shed from cells.

[0049] Exosomes may be detected in or isolated from any suitable sample type, such as, for example, a bodily fluid. As used herein, the term "isolated" refers to separation from the natural environment and is intended to include at least partial purification, and may include substantial purification. As used herein, the term "sample" refers to any sample suitable for the methods provided by the present invention. The sample may be any sample containing exosomes suitable for detection or isolation. Sample sources include blood, bone marrow, pleural fluid, ascites, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, synovial fluid, and bronchial washings. 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 containing blood cells or components thereof, such as veins, arteries, peripheries, tissues, cords, etc. For example, samples can be obtained and processed using well-known and routine clinical methods (e.g., techniques for collecting and processing whole blood). In one aspect, an exemplary sample can be peripheral blood collected from a subject with cancer.

[0050] Exosomes can 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 dissolve the cells and release exosomes.When isolating exosomes from tissue samples, it is important to select a homogenization and lysis method that does not destroy exosomes.The exosomes intended herein are preferably isolated from body fluids dissolved in a physiologically acceptable solution, such as buffered saline, growth medium, various aqueous media, etc.

[0051] 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 medium. Although not necessary, even higher purity exosomes 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 for 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).

[0052] One commonly accepted protocol for isolating exosomes involves ultracentrifugation, often combined with a sucrose density gradient or sucrose cushion to float the relatively low-density exosomes. Isolation of exosomes by differential centrifugation is complicated by the potential overlap in size distribution with other microvesicles or macromolecular complexes. Furthermore, centrifugation can provide an insufficient means of separating vesicles based on size. However, when differential centrifugation is combined with sucrose gradient ultracentrifugation, exosomes can be highly enriched.

[0053] Size-based exosome isolation using alternatives to ultracentrifugation is another option. Successful exosome purification using ultrafiltration, a method less time-consuming than ultracentrifugation and requiring no specialized equipment, has been reported. Similarly, a commercially available kit (EXOMIR™, Bio Scientific) is available that uses positive fluid pressure to remove cells, platelets, and cellular debris in one microfilter and capture vesicles larger than 30 nm in a second microfilter. However, this process does not recover exosomes; instead, their RNA content can be extracted directly from the material captured in the second microfilter and then used for PCR analysis. Using HPLC-based protocols, these processes require specialized equipment and are difficult to scale up, but potentially yield highly pure exosomes. 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 miRNAs may be contained within extracellular protein complexes rather than exosomes. However, treatment with a protease (eg, proteinase K) can be carried out to eliminate any contamination with "extraexosomal" proteins.

[0054] In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosomes in a sample, such as techniques involving immunospecific 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 a specific cell type 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 to one side. After the blood is removed, the captured cells are retained along with the beads. Many variations of this general method are known in the art and are suitable for use in isolating exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then an antibody is added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins known to be found on cancer cell-derived exosomes include ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), 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.

[0055] As used herein, analysis includes any method that allows for direct or indirect visualization of exosomes, and may be in vivo or ex vivo. For example, analysis may include, but is not limited to, ex vivo detection and visualization by microscopy or cytometry of exosomes bound to a solid support, flow cytometry, fluorescence imaging, etc. In an exemplary aspect, the cancer cell-derived exosomes contain ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), glypican-1 (GPC1), histone H2A type 2-A (HIST1H2AA), histone H2A type 1-A (HIST1H1AA), histone H3.3 (H3F3A), histone H3.1 (HIST1H3A), zinc finger protein 37 homolog (ZFP37), laminin subunit beta-1 (LAMB1), Tubulointerstitial nephritis antigen-like (TINAGL1), peroxiredeoxin-4 (PRDX4), collagen α-2(IV) chain (COL4A2), putative protein C3P1 (C3P1), hemicentin-1 (HMCN1), putative rhophilin-2-like protein (RHPN2P1), ankyrin repeat domain-containing protein 62 (ANKRD62), tripartite motif-containing protein 42 (TRIM42), junction plakoglobin (JUP), tubulin β-2B chain (TUBB2B), endoribonuclease Dicer (DICER1), E3 ubiquitin-protein ligase TRIM71 (TRIM71), and Katanin p60 ATPase-containing subunit A-like 2 (KATNAL2), protein S100-A6 (S100A6), 5'-nucleotidase domain-containingProtein 3 (NT5DC3), valine-tRNA ligase (VARS), kazrin (KAZN), ELAV-like protein 4 (ELAVL4), ring finger protein 166 (RNF166), FERM and PDZ domain-containing protein 1 (FRMPD1), 78 kDa glucose-regulated protein (HSPA5), trafficking protein particle complex subunit 6A (TRAPPC6A), squalene monooxygenase (SQLE), tumor susceptibility gene 101 protein (TSG101), vacuolar protein sorting 28 homolog (VPS28), prostaglandin F2 receptor negative regulator (PTGFRN), isobutyryl-CoA dehydrogenase, mitochondrial (ACAD8), 26S protease regulatory subunit 6B (PSMC4), elongation factor 1-gamma (EEF1G), titin (TTN), tyrosine-protein phosphatase type 13 (PTPN13), triosephosphate isomerase (TPI1), or carboxypeptidase E (CPE), and then bound to a solid support and / or visualized using microscopy or cytometric detection.

[0056] It should be noted that not all proteins expressed in a cell are found in the exosomes secreted by that cell (Figure 11). 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, one study found that 190 / 190 pancreatic ductal adenocarcinoma patients had higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015, the entire contents of which are incorporated herein by reference). Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes.

[0057] 1. Exemplary Protocol for Harvesting Exosomes from Cell Culture On day 1, seed a sufficient number of cells (e.g., approximately 5 million cells) into a T225 flask containing medium containing 10% FBS so that the cells are approximately 70% confluent the next day. On day 2, aspirate the medium from the cells, wash them twice with PBS, and then add 25–30 mL of basal medium (i.e., without PenStrep or FBS) to the cells. Incubate the cells for 24–48 hours. While a 48-hour incubation is preferred, some cell lines are sensitive to serum-free medium, so the incubation time must be reduced to 24 hours. Note that FBS contains exosomes, which can strongly skew NanoSight results.

[0058] On day 3 / 4, collect the medium and centrifuge it at 800 x g for 5 minutes 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 minutes 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 per tube. If the volume of medium per tube is less than 35 mL, fill the remainder of the tube with PBS to make 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2-4 hours at 4°C using an 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 transfer the remaining medium into an aspirator pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifuged at 28,000 rpm for 1-2 h repeatedly to further purify the exosome population.

[0059] Finally, resuspend the exosome pellet in 210 μL of PBS. If multiple ultracentrifuge tubes are used for each sample, resuspend each exosome pellet consecutively using the same 210 μL of PBS. For each sample, remove 10 μL and add it to 990 μL of HO for use in nanoparticle tracking analysis. Use the remaining 200 μL of exosome-containing suspension for downstream processing or immediately store at -80 °C.

[0060] 2. Exemplary Protocol for Extracting Exosomes from Serum Samples First, thaw the serum sample on ice. Then, dilute 250 μL of the cell-free serum sample with 11 mL of PBS and filter it 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 of PBS. Ultracentrifuge for a second time at 150,000 × g at 4 ° C for 2 hours. Finally, carefully discard the supernatant and resuspend the exosome pellet in 100 μL of PBS for analysis.

[0061] 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) were mixed with 1 μg of siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). The exosomes or liposomes were electroporated using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, the exosomes or liposomes were treated with protease-free RNase followed by the addition of 10x concentrated RNase inhibitor. Finally, the exosomes or liposomes were washed with PBS using ultracentrifugation as described above.

[0062] II. Disease Diagnosis, Prognosis, and Treatment Certain aspects of the present invention provide for treating patients with exosomes expressing or containing therapeutic or diagnostic substances. As used herein, a "therapeutic substance" refers to an atom, molecule, or compound useful in the treatment of cancer or other conditions. Examples of therapeutic substances include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, chelating agents, boron compounds, photoactive agents, and dyes. As used herein, a "diagnostic substance" refers to an atom, molecule, or compound useful for diagnosing, detecting, or visualizing disease. According to embodiments described herein, diagnostic substances may include, but are not limited to, radioactive substances (e.g., radioisotopes, radionuclides, radiolabels, or radiotracers), dyes, contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes, and enhancing agents (e.g., paramagnetic ions).

[0063] In some aspects, the therapeutic recombinant protein may be a protein having an activity missing in a patient's cells, a protein having a desired enzymatic activity, a protein having a desired inhibitory activity, or the like. For example, the protein may be a transcription factor, an enzyme, a protein toxin, an antibody, a monoclonal antibody, or the like. The monoclonal antibody may specifically or selectively bind to an intracellular antigen. The monoclonal antibody may inhibit the function of the intracellular antigen and / or disrupt a protein-protein interaction. Another aspect of the present invention provides for diagnosing a disease based on the presence of cancer cell-derived exosomes in a patient sample.

[0064] Since exosomes are known to contain the machinery necessary to complete mRNA transcription and protein translation (see PCT / US2014 / 068630, the entire contents of which are incorporated herein by reference), mRNA or DNA nucleic acids encoding therapeutic proteins can be introduced into exosomes by transfection. Alternatively, therapeutic proteins themselves can be electroporated into exosomes or directly incorporated into liposomes. Exemplary therapeutic proteins include, but are not limited to, tumor suppressor proteins, peptides, wild-type protein counterparts of mutant proteins, DNA repair proteins, protease enzymes, protein toxins, proteins that can inhibit the activity of intracellular proteins, proteins that can activate the activity of intracellular proteins, or any proteins that need to reconstitute loss of function. Specific examples of exemplary therapeutic proteins include 123F2, Abcb4, Abcc1, Abcg2, Actb, Ada, Ahr, Akt, Akt1, Akt2, Akt3, Amhr2, Anxa7, Apc, Ar, Atm, Axin2, B2m, Bard1, Bcl2l1, Becn1, Bhlhal5, Bin1, Blm, Braf, Brca1, Brca2, Brca3, Braf, Brcata, Brinp3, Brip1, Bub1b, Bwscr1a, Cadm3, Casc1, Casp3, Casp7, Casp8, Cav1, Ccam, Ccnd1, Ccr4, Ccs1, Cd28, Cdc25a, Cd95, Cdh1, Cdkn1a, Cdkn1b, Cdkn 2a, Cdkn2b, Cdkn2c, Cftr, Chek1, Chek2, Crcs1, Crcs10, Crcs11, Crcs2, Crcs3, Crcs4, Crc s5, Crcs6, Crcs7, Crcs8, Crcs9, Ctnnb1, Cts1, Cyp1a1, Cyp2a6, Cyp2b2, Cyld, Dcc, Dkc1, Di cer1, Dmtf1, Dnmt1, Dpc4, E2f1, Eaf2, Eef1a1, Egfr, Egfr4, Erbb2, Erbb4, Ercc2, Ercc6, Er cc8, Errfi1, Esr1, Etv4, Faslg, Fbxo10, Fcc, Fgfr3, Fntb, Foxm1, Foxn1, Fus1, Fzd6, Fzd7,Fzr1、Gadd45a、Gast、Gnai2、Gpc1、Gpr124、Gpr87、Gprc5a、Gprc5d、Grb2、Gstm1、Gstm5、Gstp1、Gstt1、H19、 H2afx、Hck、Lims1、Hdac、Hexa、Hic1、Hin1、Hmmr、Hnpcc8、Hprt、Hras、Htatip2、Il1b、Il10、Il2、Il6、Il8rb Inha, Itgav, Jun, Jak3, Kit, Klf4, Kras, Kras2, Kras2b, Lig1, Lig4, Lkb1, Lmo7, Lncr1, Lncr2, Lncr3, Lncr4, Ltbp4, Luca1, Luca2, Lyz2, Lzts1, Mad1l1, Mad2l1, Madr2 / Jv18, Mapk14, Mcc, Mcm4, Men1, Men2, Met, Mgat5, Mif, Mlh1, Mlh3, Mmac1, Mmp8 、Mnt、Mpo、Msh2、Msh3、Msh6、Msmb、Mthfr、Mts1、Mutyh、Myh11、Nat2、Nbn、Ncoa3、Neil1、Nf1、Nf2、Nfe2l1、Nhej1、Nkx2-1、N kx2-9、Nkx3-1、Nprl2、Nqo1、Nras、Nudt1、Ogg1、Oxgr1、p16、p 19、p21、p27、p27mt、p57、p14ARF、Palb2、Park2、Pggt1b、Pgr、P i3k、Pik3ca、Piwil2、Pl6、Pla2g2a、Plg、Plk3、Pms1、Pms2、Po ld1、Pole、Ppard、Pparg、Ppfia2、Ppm1d、Prdm2、Prdx1、Prkar 1a、Ptch、Pten、Prom1、Psca、Ptch1、Ptf1a、Ptger2、Ptpn13、P tprj、Rara、Rad51、Rassf1、Rb、Rb1、Rb1cc1、Rb12、Recgl4、Ret 、Rgs5、Rhoc、Rint1、Robo1、Rpl38、S100a4、SCGB1A1、Skp2、Smad2、Smad3、Smad4、Smarcb1、Smo、Snx25、Spata13、Srpx、Ssic 1、Sstr2、Sstr5、Stat3、St5、St7、St14、Stk11、Suds3、Tap1、T bx21、Terc、Tnf、Tp53、Tp73、Trpm5、Tsc2、Tsc1、Vhl、Wrn、Wt1、These include Wt2, Xrcc1, Xrcc5, Xrcc6, and Zac1.

[0065] One specific type of protein that may be desirable to be introduced into the intracellular space of diseased cells is antibody (for example, monoclonal antibody).Such antibody can disrupt the function of intracellular protein and / or disrupt intracellular protein-protein interaction.The exemplary target of such monoclonal antibody includes, but is not limited to, the protein involved in RNAi pathway, telomerase, transcription factor that controls disease process, kinase, phosphatase, protein that is required for DNA synthesis, protein that is required for protein translation. Specific examples of exemplary therapeutic antibody targets include proteins encoded by the following genes: Dicer, Ago1, Ago2, Trbp, Ras, raf, wnt, btk, Bcl-2, Akt, Sis, src, Notch, Stathmin, mdm2, abl, hTERT, c-fos, c-jun, c-myc, erbB, HER2 / Neu, HER3, VEGFR, PDGFR, c-kit, c-met, c-ret, flt3, API, AML1, ax1, alk, fins, fps, gip, lck, Stat, Hox, MLM, PRAD-I, and trk. In addition to monoclonal antibodies, any antigen-binding fragment thereof, e.g., scFv, Fab fragment, Fab', F(ab')2, Fv, peptibody, diabody, triabody, or minibody, are also contemplated. Any such antibody or antibody fragment may be glycosylated or aglycosylated.

[0066] Because exosomes are known to contain DICER and active RNA-processing RISC complexes (see PCT Publication WO 2014 / 152622, incorporated herein by reference in its entirety), shRNAs introduced into exosomes by transfection can be matured into RISC complex-bound siRNAs along with the exosomes themselves. Alternatively, mature siRNAs themselves can be introduced into exosomes or liposomes by transfection. Thus, by way of example, wild-type or mutant versions of developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth factors or differentiation factors and their receptors, neurotransmitters and their receptors), tumor suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTE, etc.) can be used. N, Rb, uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2 / JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1, r as, MMAC1, FCC, MCC, FUS1, Gene26(CACNA2D2), PL6, Beta*(BLU), Luca-1(HYAL1), Luca-2(HYAL2), 123F2(RASSF1), 101F6, Gene21(NPRL2), or SEMA3 Describe the details of the movie (see CD 5).スパーゼ-3、Bax、Bag-1、CRADD、TSSC3、bax、hid、Bak、MK Q-7、PARP、bad、bcl-2、MST1、bbc3、Sax、CARE、およびBID ) (including GM-CSF, G-CSF, IL-1α, IL-1β, and IL-2). IL-3、IL-4、IL-5、LET-6、LET-7、LET-8、LET-9、LET-10、LET-11、LET-12、LET-13、LET-14、LET-15、LET-16、LET-17、LET-17. 18、ART-19、ART-20、ART-21、ART-22、IC-23、IC-24、IC-25、IC-26、IC-27、IC-28、IC-29、IC-30、IC-3 IFN-α、IFN-β、IFN-γ、MIP-1α、MIP-1β、TGF-β、TNF-α TNF-β, PDGF (mda7) and activators of BLC1 and BC L6、CBFA1、CSFIR、GRASS、ERBB、EBRB2、ETS1、ETS1、ETV6、FGR、FOX、FYN、HCR、HRAS、JUN、KRAS、LCK MDM2、MLL、MYB、MYC、MYCL1、MYCN、NRAS、PIM1、PML、 RET、SRC、TAL1、TCL3、〈〈 YES) should appear in the ACP file. ADP-catalyzed activity of the hycroxylase catalyzes the activation of the ADP-catalyzed enzyme. (pyrophorylase)、ATP is metabolized by the pyrophorylase (alcoholdehycrogenases, amylases, amyloglucosidases, catalases, cellulases, cyclooxygenases, decarboxylases, dextrinases, esterases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, GTPases, helicases, hemicellulases, integrases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nucleases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, endopolygalacturonases, proteinases and peptideases, pullulanases, recombinases, reverse transcriptases, topoisomerases, xylanases) are possible classes of target genes that may be used in the methods of the present invention to modulate or attenuate target gene expression. In some cases, sh / siRNA may be designed to specifically target a mutant version of a gene expressed in cancer cells without affecting the expression of the corresponding wild-type version. In fact, any inhibitory nucleic acid can be applied in the compositions and methods of the present invention, provided that such inhibitory nucleic acid is found by any supplier to be a verified down-regulator of the protein of interest.

[0067] When designing RNAi, several factors need to be considered, such as the nature of siRNA, the durability of silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into an organism typically contains exon sequences. Furthermore, the RNAi process depends on homology. Therefore, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA sequence exhibits more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with less than about 80% identity with the target gene have significantly less effect. Therefore, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0068] Exosomes may also be engineered to contain gene editing systems, such as CRISPR / Cas systems. Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts derived from CRISPR loci. In some aspects, Cas nuclease and gRNA (comprising a fusion of a target sequence-specific crRNA with a specific tracrRNA) are introduced into cells. Generally, the target site at the 5' end of the gRNA uses complementary base pairing to target the Cas nuclease to the target site, e.g., a gene. The target site may be selected based on the position immediately 5' of the protospacer adjacent motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, the gRNA targets the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems feature elements that promote CRISPR complex formation at the target sequence site. Typically, the term "target sequence" generally refers to a sequence that the guide sequence is designed to be complementary to, and hybridization between the target sequence and the guide sequence promotes CRISPR formation. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote CRISPR complex formation. The CRISPR system in exosomes engineered to contain such a system may function to edit genomic DNA in target cells, or the system may edit DNA within the exosome itself.

[0069] In addition to protein-based and nucleic acid-based therapeutic agents, exosomes may be used to deliver small molecule drugs alone or in combination with any protein-based and nucleic acid-based therapeutic agent. Exemplary small molecule drugs contemplated for use in this embodiment include toxins, chemotherapeutic agents, agents that inhibit the activity of intracellular proteins, agents that activate the activity of intracellular proteins, agents for preventing restenosis, agents for treating kidney disease, agents used for intermittent claudication, agents used in the treatment of hypotension and shock, angiotensin-converting enzyme inhibitors, antianginal agents, antiarrhythmic agents, antihypertensive agents, anti-iotensin II receptor antagonists, antiplatelet agents, b-blockers with b1 selectivity, beta-blockers, botanicals for cardiovascular indications, calcium channel blockers, cardiovascular / diagnostic agents, central alpha-2 agonists, coronary vasodilators, diuretics and renal tubule inhibitors, neutral endopeptidase / angiotensin-converting enzyme inhibitors, peripheral vasodilators, potassium channel blockers, and the like. channel openers, potassium salts, anticonvulsants, antiemetics, antiemetics, antiparkinsonian agents, antispasmodics, stimulants, agents applicable in the treatment of trauma, agents applicable in the treatment of Alzheimer's disease or dementia, agents applicable in the treatment of migraine, agents applicable in the treatment of neurodegenerative diseases, agents applicable in the treatment of Kaposi's sarcoma, agents applicable in the treatment of AIDS, cancer chemotherapy agents, agents applicable in the treatment of immune disorders, agents applicable in the treatment of psychiatric disorders, analgesics, epidural and intrathecal anesthetics, systemic, local and regional neuromuscular blocking sedatives, pre-anesthetic adrenal / ACTH, anabolic steroids, agents applicable in the treatment of diabetes, dopamine agonists, growth hormone and analogues, hyperglycemic agent), hypoglycemic agents, oral insulin, large-volume parenteral drugs (LVP), lipid-altering agents, metabolic studies and inborn errors of metabolism, nutrients / amino acids, nutritional LVP, obesity drugs (appetite suppressants), somatostatin, thyroid agents, vasopressin, vitamins, corticosteroids, mucolytics, pulmonary anti-inflammatory agents, pulmonary surfactants, antacids, anticholinergics, antidiarrheals, antiemetics, cholelitholyticsagent), inflammatory bowel disease agents, irritable bowel syndrome agents, liver agents, metal chelators, various gastric secretory agents, pancreatitis agents, pancreatic enzymes, prostaglandins, proton pump inhibitors, sclerosing agents, sucralfate, antiprogestins, contraceptives, oral contraceptives, parenteral dopamine agonists, estrogens, gonadotropins, GNRH agonists, GHRH antagonists, labor-inducing agents, progestins, agents acting on the uterus, antianemic agents, anticoagulants, antifibrinolytic agents, antiplatelet agents, antithrombin agents, blood coagulants, fibrinolytic agents, hematology, heparin inhibitors, metal chelators, prostaglandins, vitamin K, antiandrogens, aminoglycosides, antibacterial agents, sulfonamides, cephalosporins, clindamycin These include, but are not limited to, mycins, dermatological agents, surfactants, erythromycin, anthelmintics, antifungals, antimalarials, antimycobacterial agents, antiparasitics, antiprotozoal agents, antitrichomonasal agents, antituberculous agents, immunomodulators, immunostimulants, macrolide antibiotics, antiparasitic agents, corticosteroids, cyclooxygenase inhibitors, enzyme blockers, immunomodulators for rheumatic diseases, metalloproteinase inhibitors, nonsteroidal anti-inflammatory agents, analgesics, antipyretics, alpha adrenergic agonists / blockers, antibiotics, antivirals, beta adrenergic blockers, carbonic anhydrase inhibitors, corticosteroids, immune system regulators, mast cell inhibitors, nonsteroidal anti-inflammatory agents, and prostaglandins.

[0070] Exosomes may also be used to deliver diagnostic agents, including, but not limited to, magnetic resonance imaging enhancing agents, positron emission tomography products, radioactive diagnostic agents, radioactive therapeutic agents, radiopaque contrast agents, radiopharmaceuticals, ultrasound imaging agents, and angiographic diagnostic agents.

[0071] As used herein, "subject" refers to any individual or patient that the method is carried out.Generally, the subject is human, but as will be understood by those skilled in the art, the subject can also be an animal.Therefore, other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included within the definition of subject.

[0072] "Treatment" and "treating" refer to the administration or application of a therapeutic substance to a subject, or the performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment may include the administration of chemotherapy, immunotherapy, or radiation therapy, the performance of surgery, or any combination thereof.

[0073] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that enhances or improves the well-being of a subject with respect to the medical treatment of the condition. This term includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer may include, for example, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Treating cancer may also refer to extending the survival of a subject with cancer.

[0074] As used herein, the term "cancer" may be used to describe a solid tumor, a metastatic cancer, or a non-metastatic cancer. In certain aspects, the cancer may arise in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus.

[0075] Cancers include, specifically, the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; adenocarcinoma of the auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; hemangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus melanoma in giant pigmented nevus); epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mullerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor Tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease The disease may include, but is not limited to, Hodgkin's disease, granulomatous granulomatosis, malignant lymphoma, small lymphocytic, malignant lymphoma, diffuse large cell, malignant lymphoma, follicular, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0076] The terms "contacted" and "exposed," when applied to cells, are used herein to describe the process by which a therapeutic agent is delivered to or placed in direct juxtaposition with a target cell. For example, to kill the cell, one or more agents are delivered to the cell in an amount effective to kill the cell or prevent the cell from dividing.

[0077] An effective patient response or a patient's "responsiveness" to a treatment refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder. Such benefit may include a cellular or biological response, a complete response, a partial response, stable disease (no progression or recurrence), or a response that subsequently recurs. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0078] Treatment outcomes can be predicted and monitored, and / or patients who would benefit from such treatment can be identified or selected by the methods described herein.

[0079] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of the neoplastic condition involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy. Other therapeutic regimens may be combined with the administration of anti-cancer agents, such as therapeutic compositions and chemotherapeutic agents. For example, patients who are to be treated with such anti-cancer agents may also undergo radiation therapy and / or surgery.

[0080] 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.

[0081] Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological preparations containing one or more of the active substances, or the tissues, tumors, and / or cells can be contacted 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.

[0082] Coadministration may include simultaneous administration of two or more agents in the same dosage form, simultaneous administration 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 administered simultaneously. Alternatively, the therapeutic composition and another therapeutic agent can be administered simultaneously, where both agents are present in separate formulations. Alternatively, one therapeutic agent can be administered immediately followed by the other therapeutic agent, or vice versa. In other administration protocols, the therapeutic composition and another therapeutic agent may be administered minutes apart, hours apart, or days apart.

[0083] The first anti-cancer treatment (e.g., exosomes expressing a recombinant protein or a recombinant protein isolated from an exosome) may be administered prior to, during, or after the second anti-cancer treatment, or in various combinations relative to the second anti-cancer treatment. Administration may occur simultaneously or at intervals ranging from minutes to days to weeks. In embodiments in which the first treatment is provided to the patient separately from the second treatment, the effective period will generally not expire between the respective deliveries, so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the first and second therapies may be provided to the patient within about 12 to 24 hours or 72 hours of administering either therapy, more particularly, within about 6 to 12 hours of administering either therapy. In some circumstances, significantly longer treatment periods may be desirable. In this case, the period between each administration can be from a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0084] In certain embodiments, the course of treatment lasts from 1 day to 90 days or longer (such ranges are inclusive). It is contemplated that one agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof, and another agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof. Within a single day (24-hour period), the patient may receive one or more administrations of the agent. Furthermore, it is contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last from 1 day to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or longer (such ranges are inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0085] Various combinations can be used. For the example below, the first anti-cancer therapy is "A" and the second anti-cancer therapy is "B". TIFF0007813099000001.tif18128

[0086] Administration of any compound of the invention or administration of any therapy of the invention to a patient will follow standard protocols for administering such compounds, taking into account the toxicity, if any, of the agents, and thus, in some embodiments, there will be a step of monitoring for toxicity resulting from the combination therapy.

[0087] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to the mode of activity within cells, for example, whether or at what stage of the cell cycle they affect. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or affect nucleic acid synthesis to induce chromosomal and mitotic abnormalities.

[0088] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamines, including ylolomelamime; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; Duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichi n), phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosurea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I); dynemicins, including dynemicin A; bisphosphonates, e.g., clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxo doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zino Statins, or zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, propionibacterium, thiampicillin ... Dromostanolone onate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate;Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin n); sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C") ); cyclophosphamide; taxoids, such as paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CP T-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; cisplatin, carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0089] 2. Radiation therapy Other factors that cause DNA damage and have been widely used include gamma rays, X-rays, and / or what are commonly known as specific delivery of radioisotopes to tumor cells. Other forms of DNA damage, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation, are also contemplated. All of these factors likely affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0090] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination with or in conjunction with the methods of the present invention. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is one such example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can act as an effector of therapy, or it can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) or simply act as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0091] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., that is not present on the majority of other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. Another aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0092] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0093] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either strengthen signals (e.g., costimulatory molecules) or weaken signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0094] The immune checkpoint inhibitor may be a drug, e.g., a small molecule, a recombinant ligand or receptor, or, in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both of which are incorporated herein by reference). Known immune checkpoint protein inhibitors or analogs thereof may be used, in particular chimeric, humanized, or human antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and / or equivalent names MK-3475 and pembrolizumab.

[0095] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference.

[0096] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0097] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80, also known as B7-1, and CD86, also known as B7-2, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0098] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0099] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art, or art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0100] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to an antibody described above (e.g., at least about 90%, at least about 95%, or at least about 99% variable region identity to ipilimumab).

[0101] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0102] In some embodiments, immunotherapy may involve adoptive immunotherapy, which involves the transfer of ex vivo-generated autoantigen-specific T cells. T cells used in adoptive immunotherapy can be generated by expanding antigen-specific T cells or by redirecting T cells through genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor-specific T cells have been shown to be successful in treating melanoma. Novel specificities in T cells have been successfully generated by gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety linked to one or more signaling domains in a fusion molecule. Typically, the binding moiety of a CAR consists of the light chain fragment of a monoclonal antibody and the antigen-binding domain of a single-chain antibody (scFv), in which the variable fragment is connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of first-generation CARs are derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. CARs have been successfully used to redirect T cells to antigens expressed on the surface of tumor cells from a variety of neoplasms, including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0103] In one embodiment, the present application provides a combination therapy for treating cancer comprising adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted against tumor antigens.

[0104] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventative, diagnostic, or staging surgery, curative, and palliative surgery. Curative surgery involves resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0105] When cancer cells, tissues, or tumors are partially or completely removed, a cavity may be formed in the body.Treatment can be carried out by perfusion, direct injection, or local application of additional anticancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments can also be various dosages.

[0106] 5. Other agents It is contemplated that other agents may be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that sensitize hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions enhances the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that sensitize hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present invention to improve the efficacy of treatment.

[0107] III. Pharmaceutical Compositions It is contemplated that exosomes expressing or containing therapeutic proteins, inhibitory RNA, and / or small molecule drugs can be administered systemically or locally to inhibit tumor cell growth, most preferably to kill cancer cells in cancer patients with locally advanced or metastatic cancer. Exosomes expressing or containing CRISPR systems can be administered intravenously, intrathecally, and / or intraperitoneally. Exosomes expressing or containing CRISPR systems can be administered alone or in combination with antiproliferative drugs. In one embodiment, exosomes expressing or containing CRISPR systems are administered to reduce a patient's cancer burden before surgery or other treatment. Alternatively, exosomes expressing or containing CRISPR systems can be administered after surgery to ensure that any remaining cancer (e.g., cancer not removed by surgery) does not survive.

[0108] The present invention is not intended to 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 a physiologically acceptable liquid, gel, solid carrier, diluent, or excipient.These therapeutic preparations, like other therapeutic substances, can be administered to mammals for veterinary use, such as veterinary use with livestock, and clinical use in humans.Generally, the dosage required for therapeutic effectiveness varies according to the type of use and administration method, and the specific requirements of each individual subject.

[0109] Where clinical application is intended, it may be necessary to prepare pharmaceutical compositions containing recombinant proteins and / or exosomes in a form appropriate for the intended use. Generally, pharmaceutical compositions may contain an effective amount of one or more recombinant proteins and / or exosomes or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human, as appropriate. Preparation of pharmaceutical compositions containing the recombinant proteins and / or exosomes disclosed herein, or additional active ingredients, will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Furthermore, for administration to animals (e.g., humans), it is understood that preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0110] Furthermore, according to certain aspects of the present invention, the composition suitable for administration may be provided dissolved in a pharmaceutically acceptable carrier with or without an inert diluent. As used herein, "pharmaceutically 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), vegetable oils, and injectable organic esters (e.g., ethyl oleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, etc. Carriers include agents, antioxidants, 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 delaying agents (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, fillers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquid and nutrient replenishers, and similar materials and combinations thereof. The carrier must be assimilable and include liquid, semi-solid, i.e., paste, or solid carriers. Additionally, 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.

[0111] Although pharmaceutically acceptable carriers are formulated specifically for administration to humans, in certain embodiments, it may be desirable to use pharmaceutically acceptable carriers formulated for administration to non-human animals but not acceptable for administration to humans (e.g., due to government regulations). Except where a conventional carrier is incompatible with the active ingredient (e.g., harmful to the recipient or detrimental to the therapeutic effect of the composition contained in the carrier), its use in therapeutic or pharmaceutical compositions is contemplated. According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such techniques are routine for those skilled in the art.

[0112] Certain embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether they need to be sterile for routes 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 local perfusion directly bathing target cells, via a catheter, by lavage, in lipid compositions (e.g., liposomes), or by other methods known to those of skill in the art or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[0113] Active compound can be formulated for parenteral administration, for example, can be formulated for injection via intravenous route, intramuscular route, subcutaneous route, or intraperitoneal route.Typically, such composition can be prepared in either liquid solution or suspension.Suitable solid dosage forms can also be prepared, which are used to prepare solution or suspension by adding liquid before injection.Preparation can also be emulsified.

[0114] 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.

[0115] The therapeutic agent may be formulated into the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino groups of the protein composition, or with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, for example, formulated for parenteral administration, such as injections or aerosols for pulmonary delivery, or formulated for alimentary administration, such as drug-release capsules.

[0116] In a specific embodiment of the present invention, the composition is combined with a semi-solid or solid carrier or thoroughly mixed with a semi-solid or solid carrier.Mixing can be performed by any convenient method, such as grinding.Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., from denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0117] In a further aspect, the present invention may relate to the use of pharmaceutical lipid vehicle compositions comprising one or more lipids and an aqueous solvent. The term "lipid" as used herein is defined to include a wide variety of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be natural or synthetic (i.e., engineered or produced by humans). However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that would be understood by those skilled in the art as lipids are also encompassed by the compositions and methods described above.

[0118] Those skilled in the art will be familiar with the range of techniques that can be used to disperse a composition in a lipid vehicle. For example, the therapeutic substance may be dispersed in a solution containing lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained as a suspension in lipids, contained or complexed with micelles or liposomes, or otherwise bound to lipids or lipid structures by any means known to those skilled in the art. The dispersion may or may not result in the formation of liposomes.

[0119] 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 administration, i.e., the appropriate route and treatment regimen, discussed above. The amount to be administered will depend on the desired effect, depending on the number of treatments and the unit dose. The actual dosage of the compositions of the present invention 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 efficacy, stability, and toxicity of the particular therapeutic agent. For example, dosages may also include doses from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such ranges inclusive), or higher, 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. The administering physician will, in any given situation, determine the concentration of active ingredient(s) in a composition and the dose appropriate for the individual subject.

[0120] 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 in charge of administration will, in any given situation, determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0121] In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, from about 2% to about 75% or from about 25% to about 60% of the weight of the unit, and any range derivable therein. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage amount of the compound is obtained in a particular unit dose. 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 when preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desirable accordingly.

[0122] 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.

[0123] IV. Nucleic Acids and Vectors In certain aspects of the present invention, nucleic acid sequences encoding therapeutic proteins or fusion proteins containing therapeutic proteins may be disclosed. Depending on which expression system is used, nucleic acid sequences can be selected based on conventional methods. For example, each gene or its variant may be codon-optimized for expression in a specific system. Various vectors can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0124] V. Recombinant Proteins, Inhibitory RNAs, and Gene Editing Systems A. Recombinant Protein Some embodiments relate to recombinant proteins and polypeptides. Certain embodiments relate to recombinant proteins or polypeptides that exhibit at least one therapeutic activity. In some embodiments, the recombinant proteins and polypeptides may be therapeutic antibodies. In some embodiments, the therapeutic antibody may be an antibody that specifically or selectively binds to an intracellular protein. In a further aspect, the protein or polypeptide may be modified to enhance serum stability. Thus, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide," it will be understood by those skilled in the art that this includes, for example, proteins or polypeptides that have additional advantages over an unmodified protein or polypeptide. It is specifically intended that embodiments relating to a "modified protein" may also be implemented with respect to a "modified polypeptide," and vice versa.

[0125] Recombinant proteins may have amino acid deletions and / or substitutions. Thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further comprise inserted or added amino acids, e.g., fusion proteins or proteins with linkers. A "modified deletion protein" lacks one or more residues of the native protein but may retain the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one that has amino acid residues deleted from at least one antigenic region, a region of the protein that has been shown to be antigenic in a particular organism, e.g., the type of organism to which the modified protein may be administered.

[0126] Substitution or replacement variants typically contain the replacement of one amino acid with another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may be conservative, i.e., one amino acid is replaced with an amino acid of similar shape or charge; substitutions may be non-conservative. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0127] In addition to deletions or substitutions, modified proteins may have residue insertions, which typically involve the addition of at least one residue to the polypeptide. This may include the insertion of a targeting peptide or targeting polypeptide, or may simply involve the insertion of a single residue. Terminal additions, called fusion proteins, are discussed below.

[0128] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, it includes about 70% to about 80% of the amino acid sequence, or about 81% to about 90% of the amino acid sequence, or even about 91% to about 99% of the amino acid sequence that is identical to or functionally equivalent to the amino acid sequence of a reference polypeptide, provided that the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be biologically functionally equivalent to its native counterpart.

[0129] It is also understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, as long as they meet the criteria set forth above, including the maintenance of biological protein activity associated with protein expression, but remain essentially as set forth in one of the sequences disclosed herein. The addition of terminal sequences applies particularly to nucleic acid sequences that may, for example, contain various non-coding sequences adjacent to the 5' or 3' portion of the coding region, or may contain various internal sequences, i.e., introns, which are known to exist within genes.

[0130] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full-length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0131] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous, and the amino acid residue sequence is not interrupted by non-amino acids. In other embodiments, the sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0132] Thus, the term "protein or peptide" includes amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0133] Certain aspects of the present invention relate to fusion proteins. These molecules may have the N- or C-terminus of a therapeutic protein linked to a heterologous domain. For example, fusions may also use leader sequences from other species to enable recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably a cleavable antibody epitope, to facilitate fusion protein purification. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0134] In certain embodiments, the therapeutic protein may be linked to a peptide that extends in vivo half-life, such as an XTEN polypeptide (Schellenberger et al., 2009), an IgG Fc domain, albumin, or an albumin-binding peptide.

[0135] Methods for making fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein or by attaching a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0136] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by joining the genes with a bridging DNA segment that encodes a peptide linker that is spliced ​​between the tandemly connected polypeptides, the linker being of sufficient length to allow correct folding of the resulting fusion protein.

[0137] B. Inhibitory RNA SiNA (for example, siRNA) is well known in the art.For example, siRNA and double-stranded RNA are described in United States Patent No. 6,506,559 and United States Patent No. 6,573,099 and United States Patent Application No. 2003 / 0051263, United States Patent No. 2003 / 0055020, United States Patent No. 2004 / 0265839, United States Patent No. 2002 / 0168707, United States Patent No. 2003 / 0159161 and United States Patent No. 2004 / 0064842.All of these are incorporated herein by reference in their entirety.

[0138] The nucleic acid components within a siNA do not need to be the same type or uniform throughout (e.g., a siNA may contain both nucleotides and nucleic acids or nucleotide analogs). Typically, a siNA 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 invention, a siNA may contain only a single nucleic acid (polynucleotide) or nucleic acid analog, which may form a double-stranded structure by complementing itself (e.g., forming a hairpin loop). The double-stranded structure of a siNA may contain 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 nucleic acid bases, including all ranges therein. The siNA may comprise 17 to 35 consecutive nucleic acid bases, more preferably 18 to 30 consecutive nucleic acid bases, more preferably 19 to 25 consecutive nucleic acid bases, more preferably 20 to 23 consecutive nucleic acid bases, or 20 to 22 consecutive nucleic acid bases, or 21 consecutive nucleic acid bases, that hybridize with 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.

[0139] The agent of the present invention useful for carrying out the method of the present invention includes, but is not limited to, siRNA.Typically, the introduction of double-stranded RNA (dsRNA), sometimes referred to herein as small interfering RNA (siRNA), induces strong and specific gene silencing, a phenomenon known as 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 for knocking out the activity of specific genes.

[0140] When designing RNAi, several factors must be considered, such as the nature of siRNA, the durability of the silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into an organism typically contains exon sequences. Furthermore, the RNAi process depends 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 sequence exhibits more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with less than about 80% identity with the target gene have significantly less effect. Therefore, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0141] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19 to 25 nucleotides and are capable of modulating gene expression. In the context of the present invention, siRNAs are preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, siRNAs are about 19 to about 25 nucleotides in length.

[0142] A target gene generally refers to a polynucleotide that includes a region encoding a polypeptide, or a polynucleotide region that regulates replication, transcription, or translation, or other processes important for the expression of a polypeptide, or a polynucleotide that includes both a region encoding a polypeptide and a region operably linked to the region encoding the polypeptide that regulates expression. Any gene expressed in a cell can be targeted. Preferably, the target gene is a gene involved in or related to the progression of cellular activities important for disease, or a gene of particular interest as a research subject.

[0143] 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 commercial supplier of pre-designed siRNA is Ambion®, Austin, Tex. Another commercial supplier is Qiagen® (Valencia, Calif.).The inhibitory nucleic acid that can be applied in the compositions and methods of the present invention can be any nucleic acid sequence that has been found by any supplier to be a verified down-regulator of the protein of interest.It is understood that without undue experimentation and with the disclosure of the present invention, additional siRNA can be designed and used to carry out the method of the present invention.

[0144] 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 invention may also include non-standard nucleotides, including non-natural nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide 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 (for example, 2'-O-methylribonucleotide, 2'-deoxy-2'-fluororibonucleotide, "universal base" nucleotide, 5-C-methyl nucleotide, one or more phosphorothioate internucleoside 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 herein by reference in its entirety). Collectively, all of the nucleic acid or RNA described above that have been changed are referred to as modified siRNA.

[0145] C. Gene Editing Systems Generally, "CRISPR system" refers collectively to transcripts and other elements involved in the expression or induction of activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts derived from a CRISPR locus.

[0146] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a Type I, Type II, or Type III CRISPR system, and may be derived from a particular organism that contains an endogenous CRISPR system, such as, for example, Streptococcus pyogenes.

[0147] In some aspects, Cas nuclease and gRNA (comprising a fusion of a target sequence-specific crRNA and a certain tracrRNA) are introduced into cells.Generally, the target site at the 5' end of gRNA uses complementary base pairing to target the target site, for example, a gene, to Cas nuclease.The target site can be selected based on its location immediately 5' of the protospacer adjacent motif (PAM) sequence, for example, immediately 5' of NGG or NAG, typically.In this regard, gRNA targets the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of guide RNA to correspond to the target DNA sequence.Generally, CRISPR system is characterized by elements that promote CRISPR complex formation at the target sequence site. Typically, a "target sequence" generally refers to a sequence to which a guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.

[0148] CRISPR system can induce double-strand breaks (DSB) at target sites, and then induce disruption as disclosed herein.In other embodiments, Cas9 variants, considered "nickases," are used to nick single-stranded target sites.For example, paired nickases can be used to improve specificity, and each nickase is directed by a pair of different gRNAs that target sequences, so that nicks are simultaneously introduced and 5' overhangs are introduced.In other embodiments, catalytically inactive Cas9 is fused with heterologous effector domains, such as transcription repressors or activators, to affect gene expression.

[0149] The target sequence may comprise any polynucleotide, for example, DNA or RNA polynucleotide. The target sequence may be in the nucleus or cytoplasm of a cell, for example, in an organelle of a cell. Generally, the sequence or template that can be used for recombination into a target locus that comprises a target sequence is called an "editing template" or an "editing polynucleotide" or an "editing sequence". In some aspects, an exogenous template polynucleotide may be called an editing template. In some aspects, the recombination is homologous recombination.

[0150] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). The tracr sequence may also comprise or consist of all or a portion of the wild-type tracr sequence (e.g., about 20, about 26, about 32, about 45, about 48, about 54, about 63, about 67, about 85 or more nucleotides, or more than about 20, more than about 26, more than about 32, more than about 45, more than about 48, more than about 54, more than about 63, more than about 67, more than about 85 or more nucleotides of the wild-type tracr sequence), and may become part of a CRISPR complex, e.g., by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0151] One or more vectors expressing one or more elements of the CRISPR system can be introduced into cells so that expression of the elements results in the formation of CRISPR complexes at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements in a single vector can be combined with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements in one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0152] The vector may comprise regulatory elements operably linked to an enzyme coding sequence encoding a CRISPR enzyme, e.g., a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0153] The CRISPR enzyme can be Cas9 (e.g., derived from S. pyogenes or S. pneumoniae). CRISPR enzymes can induce cleavage of one or both strands at the location of a target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme is incapable of cleaving one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 derived from S. pyogenes converts Cas9, which is derived from a nuclease that cleaves both strands, into a nickase (cleaving one strand). In some embodiments, Cas9 nickases can be used in combination with guide sequences, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows for nicks to be made on both strands and can be used to induce NHEJ or HDR.

[0154] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells.Eukaryotic cells may be or be derived from a specific organism, such as a mammalian eukaryotic cell, including but not limited to, human, mouse, rat, rabbit, dog, or non-human primate.Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with the codon that is frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence, to enhance expression in the host cell of interest.Different species exhibit unique biases for certain codons of certain amino acids.Codon bias (the difference in codon usage between organisms) is often correlated with the translation efficiency of messenger RNA (mRNA), and as a result, it is believed that the translation efficiency of messenger RNA (mRNA) depends, inter alia, on the nature of the codon to be translated and the availability of certain transfer RNA (tRNA) molecules. In a given cell, the predominance of a selected tRNA reflects that codon is the most frequently used codon in peptide synthesis, and therefore genes can be tailored based on codon optimization for optimal gene expression in a particular organism.

[0155] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of a CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99% or more, or more than about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99% or more, when properly aligned using a suitable alignment algorithm.

[0156] Optimal alignment can be determined using any suitable algorithm for aligning sequences, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available from soap.genomics.org.cn), and Maq (available from maq.sourceforge.net).

[0157] CRISPR enzymes can be part of fusion proteins that contain one or more heterologous protein domains.CRISPR enzyme fusion proteins can also contain any additional protein sequence, optionally a linker sequence, between any two domains.Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes may be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, LexA DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can be part of fusion proteins containing CRISPR enzymes are described in US20110059502, which is incorporated herein by reference.

[0158] VI. Kits and Diagnostics Various aspects of the present invention contemplate kits containing the necessary components for purifying exosomes from body fluids or tissue culture media. Other aspects contemplate kits containing the necessary components for isolating exosomes and transfecting them with therapeutic nucleic acids, therapeutic proteins, or nucleic acids encoding therapeutic proteins. The kits may include one or more sealed vials containing any of these components. In some embodiments, the kits may also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, that are compatible with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass. The kits may further include instructions outlining the procedural steps of the methods described herein, following procedures substantially similar to those described herein or known to those skilled in the art. The instruction information may be present in a computer readable medium comprising machine readable instructions that, when executed using a computer, display a real or virtual method of purifying exosomes from a sample and transfecting a therapeutic nucleic acid into the exosomes, expressing a recombinant protein in the exosomes, or introducing a recombinant protein into the exosomes by electroporation. [Example]

[0159] VII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below demonstrate techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for practicing the invention. However, in light of the present 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 like or similar results without departing from the spirit and scope of the invention.

[0160] material and method Cell culture. MCF7, MDA-MB231, E10, HDF, and BJ human cell lines and the NIH 3T3 mouse cell line were cultured in DMEM containing 10% FBS. 4T1 and 67NR mouse cell lines were cultured in RPMI containing 10% FBS. The MCF10A human mammary epithelial cell line was cultured in DMEM / F12 medium containing 5% horse serum, 20 ng / ml EGF, 0.5 mg / ml hydrocortisone, 100 ng / ml cholera toxin, and 10 μg / ml insulin. All cells were sourced from the American Type Culture Collection (ATCC).

[0161] Exosome isolation and purification. Exosomes were purified by differential centrifugation as previously described (Luga et al., 2012; Thery et al., 2006). Supernatant from cells cultured for 48 h was subjected to sequential centrifugation steps at 800 g and 2000 g. The resulting supernatant was filtered using a 0.2 μm filter. The pellet was collected after ultracentrifugation at 100,000 g for 3 h in an SW40Ti swinging bucket rotor (Beckman-Coulter). The supernatant was removed, and the pellet was resuspended in PBS, followed by another ultracentrifugation at 100,000 g for 3 h. The resulting pellet was analyzed for exosome content. Exosomes used for RNA extraction were resuspended in 500 μL of Trizol. Exosomes used for protein extraction were resuspended in urea / SDS lysis buffer (8 M urea, 2.5% SDS, 5 μg / mL leupeptin, 1 μg / mL pepstatin, and 1 mM phenylmethylsulfonyl fluoride). Exosomes used to deliver cells were resuspended in serum-free DMEM culture medium. For other applications, isolated exosomes were processed as described in the remaining experimental procedures.

[0162] Imaging flow cytometry analysis (ImageStream). Exosomes were attached to 4 μm aldehyde / sulfate latex beads (Invitrogen, Carlsbad, CA, USA) dissolved in 0.9% NaCl saline (B. Braun Medical Inc, Bethlehem, PA, USA). The reaction was stopped with 100 mM glycine and 2% BSA in saline and blocked with 10% BSA for 30 minutes at room temperature with rotation. After washing with saline / 2% BSA, the bead-bound exosomes were centrifuged at 10,000 rpm for 2 minutes and incubated with 1:200 anti-CD63 (Santa Cruz), anti-CD9 (Abcam), anti-CD81 (Abcam), anti-CD82 (Abcam), and anti-FLOT1 (Santa Cruz) for 30 minutes with rotation at 4°C. The beads were centrifuged at 10,000 rpm for 2 minutes, washed with saline / 2% BSA, and incubated with 1:400 Alexa-488 secondary antibody (Life Technologies, NY 14072) for 30 minutes with rotation at 4°C. After three washes, the beads were resuspended in saline and analyzed using ImageStream® (Merck Millipore). To avoid pixel saturation, the image acquisition gain (%) was set using the positive sample. Image processing was performed using IDEAS® (Merck Millipore) software. A gate was defined to exclude out-of-focus beads and select a single bead. The Alexa-488-positive bead gate was defined relative to the negative control sample. The percentage of positive beads is relative to the number of events analyzed per sample.

[0163] Immunogold labeling and electron microscopy. Pelleted exosomes were fixed by resuspending in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Optimally fixed specimens were placed on 300-mesh carbon / Formvar-coated grids and allowed to absorb in Formvar for a minimum of 1 minute. For immunogold staining, grids were placed in blocking buffer for the blocking / permeabilization step for 1 hour. Without rinsing, grids were immediately placed in the appropriate dilution of primary antibody (polyclonal anti-GFP 1:10, Abcam) overnight at 4°C. As a control, some grids were not exposed to the primary antibody. The next day, all grids were rinsed with PBS and floated on multiple drops of the appropriate secondary antibody conjugated with 10 nm gold particles (AURION, Hatfield, PA) for 2 hours at room temperature. The grids were then rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes. After rinsing with PBS and distilled water, the grids were dried and stained with uranyl acetate for contrast. Samples were observed under a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR), and images were captured with an AMT CCD camera (Advanced Microscopy Techniques, Danvers, MA).

[0164] EGF stimulation of exosomes. Exosomes were collected from MBA-MB-231 cells as described above. 1 x 10 9 pieces~3×10 9Exosomes were resuspended in 1 mL PBS and various concentrations of rEGF were added. Exosome suspensions with or without EGF were incubated at 37°C with 5% CO2 for 15 minutes and then placed on ice. Three replicates were pooled, PBS was added to a total volume of 11 mL, and stimulated exosomes were collected by ultracentrifugation at 100,000g for 3 hours in an SW40Ti swinging bucket rotor as described above. Protein extracts were collected from pelleted stimulated exosomes in urea / SDS lysis buffer (containing 100 mM NaF and 1 mM NaOV4) for immunoblot analysis or Triton X-100 buffer (150 mM NaCl, 1% (v / v) Triton X-100, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 50 mM 6-aminohexanoic acid, 10 mM EDTA, 5 mM N-ethylmaleimide, 5 mM benzamidine, 5 μg / mL leupeptin, 1 μg / mL pepstatin, 1 mM phenylmethylsulfonyl fluoride, 100 mM NaF, and 1 mM NaOV4) for immunoprecipitation assays.

[0165] Protein Western Blot and Antibodies. Exosome protein extracts were loaded onto an acrylamide gel according to the bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific) and transferred to a PVDF membrane (Immobilon P) by wet electrophoretic transfer. Blots were blocked with 5% nonfat dry milk in TBS / 0.05% Tween 20 for 1 h at RT and incubated with the following primary antibodies: 1:300 anti-CD9 ab92726 (Abcam); 1:300 anti-TSG101 ab83 (Abcam); 1:1000 anti-EGFR 4267S (CST); 1:300 anti-CD63 sc-365604 (Santa Cruz); 1:200 anti-CD81 cs-166029 (Santa Cruz); 1:1000 anti-pEGFR Tyr1068 3777S (CST); 1:2000 anti-GRB2 610111 (BD Biosciences); 1:1000 anti-Shc 06-203 (Millipore); and 1:5000 anti-GFP. The sections were incubated overnight at 4°C with Ab13970 (Abcam); 1:1000 GAPDH ab9483 (Abcam); 1:10,000 HRP-conjugated β-actin a3854 (Sigma); 1:400 anti-RNA Pol II catalog number 39097 (Active Motif); 1:500 anti-Hsp90 ab1429 (Abcam); 1:500 anti-eIF3A ab86146 (Abcam); and 1:500 anti-eIF4A1 ab31217 (Abcam). HRP-conjugated secondary antibodies (Sigma, 1:2000) were incubated for 1 hour at room temperature. After antibody incubation, the sections were washed four times with 1x TBS 0.05% Tween 20 on an orbital shaker at 10-minute intervals. The blots were developed using Pierce's chemiluminescent reagent.

[0166] Immunoprecipitation. Exosome and cellular protein extracts were gently rocked at 4°C for 2 hours. The lysates were centrifuged at 14,000g for 15 minutes in a pre-cooled centrifuge, and the pellet was discarded. Protein A or G agarose / Sepharose beads were washed twice with PBS and reconstituted to a 50% slurry in PBS. The bead / slurry mix (100 μl) was added to 100 μg of exosome protein extract or 20 μg of cellular protein extract and incubated at 4°C for 10 minutes. The beads were removed by centrifugation at 14,000g for 10 minutes at 4°C, and the pellet was discarded. 10 μg of anti-eIF4A1 antibody was added to 100 μL of exosome lysate and incubated overnight at 4°C on an orbital shaker. 100 μL of protein A or G agarose / Sepharose bead slurry was added and left overnight at 4°C. After centrifugation, the supernatant was discarded and the beads were washed three times with ice-cold urea / SDS buffer. The agarose / sepharose beads were boiled for 5 minutes to dissociate immune complexes from the beads. The beads were collected by centrifugation, and immunoblots were performed on the supernatant.

[0167] Identification of amino acids using UPLC-MS. Exosomes were mixed with 200 μL of methanol containing the internal standard tryptophan-d5 and incubated at -20°C for 1 hour. After centrifugation at 16,000 g and 4°C for 15 minutes, 190 μL of the supernatant was collected and the solvent removed. The dried extract was reconstituted in 15 μL of methanol, of which 10 μL was transferred to a microtube for derivatization. The chromatographic separation and mass spectrometry detection conditions used are summarized in Table 1. The mass range, 50–1000 m / z, was calibrated using sodium formate cluster ions. To examine the retention time stability and sensitivity of the LC / MS system throughout the course of the run, appropriate test mixtures of standard compounds were analyzed before and after every set of two randomized identical sample injections.

[0168] Table 1. Chromatography conditions for the amino acid platform TIFF0007813099000002.tif106164

[0169] Data were processed using the TargetLynx application manager in MassLynx 4.1 software (Waters Corp., Milford, USA). A set of predefined retention times, mass-to-charge ratio pairs, and Rt-m / z values ​​corresponding to the metabolites included in this analysis were fed into the program. The associated extracted ion chromatograms (mass tolerance window = 0.05 Da) were then peak-detected and noise-reduced in both the LC and MS domains to ensure that only true metabolite-related features were processed by the software. A list of chromatographic peak areas was then generated for each sample injection using Rt-m / z data pairs (retention time tolerance = 6 s) as identifiers. For each metabolite, a normalization factor was calculated by dividing its intensity in each sample by the recorded internal standard intensity in the same sample.

[0170] Digital qPCR. Digital RNA reactions were performed using 3 ng of cDNA, TaqMan® Universal Master mix, and QuantStudio™ 3D Digital PCR Master Mix v1 (Applied Biosystems) according to the manufacturer's recommendations. A total of 14.5 μL of the mix was loaded onto a QuantStudio™ 3D Digital PCR 20K Chip Kit v1 (Applied Biosystems) using a QuantStudio™ 3D Digital PCR Chip Loader (Applied Biosystems). PCR reactions were performed on a GeneAmp® 9700 (Applied Biosystems) according to the manufacturer's protocol. The chip was imaged using a QuantStudio™ 3D Digital PCR Instrument (Applied Biosystems).

[0171] Table 2: Digital PCR primers TIFF0007813099000003.tif50160

[0172] Exosomes 35 S] methionine labeling. Exosomes were isolated as described above and diluted with 0.1-1.0 mCi / ml trans label [ 35 The exosomes were resuspended in methionine-free culture medium containing [S]-L-methionine (Amersham Biosciences) but without FBS and incubated overnight. Alternatively, exosomes were incubated in the presence of cycloheximide (Sigma, 100 μg / mL). Exosomes were pelleted, washed with ice-cold PBS, and resuspended in urea / SDS lysis buffer as described above. Protein extracts were quantified using a BCA protein assay kit, run on an acrylamide gel, and transferred to a PVDF membrane (Immobilon P) by wet electrophoretic transfer. The membrane was then analyzed by autoradiography using the EN3HANCE® autoradiography enhancer according to the manufacturer's instructions (Perkin-Elmer).

[0173] Real-time PCR analysis. DNase-treated RNA was retrotranscribed using MultiScribe Reverse Transcriptase (Applied Biosystems) and oligo-d(T) primers after total exosomal RNA purification using Trizol (Invitrogen). Real-time PCR was performed on an ABI PRISM 7300HT Sequence Detection System Instrument using SYBR Green Master Mix (Applied Biosystems) and β-actin as a control. The 28S rRNA primer pair (QF00318857) and 18S rRNA primer pair (QF00530467) were purchased from Qiagen as ready-to-use specific primer pairs. Other primers are listed below. Each assay was performed in triplicate. The threshold cycle (Rothstein et al.), the fraction of cycles at which the amount of amplified target reaches a certain threshold, was determined, and expression was measured using the 2-ΔCt formula as previously reported (Livak and Schmittgen, 2001).

[0174] Table 3: qPCR probes TIFF0007813099000004.tif36161

[0175] Lysate preparation for in vitro transcription and translation. Exosome and cell pellets were washed once with ice-cold PBS and resuspended in an equal volume of ice-cold 20 mM HEPES (pH 7.5), 100 mM potassium acetate, 1 mM magnesium acetate, 2 mM dithiothreitol, and 100 μg / mL lysolecithin. After 1 minute on ice, they were repelleted and resuspended in an equal volume of ice-cold hypotonic extraction buffer. After 5 minutes on ice, the lysate was disrupted by passing it 10 times through a 26-gauge needle attached to a 1 mL syringe. The resulting homogenate was centrifuged at 1000 g for 5 minutes at 4°C. The supernatant was collected, and aliquots were frozen in liquid nitrogen and stored at -80°C for use in in vitro translation assays.

[0176] In vitro coupled transcription and translation. Lysates obtained from cells and exosomes as described above were used for in vitro translation in a 12 μL reaction volume. Standard reaction conditions were as follows: cell lysate (final protein concentration 10 μg) or exosome lysate (final concentration 100 μg), 1 μg of pEMT7-GFP cDNA expression plasmid, 20 mM HEPES-KOH (pH 7.6), 80 mM potassium acetate, 1 mM magnesium acetate, 1 mM ATP, 0.12 mM GTP, 17 mM phosphocreatine, 0.1 mg / mL creatine phosphokinase, 2 mM dithiothreitol, 40 μM of each of the 20 amino acids, 0.15 mM spermidine, and 400 U / mL RNAsin (Promega). Incubation was performed at 37°C for 3 h.

[0177] Exosome electroporation and culture. Exosomes were pelleted and resuspended in 400 μL of electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25 mM potassium chloride, 21% Optiprep) along with 20 μg of plasmids (pCMV-GFP, pEGFP-p53 Addgene plasmid 12091, pcDNA3-RLUC-POLIRES-FLUC, and pcDNA-FLUC). Exosomes were electroporated using a Gene Pulser Xcell Electroporation System (BioRad) in a 4 mm cuvette, as previously described (Alvarez-Erviti et al., 2011). Where necessary, exosomes were electroporated in the presence of cycloheximide (Sigma, 100 μg / mL) for translation inhibition or α-amanitin (Sigma, 30 μg / mL) for transcription inhibition. Electroporated exosomes were cultured in serum-free DMEM at 37°C for the indicated time points.

[0178] Flow cytometry analysis of electroporated exosomes. Exosome preparations (5–10 μg) were incubated with 5 μL of 4 μm diameter aldehyde / sulfate latex beads (Interfacial Dynamics, Portland, OR) and resuspended in 600 μL. Exosome-coated beads were analyzed on a FACS Calibur flow cytometer (BD Biosciences) and analyzed for green fluorescence.

[0179] Exosome delivery and confocal microscopy. MCF10A cells were plated at appropriate confluency onto glass coverslips in 12-well plates and cultured overnight. The following day, cells were incubated with MDA-MB-231 exosomes resuspended in serum-free DMEM for 2 hours, washed with 1x cold PBS, and fixed with 4% PFA / PBS for 20 minutes at room temperature. Slides were permeabilized with 0.5% Triton X-100 in PBS for 10 minutes at room temperature and counterstained with DAPI. Images were acquired using a Zeiss LSM510 Upright Confocal System, using the recycle tool to maintain identical settings. For data analysis, images were selected from a pool drawn from at least two independent experiments. Figures show representative fields.

[0180] Reverse transwell assay. Exosomes were isolated from MDA-MB-231 cells as described above, resuspended in PBS, and quantified using Nanosight NTA. 10 x 10 exosomes dissolved in 150 μL of PBS were used. 9Exosomes were added to each bottom well of a 96-well Corning™ HTS Transwell® system. PBS alone was added to the bottom wells as a negative control. Inserts with polycarbonate membranes with 40 nm pores were added to each well, and 100 μL of PBS alone, PBS containing 20% ​​FBS, or PBS containing 10,000 ng / ml EGF was added to the inserts. The Transwell plates were incubated at 37°C with 5% CO2, and samples were collected from the top inserts after 4 and 24 hours of incubation for exosome quantification using a Nanosight NTA.

[0181] Statistics. Error bars indicate ±s.d. between biological replicates. Technical triplicates and biological triplicates of each experiment were performed. Statistical significance was calculated by Student's t-test, ANOVA, or Mann-Whitney test, as appropriate and indicated in the figure legends.

[0182] Example 1 – EGFR phosphorylation is detected in exosomes derived from MDA-MB-231, triple-negative human breast cancer cells Using established ultracentrifugation methods (Melo et al., 2015; Melo et al., 2014), exosomes were isolated from various mouse and human cell lines. Isolated exosomes are heterogeneous mixtures, with the same size distribution consistently observed between preparations. NanoSight nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM) revealed particles with an average diameter of 104 ± 1.5 nm and a size distribution ranging from approximately 30 to 200 nm. This was confirmed by transmission electron microscopy (TEM), which showed extracellular vesicles surrounded by a lipid bilayer (Figure 8A-C). Isolated exosomes were further shown to possess known exosome markers by immunogold / TEM imaging, immunoblot analysis, and imaging flow cytometry (Raposo and Stoorvogel, 2013) (Figure 8D-F). To further confirm purity, exosome samples were plated onto solidified LB plates. No colonization was observed when compared to bacterial controls obtained from oral swabs, demonstrating the absence of bacterial contamination in the isolated exosomes (Figure 8G).

[0183] The EGFR content of exosomes obtained from various cell lines was examined by immunoblotting. While exosomes derived from all cell lines showed low levels of EGFR expression, exosomes derived from the BJ fibroblast cell line and the MDA-MB-231 triple-negative breast cancer cell line showed strong expression of this receptor. The known exosome marker CD81 was shown as a loading control (Figure 1A). Given that EGFR is important for the progression of triple-negative breast cancer (Lim et al., 2016; Liu et al., 2012; Nakai et al., 2016), we further explored the functional role of EGFR in MDA-MB-231-derived exosomes. Exosomes were obtained from MDA-MB-231 and MCF10A cells, and one billion exosomes were incubated with 500 ng / ml recombinant human EGF (rhEGF) in serum-free culture medium at 37°C for 15 minutes. Immunoblotting of protein extracts obtained from these exosomes using an antibody specific for the Tyr1068 residue of EGFR revealed increased phosphorylation levels of this receptor in exosomes derived from MDA-MB-231, but not nontumorigenic MCF10A, breast epithelial cells (Figure 1B). Baseline levels of EGFR were unchanged in all samples, confirming the specificity of the observed increased phosphorylation. Stimulation with recombinant human EGF (rhEGF) also increased phosphorylated ERK levels, suggesting that the observed EGFR phosphorylation triggers downstream signaling events within exosomes (Figure 1C). Furthermore, examination of the protein content of MDA-MB-231 exosomes revealed that MDA-MB-231 exosomes also contain downstream effectors of EGFR, namely, GRB2 and Shc (Figure 1C).

[0184] We next examined whether exosomal EGFR could bind to its downstream adaptor upon stimulation with rhEGF. Exosomes were stimulated with rhEGF for 15 min at 37°C. Exosomal protein extracts were subjected to pull-down assays using specific antibodies for GRB2 and Shc. Upon stimulation with EGF, increased co-immunoprecipitation with EGFR was detected (Figure 1D,E). Isotype IgG was used as a negative control for the pull-down and showed no EGFR co-immunoprecipitation. Furthermore, by reversing this assay and pulling down EGFR, we were able to detect co-immunoprecipitated GRB2 exclusively in EGF-stimulated exosomes (Figure 8B). Collectively, these results demonstrate that exosomes derived from MDA-MB-231 cells contain EGFR, which can be phosphorylated by incubation with ligands under cell-free conditions, thereby triggering putative downstream signaling events within the exosome.

[0185] Example 2 - EGF stimulation of exosomes alters the protein content of exosomes Receptor tyrosine kinases require ATP as a substrate for kinase activity, and prostate-derived exosomes have been shown to be capable of generating ATP (Ronquist et al., 2013a). To further confirm the presence of phosphorylation activity in the absence of cells, an ATP quantification assay was performed on exosomes stimulated with or without rhEGF. Using a luminescence-based kit, ATP was detected in exosomes derived from MDA-MB-231 and MCF10A cells, although at lower concentrations in the latter. Upon stimulation with EGF, exosomes derived from MDA-MB-231 cells showed a slight decrease in ATP content, whereas exosomes derived from MCF10A cells did not (Figure 2A). To further examine the effect of EGF stimulation on exosomes, exosomes were incubated with EGF for 48 hours in a cell-free condition. Compared to unstimulated exosomes, GRB2 protein levels were consistently higher in exosomes stimulated with EGF for 48 hours (Figure 2B). This raised the intriguing possibility that growth factor stimulation altered the protein content of exosomes. To further investigate this possibility, mass spectrometry was performed on protein extracts obtained from exosomes unstimulated or stimulated with rhEGF. Protein extracts were subjected to trypsin digestion and evaluated using an ESI-TRAP mass spectrometer to obtain MS / MS peptide spectra for each sample. The resulting spectra were then evaluated against the SwissProt database to identify peptides, with the goal of obtaining a list of proteins for each exosome sample. Using the open-access FunRich functional enrichment analysis tool (Pathan et al., 2015), we observed that the majority of hits identified in unstimulated and stimulated exosomes matched proteins previously identified in exosomes (Vesiclepedia database) (Figure 2C). A larger number of proteins were identified in rhEGF-stimulated exosomes compared to unstimulated exosomes (491 vs. 371, Figure 2D).While the majority of these proteins were found in both stimulated and unstimulated exosomes, 224 of the 491 proteins were detected only after rhEGF stimulation. EGFR was identified in both samples, whereas GRB2 was only identified in rhEGF-stimulated exosomes (Figure 2E). However, it must be emphasized that this does not mean that GRB2 is absent from unstimulated exosomes; it may be present at levels below the detectable threshold for this type of analysis. We used emPAI (Exponentially Modified Protein Abundance Index), which allows label-free quantification of relative changes in protein abundance based on observable peptide matches (Ishihama et al., 2005). The top 15 proteins that showed a strong increase in rhEGF-stimulated exosomes compared to their unstimulated counterparts included several proteins involved in actin remodeling and membrane dynamics, such as α-actinin, MARCKS, ezrin, moesin, and integrin α-2 (Tables 4 and 5). Gene Ontology (GO) analysis was then performed using the PANTHER overrepresentation test. Interestingly, among the top GO biological processes enriched in rhEGF-stimulated exosomes, several were related to actin remodeling and migration (5 of the top 20 pathways, Table 6).

[0186] Table 4. Top 15 proteins identified as upregulated in exosomes derived from MDA-MB-231 cells incubated with 500 ng / ml EGF at 37°C for 48 hours compared to control exosomes, based on protein scores using the emPAI method (Ishihama et al., 2005). TIFF0007813099000005.tif218164

[0187] Table 5. Top 15 proteins identified as down-regulated in exosomes derived from MDA-MB-231 cells incubated with 500 ng / ml EGF at 37°C for 48 hours compared to control exosomes, based on protein scores using the emPAI method (Ishihama et al., 2005). TIFF0007813099000006.tif198164

[0188] Table 6: Top 20 Gene Ontology (GO) pathways identified based on differential protein scores between control exosomes and exosomes incubated with 500 ng / ml EGF at 37°C for 48 hours. A list of differentially expressed proteins was obtained using the emPAI method and used as input for GO analysis using the PANTHER overrepresentation test. TIFF0007813099000007.tif26168TIFF0007813099000008.tif241168TIFF0007813099000009.tif207168

[0189] Collectively, these mass spectrometry data suggest that MDA-MB-231 exosomes can alter their protein content upon stimulation with rhEGF. These data also suggest that the same exosomes stimulated with rhEGF undergo actomyosin remodeling and migration, demonstrating a motile phenotype in response to rhEGF stimulation. A bicinchoninic acid (BCA) assay for protein quantification confirmed that protein content increased in exosomes stimulated with rhEGF compared with unstimulated controls (Figure 2F). β-actin immunoblotting also indicated that the level of polymerized actin in exosomes stimulated with various amounts of rhEGF increased compared with control unstimulated exosomes (Figure 2G). Collectively, these observations indicate an unexpected degree of biological activity in exosomes. Therefore, we further investigated the possibility that exosomes can synthesize proteins de novo under permissive conditions and explored the potential induction of exosome motility by growth factor stimulation.

[0190] Example 3 - Exosomes derived from various cell types contain functional components required for transcription and translation Analysis of proteomic data derived from exosomes of various cellular origins revealed the presence of several components of the protein synthesis machinery, such as eukaryotic initiation factors, ADP-ribosylation factors, and ribosomal proteins (Choi et al., 2012; Melo et al., 2015; Pisitkun et al., 2004; Valadi et al., 2007) (Figures 10, 11A,B). This information, taken together with the knowledge that mRNAs and their corresponding proteins are found in exosomes, further suggests that isolated exosomes may have the ability to translate mRNAs into proteins.

[0191] Quantitative PCR (qPCR) analysis confirmed the presence of both 18S and 28S rRNA, as well as tRNAs for methionine, glycine, leucine, serine, and valine, in all analyzed exosomes (Figure 12A,B). Furthermore, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of exosomes revealed the presence of all free amino acids (Figure 3A). Immunoblotting analysis identified the presence of distinct members of the translation initiation complex, including eIF4A, eIF3A, and eIF1A, in exosomes (Figure 3B). This confirmed the observations made by mass spectrometry. Furthermore, initiation factors eIF4A and eIF3A co-immunoprecipitated in protein extracts obtained from exosomes (Morino et al., 2000) (Figure 12C).

[0192] To functionally address the relevance of exosome-resident components for protein production, total protein extracts from exosomes isolated from MCF10A and MDA-MB-231 cells were incubated with a cDNA expression plasmid for green fluorescent protein (GFP plasmid). A coupled in vitro transcription-translation assay was performed. Western blot analysis of the extracts after incubation with a GFP-encoding plasmid revealed GFP protein production (Figure 3C). The fact that exosome lysates from both MDA-MB-231 and MCF10A cells allowed protein synthesis from the GFP-expressing plasmid supports the belief that exosomes derived from various cell sources likely contain all the functional components required for both DNA transcription and mRNA translation. Consistent with their DNA transcription capacity, further immunoblot analysis of protein extracts derived from exosomes isolated from various cell sources identified the presence of both phosphorylated and unphosphorylated forms of RNA polymerase II subunits (Figure 3D).

[0193] Example 4 - Exosomes are capable of cell-independent protein synthesis To further validate the finding that exosomes can autonomously carry out de novo mRNA translation, we used isolated exosomes obtained from MDAMB-231 cells and the mouse lung cancer E10 cell line to label newly synthesized proteins. 35 The assay was performed at 37°C to activate putative biosynthetic processes and potential autocrine stimulation. 35 Autoradiography of protein extracts derived from exosomes incubated for 72 hours in the presence of S-methionine showed that the radioactive amino acid was incorporated into several proteins in the 40–300 kDa range, primarily those that comprise the exosomes. 35 This was demonstrated when exosomes derived from various cancer cells were incubated with the protein translation inhibitor cycloheximide together with S-methionine (Figure 3E). 35 A different labeled protein pattern was observed when exosomes were incubated with S-methionine. Furthermore, we quantified the total protein content from freshly isolated exosomes incubated in cell-free culture medium. After 48 hours of incubation, the total exosomal protein content significantly increased (Figure 3F).

[0194] Next, we established an exosome in vitro translation protocol to confirm whether transcription and translation occur in intact exosomes rather than in exosome lysates. We directly electroporated the pCMV-GFP expression plasmid into exosomes derived from MDA-MB-231 cells (Borges et al., 2013; El-Andaloussi et al., 2012; Kamerkar et al., 2017), and then incubated the electroporated exosomes in serum-free culture medium at 37°C for 48 hours. qPCR analysis of exosomal RNA isolated after DNase digestion revealed the presence of GFP mRNA in exosomes electroporated with the pCMV-GFP expression plasmid (Figure 4A). Transmission electron microscopy revealed that the structure of exosomes electroporated with the pCMV-GFP plasmid was intact, and immunogold labeling using an anti-GFP antibody revealed that the protein was only detectable in exosomes containing the GFP plasmid (Figure 4B). Immunoblot analysis of exosome protein extracts using a GFP antibody further confirmed the presence of GFP in pCMV-GFP plasmid-electroporated exosomes, which was observed as early as 12 h after electroporation (Figure 4C). GFP did not increase above the levels observed at 24 h, but could still be observed in exosomes electroporated with the expression plasmid after 1 week and even 1 month (Figure 4C,D). The same pattern was observed in exosomes derived from MCF10A cells. This confirms that exosomes derived from various cells, not just tumorigenic cells, contain all necessary components and are capable of de novo protein synthesis (Figure 13A).

[0195] Immunoblot analysis of exosomes electroporated with the GFP plasmid revealed that GFP levels were reduced by approximately 80% when incubated in the presence of cycloheximide, a protein translation inhibitor (Figure 4E). GFP production was also reduced in the presence of α-amanitin, a transcription inhibitor of RNA polymerase II (Figure 4E,F). NanoSight NTA of electroporated exosomes using a 488 nm laser also detected green fluorescence in exosomes electroporated with the pCMV-GFP plasmid, but not in mock-electroporated exosomes or exosomes electroporated with the plasmid plus cycloheximide or α-amanitin (Figure 13B). Furthermore, the presence of GFP was detected by bead-based flow cytometry analysis of plasmid-containing exosomes using various electroporation conditions (Figure 13C). Next, the exosomes were incubated at 37°C for 24 hours to initiate biological processes, and then electroporated with the pCMV-GFP plasmid. GFP production was reduced as detected by immunoblotting, suggesting that the necessary components for transcription and translation were depleted in exosomes preincubated at 37°C (Figure 4G).

[0196] To confirm that these results were not unique to GFP, we used an ovalbumin-expressing plasmid (pCMV-Ova), a protein not expressed in mammalian cells. As with GFP, immunoblotting analysis of exosomes after electroporation and incubation at 37°C for 48 hours demonstrated that ovalbumin was produced only in exosomes electroporated with the pCMV-Ova plasmid (Figure 13D).

[0197] In eukaryotes, initiation of protein translation of the majority of mRNAs requires recognition of the 5' cap structure by the eIF4F complex (Merrick, 2004). To determine whether protein translation in exosomes is cap-dependent, we used a cDNA bicistronic construct consisting of two distinct luciferase cistrons separated by an internal ribosome entry site (Figure 4H) (Poulin et al., 1998). In this system, Renilla luciferase translation is cap-dependent, whereas firefly luciferase translation is driven by a poliovirus IRES and is therefore cap-independent (Figure 4H). Direct introduction of the plasmid into exosomes by electroporation increased Renilla luciferase activity, while firefly luciferase activity remained apparently unchanged (Figure 4I) (Poulin et al., 1998). This suggests that protein translation in exosomes is cap-dependent. Because the requirements for activity of the firefly and Renilla luciferase enzymes are different, we repeated this assay using a plasmid expressing firefly luciferase under the control of the CMV promoter, and luciferase activity was also observed in pCMV-Fluc electroporated exosomes (Figure 4J).

[0198] Example 5 - mRNA translation in exosomes produces functional proteins and can be stimulated by growth factors MCF10A cells pretreated with cycloheximide were incubated with MDA-MB-231 exosomes directly electroporated with pCMV-GFP plasmid. Confocal microscopy revealed green fluorescence in MCF10A cells, likely due to the GFP protein delivered by MDA-MB-231 exosomes (after transcription and translation) (Figure 5A, upper and lower panels). Interestingly, cells directly electroporated with pCMV-GFP plasmid exhibited a different GFP fluorescence pattern from that observed in cells incubated with pCMV-GFP plasmid-containing exosomes (Figure 5A, middle and lower panels).

[0199] MDA-MB-231 cells overexpress an inactive mutant form of the tumor suppressor protein p53, which is therefore unable to activate the p21 promoter (Gartel et al., 2003). Wild-type (wt) p53 typically responds to DNA damage by directly inducing p21 and promoting cell cycle arrest (Zilfou and Lowe, 2009). Exosomes isolated from MDA-MB-231 cells were electroporated with a plasmid encoding wt p53 fused to GFP. The electroporated exosomes were incubated in culture medium for 48 hours to allow transcription and translation to occur, producing wt p53 protein (Figure 5B). The newly formed wt p53-containing exosomes were then incubated with recipient MDA-MB-231 cells under the influence of cycloheximide. The recipient MDA-MB-231 cells showed significantly increased p21 expression (Figure 5C). This confirmed the functionality of the wt p53 protein synthesized exclusively by exosomes (Figure 5B). To further confirm that this increase in p21 expression was indeed due to newly synthesized wt p53 protein by exosomes and not due to plasmid delivery, MDA-MB-231-derived exosomes were electroporated with p53-GFP plasmid and incubated for 48 hours to synthesize wt p53 protein before incubation with recipient MDA-MB-231 cells (48 hours), or immediately delivered to recipient MDA-MB-231 cells without producing wt p53 protein (0 hours, Figure 13E). Exosomes delivered after 48 hours of active synthesis of wt p53 protein induced greater p21 expression in recipient MDA-MB-231 cells as early as 30 minutes after exosome incubation than exosomes containing only the plasmid. Plasmid-only exosomes show the same baseline p21 expression as observed in control MDA-MB-231 cells (Figure 13E).

[0200] To further demonstrate that MDA-MB-231 cell-derived exosomes exhibit a baseline capacity for endogenous protein synthesis, exosomes were incubated at 37°C in the presence and absence of cycloheximide. Immunoblotting of protein extracts from these exosomes revealed that incubation with cycloheximide consistently reduced the expression levels of the small cytoplasmic proteins β-actin and GAPDH, again demonstrating the presence of a baseline level of protein synthesis in these exosomes (Figure 5D).

[0201] To determine which proteins are produced by MDA-MB-231 exosomes in the absence of external stimuli, we performed a modified version of stable isotope labeling with amino acids in culture (SILAC). MDA-MB-231 cell-derived exosomes were cultured in a heavily labeled (heavy labeled) 13 C-lysine and 15 MDA-MB-231 exosomes were incubated in SILAC medium supplemented with N-arginine. MDA-MB-231 exosomes were incubated in heavy-labeled SILAC medium for 5 days, and protein extracts were obtained, trypsin-digested, and subjected to mass spectrometry. Although only a few heavy-labeled peptides matched the MS / MS spectra obtained, 11 proteins were identified, each matching one or two peptides containing heavy-labeled amino acids (Table 7). This confirms that low-level, baseline mRNA translation occurs in exosomes, resulting in the formation of very small amounts of newly synthesized proteins.

[0202] (Table 7) 13 C-lysine and 15 A list of proteins containing peptides matching the heavy isotope-containing spectra obtained from mass spectrometry of protein extracts from MDA-MB-231 exosomes incubated with N-arginine SILAC medium for 5 days. Each protein listed is 13 C-lysine or 15Contains at least one peptide that matches the N-arginine heavy labeling spectrum. TIFF0007813099000010.tif251138TIFF0007813099000011.tif251153TIFF0007813099000012.tif25145

[0203] Next, the protein translation assay was repeated using exosomes derived from MDA-MB-231 cells harboring electroporated pCMV-GFP plasmid. The exosomes were incubated in serum-free culture medium with or without rhEGF stimulation at 37°C for 48 hours. Unstimulated exosomes showed baseline levels of GFP production, but incubation with various concentrations of rhEGF increased GFP levels (Figure 5E). This again confirmed that all exosomes are capable of synthesizing proteins, whereas growth factor stimulation can alter production rates by increasing protein synthesis levels.

[0204] Example 6 - Exosomes actively migrate in response to stimulation with rhEGF and serum factors To determine whether growth factors can induce a motile phenotype in exosomes, we designed a reverse migration assay based on a Boyden chamber system containing rhEGF and serum factors. Ten billion exosomes isolated from MDA-MB-231 cells were placed in culture wells of a 96-well plate and covered with polycarbonate surface inserts with 400 nm pores. The inserts contained PBS, PBS containing 10 μM rhEGF, or PBS containing 20% ​​exosome-depleted FBS (Figure 6A). Because exosome-depleted FBS may still contain trace amounts of exosomes (data not shown), as a control, 20% FBS was placed on the surface of the top insert without exosomes in the bottom well. After incubation at 37°C, samples were obtained from the top insert at various time points, and exosomes were quantified using a Nanosight NTA.

[0205] After 4 hours of incubation, the levels of exosomes on the top inserts were comparable across all experimental groups (Figure 6B). After 24 hours of incubation, 20% FBS significantly increased exosome migration from bottom to top, suggesting a sustained chemotactic effect on MDA-MB-231 exosomes toward the high serum growth factor gradient (Figure 6C). Inserts containing 20% ​​FBS but no exosomes on the top well contained significantly fewer exosomes after 24 hours, confirming the identity of the migrating exosomes as derived from MDA-MB-231 cells (Figure 6C). While PBS resulted in negligible amounts of exosome migration, rhEGF alone also induced exosome motility, albeit at a low level compared to full serum-associated growth factors (Figure 6C). Collectively, these results suggest that exosomes exhibit functional chemotactic capabilities that can be induced by growth factors.

[0206] Example 7 – Exosomes specifically enhance protein production in tumor-bearing mice To address whether the ability of exosomes to respond to tumor-induced growth factor gradients involves the endogenous production of novel proteins with functional significance, we created a reference mouse model. Mice with established 4T1 mammary tumors were injected with 5 billion MDA-MD 231 / CD63-mCherry exosomes electroporated with GFP or ovalbumin-expressing plasmids. Control arms of this study included CD63-mCherry exosomes without the above plasmids and CD63-mCherry exosomes electroporated with the plasmids and cyclohexamide. Tumors, serum, and several other organs were collected 24 hours after IP injection of exosomes into tumor-bearing or non-tumor-bearing mice. Exosomes were FACS-isolated using the CD63-mCherry tag and assayed for GFP or ovalbumin protein. GFP and ovalbumin were detected primarily in the tumors, lungs, bones, brains, and serum of tumor-bearing mice, but were found at very low levels in tissues from non-tumor-bearing mice and tumor-bearing mice injected with cyclohexamide-containing exosomes. These results demonstrate that exosomes can be detected in the liver, lungs, and brains of non-tumor-bearing mice, but that exosomes actively invade these organs, including tumor tissue (presumably through enhanced motility), and respond biologically by producing novel proteins. Furthermore, serum-derived exosomes from tumor-bearing mice exhibit protein production, suggesting that tumors biologically affect exosomes at a systemic level.

[0207] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or order of steps thereof without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. 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 invention as defined by the appended claims.

[0208] 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. TIFF0007813099000013.tif201160TIFF0007813099000014.tif245160TIFF0007813099000015.tif238160TIFF0007813099000016.tif245160 TIFF0007813099000017.tif238160TIFF0007813099000018.tif231160TIFF0007813099000019.tif238160TIFF0007813099000020.tif216160

Claims

1. 1. A pharmaceutical composition for use in a method of treating a cancer that produces an epidermal growth factor (EGF) gradient at the site of the cancer in a patient in need thereof, comprising: The pharmaceutical composition comprises a transfected exosome, The method includes administering the transfected exosome to the patient; the exosomes contain epidermal growth factor receptor (EGFR) on their surface, are isolated from a body fluid sample obtained from the patient, and are transfected with a nucleic acid encoding a therapeutic protein; the EGFR can be stimulated and phosphorylated by contact with EGF; the exosomes containing EGFR on their surface have been stimulated by contact with EGF before the transfected exosomes are administered to the patient; and Stimulation of the exosomes containing EGFR on their surface induces translation of the nucleic acid encoding the therapeutic protein and protein synthesis. The pharmaceutical composition.

2. The pharmaceutical composition of claim 1, wherein the nucleic acid is mRNA.

3. 10. The pharmaceutical composition of claim 1, wherein the nucleic acid is a plasmid.

4. The pharmaceutical composition of claim 1, wherein the nucleic acid is a cDNA.

5. 2. The pharmaceutical composition of claim 1, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

6. The pharmaceutical composition of claim 1, wherein the cancer comprises a tumor.

7. 10. The pharmaceutical composition of claim 1, wherein the cancer is metastatic.

8. 8. The pharmaceutical composition of claim 7, wherein the cancer site is a metastatic nodule.

9. 10. The pharmaceutical composition of claim 1, wherein the therapeutic protein is a kinase, phosphatase, or transcription factor.

10. 10. The pharmaceutical composition of claim 1, wherein the therapeutic protein corresponds to a wild-type version of a protein that is mutated or inactivated in cells at the site of the cancer.

11. 10. The pharmaceutical composition of claim 1, wherein the therapeutic protein corresponds to a dominant-negative version of a protein that is overactive in cells at the site of the cancer.

12. 10. The pharmaceutical composition of claim 1, wherein the therapeutic protein is a tumor suppressor.

13. 10. The pharmaceutical composition of claim 1, wherein the exosomes are transfected by electroporation.

14. 10. The pharmaceutical composition of claim 1, wherein the method further comprises administering at least a second therapy to the patient.

15. 15. The pharmaceutical composition of claim 14, wherein the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

16. 10. The pharmaceutical composition of claim 1, wherein the exosomes are contained in a tissue scaffold matrix.

17. 10. The pharmaceutical composition of claim 1, wherein the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum.

18. 18. The pharmaceutical composition of claim 17, wherein the bodily fluid sample comprises bone marrow aspirate.

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