CN112533643A - Use of exosomes for targeted delivery of therapeutic agents - Google Patents

Abstract

Provided herein are methods of using exosomes that function like minicells to deliver therapeutic agents to diseased or dysregulated cells. In particular, exosomes may be targeted to specific regions of the body using a gradient of growth factors. These gradients are also used to trigger protein expression in exosomes at the desired target from the transfected nucleic acids.

Description

Use of exosomes for targeted delivery of therapeutic agents

Reference to related applications

This application claims priority to U.S. provisional application No. 62/649,057 filed on 28/3/2018, the entire contents of which are incorporated herein by reference.

Reference to sequence listing

This application contains a sequence listing, which has been filed in ASCII format by EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on 21.3.2019, named utfc.p1363wo _ st25.txt and has a size of 3 kilobytes.

Background

1. Field of the invention

The present invention relates generally to the fields of biology, medicine and oncology. More particularly, it relates to the use of exosomes to target therapeutic agents to diseased or dysregulated cells.

2. Description of the related Art

Exosomes are small Extracellular Vesicles (EVs) with lipid bilayers that contain proteins and polynucleotides, including messenger RNA (mrna), non-coding RNA and double-stranded genomic DNA (Kalluri, 2016; Raposo and Stoorvogel, 2013). After initial discovery that exosomes were byproducts of reticulocyte differentiation (Harding et al, 1984; Raposo and Stoorvogel, 2013), it is now generally accepted that exosomes are actually secreted by all mammalian cells and are found in all body fluids (El-Andaloussi et al, 2013; Kalluri, 2016).

Exosomes are part of a larger population of extracellular vesicles that also include microvesicles and apoptotic bodies (Colombo et al, 2014). In extracellular vesicles, exosomes are usually distinguished by their unique biogenesis (biogenesis) via the endocytic pathway. Endocytic vesicles mature into late endosomes, also known as polyvesicles, which contain a number of intracellular vesicles (ILVs) produced by the invagination of the endosomal membrane. Through possible fusion of these vesicles with the plasma membrane, exosomes are released into the extracellular space and enter the circulation (Bastos et al, 2017; Colombo et al, 2014). Due to their endocytic origin, the exosome membranes have a polarity similar to the cell membrane, which comprises a membrane protein anchored with its intracellular domain facing the lumen (lumen) and its extracellular domain facing the extracellular space. Although the protein content of exosomes varies depending on their cellular origin, several proteins show a general enrichment. 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, different proteomic studies of exosomes derived from many different cell types have identified many components associated with the protein translation machinery, such as eukaryotic initiation factors, ADP-ribosylation factors, ribosomal proteins (Pisitkun et al, 2004; Valadi et al, 2007). In addition, it has been proposed to deliver a subset of transcriptional and translational regulators identified in exosomes by proteomic analysis to recipient cells, thereby altering their gene and protein expression patterns (Ung et al, 2014).

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 (seshacharyuuetal, 2012). Upon binding to one of its ligands, such as Epidermal Growth Factor (EGF), the receptor dimerizes, forming either a homodimer or a heterodimer with other members of the ErbB family (seshacharyuu et al, 2012). This dimerization activates the intrinsic kinase activity of the receptor, resulting in autophosphorylation of different key tyrosine residues on its cytoplasmic domain. This autophosphorylation reaction recruits different adaptor proteins containing SH2 and PTB (phosphotyrosine binding) domains, such as Shc and GRB2, which mediate different downstream signaling activities, including the synthesis of related proteins (normano et al, 2006; Tomas et al, 2014). Phosphorylated EGFR is eventually ubiquinated and transported to the endosomal pathway, from which it is either recycled back to the membrane, or remains in the late endosomal pathway, leading to integration into the multivesicular body or lysosomal degradation (Tomas et al, 2014). Since the vesicles originate from exosomes, post-phosphorylation recycling of EGFR (and other growth factors) may contribute to their membrane localization in these extracellular vesicles.

EGFR signaling has been shown to be important for the progression of different malignancies (e.g., 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 were performed in the context of carcinogenesis. EGFR signaling is particularly involved in the pattern of cellular uptake and secretion of exosomes from different sources. In mantle cell (mantle cell) cancer cells, incubation with gefitinib (an EGFR inhibitor) has been shown to significantly reduce exosome uptake (Hazan-Halevy et al 2015). Treatment of lung cancer cells with gefitinib resulted in increased secretion of exosomes, which mediate horizontal transfer of cisplatin resistance (Li et al, 2016). It is also known that the metastasis of EGFR by cancer cell-derived exosomes causes alterations in the components of the microenvironment (e.g., endothelial cells and T cells) (Al-Nedawi et Al, 2009; Huang et Al, 2013). Recently, EGFR-containing exosomes derived from gastric cancer cells were shown to be delivered to stromal cells in the liver, mediating metastasis (Zhang et al, 2017). Finally, exosomes derived from breast cancer cells were shown to contain a functionally phosphorylated form of EGFR, which can be transferred to monocytes, mediating their survival by activating the ERK pathway (Song et al, 2016).

While the delivery of EGFR and members of the protein translation machinery by exosomes in the context of cell-cell communication appears to be of significant biological importance, these properties can be exploited to target the delivery of therapeutic agents to certain tissues and induce the production of therapeutic proteins at the desired site of delivery.

Summary of The Invention

Here, protein synthesis is induced in exosomes by growth factor stimulation. Exosomes comprising DNA, RNA, and proteins may respond to biological stimuli and initiate properties such as: migration, reproduction, initiation of signaling networks/cascades, transcription and protein translation. Thus, in one embodiment, provided herein are exosomes having the ability to function like minicells (minicells). As discussed further below, these minicell-like exosomes may be used in a variety of therapeutic ways to treat a variety of diseases and/or disorders.

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

In some aspects, the method is further defined as a method of administering a therapeutic protein to diseased cells in a patient. In some aspects, the exosomes obtained in step (a) are obtained from a bodily fluid sample obtained from a patient. In some aspects, the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate/tears, or serum. In some aspects, the nucleic acid is mRNA, plasmid, or cDNA.

In some aspects, the disease or disorder is cancer, injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a renal disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a genitourinary disorder, or a 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.

In some aspects, the therapeutic protein is a kinase, a phosphatase, or a transcription factor. In certain aspects, the therapeutic protein corresponds to a wild-type form of the protein that is mutated or inactivated in cells at the site of the disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant negative form (dominant negative version) of a protein that is overactive in cells at the site of the disease or disorder. In certain aspects, the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor. In some aspects, the exosomes comprise CD47 on their surface. In some aspects, the transfection comprises electroporation.

In some aspects, the method further comprises administering at least a second treatment to the patient. In some aspects, the second treatment comprises surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

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

In some aspects, the method is further defined as a method of administering a therapeutic agent to diseased cells in a patient. In some aspects, the exosomes obtained in step (a) are obtained from a bodily fluid sample obtained from a patient. In certain aspects, the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate/tears, or serum.

In some aspects, the therapeutic agent is a therapeutic protein, antibody, inhibitory RNA, gene editing system, or 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, a phosphatase, or a transcription factor. In certain aspects, the therapeutic protein corresponds to a wild-type form of the protein that is mutated or inactivated in cells at the site of the disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant negative form of the protein that is overactive in cells at the site of the disease or disorder. In some aspects, the small molecule drug is an imaging agent.

In some aspects, the disease or disorder is cancer, injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a renal disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a genitourinary disorder, or a 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, wherein the therapeutic protein is a tumor suppressor. In some aspects, the disease or disorder is cancer, wherein the therapeutic agent is an inhibitory RNA that targets an oncogene.

In some aspects, the exosomes comprise CD47 on their surface. In some aspects, the transfection comprises electroporation. In some aspects, the method further comprises administering at least a second treatment to the patient. In some aspects, the second treatment comprises surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

In other aspects, exosomes used according to some embodiments are contained in a tissue scaffold matrix. For example, such a matrix may be a synthetic matrix, such as a degradable or absorbable in tissue matrix. In other aspects, the matrix can be a living tissue matrix. In some aspects, the exosomes of some embodiments are cultured in a matrix.

As used herein, "substantially free" with respect to a specified component is used herein to mean that no specified component is purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified components resulting from any unintended contamination of the composition is well below 0.01%. Most preferred are compositions wherein the amount of the specified component is not detectable by standard analytical methods.

As used herein in the specification, a noun without a quantitative term change may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the nouns without the numerical modification may mean one or more than one.

The use of the term "or/and" in the claims is used to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive, although the disclosure supports definitions that refer only to alternatives and "and/or". "additional," as used herein, may mean at least a second or more.

Throughout this application, the term "about" is used to indicate a value that includes inherent variations in the error of the apparatus, method used to determine the value, or variations that exist between study subjects.

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 the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of Drawings

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.

FIGS. 1A to 1E. EGFR phosphorylation in exosomes. Figure 1A-immunoblots of EGFR expression on exosomes obtained from different human and murine cell lines. The exosome marker CD81 was used as a loading control and to confirm the exosome source of the protein extract. FIG. 1B-immunoblot showing EGFR phosphorylation on exosomes derived from MDA-MB-231 cells but not MCF10A cells after incubation with 500ng/ml rhEGF for 15 min at 37 ℃. Phosphorylation levels were detected using an antibody specific for the Tyr1068 residue of EGFR. EGFR levels are shown as loading controls to determine differences in phosphorylation. Tape density measurement quantification was performed using ImageJ software. FIG. 1C-immunoblot showing the presence of EGFR adaptor proteins Shc and GRB2 in exosomes derived from MDA-MB-231 cells with and without stimulation with rhEGF for 15 min at 37 ℃ and increased levels of phosphorylated ERK protein. The exosome marker CD81 was used as a loading control and to confirm the exosome source of the protein extract. FIG. 1D-GRB immune complexes were obtained from protein extracts of MDA-MB-231 exosomes using GRB2 specific antibodies, with and without incubation with 500ng/ml rhEGF at 37 ℃ for 15 minutes. Immunoblot analysis of immune complexes showed association of GRB2 with EGFR only after rhEGF stimulation. Non-specific isotype control IgG was used as a negative control for GRB2 pull down (pulldown). Equal volumes of stimulated and unstimulated extracts were probed for β -actin as an input control. Figure 1E-experiments similar to pull-down experiments (in duplicate) with Shc antibody, which also shows association with EGFR only after stimulation with 500ng/ml rhEGF at 37 ℃ for 15 minutes. Non-specific isotype control IgG was used as a negative control for Shc pull-down. Equal volumes of stimulated and unstimulated extracts were probed for β -actin as an input control.

Fig. 2A to 2G. EGFR phosphorylation alters exosome content. Figure 2A-luciferase-based ATP assay was run on protein extracts obtained from exosomes either unstimulated or after rhEGF stimulation at 37 ℃ for 15 minutes. The luminescence of the luciferin source is measured in a plate reader and expressed in arbitrary units. Significance was determined by the Mann-Whitney (Mann-Whitney) test (n ═ 4). Figure 2B-immunoblot analysis of exosomes from MDA-MB-231 cells incubated with 500ng/ml rhEGF at 37 ℃ for 48 hours, showing increased levels of both pEGFR and GRB2 when compared to unstimulated exosomes. FIG. 2C-cellular component correlation analysis of mass spectral data obtained from MDA-MB-231 exosomes after unstimulation or incubation with 500ng/ml EGF for 48 hours at 37 ℃. A list of important proteins identified for stimulated and unstimulated exosomes was obtained and used as input to an open access FunRich assay tool to identify subcellular origins of the identified proteins. FIG. 2D-Venn diagram (Venn diagram) depicting the overlap of proteins identified in control MDA-MB-231 exosomes and exosomes incubated with 500ng/ml rhEGF for 48 hours at 37 ℃. FIG. 2E-separate EGFR and GRB2 protein scores obtained from mass spectrometric analysis of MDA-MB-231 exosomes with or without incubation for 48 hours with 500ng/ml rhEGF. FIG. 2F-BCA analysis of protein extracts obtained from control MDA-MB-231 exosomes and exosomes incubated with 500ng/ml rhEGF for 48 hours at 37 ℃. Significance was determined by the mann-whitney test (n ═ 3). FIG. 2G-immunoblot analysis of β -actin expression on protein extracts obtained from control MDA-MB-231 exosomes and exosomes incubated with 500ng/ml and 1000ng/ml rhEGF for 48 hours at 37 ℃. (. p < 0.05,. p < 0.01,. p < 0.005,. p < 0.0001).

Fig. 3A to 3F. Exosomes contain functional components for transcription and translation. FIG. 3A-ultra-high performance liquid chromatography-mass spectrometry (UPLCMS) for the detection of free amino acids in MCF10A, MDA-MB-231, HDF, E10 and NIH-3T3 derived exosomes. The data was represented as a heat map (HeatMap) using normalized signal intensity (log10), with hotter colors corresponding to higher intensity levels, as shown in the color legend. Figure 3B-immunoblot of eIF4a1, eIF3A and eIF1A in protein extracts of exosomes obtained from E10, NIH-3T3, MCF10A, HDF and MDA-MB-231 derived exosomes. CD9 was used as a loading control. FIG. 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. FIG. 3D-immunoblot analysis of RNA polymerase II in exosome protein extracts, whichMiddle CD9 is shown as loading control. FIG. 3E-is derived from³⁵Autoradiography of exosomes of cultured MDA-MB-231 and E10 cells in the presence of S-methionine. Will be discharged only to the body and in³⁵Exosomes cultured in the presence of S-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 under cell-free conditions. Significance was determined by one-way ANOVA followed by Tukey multiple comparison test (× p < 0.05, × p < 0.01, × p < 0.005, × p < 0.0001, n ═ 3).

Fig. 4A to 4J. Exosomes synthesize novel 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 electroporated either without electroporation, mock electroporation or electroporation with the pCMV-GFP plasmid in the presence or absence of alpha-amanitin. Expression levels were normalized to GAPDH. Figure 4B-transmission electron microscopy images of exosomes electroporated with GFP plasmid and incubated for 48 hours in cell-free conditions with immunogold labeling with anti-GFP antibody (lower panel). Secondary antibody alone was used as negative control (upper row). The gold particles are depicted as black dots. Scale bar, 100 nm. FIG. 4C-immunoblot of GFP protein expression in exosomes electroporated with pCMV-GFP plasmid and incubated at 37 ℃ for 12 hours, 2 days, or 1 week. Exosome only and exosomes that mimic electroporation 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). The non-electroporated exosomes were used as negative controls. 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 either immediately after isolation (0 hours) or after incubation under cell-free conditions (24 hours) and culture as previously described. Exosomes mimicking electroporation were used as negative controls. The exosome marker TSG101 was used as a loading control for the presence of exosomes. FIG. 4F-immunoblot of GFP protein expression in exosomes electroporated with the pCMV-GFP plasmid and cultured with the translation inhibitor cycloheximide. Exosome only and exosomes that mimic electroporation were used as negative controls. TSG101 was used as a loading control for the presence of exosomes. Tape density measurements were performed using ImageJ software. FIG. 4G-immunoblot of GFP protein expression in exosomes electroporated with GFP plasmid and cultured with the transcription inhibitor α -amanitine. Exosome only and exosomes that mimic electroporation were used as negative controls. TSG101 was used as a loading control for the presence of exosomes. Tape density measurements were performed using ImageJ software. FIG. 4H-schematic representation of a dicistronic plasmid (pCDNA3-rLuc-polIRESfLuc) used as a reporter for cap-dependent or cap-independent translation. FIG. 4I-Renilla luciferase (r-Luc) and firefly luciferase (f-Luc) activity measured by bioluminescence after 48 hours incubation with bicistronic plasmid electroporated exosomes. The non-electroporated exosomes were used as negative controls. FIG. 4J-luminescence counts measured from exosomes incubated for 48 hours after electroporation with or without plasmid with firefly luciferase expressed under the CMV promoter.

Fig. 5A to 5E. Protein translation in exosomes produces functional proteins and can be enhanced by growth factor stimulation. FIG. 5A-confocal microscopy showing the presence of GFP in MCF10A electroporated cells and MCF10A cells pretreated with cycloheximide and incubated with MDA-MB-231 derived exosomes electroporated with the pCMV-GFP plasmid and preincubated for 48 hours. MCF10A cells treated with non-electroporated MDA-MB-231 derived exosomes were used as negative controls. FIG. 5B-immunoblot analysis of GFP expression on protein lysates from MDA-MB-231 derived exosomes electroporated with p53-GFP expression plasmid. The non-electroporated exosomes were used as negative controls. TSG101 was used as a loading control for the presence of exosomes. FIG. 5C-p 21 mRNA expression in MDA-MB-231 cells treated with: exosomes derived from electroporated MDA-MB-231 were simulated, or electroporated with the p53-GFP plasmid in the presence or absence of cycloheximide. Expression levels were normalized to housekeeping gene GAPDH. FIG. 5D-immunoblot of exosomes isolated from MDA-MB-231 cells incubated with 100. mu.g/ml of the translation inhibitor cycloheximide. The exosome lysates were probed for β -actin and GAPDH. Tape density measurement quantification was performed using ImageJ software. FIG. 5E-immunoblot of GFP protein expression in exosomes electroporated with pCMV-GFP plasmid and subsequently incubated for 48 hours at 37 ℃ in the presence of varying concentrations of rhEGF. Both exosomes mimicking electroporation and exosomes not incubated with rhEGF were shown as negative controls. The exosome marker CD81 was used as a loading control. Tape density measurement quantification was performed using ImageJ software.

Fig. 6A to 6C. Exosomes derived from MDA-MB-231 cells showed chemotaxis towards a gradient of growth factors. Figure 6A-schematic showing setup of efflux retrograde migration assay. Briefly, 10X 10 cells isolated from MDA-MB-231 cells⁹Placing the individual exosomes in Corning

In the bottom hole of the system. HTS with 400nm pores

The insert was placed on top of an exosome suspension containing PBS, 20% FBS or 10,000ng/ml rhEGF and incubated at 37 ℃. After different time points, the number of exosomes on the apical insert was measured by Nanosight NTA to assess the movement of exosomes (motility). FIG. 6B&6C-quantification of MDA-MB-231 exosomes on the apical insert determined by retrograde migration by Nanosight NTA after 4 hours (FIG. 6B) and 24 hours (FIG. 6C) incubation at 37 ℃. Significance was determined by a one-way ANOVA followed by a Newman-Keuls (Newman-Keuls) multiple comparison test. (. p < 0.05,. p < 0.01,. p < 0.005,. p < 0.0001, n ═ 3).

Fig. 7A to 7D. Tumor-bearing mice showed increased protein synthesis in the delivered exosomes. FIG. 7A-schematic drawing depicting the experimental plan of in vivo translation experiments. Briefly, female Balb/C mice were injected in situ with 4T1 tumor and tumors were grown to 500mm³Thereafter, mice were injected with 300 billion MDA-MB-231 exosomes electroporated with pCMV-mCherry plasmid. Mice were euthanized 12 hours after exosome injection and sera were collected for exosome extraction. FIG. 7B-shows 4T1 tumor and menses injectedGraphs of tumor growth in mice with electroporated exosomes or 4T1 tumor alone, showing comparable growth kinetics. Figure 7C-Nanosight NTA analysis of exosomes extracted from sera of healthy mice injected with electroporated exosomes, and 4T1 tumor-bearing mice injected with electroporated exosomes and 4T1 tumor-bearing mice without exosome injection. All exosomes showed similar size peaks, about 100 nm. Figure 7D-Nanosight NTA quantification of serum exosomes shown in figure 7C, which shows no significant difference in the amount of exosomes obtained from sera of different animals, but a trend towards more exosomes in 4T1 tumor-bearing mice injected with MDA-MB-231 electroporated exosomes.

Fig. 8A to 8G. And (5) characterizing an exosome. 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 the particles in solution, which shows an average size of 104nm and also shows no peaks at the larger size. The right graph shows the concentration and size distribution of the particles in the solution. Figure 8B-atomic force microscopy image of exosomes (left panel). The right graph shows the distribution of particles in the analyzed area. FIG. 8C is a transmission electron micrograph of an exosome of MDA-MB-231. Scale bar-100 nm. FIG. 8D-Transmission Electron micrograph of immunogold labeled MDA-MB-231 exosomes using anti-CD 9 antibody. The gold particles are depicted 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 from MDA-MB-231 cells conjugated to 0.4 μm beads using antibodies against markers CD9, CD81, CD82, and CD 63. Fig. 8G-representative image of LB plates incubated with escherichia coli (e.coli) and either MDA-MB-231 (top) or MCF10A (bottom) exosomes, showing colony formation on the escherichia coli inoculation side (left) but not on the exosome inoculation side (right).

Fig. 9A to 9B. EGFR phosphorylation and downstream biological activity in exosomes from MDA-MB-231 cells. FIG. 9A-immunoblot of protein extracts obtained from MDA-MB-231 cells and probed for p-EGFR and GRB 2. Beta-actin was used as loading control. FIG. 9B-immunoblot of immunocomplexes obtained with anti-EGFR antibody pull-down from protein lysates of MDA-MB-231 exosomes (with or without incubation with 500ng/ml for 15 min at 37 ℃). The immune complexes were probed for GRB 2. Non-specific isotype control IgG was used as a negative control pulled down by GRB 2. Equal volumes of stimulated and unstimulated extracts were probed for β -actin as an input control.

Fig. 10. Proteomic analysis of exosomes derived from a variety of cells. The heatmap represents the binary identification (binding identification) of all individual proteins contained in the protein translation pathway in the Reactome (Croft et al, 2014) database, obtained from the mass spectral data below: mouse hepatocytes (valdi 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 presence of each protein in each dataset and white indicates its absence. The summary column indicates the degree of ubiquity of each protein in all the analyzed datasets, with warmer colors indicating a more extensive distribution among the different types of exosomes.

Fig. 11A to 11B. Proteomic analysis of exosomes from a variety of sources. Figure 11A-heatmap, which represents the number of proteins associated with different pathways associated with protein translation in the Reactome (Croft et al, 2014) database identified in mass spectral data obtained from: mouse hepatocytes (valdi 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). Warmer colors indicate a higher number of proteins identified per pathway. FIG. 11B-heatmap, which represents protein scores for proteins associated with protein translation identified in mass spectra obtained from exosomes isolated from HDF, NIH 3T3, MDA-MB231, MCF10A, and E10 cells. Warmer colors indicate higher protein scores.

Fig. 12A to 12C. Exosomes comprise nucleic acids and proteins associated with the protein translation machinery. FIG. 12A-RNA extracted from exosomes of NIH-3T3, E10, 67NR, 4T1, HDF, MCF10A, MCF7 and MDA-MB-231 cell lines was used to quantify 18S and 28S rRNA by qPCR. Expression levels of rRNA were normalized to U6 snRNA expression. The bars in each group represent, from left to right, NIH 3T3, E10, 67NR, 4T1, HDF, MCF10A, MCF7 and MDA-MB-231. Figure 12B-RNA extracted from 4T1 exosomes and cells were used to identify the presence of tRNAMet, tRNAGly, tRNALeu, tRNASer and tRNAVal by digital qPCR. The bars in each group represent, from left to right, Leu, Met, Val, Ser and Gly. FIG. 12C-immunoprecipitation of eIF4A1, showing 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.

Fig. 13A to 13E. 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 ℃ for different time periods. Exosome only and exosomes that mimic electroporation were used as negative controls. CD63 was used as a loading control and to confirm the presence of exosomes. Figure 13B-depicts a graph depicting the amount of green exosomes detected by NanoSight in MCF 10A-derived exosomes electroporated with GFP plasmid. Exosomes from MCF10A, mock-electroporated exosomes from MCF10A, and exosomes electroporated with α -amanitine and cycloheximide were used as negative controls. Figure 13C-flow cytometry analysis of beads attached to exosomes after electroporation with GFP plasmid using elevated voltage, showing the percentage of beads with green fluorescent signal. FIG. 13D-immunoblot of ovalbumin protein levels in MDA-MB-231 exosomes electroporated with pCMV-Ova plasmid and incubated for 48 hours at 37 ℃. Beta-actin was used as loading control. FIG. 13E-p 21 mRNA expression in MDA-MB-231 cells treated with: exosomes derived from MDA-MB-231 that mimic electroporation, or were electroporated with the p53-GFP plasmid and added to the cells immediately (0 hours) or were allowed to incubate at 37 ℃ for 48 hours (48 hours) under cellular conditions prior to treatment. Exosomes were added to cells for 30 minutes or 48 hours, followed by RNA extraction. Expression levels were normalized to housekeeping gene GAPDH.

Detailed Description

Extracellular Vesicles (EVs), including exosomes, are nanoscale intercellular communication carriers with lipid bilayers that encapsulate cytosolic-like substances. Exosomes are involved in several physiological processes and comprise DNA, RNA and proteins. It is generally assumed that all of the contents of exosomes are derived from cells, and that they remain intact (remain as suc) in exosomes before they enter other cells and deposit their contents. Exosomes are released in large quantities by all cells and are considered to be garbage bags (garbages) that carry cellular components as payloads to the extracellular space without any biological significance to the exosomes themselves.

Provided herein are exosomes that behave like minicells and exhibit the following capabilities: biologically in response to stimuli and to proliferation and migration, just like cells, but without a well-defined nucleus. These exosomes exhibit chemotaxis towards serum factors and, upon stimulation with growth factors (e.g., EGF), will phosphorylate EGFR receptors on their surface and initiate signaling cascades that lead to transcription and translation of new proteins. When these exosomes are injected into tumor-bearing mice, they preferentially accumulate in the tumor. The ability of these exosomes to protein translation and growth factor response together provide them with a functional role in tissue homeostasis and disease regulation.

I. Aspects of the invention

In the past years, extracellular vesicles, and in particular exosomes, have gained widespread attention, where several components, such as DNA, RNA and proteins, have been identified. In addition, exosomes have been implicated in affecting many different biological processes by transferring their contents into recipient cells of different tissues, as well as in unique forms that promote cell-cell communication (bases et al, 2017). Alternatively, the potential of exosomes as delivery vehicles for therapy has also been reported, particularly in the context of cancer or neuropathology (El-Andaloussi et al, 2012; Kamerkar et al, 2017). However, the exact pattern of systemic distribution and organotropism (tropism) of exosomes is not yet fully understood.

However, the nuclear and cytoplasmic components of exosomes are not only used for passive transfer to recipient cells, but can also respond to external stimuli to phosphorylate growth factor receptors (e.g., EGFR) and produce new proteins by active transcription and translation. External stimulation of exosomes may initiate de novo biological activities such as retrograde migration. It is envisioned that exosomes may function like minicells, although primitive in terms of their fine-tuning operation in response to external stimuli. Indeed, it has recently been proposed that exosomes could potentially constitute an existing characterization of pro-cellular (protocellular) ribosomes for which they need to contain rRNA, as demonstrated in this study (Sinkovics, 2015). Exosomes have a biological response and migrate in an active manner towards a growth factor gradient. Actin remodeling may be involved. The mode of actin polymerization may therefore constitute an interesting target in the regulation of exosome biodistribution.

Vesicles obtained from different species, such as prostasomes (prostasomes), have been shown to contain different components of the glycolytic pathway that allow them to produce ATP under cell-free conditions (Ronquist et al, 2013 a; Ronquist et al, 2013 b). Although direct transcription in exosomes has not been previously reported, one study showed that exosomes from bovine milk infected with bovine leukemia virus have been shown to exhibit reverse transcriptase activity (Yamada et al, 2013). It has also recently been demonstrated that mature mirnas can be independently produced in exosomes isolated from cancer cells (mlo et al, 2014). Here, exosomes are demonstrated to have the inherent ability to synthesize functional proteins de novo through DNA transcription coupled with mRNA translation. Following megakaryocyte differentiation, platelets can translate proteins from mRNA molecules retained therein (weyroch et al, 2004). Nevertheless, no DNA transcription of new mRNA molecules in platelets has been reported. Furthermore, a focus of mRNA translation activity (foci) associated with polysomes and mRNA binding proteins was observed in dendritic spines (even when cleaved from the bulk of the cell) (Aakalu et al, 2001; Smith et al, 2001; Steward and Levy, 1982). It is therefore clear that some cellular structures retain the capacity of protein biosynthesis in the absence of nuclei, perhaps in order to support their specific biological functions in a rapid manner. In addition to protein translation, exosomes are capable of DNA transcription by RNA polymerase II. It is well recognized that naked DNA can undergo basic transcription in the anucleate domain with minimal transcription machinery components (i.e., RNA Pol II alone and a mixture of six General Transcription Factors (GTFs)) (Lorch et al, 2014; Nagai et al, 2017). Exosomes have also been shown to contain a plethora of transcription factors that can be delivered to cells to alter their protein expression pattern (Ung et al, 2014). It is therefore conceivable that exosomes contain naked DNA residues not bound by chromatin, which can undergo transcription in the presence of these minimal transcription components.

The study also showed that components required for transcription/translation may be consumed within 24 hours, resulting in limited rates of transcription and translation. Since it has been suggested that different subsets of exosomes may have different molecular characteristics (Willms et al, 2016), it is possible that only a small subset of exosomes have the ability to synthesize from a head protein. Newly synthesized proteins in exosomes are functionally active, suggesting an appropriate protein conformation. Exosomes have been shown to contain not only components of ribosomes, but also several molecular chaperones, such as Hsp60 and Hsp 70. Ribosomes themselves play an important role in co-translational protein folding, for example, they can promote the formation of secondary structures in newly formed proteins. Ribosomes also serve as chaperone-associated platforms that can assist proper folding of nascent proteins (Kramer et al, 2009). It is envisioned that these exosome components may contribute to the stabilization of newly formed proteins. Physical constraints (as in the exosomal cavity) can also have a stabilizing effect on protein folding (Rao and Cruz, 2013). However, the possibility that many proteins may exhibit an inappropriate conformation or misfolding cannot be excluded. These may still have important biological implications, as evidenced by the recently revealed unexpected characteristics of the "dark proteome" (dark proteome) (Perdigao et al, 2015), which suggests that proteins with unknown structure or regions of intrinsic disorder may have important physiological functions.

Careful and quantitative identification of naturally synthesized proteins in exosomes is essential to fully understand the biological significance of the process. However, it is clear that this can have a significant impact on the understanding of eukaryotic biology for re-evaluation. Recent studies have shown that cells can selectively incorporate mRNA into exosomes (Raposo and Stoorvogel, 2013). This increases the likelihood that mRNA selectively packaged into exosomes may be translated into a protein whose expression is inhibited in its cell of origin, as shown in this study as proof of concept. The identification of newly synthesized proteins in exosomes up to one month after translation suggests that exosome-mediated protein production may result in a significant extension of protein half-life, probably due to lower levels of protein-degrading enzymes.

Taken together, these results demonstrate that exosomes have previously unrecognized biological activity, with potentially profound effects on body homeostasis and tissue pathogenesis. It is speculated that the growth factor gradient may play a role in the systemic tropism of exosomes in vivo. Disruption of the naturally occurring growth factor production pattern can have a direct impact on both the redistribution of exosomes and the delivery pattern. These response patterns can have potential impact on determining cell-cell communication, particularly between distant body parts. The fact that they can alter their protein expression patterns in response to these extracellular signals suggests that exosomes may serve as the primary responders to tissue injury. Taken together, these findings provide novel insights into the basic biology of exosomes and information about their biological function in homeostasis and their potential impact on disease state.

I. Lipid-based nanoparticles

In some embodiments, the lipid-based nanoparticle is a liposome, exosome, lipid preparation, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP: cholesterol vesicle). Lipid-based nanoparticles can be positively charged, negatively charged, or neutral. Lipid-based nanoparticles may contain the necessary components to allow transcription and translation, signal transduction, chemotaxis, or other cellular functions.

A. Liposomes

"liposomes" is a generic term that encompasses a variety of mono-and multilamellar lipid carriers formed by the production of closed lipid bilayers or lipid aggregates. Liposomes can be characterized as having a vesicular structure with a bilayer membrane, typically comprising phospholipids, and an internal medium, typically comprising an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposome is charge neutral.

Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Such liposomes form spontaneously when phospholipids-containing lipids are suspended in an excess of aqueous solution. The lipid component undergoes self-rearrangement and entraps water and dissolved solutes between the lipid bilayers prior to forming the closed structure. Lipophilic molecules or molecules with lipophilic regions may also be dissolved in or associated with the lipid bilayer.

In some particular aspects, the polypeptide, nucleic acid, or small molecule drug can be, for example, encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, linked to a liposome by a linker molecule associated with both the liposome and the polypeptide/nucleic acid, embedded in a liposome, complexed with a liposome, and the like.

As known to those of ordinary skill in the art, liposomes for use in accordance with embodiments of the present invention can be prepared by various methods. For example, a phospholipid, such as, for example, the neutral phospholipid Dioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol. The lipids are then mixed with the polypeptides, nucleic acids, and/or other components. Tween 20 was added to the lipid mixture such that tween 20 was about 5% by weight of the composition. Excess t-butanol is added to the mixture such that the volume of t-butanol is at least 95%. The mixture was vortexed, frozen in a dry ice/acetone bath and lyophilized overnight. The lyophilized formulation is stored at-20 ℃ and can be used for up to three months. When needed, the lyophilized liposomes were reconstituted in 0.9% saline.

Alternatively, liposomes can be prepared by mixing the lipids in a solvent in a container (e.g., glass, pear-shaped flask). The volume of the container should be ten times greater than the volume of the intended liposomal suspension. The solvent was removed using a rotary evaporator under negative pressure at about 40 ℃. The solvent is typically removed in about 5 minutes to 2 hours, depending on the desired liposome volume. The composition may be further dried in a desiccator under vacuum. Dried lipids are typically discarded after about 1 week due to a tendency to deteriorate over time.

The dried lipids can be hydrated in sterile and pyrogen-free water by shaking at about 25mM to 50mM phospholipid until all lipid membranes are resuspended. The aqueous liposomes can then be divided into aliquots, each placed in a vial, lyophilized and sealed under vacuum.

The dried lipids or lyophilized liposomes prepared as described above can be dehydrated and reconstituted in a solution of the protein or peptide and diluted to a suitable concentration with a suitable solvent (e.g., DPBS). The mixture was then vigorously shaken in a vortex mixer. Other materials not encapsulated (e.g., reagents including but not limited to hormones, drugs, nucleic acid constructs, etc.) were removed by centrifugation at 29,000 × g and the liposome particles were washed. The washed liposomes are resuspended at a suitable total phospholipid concentration, for example, about 50mM to 200 mM. The amount of other materials or active agents encapsulated can be determined according to standard methods. After determining the amount of other materials or active agents encapsulated in the liposome formulation, the liposomes can be diluted to the appropriate concentration and stored at 4 ℃ until use. Pharmaceutical compositions comprising liposomes typically comprise a sterile pharmaceutically acceptable carrier or diluent, for example, water or saline solution.

Other liposomes that can be used with embodiments of the invention include cationic liposomes, for example, as described in WO02/100435A1, U.S. patent 5,962,016, U.S. application 2004/0208921, WO03/015757A1, WO04029213A2, U.S. patent 5,030,453, and U.S. patent 6,680,068, all of which are incorporated by reference herein in their entirety without disclaimer.

In preparing such liposomes, any of the protocols described herein, or as known to those of ordinary skill in the art, can be used. Additional non-limiting examples of preparing liposomes are described in U.S. patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; international applications PCT/US85/01161 and PCT/US89/05040, each of which is incorporated herein by reference.

In certain embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., a DOPC liposome). As used herein, "neutral liposomes" or "uncharged liposomes" are defined as liposomes having one or more lipid components that produce a substantially neutral net charge (substantially uncharged). By "substantially neutral" or "substantially uncharged" is meant that a minority, if any, of the lipid components in a given population (e.g., a population of liposomes) comprises a charge that is not eliminated by the opposite charge of the other component (i.e., less than 10% of the components comprise the unabated charge, more preferably less than 5%, and most preferably less than 1%). In certain embodiments, neutral liposomes can comprise primarily lipids and/or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

Liposomes and/or lipid-based nanoparticles of embodiments of the invention may comprise phospholipids. In certain embodiments, a single phospholipid may be used to produce liposomes (e.g., a neutral phospholipid (e.g., DOPC), may be used to produce neutral liposomes). In other embodiments, more than one phospholipid may be used to produce liposomes. The phospholipids may be derived from natural or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine; because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), these compounds are particularly useful for producing neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, lipids other than phospholipids (e.g., cholesterol) may be used.

Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauroyl phosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauroyl phosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG") (DOPC), Distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelin ("DSSP"), distearoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), cephalitoylphosphatidylserine ("BPS"), dipalmitoylphosphatidylcholine ("DPSP"), dimyristoylphosphatidylcholine ("DMPC"), 1, 2-distearoylsn-glycero-3-phosphocholine ("DAPC"), 1, 2-diaroylsn-glycero-3-phosphocholine ("DBPC") (snsn), snsn-glycero-3-phosphocholine ("DBPC") (DMPC), 1, 2-docosadienoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloxy phosphatidylcholine ("POPC"), palmitoyloxy phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

B. Efflux body

The terms "microvesicle" and "exosome" as used herein refer to a membranous particle having a diameter (or largest dimension when the particle is not spherical) of about 10nm to about 5000nm, more typically 30nm to 1000nm, and most typically about 50nm to 750nm, wherein at least a portion of the exosome membrane is obtained directly from the cell. Most commonly, the size (mean diameter) of exosomes is up to 5% of the size of the donor cells. Thus, exosomes of particular concern include exosomes shed from cells.

Exosomes may be detected or isolated from any suitable sample type, such as, for example, a bodily fluid. The term "isolated" as used herein means separated from its natural environment and is intended to include at least partial purification, and may include substantial purification. The term "sample" as used herein refers to any sample suitable for the methods provided herein. The sample may be any sample comprising an exosome suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid, and bronchial washes (bronchial wash). 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 in the present invention may be extracted from any source known, including blood cells or components thereof, e.g., venous, arterial, peripheral, tissue, tape, etc. For example, the sample may be obtained and processed using well-known and conventional clinical methods (e.g., methods for drawing and processing whole blood). In one aspect, an exemplary sample can be peripheral blood drawn from a subject having cancer.

Exosomes may also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When exosomes are isolated from a tissue source, it may be desirable to homogenize the tissue to obtain a single cell suspension, and then lyse the cells to release the exosomes. When exosomes are isolated from tissue samples, it is important to select homogenization and lysis methods that do not result in destruction of exosomes. Exosomes contemplated herein are preferably isolated from a bodily fluid in a physiologically acceptable solution, e.g., buffered saline, growth medium, various aqueous media, and the like.

Exosomes may be isolated from freshly collected samples or from samples that have been frozen or stored under refrigeration. In some embodiments, exosomes may be isolated from cell culture medium. Although not required, if the fluid sample is clarified to remove any debris from the sample prior to precipitation with the volume exclusion polymer, an exosome of higher purity can be obtained. Clarification methods include centrifugation, ultracentrifugation, filtration or ultrafiltration. Most typically, exosomes may be isolated by a variety of methods known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatants. Exemplary methods for isolating exosomes 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).

One accepted protocol for isolating exosomes includes ultracentrifugation, which is typically combined with a sucrose density gradient or sucrose cushion (cushion) to float relatively low density exosomes. Separation of exosomes by continuous differential centrifugation is complicated by the possibility of overlapping with other microvesicle or macromolecular complex size distributions. Furthermore, centrifugation may not provide a sufficient means to separate vesicles according to their size. However, continuous centrifugation, when combined with sucrose gradient ultracentrifugation, can provide high exosome enrichment.

Using an alternative to the ultracentrifugation route, separation of exosomes based on size is another option. Successful purification of exosomes has been reported using ultrafiltration methods, which are less time consuming than ultracentrifugation and do not require the use of special equipment. Similarly, commercial kits (EXOMIR) are available^TMBio Scientific) which allows the removal of cells, platelets and cell debris on one microfilter and the capture of vesicles larger than 30nm on a second microfilter using positive pressure driving fluid. However, for this process, exosomes were not recovered and their RNA content was extracted directly from the material captured on the second microfilter and subsequently available for PCR analysis. HPLC-based protocols can potentially enable one to obtain high purity exosomes, although these methods require specialized equipment and are difficult to scale up. An important problem is that both blood and cell culture media contain a large number of nanoparticles (some non-vesicles) in the same size range as the exosomes. For example, some mirnas may be contained in extracellular protein complexes, rather than exosomes; however, protease (e.g., proteinase K) treatment can be performed to eliminate "exosome" eggsAny possible contamination of the white matter.

In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosome samples, such as techniques involving immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, typically involves attaching antibodies to proteins found on specific cell types to small paramagnetic beads. When the antibody-coated beads are mixed with a sample (e.g., blood), they attach to and surround specific cells. The sample was then placed in a strong magnetic field, causing the beads to settle to one side. After removal of the blood, the captured cells remain with the beads. Many variations of this general method are well known in the art and are suitable for isolating exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde/sulfate beads), and then antibodies are added to the mixture to recognize epitopes on the exosome surface attached to the beads. Exemplary proteins known to be found on exosomes of cancer cell origin include ATP-binding cassette subfamily 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 (GPC 1). Cancer cell-derived exosomes may be isolated using, for example, antibodies or aptamers to one or more of these proteins.

The assays used herein include any method that allows direct or indirect visualization of exosomes, and may be in vivo or ex vivo. For example, the analysis may include, but is not limited to: ex vivo microscopy or cytometric detection and visualization of exosomes bound to solid substrates, flow cytometry, fluorescence imaging, and the like. In one exemplary aspect, cancer cell-derived exosomes are detected using antibodies to one or more of the following and then bound to a solid substrate and/or visualized using microscopy or cytometric detection methods: ATP-binding cassette subfamily 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 A (HIST1H2AA), histone H2A-type A (HIST1H1AA), histone H3.3(H3F A), histone H3.1(HIST1H3A), histone homolog 37 (ZFP37), laminin subunit beta-1 (LAMB1), tubulointerstitial nephritis antigen-like protein (TINAGL1), peroxide oxidoreductase 4(PRDX 5), collagen alpha-2 (KRIV) chain (COL4A2), protein of Hemoc 3C 24 (RHP 1C 1), putative protin-like protein containing putative polypeptide PN 592 PN2, Rhone PN 2-like protein 4 (HN 599), Rhor-like protein 4 (HCP 598), Rhor-like protein, Triplex motif-containing protein 42(TRIM42), connexoazurin (JUP), tubulin beta-2B chain (TUBB2B), endoribonuclease (DICER1), E3 ubiquitin protein ligase TRIM71(TRIM71), swordin p60 ATPase-containing subunit A-like 2(KATNAL2), protein S100-A6(S100A6), 5' nucleotidase domain-containing protein 3(NT5DC3), valine-tRNA ligase (VARS), Kazrin (KAZN), ELAV-like protein 4(ELAVL4), cyclofetin 166(RNF166), FERM and PDZ domain-containing protein 1(FRMPD1), glucose regulatory protein (HSPA5) of 78, transporter granule complex subunit 6A (TRAPPC6A), nonene monokine (RNLE), tumor susceptibility gene 101 (SQTSG 101), Squalog protein 5828), regulin (ACAG receptor for Sfyas), prostaglandin A2 receptor homolog (PTRN 2), prostaglandin receptor homolog of regula, and prostaglandin receptor for protein, 26S protease regulatory subunit 6B (PSMC4), elongation factor 1-gamma (EEF1G), Myoglobin (TTN), tyrosine protein phosphatase type 13 (PTPN13), triose phosphate isomerase (TPI1), or carboxypeptidase E (CPE).

It should be noted that not all proteins expressed in a cell are found in exosomes secreted by the cell (see figure 11). For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells but not found in exosomes secreted by MCF-7 cells (Baietti et al, 2012). As another example, one study found 190/190 pancreatic ductal adenocarcinoma patients to have higher GPC1+ exosome levels than healthy controls (mlo et al, 2015, which is incorporated herein by reference in its entirety). Notably, on average only 2.3% of healthy controls had GPC1+ exosomes.

1. Exemplary protocols for collection of exosomes from cell cultures

On day 1, enough cells (e.g., about 500 ten thousand cells) were seeded in a T225 flask in medium containing 10% FBS so that the cells were about 70% confluent (confluent) the next day. On day 2, the media on the cells was aspirated, the cells were washed twice with PBS, and then 25 to 30mL of minimal media (i.e., no PenStrep or FBS) was added to the cells. Cells were incubated for 24 to 48 hours. 48 hours incubation is preferred, but some cell lines are more sensitive to serum-free medium, so the incubation time should be reduced to 24 hours. Note that FBS contains exosomes that would severely affect (skew) NanoSight results.

At day 3/4, the medium was collected and centrifuged at 800 × g for 5 minutes at room temperature to pellet dead cells and large debris. The supernatant was transferred to a new conical tube and the medium was centrifuged again at 2000 × g for 10 min to remove other large debris and large vesicles. The medium was passed through a 0.2 μm filter and then aliquoted into ultracentrifuge tubes (e.g., 25 × 89mm Beckman Ultra-Clear) using 35mL per tube. If the media volume of each tube is less than 35mL, fill the rest of the tube with PBS to reach 35 mL. The medium was ultracentrifuged using SW 32Ti rotor (k-factor 266.7, RCF max 133,907) at 28,000rpm for 2 to 4 hours at 4 ℃. The supernatant was carefully aspirated until approximately 1 inch of liquid remained. The tube is tilted and the remaining media is allowed to slowly enter the pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifuged at 28,000rpm for 1 to 2 hours in order to further purify the exosome population.

Finally, the exosome pellet was resuspended in 210 μ Ι _ PBS. If there were multiple ultracentrifuge tubes per sample, each exosome pellet was resuspended in series using the same 210 μ Ι _ PBS. For each sample, 10 μ L was taken and added to 990 μ L H₂O for nanoparticle tracking analysis. The remaining 200. mu.L of the exosome-containing suspension was used for downstream processes or immediately stored at-80 ℃.

2. Exemplary protocols for extraction of exosomes from serum samples

First, serum samples were thawed on ice. Then, 250 μ L of the cell-free serum sample was diluted in 11mL PBS; filtration was performed through a 0.2 μm pore filter. The diluted samples were ultracentrifuged at 150,000 Xg overnight at 4 ℃. The next day, the supernatant was carefully discarded, and the exosome pellet was washed with 11mL PBS. A second round of ultracentrifugation was performed at 150,000 Xg for 2 hours at 4 ℃. Finally, the supernatant was carefully discarded and the exosome pellet was resuspended in 100 μ Ι _ PBS for analysis.

C. Exemplary protocols for electroporation of exosomes and liposomes

Will be 1 × 10⁸Individual exosomes (measured by NanoSight analysis) or 100nm liposomes (e.g., purchased from encapula Nano Sciences) and 1 μ g of sirna (qiagen) or shRNA were mixed in 400uL of electroporation buffer (1.15mM potassium phosphate, pH 7.2, 25mM potassium chloride, 21% Optiprep). Exosomes or liposomes are electroporated using 4mm cuvettes (see, e.g., Alvarez-Erviti et al, 2011; El-Andaloussi et al, 2012). After electroporation the exosomes or liposomes were treated with RNAse without protease followed by the addition of 10 x concentrated RNAse inhibitor. Finally, exosomes or liposomes were washed with PBS according to the ultracentrifugation method, as described above.

Diagnosis, prognosis and treatment of diseases

Certain aspects of the invention provide for treating a patient with an exosome expressing or comprising a therapeutic or diagnostic agent. As used herein, a "therapeutic agent" is an atom, molecule or compound that can be used to treat cancer or other disorders. Some examples of therapeutic agents include, but are not limited to: drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, chelators, boron compounds, photosensitizers and dyes. As used herein, a "diagnostic agent" is an atom, molecule or compound that can be used to diagnose, detect or visualize a disease. According to some embodiments described herein, diagnostic agents 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 enhancers (e.g., paramagnetic ions).

In some aspects, the therapeutic recombinant protein can be a protein having activity that has been lost in a patient's cell, 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. Monoclonal antibodies can specifically or selectively bind to intracellular antigens. Monoclonal antibodies can inhibit the function of intracellular antigens and/or disrupt protein-protein interactions. Other aspects of the invention provide for diagnosing a disease based on the presence of cancer cell-derived exosomes in a patient sample.

Because exosomes are known to contain the machinery necessary to complete mRNA transcription and protein translation (see PCT/US2014/068630, which is incorporated herein by reference in its entirety), mRNA or DNA nucleic acids encoding therapeutic proteins can be transfected into exosomes. Alternatively, the therapeutic protein itself may be electroporated into exosomes or incorporated directly into liposomes. Exemplary therapeutic proteins include, but are not limited to: tumor suppressor proteins, peptides, wild-type protein counterparts to mutant proteins, DNA repair proteins, proteolytic enzymes, protein toxins, proteins that can inhibit intracellular protein activity, proteins that can activate intracellular protein activity, or any protein for which it is desirable to reconstitute its lost function. Some specific examples of exemplary therapeutic proteins include: 123F2, Abcb4, Abcc 4, Abcg 4, Actb, Ada, Ahr, Akt 4, Amhr 4, Anxa 4, Apc, Ar, Atm, Axin 4, B2 4, Bard 4, Bcl211, Becn 4, Bhlhal 4, Bin 4, Blm Braff, Brca 4, Brcatf, Brip 4, Buchb 14, Bwscra, Cadm 4, Casc 4, p4, Ccp 4, Casp 4, Cap 4, Cav 4, Cdcr 4, Fastcs 3636363672, Cdccs 363672, Cdccs 36k 4, Cdcr 4, Cdccs 36k 4, Cdcr 36k 4, Cdcr 4, Cdcs 4, Cdcr 36k 4, Cdcr 4, Cdcs 36k 4, Cdcr 36k 4, Cdcr 4, Cdcs 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 4, Cdcs 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 36k 4, Cdcr 4, C, Htatip2, Ill 1b, Ill 10, Ill 2, Ill 6, Ill 8Rb Inha, Itgav, Jun, Jak3, Kit, Klf4, Kras2, Kras 22, Lig 2, Lig 2, Lmo 2, Lncrl, Lncr2, Ppcr 2, Lncr2, Ltbp 2, Luca2, Lzts 2, Mad1l 2, Mad211, Ppmar 2/Jv 2, Mapk 2, Mcc, Mcm 2, Men2, Pmtp 2, Mg3672, Mif, Mlh 2, Mmamac 2, Nbmc 2, Nbpr 2, Ptnrgr3672, Ptnrgrp 2, PtNprNprl 2, Ptnrbl 2, Ptnrgr3672, Ptnrgrp 2, Ptnrgr3672, Ptnrgr36k 2, Ptnrgr3672, Ptnrg 2, Ptnrgr3672, Ptnrb 2, Ptnrg 2, Pfpr 2, Pprnrfc 2, PprNprnrfc 2, PprNprNprnrfc 2, Pfpr 2, Pfp3672, Pfpr 2, Pfp3672, PprNprNprNprNprNprNprNprNprNprNprNprNprnrf 2, Pfp3672, 2, Pfpr 2, Pfp3672, Rpl38, S100a4, SCGB1A1, Skp2, Smad2, Smad3, Smad4, Smrcb 1, Smo, Snx25, Spata13, Srpx, Ssic1, Sstr2, Sstr5, Stat3, St5, St7, St14, Stk11, Suds3, Tap1, Tbx21, Terc, Tnf, Tp53, Tp73, Trpm5, Tsc2, Tsc1, Vhl, Wrn, Wt1, Wt2, Xrcc1, Xrcc5, Xrcc6, and Zac 1.

One particular type of protein that may be expected to be introduced into the intracellular space of diseased cells is an antibody (e.g., a monoclonal antibody). Such antibodies may disrupt the function of intracellular proteins and/or disrupt intracellular protein-protein interactions. Exemplary targets for such monoclonal antibodies include, but are not limited to, proteins involved in the RNAi pathway, telomerase, transcription factors controlling disease processes, kinases, phosphatases, proteins required for DNA synthesis, proteins required for protein translation. Some 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, AMLl, axl, alk, fins, fps, gip, lck, Stat, Hox, MLM, PRAD-I and trk. In addition to monoclonal antibodies, any antigen binding fragment thereof is contemplated, such as scFv, Fab fragments, Fab ', F (ab') 2, Fv, peptibodies, diabodies, triabodies, or minibodies. Any such antibody or antibody fragment may be glycosylated or aglycosylated.

Since exosomes are known to contain DICER and an active RNA processing RISC complex (see PCT publn. wo 2014/152622, incorporated herein by reference in its entirety), shrnas transfected into exosomes can mature into RISC complexes that bind siRNA to the exosomes themselves. Alternatively, the mature siRNA itself may be transfected into exosomes or liposomes. Thus, for example, the following are possible target gene classes that may be used in the methods of the invention to modulate or attenuate target gene expression: wild-type or mutant forms of developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix (Winged helix) family members, Hox family members, cytokines/lymphokines and their receptors, growth 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, PTEN, Rb, uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN 22, DCC, DPC 2, MADR2/JV 2, MEN2, MTS 2, NF2, VHL, WRN, WT2, CFTR, CFZA-1, CRAC-72, FACS 2, LUCA2, LUCA 72, MCA 2, MCAS 2, MC, 101F6, gene 21(NPRL2), or a gene encoding SEM A3 polypeptide), a pro-apoptotic gene (e.g., CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, Bax, hid, Bak, MKP-7, PARP, bad, bcl-2, MST1, bbc3, Sax, BIK, and BID), a cytokine (e.g., GM-CSF, G-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-19, IL-3, and BID), a cytokine (e.g., GM-CSF, G-CSF, IL-1, IL-, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 IFN-alpha, IFN-beta, IFN-gamma, MIP-1 alpha, MIP-1 beta, TGF-beta, TNF-alpha, TNF-beta, PDGF, and mda7), oncogenes (e.g., ABLI, BLC1, BCL6, CBFA1, CBL, CSUNA, ERBA, ERBB, RB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, TAL1, TCL3, and YES), and enzymes (e.g., desaturase, ACP, hydroxylase, dehydrogenase, ADP, peroxidase, DNA decarboxylase, DNA polymerase, RNA, cellulase, DNA polymerase, DNA, Galactosidase, glucanase, glucose oxidase, gtpase, helicase, hemicellulase, integrase, invertase, isomerase, kinase, lactase, lipase, lipoxygenase, lysozyme, nuclease, pectinesterase, peroxidase, phosphatase, phospholipase, phosphorylase, polygalacturonase, protease and peptidase, pullulanase, recombinase, reverse transcriptase, topoisomerase, xylanase). In some cases, the sh/siRNA can be designed to specifically target mutant forms of genes expressed in cancer cells, while not affecting expression of the corresponding wild-type forms. Indeed, if any inhibitory nucleic acid has been found by any source to be a validated down-regulator of a protein of interest, such inhibitory nucleic acids may be applied to the compositions and methods of the invention.

In designing RNAi, several factors need to be considered, such as the nature of the siRNA, the persistence of the silencing effect and the choice of the delivery system. To produce an RNAi effect, the siRNA introduced into the organism will typically contain an exon sequence. Furthermore, the RNAi process is homology dependent, and therefore the sequences 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 shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity between the siRNA and the sequence of the gene to be inhibited. Sequences having less than about 80% identity to the target gene are substantially less effective. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of the unrelated gene will be affected.

Exosomes may also be engineered to comprise a gene editing system, such as a CRISPR/Cas system. In general, a "CRISPR system" refers generally to transcripts and other elements involved in or directing the expression or activity of a CRISPR-associated ("Cas") gene, including sequences encoding a Cas gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr chaperone (tracr-mate) sequences (including "direct repeat"), and portions of the same directional repeat processed by tracrRNA in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. In some aspects, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and an immobilized tracrRNA) are introduced into a cell. Generally, using complementary base pairing, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene. The target site may be selected based on its position immediately adjacent to the 5' of a pro-spacer adjacent motif (PAM) sequence, such as typically NGG or NAG. In this aspect, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote CRISPR complex formation at target sequence sites. Generally, a "target sequence" refers generally to a sequence for which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of a CRISPR complex. Complete complementarity is not necessarily required if sufficient complementarity exists to cause hybridization and promote formation of a CRISPR complex. The CRISPR system in an exosome engineered to comprise such a system may function to edit genomic DNA inside the target cell, or the system may edit DNA inside the exosome itself.

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 or nucleic acid-based therapeutic agent. Exemplary small molecule drugs contemplated for use in embodiments of the invention include, but are not limited to: toxins, chemotherapeutic agents, agents inhibiting intracellular protein activity, agents activating intracellular protein activity, agents for preventing restenosis, agents for treating kidney disease, agents for intermittent claudication, agents for treating hypotension and shock, angiotensin converting enzyme inhibitors, anti-angina agents, antiarrhythmics, anti-hypertension agents, angiotensin ii receptor antagonists, anti-platelet agents, b1 selective b blockers (b-blockers b1 selective), beta blockers, botanicals for cardiovascular indications, calcium channel blockers, cardiovascular/diagnostic agents, central alpha-2 agonists, coronary vasodilators, diuretics and tubular inhibitors, neutral endopeptidase/angiotensin converting enzyme inhibitors, peripheral vasodilators, potassium channel openers, potassium salts, anticonvulsants, antiemetics, antinociceptives, antiparkinson agents, antispasmodics, cerebral stimulants, agents applicable to the treatment of wounds, agents applicable to the treatment of alzheimer's disease or dementia, agents applicable to the treatment of migraine, agents applicable to the treatment of neurodegenerative diseases, agents applicable to the treatment of kaposi's sarcoma, agents applicable to the treatment of AIDS, cancer chemotherapeutics, agents applicable to the treatment of immune disorders, agents applicable to the treatment of psychiatric disorders, analgesics, epidural and intrathecal anesthetics, general, local, regional neuromuscular blockers, sedatives, pre-anesthesia adrenals/acth, anabolic steroids, agents applicable to the treatment of diabetes, dopamine agonists, growth hormones and analogues, hyperglycemic agents, hypoglycemic agents, oral insulin, bulk parenteral solutions (1 image volume parenteral, lvp), lipid altering agents, metabolic studies and congenital metabolic errors, nutrients/amino acids, nutritional lvp, obesity drugs (anorexics), somatostatin, thyroid agents, vasopressin, vitamins, steroid corticosteroids, mucolytic agents, pulmonary anti-inflammatory agents, lung surfactants, antacids, anticholinergics, antidiarrheals, antiemetics, cholelithiasis agents, gallstone lytic agents, inflammatory bowel disease agents, irritable bowel syndrome agents, liver agents, metal chelators, miscellaneous (miscellaneous) gastric secretion agents, pancreatitis agents, pancreatic enzymes, prostaglandins, proton pump inhibitors, sclerosing agents, sucralfate, antiprogestins, contraceptives, oral contraceptives, non-oral dopamine agonists, estrogens, gonadotropins, GNRH agonists, GHRH antagonists, oxytocics, progestogens, utero agents, antianemia agents, anticoagulants, antifibrinolytics, antiplatelet agents, antithrombin agents, procoagulants, fibrinolytics, hematology, heparin inhibitors, metal chelators, prostaglandins, vitamin K, antiandrogens, aminoglycosides, antibacterial agents, sulfonamides, cephalosporins, clindamycin, dermatological agents (dermatitics), detergents, erythromycin, anthelmintic agents (anthelmintic agents), antifungal agents, antimalarials, antimycobacterial agents, antiparasitic agents, antiprotozoal agents, trichomonad agents, antitubercular agents, immunomodulators, immunostimulants, macrocyclic lactones, antiparasitic agents, steroid corticosteroids, cyclooxygenase inhibitors, enzyme blockers, rheumatic disease immunomodulators, metalloproteinase inhibitors, non-steroidal anti-inflammatory agents, analgesics, antipyretics, alpha adrenergic agonists/blockers, antibiotics, antivirals, beta adrenergic blockers, carbonic anhydrase inhibitors, steroidal corticosteroids, immune system modulators, mast cell inhibitors, non-steroidal anti-inflammatory agents, and prostaglandins.

Exosomes may also be used to deliver diagnostic agents. Exemplary diagnostic agents include, but are not limited to: magnetic resonance image enhancing agents, positron emission tomography products, radiodiagnostic agents, radiotherapeutic agents, radiopaque contrast agents, radiopharmaceuticals, ultrasound imaging agents, and angiographic diagnostic agents.

The term "subject" as used herein refers to any individual or patient for whom the subject method is performed. Typically, the subject is a human, although those skilled in the art will appreciate that the subject may be an animal. Thus, other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, and the like), and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

"treating" and variations thereof refer to administering or applying a therapeutic agent to a subject or programming or modeling a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, the treatment may include administration of chemotherapy, immunotherapy or radiation therapy, performing surgery, or any combination thereof.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of a subject for the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of disease. For example, treatment of cancer may involve, for example, reducing the invasiveness of the tumor, reducing the growth rate of the cancer, or preventing metastasis. Treatment of cancer may also refer to prolonging survival of a subject having cancer.

The term "cancer" as used herein may be used to describe a solid tumor, a metastatic cancer or a non-metastatic cancer. In certain embodiments, the cancer may originate from the bladder, blood, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum (gum), head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

Cancer may specifically be of the following histological types, although it is not limited to these: tumor, malignant; cancer; cancer, undifferentiated; giant cell and spindle cell cancers; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinomas, malignant; bile duct cancer; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; a solid cancer; carcinoid tumors, malignant; bronchoalveolar carcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic granulosa cancer; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped sclerosing cancers; adrenocortical carcinoma; endometrioid carcinoma (endometrid carcinoma); skin appendage cancer; adenocarcinoma of the apocrine gland; sebaceous gland cancer; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma W/squamous metaplasia; thymoma, malignant; ovarian stromal tumors, malignant; thecal cell tumor, malignant; granulosa cell tumor, malignant; male blastoma, malignant; sertoli cell carcinoma; leydig cell tumor (leydig cell tumor), malignant; lipocytoma, malignant; ganglioneuroma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; cutaneous silk ball sarcoma (glomangiospora); malignant melanoma; melanoma-free melanoma; superficial invasive melanoma; malignant melanoma within giant pigmented nevi; epithelial-like cell melanoma; blue nevus, malignant; a sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; mullerian mixed tumor (mullerian mixed tumor); renal blastoma; hepatoblastoma; a carcinosarcoma; phyllomas, malignant; brenner tumor (brenner tumor), malignant; phylloid tumors, malignant; synovial sarcoma; mesothelioma, malignant; clonal cell tumors; embryonal carcinoma; teratoma, malignancy; ovarian goiter, malignant; choriocarcinoma; middle kidney tumor, malignant; angiosarcoma; vascular endothelioma, malignant; kaposi's sarcoma; vascular endothelial cell tumor, malignant; lymphangioleiomyosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; interstitial chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumors, malignant; amelogenic cell dental sarcoma; ameloblastoma, malignant; amelogenic cell fibrosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; primary plasma astrocytoma; fibroastrocytoma; astrocytomas; a glioblastoma; oligodendroglioma; oligodendroglioma; primary neuroectoderm; cerebellar sarcoma; a ganglioblastoma; neuroblastoma; retinoblastoma; olfactive neurogenic tumors; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; hodgkin's accessory granulomatous lesions; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-hodgkin lymphomas; malignant tissue cell proliferation; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid 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.

The terms "contacted" and "exposed" when applied to a cell are used herein to describe the process of delivering a therapeutic agent to a target cell or placing it in direct juxtaposition with a target cell. To achieve cell killing, for example, one or more agents are delivered to the cells in an amount effective to kill the cells or prevent them from dividing.

An effective response to treating a patient or "responsiveness" of a patient refers to the clinical or therapeutic benefit administered to a patient at risk of or suffering from a disease or disorder. Such benefits may include cellular or biological responses, complete responses, partial responses, stable disease (no progression or relapse) or responses with subsequent relapses. For example, an effective response may reduce tumor size or progression-free survival in a patient diagnosed with cancer.

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.

With respect to the treatment of neoplastic disorders, depending on the stage of the neoplastic disorder, treatment of neoplastic disorders involves one or a combination of the following treatments: surgery to remove tumor tissue, radiation therapy, and chemotherapy. Other treatment regimens may be combined with administration of anti-cancer agents, e.g., therapeutic compositions and chemotherapeutic agents. For example, a patient to be treated with such an anti-cancer agent may also receive radiation therapy and/or may undergo surgery.

For the treatment of disease, the appropriate dosage of the therapeutic composition will depend on the type of disease to be treated, as defined above, the severity and course of the disease, the clinical history and response to the agent of the patient, and the discretion of the attendant physician. The medicament is suitable for administration to a patient in one or a series of treatments.

Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect. The tissue, tumor, or cell may be contacted with one or more compositions or pharmacological agents comprising one or more agents, or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or agents. Likewise, it is contemplated that such combination therapy may be used in conjunction with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

Combined administration may include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administrations. That is, the subject therapeutic composition and the other therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, the subject therapeutic composition and another therapeutic agent may be administered simultaneously, wherein the two agents are present in separate formulations. In another alternative, the other therapeutic agent may be administered immediately after the administration of the therapeutic agent, or vice versa. In separate administration regimens, the subject therapeutic composition and the other therapeutic agent may be administered several minutes apart, or hours apart or days apart.

The first anti-cancer therapy (e.g., an exosome expressing a recombinant protein or isolated from a recombinant protein) can be administered before, during, after, or in various combinations thereof relative to the second anti-cancer therapy. Administration may be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where the first and second treatments are provided to the patient separately, it will generally be ensured that there is no significant period of time between the time of each delivery to terminate so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the first and second treatments may be provided to the patient within about 12 to 24 or 72 hours of each other, and more particularly within about 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the treatment period, with intervals between separate administrations of days (2, 3, 4,5, 6, or 7) to weeks (1, 2, 3, 4,5, 6, 7, or 8).

In certain embodiments, the course of treatment will last from 1 to 90 days or longer (such a range includes the middle days). It is contemplated that one agent may be administered on any day from day 1 to day 90 (such range includes the middle of the days) or any combination thereof, and another agent may be administered on any day from day 1 to day 90 (such range includes the middle of the days) or any combination thereof. The patient may be given one or more administrations of the agent over a single day (24 hour period). Furthermore, following a course of treatment, it is expected that there will be periods of time during which no anti-cancer therapy is administered. This period may last from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months or longer (such ranges include intermediate days), depending on the condition of the patient, e.g. his prognosis, physical strength (strength), health, etc. It is desirable that the treatment cycle be repeated as needed.

Various combinations may be employed. For the following examples, the first anti-cancer therapy is "a" and the second anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

administration of any compound or treatment of the present invention to a patient will follow the general protocol for administering such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring toxicity due to the combination therapy.

1. Chemotherapy

A variety of chemotherapeutic agents may 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 refer to a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified by the way they are active in the cell, e.g., whether they affect the cell cycle and at what stage. Alternatively, agents can be characterized based on their ability to directly cross-link DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Some examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa, carboquone, meturedpa and uredepa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethamel; annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (including the synthetic analogue topotecan); bryodin; a caristatin (callystatin); CC-1065 (including its aldorexin, kazelaixin, and bizelaixin synthetic analogs); nostoc (especially nostoc 1 and nostoc 8); dolastatin; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM 1); eleutherobin; (ii) coprinus atramentarius alkali; alcohols of coral of the species Adina stolonifera; spongistatin; nitrogen mustards such as chlorambucil, naphazel, chlorophosphamide, estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, neonebixin, benzene mustard cholesterol, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediynes (e.g., calicheamicin, particularly calicheamicin γ 1I and calicheamicin ω I1); daptomycin, including daptomycin a; diphosphonates, such as clodronate; epothilones; and neocarzinostain chromophores and related chromoproteenediyne antibiotic chromophores, aclarubicin, actinomycin, antromycin, azaserine, bleomycin, actinomycin C (cactinomycin), carrubicin, carminomycin, carcinomycin, tryptomycin, dactinomycin, daunorubicin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin, and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, macromycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogaxomycin, olivomycin, pelomomycin, puromycin, doxorubicin, roxydicin, streptonigrin, doxorubicin, Streptozotocin, tubercidin, ubenimex, setastatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as carroterone, drostandrosterone propionate, epitioandrostanol, meindroxane and testolactone; anti-adrenal agents, such as mitotane and trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; eniluracil; amsacrine; besubbs; a bisantrene group; edatrexae; desphosphamide (defofamine); colchicine; diazaquinone; (ii) nilotinib; ammonium etiolate; epothilones; etoglut; gallium nitrate; a hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol; nisridine; pentostatin; methionine mustard (phenamett); pirarubicin; losoxanthraquinone; podophyllinic acid; 2-acethydrazide; procarbazine; PSK polysaccharide complex; lezoxan; rhizomycin; a texaphyrin; germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); a tri-imine quinone; 2, 2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucomicin A, fisetin A and serpentin); uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; adding the star of tussingo; arabinoside ("Ara-C"); cyclophosphamide; taxanes, 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; norfloxacin (novantrone); (ii) teniposide; edatrexae; daunomycin; aminopterin; (ii) Hirodad; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine, carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, antiplatin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

2. Radiation therapy

Other factors that cause DNA damage and have been widely used include those commonly referred to as gamma rays, X-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and UV irradiation. It is likely that all of these factors produce extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose of X-rays ranges from a daily dose of 50 to 200 roentgens for a prolonged period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely, and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

3. Immunotherapy

The skilled person will appreciate that additional immunotherapies may be combined or used in conjunction with the methods of the invention. In the context of cancer treatment, immunotherapy generally relies on the use of an immunological effectCells and molecules are used to target and destroy cancer cells. Rituximab

Is one such example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may be used as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin a chain, cholera toxin, pertussis toxin, etc.) and used only as a targeting agent. Alternatively, the effector may be a surface molecule-bearing lymphocyte that interacts directly or indirectly with the tumor cell target. A variety of effector cells include cytotoxic T cells and NK cells.

In one aspect of immunotherapy, tumor cells must have some markers suitable for targeting (i.e., not present on most other cells). There are many tumor markers and any of these 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, erb B and p 155. Another aspect of immunotherapy is the combination of an anti-cancer effect with an immunostimulating effect. Immunostimulatory molecules also exist, 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.

Some examples of immunotherapies currently being studied or applied are immunological adjuvants, such as Mycobacterium bovis (Mycobacterium bovis), Plasmodium falciparum (Plasmodium falciparum), dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapies such as alpha, beta and gamma interferons, 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. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD 20, anti-ganglioside GM2 and anti-p 185(Hollander, 2012; Hanibuchi et al, 1998; U.S. Pat. No.5,824,311). It is contemplated that one or more anti-cancer treatments may be used with the antibody treatments described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either up signal (e.g., co-stimulatory molecules) or down signal. Inhibitory immune checkpoints that can be targeted by blockade of immune checkpoints include adenosine A2A receptor (A2A receptor, A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (B and T lymphocyte attenuator, BTLA), cytotoxic T lymphocyte-associated protein 4(cytotoxic T-lymphocyte-associated protein, CTLA-4, also known as CD152), indoleamine 2, 3-dioxygenase (indoleamine 2, 3-dioxygenase, IDO), killer-cell immunoglobulin (killer-cell immunoglobulin, KIR), lymphocyte activation gene 3(lymphocyte activation gene-3, LAG3), programmed death 1(programmed death 1, PD-1), T cell immunoglobulin domain and protein domain (killer-cell-activation domain of T-cell-3, LAG3), and adhesion-activation domain of T cell V (Ig-activation domain of T cell-activation V), VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and/or CTLA-4.

The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or in particular an antibody, such as a human antibody (e.g., international patent publication WO 2015016718; pardol, Nat Rev Cancer, 12 (4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular antibodies in chimeric, humanized or human form may be used. As the skilled artisan will appreciate, alternative and/or equivalent designations may be used for certain antibodies mentioned in the present disclosure. In the context of the present disclosure, such alternative and/or equivalent designations are interchangeable. For example, it is known that ramolizumab (lambrolizumab) is also known by the alternative and equivalent names MK-3475 and pembrolizumab (pembrolizumab).

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and/or PDL 2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, 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 binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide. Exemplary antibodies are described in U.S. Pat. nos. 8,735,553, 8,354,509, and 8,008,449, which are all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, for example, as described in U.S. patent publication nos. 20140294898, 2014022021, and 20110008369, which are all incorporated herein by reference.

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 nivolumab (nivolumab), pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 or a PD-1 binding moiety fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and

is an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, Ralizumab,

And SCH-900475, are anti-PD-1 antibodies described in WO 2009/114335. CT-011, also known as hBAT or hBAT-1, is described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

Another immune checkpoint that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4(CTLA-4), also known as CD 152. The Genbank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is present on the surface of T cells and acts as an "off" switch when bound 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 T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen presenting cells, also referred to as B7-1 and B7-2, respectively. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and may be important for regulatory T cell function. T cell activation through the T cell receptor and CD28 results in increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

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.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the methods of the invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies may be used. For example, anti-CTLA-4 antibodies disclosed in the following may be used in the methods disclosed herein: U.S. Pat. Nos. 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504(CP675,206, also known as tremelimumab; original name tremelimumab), U.S. Pat. No.6,207,156; hurwitz et al (1998) Proc Natl Acad Sci USA 95 (17): 10067-10071; camacho et al, (2004) J Clin Oncology22 (145): digest No.2505 (antibody CP-675206); and Mokyr et al (1998) Cancer Res 58: 5301-5304. The teachings of each of the foregoing publications are 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; which is incorporated herein by reference in its entirety.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and

) 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 antibody described above. 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%, 95%, or 99% variable region identity to ipilimumab).

Other molecules that are useful for modulating CTLA-4 include CTLA-4 ligands and receptors, for example, as described in U.S. patent nos. 5844905, 5885796 and international patent application nos. WO1995001994 and WO1998042752, which are all incorporated herein by reference; and immunoadhesins, such as described in U.S. patent No.8329867, which is incorporated herein by reference.

In some embodiments, the immunotherapy may be an adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo. T cells for adoptive immunotherapy can be generated by expansion of antigen-specific T cells or by redirection of T cells by genetic engineering (Park, Rosenberg et al.2011). The isolation and metastasis of tumor-specific T cells has been shown to successfully treat melanoma. Genetic transfer through transgenic T cell receptors or Chimeric Antigen Receptors (CARs) has successfully generated new specificities in T cells (Jena, Dotti et al 2010). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding portion of a CAR consists of the antigen binding domain of a single chain antibody (scFv), which comprises a light fragment and a variable fragment of a monoclonal antibody, connected by a flexible linker. Receptor or ligand domain based binding moieties have also been used successfully. The signaling domain of the first generation CARs was derived from either the cytoplasmic region of CD3 ζ or the Fc receptor gamma chain. CARs have successfully redirected T cells against antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors (Jena, Dotti et al 2010).

In one embodiment, the present application provides a combination therapy for treating cancer, wherein the combination therapy comprises 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 to a tumor antigen.

4. Surgery

About 60% of people with cancer will undergo some type of surgery, including prophylactic, diagnostic or staged, therapeutic and palliative surgery. Therapeutic surgery includes resection in which all or part of cancerous tissue is physically removed, resected, and/or destroyed, and may be used in conjunction with other treatments, such as the treatments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or replacement therapy. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

After resection of some or all of the cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local administration of an additional anti-cancer therapy in the area. Such treatment may 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 may also be at different dosages.

5. Other agents

It is contemplated that other agents may be used in combination with certain aspects of the invention to increase the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatics and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiating agents may be used in combination with certain aspects of the invention to increase the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the efficacy of the present invention. Some examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and Lovastatin (Lovastatin). It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the invention to increase the efficacy of the treatment.

Pharmaceutical composition

It is contemplated that exosomes expressing or comprising therapeutic proteins, inhibitory RNAs, and/or small molecule drugs may be administered systemically or locally to inhibit tumor cell growth, and most preferably, kill cancer cells in cancer patients with locally advanced or metastatic cancer. They may be administered intravenously, intrathecally and/or intraperitoneally. They may be administered alone or in combination with antiproliferative agents. In one embodiment, they are administered prior to surgery or other procedure to reduce the cancer burden in the patient. Alternatively, they may be administered after surgery to ensure that any remaining cancer (e.g., cancer that has not been eliminated by surgery) cannot survive.

It is not intended that the present invention be limited by the specific nature of the therapeutic formulation. For example, such compositions may be provided in a formulation with a physiologically tolerable liquid, gel, solid carrier, diluent or excipient. These therapeutic formulations can be administered to mammals, such as domestic animals, for veterinary use, and humans for clinical use in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary depending on the type of use and the mode of administration and the particular needs of the individual subject.

In the case of the intended clinical application, it may be necessary to prepare a pharmaceutical composition comprising the recombinant protein and/or exosomes in a form suitable for the intended application. Generally, the pharmaceutical composition may comprise 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 an adverse, allergic, or other untoward reaction when administered (as the case may be) to an animal, such as a human. In light of the present disclosure, those skilled in the art will be aware of the preparation of Pharmaceutical compositions comprising recombinant proteins and/or exosomes or additional active ingredients as disclosed herein, as exemplified by Remington's Pharmaceutical Sciences, 18th ed., 1990, which is incorporated herein by reference. Further, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, general safety and purity Standards as required by the FDA Office of Biological Standards.

Further in accordance with certain aspects of the present invention, compositions suitable for administration may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol/water solutions, ethanol, saline solutions, parenteral carriers such as sodium chloride, Ringer's dextrose, and the like); non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), vegetable oils, and injectable organic esters, such as ethyl oleate); a lipid; a liposome; a dispersion medium; coatings (e.g., lecithin); a surfactant; an antioxidant; preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof); isotonic agents (e.g., sugars and sodium chloride); absorption retarders (e.g., aluminum monostearate and gelatin); salt; a drug; a drug stabilizer; gelling agent; a resin; a filler; a binding agent; an excipient; a disintegrant; a lubricant; a sweetener; a flavoring agent; a dye; fluids and nutritional supplements; such as substances and combinations thereof, as known to those of ordinary skill in the art. The carrier should be absorbable and include liquid, semi-solid, i.e., paste, or solid carriers. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, for example 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. Suitable 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.

Pharmaceutically acceptable carriers are specifically formulated for administration to humans, although in certain embodiments it may be desirable to use pharmaceutically acceptable carriers that are formulated for administration to non-human animals but are not acceptable for administration to humans (e.g., due to government regulations). Unless any conventional carrier is incompatible with the active ingredient (e.g., detrimental to the recipient or therapeutic efficacy of the composition contained therein), it is contemplated that it may be used in therapeutic or pharmaceutical compositions. According to certain aspects of the present invention, the compositions are combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, and the like. Such operations are conventional to those skilled in the art.

Certain embodiments of the invention may include different types of carriers depending on whether they are administered as a solid, liquid or aerosol, and whether the route of administration (e.g., injection) requires sterility. The composition may be applied as follows: intravenous, intradermal, transdermal, intrathecal, intraarterial, intraperitoneal, intranasal, intravaginal, intrarectal, intramuscular, subcutaneous, mucosal, oral, topical, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, bathing target cells by direct local perfusion, by catheter, by lavage, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing methods, as known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., 1990, incorporated herein by reference).

The active compounds can be formulated for parenteral administration, for example, formulated for injection by the intravenous, intraarterial, intramuscular, subcutaneous or even intraperitoneal routes. Generally, such compositions may be prepared as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions after addition of liquid prior to injection may also be prepared; and the formulation may also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations comprising 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 form must be sterile and must be fluid to the extent that it can be easily injected. It should also be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

The therapeutic agents may be formulated into compositions as free bases, neutral or salt forms. 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, for example, hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, or mandelic acid, and the like. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine or procaine and the like. After formulation, the solution will be administered in a manner compatible with the dosage formulation and in, for example, a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, e.g., formulated for parenteral administration, e.g., injectable solutions, or aerosols for delivery to the lung, or formulated for digestive administration, e.g., drug release capsules and the like.

In one embodiment of the invention, the composition is intimately combined or admixed with a semi-solid or solid carrier. The mixing may be carried out in any convenient manner, for example, milling. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Some examples of stabilizers for use in the compositions include buffers, amino acids (e.g., glycine and lysine), carbohydrates (e.g., dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like).

In other embodiments, the invention may relate to the use of a pharmaceutical lipid carrier composition comprising one or more lipids and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with organic solvents. This broad class of compounds is well known to those skilled in the art, and when the term "lipid" is used herein, it is not limited to any particular structure. Some examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are typically biological substances. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, thioesters, lipids with ether and ester linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds understood by those of skill in the art to be lipids other than those specifically described herein are also encompassed by the compositions and methods.

One of ordinary skill in the art will be familiar with a range of techniques that can be used to disperse the composition in a lipid carrier. For example, the therapeutic agent may be dispersed in a solution comprising a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bound to a lipid, contained as a suspension in a lipid, contained or complexed with micelles or liposomes, or otherwise associated with a lipid or lipid structure by any means known to one of ordinary skill in the art. Dispersion may or may not result in the formation of liposomes.

The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response discussed above in relation to its administration (i.e., the appropriate route and treatment regimen). The amount to be administered depends on the desired effect, both in terms of the amount treated and the unit dose. The actual dosage amount of the composition of the present invention to be administered to a patient or subject may be determined by physical and physiological factors such as the weight, age, health condition and sex of the subject, the type of disease to be treated, the degree of disease penetration, previous or concurrent therapeutic intervention, the specific disease of the patient, the route of administration, and the efficacy, stability and toxicity of the particular therapeutic agent. For example, a dose may also comprise from about 1 μ g/kg/body weight to about 1000 mg/kg/body weight per administration (such ranges include intervening doses) or more, and any ranges derivable therein. In some non-limiting examples of ranges that can be inferred from the numbers listed herein, a range of about 5 μ g/kg/body weight to about 100 mg/kg/body weight, about 5 μ g/kg/body weight to about 500 mg/kg/body weight, and the like can be administered. In any event, the medical personnel responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

The actual dosage amount of the composition administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, specific disease state of the patient, and the route of administration. Depending on the dose and route of administration, the preferred dose and/or the number of administrations of the effective amount may vary according to the response of the subject. In any event, the medical personnel responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

In certain embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise from about 2% to about 75%, or such as from about 25% to about 60%, by weight of the unit, and any range derivable therein. Naturally, the amount of active compound in each therapeutically useful composition can be prepared in such a way that: the appropriate dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life and other pharmacological considerations, and thus, a wide variety of dosages and treatment regimens may be desired.

In other non-limiting examples, the dose can further comprise about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 milligram/kg/body weight or more per administration, and any range derivable therein. In some non-limiting examples of ranges that can be inferred from the numbers listed herein, based on the numbers above, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 mg/kg/body weight, etc., can be administered.

Nucleic acids and vectors

In certain aspects of the invention, nucleic acid sequences encoding therapeutic proteins or fusion proteins containing therapeutic proteins may be disclosed. The nucleic acid sequence may be selected based on conventional methods, depending on the expression system used. For example, individual genes or variants thereof may be codon optimized for expression in a certain system. A variety of 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.

Recombinant proteins, inhibitory RNAs and Gene editing systems

A. Recombinant proteins

Some embodiments relate to recombinant proteins and polypeptides. Some embodiments relate to recombinant proteins or polypeptides that exhibit at least one therapeutic activity. In some embodiments, the recombinant protein or polypeptide may be a therapeutic antibody. In some aspects, the therapeutic antibody can be an antibody that specifically or selectively binds to an intracellular protein. In other aspects, the protein or polypeptide may be modified to improve serum stability. Thus, when reference is made herein to a function or activity of a "modified protein" or a "modified polypeptide", those of ordinary skill in the art will understand that this includes, for example, proteins or polypeptides that have additional advantages over the unmodified protein or polypeptide. It is specifically contemplated that embodiments relating to "modified proteins" may be practiced with respect to "modified polypeptides" and vice versa.

The recombinant protein may have deletions and/or substitutions of amino acids; thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may also comprise inserted or added amino acids, such as, for example, proteins with fusion proteins or with linkers. A "modified deleted protein" lacks one or more residues of the native protein, but may have 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 deleted protein is a protein having amino acid residues deleted from at least one antigenic region (i.e., a region of the protein that is determined to be antigenic in a particular organism, such as the type of organism to which the modified protein may be administered).

A substitution or substitution variant typically comprises the exchange of one amino acid for another at one or more positions within a protein, and may be designed to modulate one or more characteristics of the polypeptide, particularly its effector function and/or bioavailability. Substitutions may or may not be conservative, i.e., a substitution of an amino acid by a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; glutamic to aspartic acids; 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.

In addition to deletions or substitutions, the modified protein may have an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include insertion of a targeting peptide or polypeptide or only a single residue. End additions referred to as fusion proteins are discussed below.

The term "biologically functional equivalent" is well known in the art and is defined herein in further detail. Thus, sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to those of a control polypeptide are included, provided that the biological activity of the protein is maintained. In certain aspects, a recombinant protein may be biologically functionally equivalent to its natural counterpart.

It will also be understood that the amino acid and nucleic acid sequences may comprise additional residues, for example additional N-or C-terminal amino acids, or 5 'or 3' sequences, and still be substantially as shown by one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including maintaining biological protein activity where protein expression is involved. The addition of terminal sequences applies in particular to nucleic acid sequences which may, for example, comprise various non-coding sequences flanking the 5 'or 3' part of the coding region or may comprise various internal sequences, i.e.introns, which are known to be present within genes.

As used herein, a protein or peptide generally refers to, but is not limited to, proteins of greater than about 200 amino acids up to the full-length sequence translated from a gene; a polypeptide of greater 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.

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 the protein or peptide are contiguous without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid portions. In some embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.

Thus, the term "protein or peptide" encompasses an amino acid sequence comprising at least one of the 20 common amino acids found in naturally occurring proteins or at least one modified or abnormal amino acid.

Certain embodiments of the present invention relate to fusion proteins. These molecules may have a therapeutic protein linked at the N-or C-terminus to a heterologous domain. For example, fusion may also use leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Another useful fusion includes the addition of a protein affinity tag, e.g., a serum albumin affinity tag or six histidine residues, or an immunologically active domain, e.g., an antibody epitope, preferably cleavable to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), and glutathione-S-transferase (GST).

In a particular embodiment, the therapeutic protein may be linked to a peptide that increases half-life in vivo, such as an XTEN polypeptide (Schellenberger et al, 2009), an IgG Fc domain, albumin, or an albumin binding peptide.

Methods for producing 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 complete fusion protein.

The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by linking the gene to bridging DNA segments encoding peptide linkers (splicing between the tandemly linked polypeptides). The linker will be of sufficient length to allow proper folding of the resulting fusion protein.

B. Inhibitory RNA

siNA (e.g., siRNA) are well known in the art. For example, siRNA and double stranded RNA have been described in U.S. patent nos. 6,506,559 and 6,573,099 and U.S. patent applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161 and 2004/0064842, which are all incorporated herein by reference in their entirety.

Within a siNA, the components of the nucleic acid need not all be of the same type or homologous (e.g., a siNA may comprise nucleotides and nucleic acids or nucleotide analogs). Generally, siNA forms a double-stranded structure; the double-stranded structure may be produced from two separate nucleic acids that are partially or fully complementary. In certain embodiments of the invention, a siNA may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing itself (e.g., forming a hairpin loop). The double-stranded structure of the siNA may comprise 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive nucleobases, including all ranges therein. The siNA may comprise 17 to 35 consecutive nucleobases, more preferably 18 to 30 consecutive nucleobases, more preferably 19 to 25 nucleobases, more preferably 20 to 23 consecutive nucleobases, or 20 to 22 consecutive nucleobases, or 21 consecutive nucleobases, which hybridize to a complementary nucleic acid (which may be another portion of the same nucleic acid or a separate complementary nucleic acid) to form a double stranded structure.

Agents of the invention that may be used to practice the methods of the invention include, but are not limited to, siRNA. Generally, the introduction of double-stranded RNA (dsrna), which may alternatively be referred to herein as small interfering RNA (siRNA), induces potent and specific gene silencing, a phenomenon known as RNA interference or RNAi. RNA interference is referred to as "cosuppression", "post-transcriptional gene silencing", "sense suppression" and "suppression" (silencing). RNAi is an attractive biotechnological tool because it provides a means to knock out specific gene activities.

In designing RNAi, several factors need to be considered, such as the nature of the siRNA, the persistence of the silencing effect and the choice of the delivery system. To produce an RNAi effect, the siRNA introduced into the organism will typically contain an exon sequence. Furthermore, the RNAi process is homology dependent, and therefore the sequences 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 shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity between the siRNA and the sequence of the gene to be inhibited. Sequences having less than about 80% identity to the target gene are substantially less effective. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of the unrelated gene will be unaffected.

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

A target gene generally means a polynucleotide comprising a region encoding a polypeptide, or a region of a polynucleotide that modulates replication, transcription or translation or other processes important for expression of a polypeptide, or a polynucleotide comprising both a region encoding a polypeptide and a region operably linked thereto that modulates expression. Any gene expressed in the cell can be targeted. Preferably, the target gene is a target gene involved in or associated with the progression of cellular activity important to the disease or of particular interest as a subject of study.

The siRNA may be obtained from commercial sources, natural sources, or may be synthesized using any of a number of techniques well known to those of ordinary skill in the art. For example, one commercial source of pre-designed siRNA is

Austin, Tex. The other is

(Valencia, Calif.). Inhibitory nucleic acids that may be employed in the compositions and methods of the invention may be any nucleic acid sequence found by any source that is a verified down-regulator of a protein of interest. Without undue experimentation and using the present disclosure, it will be appreciated that additional siRNAs can be designed and used to practice the methods of the present invention.

The siRNA may further comprise alterations of one or more nucleotides. Such alterations may include the addition of non-nucleotide species, for example, to the end or within (at one or more nucleotides of the RNA) 19 to 25 nucleotides of RNA. In certain aspects, the RNA molecule comprises a 3' -hydroxyl group. The nucleotides in the RNA molecules of the invention may also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may comprise a modified backbone, such as a phosphorothioate, phosphorodithioate, or other modified backbone known in the art, or may comprise non-natural internucleoside linkages. Additional modifications of sirnas (e.g., 2 ' -O-methyl ribonucleotides, 2 ' -deoxy-2 ' -fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxy abasic residues (incorporated)) can be found in U.S. application publication 2004/0019001 and U.S. patent No 6,673,611 (each of which is incorporated by reference in its entirety). All such altered nucleic acids or RNAs described above are collectively referred to as modified sirnas.

C. Gene editing system

In general, a "CRISPR system" refers generally to transcripts and other elements involved in or directing the expression or activity of a CRISPR-associated ("Cas") gene, including sequences encoding a Cas gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr chaperone (tracr-mate) sequences (including "direct repeats," and partial direct repeats processed by tracrRNA in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from CRISPR loci.

A CRISPR/Cas nuclease or CRISPR/Cas nuclease system can comprise a non-coding RNA molecule (guide) RNA whose sequence specifically binds to DNA; and a Cas protein (e.g., Cas9) having 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, e.g. from a specific organism comprising an endogenous CRISPR system, e.g. Streptococcus pyogenes (Streptococcus pyogenes).

In some aspects, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and an immobilized tracrRNA) are introduced into a cell. Generally, using complementary base pairing, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene. The target site may be selected based on its location immediately adjacent to the 5' of the Protospacer Adjacent Motif (PAM) sequence (e.g. typically NGG or NAG). In this aspect, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote CRISPR complex formation at target sequence sites. Generally, a "target sequence" refers generally to a sequence for which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of a CRISPR complex. Complete complementarity is not necessarily required if sufficient complementarity exists to cause hybridization and promote formation of a CRISPR complex.

The CRISPR system can induce Double Stranded Breaks (DSBs) at the target site, followed by disruption as described herein. In other embodiments, a Cas9 variant, which is considered a "nickase," is used to nick a single strand at a target site. Pairs of nicking enzymes can be used, for example to improve specificity, each enzyme being directed by a different pair of gRNA targeting sequences such that when nicks are introduced simultaneously, 5' overhangs are introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of the cell, for example within an organelle of the cell. Generally, sequences or templates that are useful for recombination into a target locus comprising a target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In some aspects, the exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

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

One or more vectors that drive expression of one or more elements of the CRISPR system can be introduced into a cell such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites. The module may also be delivered to the cell as a protein and/or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr partner sequence, and the tracr sequence may each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed by the same or different regulatory elements may be combined in a single vector, while one or more additional vectors provide any components of the CRISPR system that are not comprised in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, the one or more insertion sites are located upstream and/or downstream of one or more sequence elements of 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 within a cell.

The vector may comprise regulatory elements operably linked to an enzyme coding sequence encoding a CRISPR enzyme (e.g., a Cas protein). Some non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 (also known as Csn 7 and Csx 7), Cas7, Csy 7, Cse 7, Csc 7, Csa 7, Csn 7, Csm 7, Cmr 7, Csb 7, Csx 7, CsaX 7, csaf 7, Csx 36f 7, Csx 36f 7. These enzymes are known; for example, the amino acid sequence of the streptococcus pyogenes (s.pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW 2.

The CRISPR enzyme may be Cas9 (e.g. from streptococcus pyogenes or streptococcus pneumoniae (s.pneumonia)). CRISPR enzymes can direct cleavage of one or both strands at a location of a target sequence, e.g., within the target sequence and/or within a complementary sequence of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising the target sequence. For example, an aspartate to alanine substitution in the RuvC I catalytic domain of Cas9 from streptococcus pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves single strand). In some embodiments, Cas9 nickase may 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 both strands to be nicked and used to induce NHEJ or HDR.

In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in a particular cell, e.g., a eukaryotic cell. Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a more or most frequently used codon in the gene of the host cell while maintaining the native amino acid sequence. Various species exhibit specific biases for certain codons for particular amino acids. Codon bias (difference in codon usage between organisms) is usually related to the translation efficiency of messenger rna (mrna), which in turn is believed to depend inter alia on the identity of the translated codons and the availability of specific transfer rna (trna) molecules. The predominance of the selected tRNA in the cell typically reflects the most frequently used codon in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

In general, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and to directly sequence-specifically bind the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher when optimally aligned using a suitable alignment algorithm.

Optimal alignment can be determined by using any suitable algorithm for aligning sequences, some non-limiting examples of which include the Smith-Waterman algorithm (Smith-Waterman algorithm); Needleman-Wenchet Algorithm (Needleman-Wunsch Algorithm); an algorithm based on a bernous-Wheeler Transform (e.g., a bernous-Wheeler Aligner); clustal W; clustal X; BLAT; novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.)); SOAP (available on SOAP. genetics. org. cn) and Maq (available on maq. sourceform. net).

The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein can comprise any additional protein sequence, and optionally a linker sequence between any two domains. Some 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 having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Some non-limiting examples of epitope tags include a histidine (His) tag, a V5 tag, a FLAG tag, an influenza Hemagglutinin (HA) tag, a Myc tag, a VSV-G tag, and a thioredoxin (Trx) tag. Some examples of reporter genes include, but are not limited to, glutathione 5 transferase (GST), horseradish peroxidase (HR), Chloramphenicol Acetyltransferase (CAT) beta galactosidase, beta glucuronidase, luciferase, Green Fluorescent Protein (GFP), HcRed, DsRed, Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and autofluorescent proteins, including Blue Fluorescent Protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or to other cellular molecules, including but not limited to Maltose Binding Protein (MBP), S-tags, Lex a DNA Binding Domain (DBD) fusions, GAL4A DNA binding domain fusions, and Herpes Simplex Virus (HSV) BP16 protein fusions. Further domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

Kits and diagnostic agents

In various aspects of the invention, kits comprising components necessary for purification of exosomes from a bodily fluid or tissue culture medium are contemplated. In other aspects, kits are contemplated that comprise components necessary for isolation of exosomes and are transfected with a therapeutic nucleic acid, a therapeutic protein, or a nucleic acid encoding a therapeutic protein therein. The kit may comprise one or more sealed vials containing any such components. In some embodiments, the kit may further comprise a suitable container means, which is a container that does not react with the components of the kit, such as an eppendorf tube, assay plate, syringe, vial, or tube. The container may be made of a sterilizable material, such as plastic or glass. The kit may also contain an instruction sheet that outlines the procedural steps of the methods set forth herein and will follow substantially the same procedures as described herein or known to one of ordinary skill in the art. The instruction information can be in a computer readable medium comprising machine readable instructions that, when executed using a computer, result in displaying a real or virtual program that purifies an exosome from a sample and transfects a therapeutic nucleic acid therein, expresses a recombinant protein therein, or electroporates a recombinant protein therein.

VII. examples

The following examples are included to illustrate some preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Materials and methods

And (5) culturing the cells. MCF7, MDA-MB231, E10, HDF and BJ human cell lines, and NIH 3T3 murine cell line were cultured in DMEM containing 10% FBS. 4T1 and 67NR murine cell lines were cultured in RPMI containing 10% FBS. MCF10A human mammary epithelial cell lines were cultured in DMEM/F12 medium containing 5% horse serum, 20ng/ml EGF, 0.5mg/ml hydrocortisone, 100ng/ml cholera toxin and 10. mu.g/ml insulin. All cells were derived from American Type Culture Collection (American Type Culture Collection) -ATCC.

And (4) separating and purifying the exosome. Exosomes were purified by differential centrifugation as previously described (Luga et al, 2012; Thery et al, 2006). The supernatant from the cells cultured for 48 hours was subjected to sequential centrifugation steps of 800g and 2000 g. The resulting supernatant was filtered using a 0.2 μm filter. After 3 hours of ultracentrifugation at 100,000g in a SW40Ti hanging basket rotor (shocking rotor), the precipitate was recovered. The supernatant was removed and the pellet was resuspended in PBS and then subjected to a second ultracentrifugation at 100,000g for 3 hours. The resulting precipitate was analyzed for exosome content. The exosomes for RNA extraction were resuspended in 500 μ Ι _ Trizol; the exosomes for protein extraction were resuspended in Urea/SDS lysis buffer (8M Urea, 2.5% SDS, 5. mu.g/mL leupeptin, 1. mu.g/mL pepsin inhibitor (pepstatin) and 1mM phenylmethylsulfonyl fluoride); and the exosomes for delivery to the cells were resuspended in serum-free DMEM medium. For other applications, the isolated exosomes were processed as described in the rest of the experimental procedures.

Imaging flow cytometry analysis (ImageStream). Exosomes were attached to 4 μm aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) in 0.9% NaCl saline solution (b. The reaction was stopped with 100mM glycine in saline and 2% BSA and blocked with 10% BSA with rotation at room temperature for 30 min. After washing in saline/2% BSA, the bead-bound exosomes were centrifuged at 10,000rpm for 2 minutes and incubated with 1: 200 anti-CD 63(Santa Cruz), anti-CD 9(Abcam), anti-CD 81(Abcam), anti-CD 82(Abcam) and anti-FLOT 1(Santa Cruz) at 4 ℃ for 30 minutes under rotation. The beads were centrifuged at 10,000rpm for 2 minutes, washed in saline/2% BSA, and incubated with a 1: 400Alexa-488 secondary antibody (Life Technologies, NY 14072) at 4 ℃ for 30 minutes with rotation. After 3 washes, the beads were resuspended in saline solution and washed

The analysis was performed on (Merck Millipore). To avoid pixel saturation, image acquisition gain (%) was set using positive samples. Use of

(Merck Millipore) software for image processing. The portal is defined to exclude out-of-focus (out-of-focus) beads and to select single beads. Alexa-488 positive bead gates were defined on the negative control samples. The percentage of positive beads is relative to the number of events analyzed per sample.

Immunogold labeling and electron microscopy. Precipitated exosomes were immobilized by resuspension in 2.5% glutaraldehyde in 0.1M phosphate buffer. The optimum concentration of the fixed sample was placed on a 300 mesh carbon/poly (methyl vinyl acetate) (formvar) coated grid and allowed to absorb the poly (methyl vinyl acetate) for at least 1 minute. For immunogold staining, the grid was placed in blocking buffer for the blocking/permeabilization step for 1 hour. Without washing, the grids were immediately placed in primary antibody at appropriate dilution overnight at 4 ℃ (polyclonal anti-GFP 1: 10, Abcam). As a control, some of the grids were not exposed to primary antibodies. The next day, all grids were washed with PBS and floated on the appropriate dilpend with 10nm gold particles (AURION, Hatfield, PA) for 2 hours at room temperature. The grid was washed with PBS and placed in 2.5% glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After washing in PBS and distilled water, the grids were dried and stained with uranyl acetate for comparison. The samples were observed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD camera (Advanced microscope Techniques, Danvers, Mass.).

EGF stimulation of exosomes. Exosomes were collected from MBA-MB-231 cells as described above. 1 to 3X 10⁹Individual exosomes were resuspended in 1mL PBS and various concentrations of egf were added. The exosome suspension with or without EGF was incubated at 37 ℃ in 5% CO₂Incubate for 15 minutes, and then place on ice. Combine three replicates and add PBS to a total volume of 11mL and stimulated exosomes were collected by ultracentrifugation at 100,000g for 3 hours in a SW40Ti basket rotor as before. In urea/SDS lysis buffer (containing 100mM NaF and 1mM NaOV)₄) The protein extract was collected from the precipitated stimulated exosomes for immunoblot analysis or in Triton X-100 buffer (150mM NaCl, 1% (v/v) Triton X-100, 10mM Na2HPO4, 2mM KH2PO4, pH 7.4, 50mM 6-aminocaproic acid, 10mM EDTA, 5mM N-ethylmaleimide, 5mM benzamidine, 5. mu.g/mL leupeptin, 1. mu.g/mL pepsin inhibitor, 1mM phenylmethylsulfonyl fluoride, 100mM NaF and 1mM NaOV₄) Wherein a protein extract is collected from the precipitated stimulated exosomes for use in an immunoprecipitation assay.

Western blot and antibody. The exosome protein extracts were loaded onto acrylamide gels according to Bicinchoninic Acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific) and transferred onto PVDF membranes (immibilonp) by wet electrophoretic transfer. Blots were blocked with 5% dried skim milk in TBS/0.05% tween 20 for 1 hour at RT and incubated with the following primary antibody overnight at 4 ℃: 1: 300 anti-CD 9 ab92726 (Abcam); 1: 300 anti-TSG 101 ab83 (Abcam); 1: 1000 anti-EGFR 4267S (CST); 1: 300 anti-CD 63 sc-365604, (Santa Cruz); 1: 200 anti-CD 81 cs-166029(Santa Cruz); 1: 1000 anti-pEGFR Tyr 10683777S (CST); 1: 2000 GRB resistant 2610111(BD Biosciences); 1: 1000 anti-Shc 06-203 (Millipore); 1: 5000 anti-GFP ab13970 (Abcam); 1: 1000GAPDH ab9483 (Abcam); 1: 10,000 HRP-conjugated β -actin a3854 (Sigma); 1: 400 anti-RNA Pol II cat #39097(Active Motif); 1: 500 anti-Hsp 90 ab1429 (Abcam); 1: 500 anti-eIF 3A ab86146 (Abcam); 1: 500 anti-eIF 4A1 ab31217 (Abcam). HRP-conjugated secondary antibodies (Sigma, 1: 2000) were incubated at room temperature for 1 hour. After antibody incubation, four washes were performed on an orbital shaker at 1 × TBS 0.05% tween 20 at 10 min intervals. Blots were developed with chemiluminescent reagents from Pierce.

And (4) performing immunoprecipitation. The exosome and cell protein extracts were gently shaken at 4 ℃ for 2 hours. The lysate was centrifuged at 14,000g for 15 minutes in a precooled centrifuge and the precipitate was then discarded. Protein a or G sepharose/sepharose (sepharose) beads were washed twice with PBS and restored to 50% slurry with PBS. The bead/slurry mixture (100. mu.l) was added to 100. mu.g exosome protein extract or 20. mu.g cellular protein extract and incubated at 4 ℃ for 10 min. The beads were removed by centrifugation at 14,000g for 10 minutes at 4 ℃ and the pellet was discarded. Mu.g of anti-eIF 4A1 antibody was added to 100. mu.L of exosome lysate and incubated overnight at 4 ℃ on an orbital shaker. Add 100. mu.L of protein A or G Sepharose/Sepharose bead slurry and let stand at 4 ℃ overnight. After centrifugation, the supernatant was discarded, and the beads were washed 3 times with ice-cold urea/SDS buffer. The agarose/sepharose beads were boiled for 5 minutes to dissociate the immune complexes from the beads. The beads were collected by centrifugation and immunoblotted on the supernatant.

Amino acids were identified using UPLC-MS. The exosomes were mixed with 200 μ L of methanol spiked with internal standard tryptophan-d 5 and incubated for one hour at-20 ℃. After centrifugation at 16,000g for 15 minutes at 4 ℃, 190 μ L of supernatant was collected and the solvent was removed. The dried extract was reconstituted in 15. mu.L of methanol, 10. mu.L of which was transferred to a microtube and derivatized. The chromatographic separation and mass spectrometric detection conditions used are summarized in table 1. The mass range is 50 to 1000m/z calibrated with cluster ions of sodium formate. An appropriate test mixture of standard compounds was analyzed before and after a whole set of random two-time replicates to check the retention time stability and sensitivity of the LC/MS system throughout the run.

Table 1 chromatographic conditions for the amino acid platform.

Data was processed using a TargetLynx application manager for MassLynx 4.1 software (Waters corp., Milford, USA). The set of predefined retention times, mass-to-charge ratio pair (Rt-m/z), corresponding to the metabolites contained in the analysis, is fed into the program. The relevant extracted ion chromatograms (mass tolerance window 0.05Da) were then peak detected and noise reduced in both the LC and MS domains, so that the software only processed the true metabolite-related features. Then, using Rt-m/z data pairs (retention time tolerance ═ 6s) as identifiers, a list of chromatographic peak areas was generated for each sample run. The normalization factor for each metabolite was calculated by dividing the intensity of each metabolite in each sample by the recorded intensity of the internal standard in that same sample.

Digital qPCR. The use of 3ng of cDNA was performed,

universal Master mix and QuantStaudio^TM3D digital PCR Master Mix v1(Applied Biosystems) digital RNA reactions were performed according to the manufacturer's recommendations. Using Quantstrudio^TM3D digital PCR chip loader (Applied Biosystems) A total of 14.5. mu.L of the mixture was loaded to QuantStaudio^TM3D digital PCR 20K chip kit v1(Applied Biosystems). In that

9700(Applied Biosystems) the PCR reaction was performed according to the manufacturer's protocol. Using Quantstrudio^TMThe chip was imaged by a 3D digital PCR instrument (Applied Biosystems).

TABLE 2 digital PCR primers.

The term of the exosome³⁵S]And (3) marking by methionine. Exosomes were isolated as before and resuspended in a suspension containing 0.1 to 1.0mCi/ml of the trans label [ 2 ]³⁵S]L-methionine, FBS-free methionine-free medium (Amersham Biosciences), and incubated overnight. Alternatively, exosomes were incubated in the presence of cycloheximide (Sigma, 100. mu.g/mL). Exosomes were pelleted, washed in ice-cold PBS, and resuspended in urea/SDS lysis buffer as previously described. Protein extracts were quantified using the BCA protein assay kit, run on acrylamide gels, and passed wetElectrophoretic transfer to PVDF membrane (Immobilon P), after which it is transferred by use of

The autoradiogram enhancing agents the membranes were analyzed by autoradiography according to the manufacturer's instructions (Perkin-Elmer).

Real-time PCR analysis. After purification of all exosome RNA with trizol (invitrogen), the dnase treated RNA was reverse transcribed with multislice reverse transcriptase (Applied Biosystems) and oligo-d (t) primers. 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 controls. 28S rRNA primer pair (QF00318857) and 18S rRNA primer pair (QF00530467) were purchased from Qiagen as ready-made specific primer pairs. Other primers are listed below. Each measurement was performed in triplicate. Threshold cycles were determined (Rothstein et al), which is the number of fractional cycles at which the amount of amplified target reached a fixed threshold (fractional cycle number), and expression was measured using the 2- Δ Ct equation, as previously reported (Livak and Schmittgen, 2001).

TABLE 3.qPCR probes

Name (R)	Sequence of	SEQ ID NO
			p21 F	5’TACCCTTGTGCCTCGCTCAG3’	5
p21 R	5’GAGAAGATCAGCCGGCGTTT3’	6
			hsa-actin F	5’CATGTACGTTGCTATCCAGGC3’	7
hsa-actin R	5’CTCCTTAATGTCACGCACGAT3’	8
			mmu-actin F	5’GGCTGTATTCCCCTCCATCG3’	9
mmu-actin R	5’CCAGTTGGTAACAATGCCATGT3’	10

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

Coupled in vitro transcription and translation. Lysates obtained from cells and exosomes were used for in vitro translation, as described previously, with a reaction volume of 12 μ Ι _. Standard reaction conditions are as follows: cell lysate (final protein concentration 10. mu.g) or exosome lysate (final concentration 100. mu.g), 1. mu.g of pEMT7-GFP cDNA expression plasmid, 20mM HEPES-KOH (pH 7.6), 80mM potassium acetate, 1mM magnesium acetate, 1mM MATP, 0.12mM GTP, 17mM phosphocreatine, 0.1mg/mL creatine phosphokinase, 2mM dithiothreitol, 40. mu.M of each of the 20 amino acids, 0.15mM spermidine and 400U/mL RNAsin (Promega). Incubation was performed at 37 ℃ for 3 hours.

Electroporation and culture of exosomes. The exosomes were pelleted and resuspended in 400. mu.L of electroporation buffer (1.15mM potassium phosphate pH 7.2, 25mM potassium chloride, 21% Optiprep) with 20. mu.g of plasmid (pCMV-GFP, pEGFP-p53 Addge plasmid 12091, pcDNA3-RLUC-POLIRES-FLUC and pcDNA-FLUC). Exosomes were electroporated using a Gene Pulser Xcell electroporation system (BioRad) using 4mm cuvettes as previously described (Alvarez-ervi et al, 2011). Where appropriate, exosomes were electroporated in the presence of cycloheximide (Sigma, 100. mu.g/mL) or alpha-amanitine (Sigma, 30. mu.g/mL) for inhibition of translation and transcription, respectively. Electroporated exosomes were cultured in serum-free DMEM at 37 ℃ for the indicated time points.

Flow cytometry analysis of electroporated exosomes. The exosome formulation (5 to 10 μ g) was incubated with 5 μ L of 4 μm diameter aldehyde/sulfate latex beads (Interfacial Dynamics, Portland, OR) and resuspended to 600 μ L. Exosome-coated beads were analyzed on a FACS Calibur flow cytometer (BD Biosciences) and for green fluorescence.

Exosome delivery and confocal microscopy. MCF10A cells were plated on inserted coverslips in 12-well plates with appropriate confluency and cultured overnight. The next day, cells were incubated with MDA-MB-231 exosomes resuspended in serum-free medium, DMEM, for 2 hours, washed with cold PBS 1X, and fixed with 4% PFA/PBS for 20 minutes at room temperature. Slides were permeabilized with PBS 0.5% Triton X-100 for 10 min at RT and counterstained with DAPI. Images were acquired using a Zeiss LSM510Upright confocal system using a recycling tool to maintain the same settings. For data analysis, images were selected from a library extracted from at least two independent experiments. The figures show representative fields of view.

Reverse transwell assay. As described previouslyExosomes were isolated from MDA-MB-231 cells and resuspended in PBS and quantified using Nanosight NTA. mu.L of 10X 10 in PBS⁹Individual exosomes were added to 96-well Corning^TM

Each bottom hole of the system. PBS alone was added to the bottom well as a negative control. Inserts containing polycarbonate membranes with 40nm wells were added to each well and 100 μ Ι _ of PBS alone, PBS with 20% FBS or PBS with 10,000ng/ml EGF were added to the inserts. Transswell plates were incubated at 37 ℃ with 5% CO₂Following incubation, samples were collected from the upper insert after 4 and 24 hours of incubation for exosome quantification using Nanosight NTA.

And (6) counting. Error bars represent ± s.d. between biological replicates. The technique and biological triplicates for each experiment were performed. Statistical significance was calculated by Student's t-test (Student's t-test), ANOVA or Mann-Whitney test (Mann-Whitney test), as the case may be, and is indicated in the figure description.

Example 1 detection of EGFR phosphorylation in exosomes derived from MDA-MB-231 (triple negative human breast cancer cells)

Exosomes were isolated from different murine and human cell lines using established ultracentrifugation techniques (Melo et al, 2015; Melo et al, 2014). The isolated exosomes appeared as heterogeneous mixtures, with the same size distribution consistently observed between formulations. NanoSight Nanoparticle Tracking Analysis (NTA) and Atomic Force Microscopy (AFM) revealed particles with size distributions averaging 104 ± 1.5nm in diameter and ranging roughly from 30 to 200 nm. This was confirmed by Transmission Electron Microscopy (TEM) showing extracellular vesicles surrounded by lipid bilayers (fig. 8A to 8C). Isolated exosomes were further shown to have known exosome markers (Raposo and Stoorvogel, 2013) by immunogold/TEM imaging, immunoblot analysis and imaging flow cytometry (fig. 8D to 8F). To further confirm its purity, exosome samples were inoculated onto coagulated LB plates, which showed no colony formation when compared to bacterial controls obtained from mouth swabs. This indicates that there was no bacterial contamination in the isolated exosomes (fig. 8G).

The EGFR content of exosomes obtained from different cell lines was probed by immunoblotting. While exosomes from all cell lines showed low levels of EGFR expression, exosomes derived from BJ fibroblast cell line and MDA-MB-231 triple negative breast cancer cell line showed strong expression of this receptor. The known exosome marker CD81 is shown as a loading control (fig. 1A). Given the importance of EGFR for the progression of triple negative breast cancer (Lim et al, 2016; Liu et al, 2012; Nakai et al, 2016), its functional role in MDA-MB-231 derived exosomes was further explored. Exosomes were derived from MDA-MB-231 and MCF10A cells, and 10 million exosomes were incubated with 500ng/ml recombinant human EGF (rhEGF) in serum-free medium at 37 ℃ for 15 minutes. Immunoblotting of protein extracts obtained from these exosomes with an antibody specific for the Tyr1068 residue of EGFR showed an increased level of phosphorylation of this receptor in exosomes derived from MDA-MB-231, but not for non-tumorigenic MCF10A mammary epithelial cells (fig. 1B). In any sample, the baseline level of EGFR was unchanged, confirming the specificity of the observed increase in phosphorylation. Stimulation with recombinant human egf (rhegf) also resulted in increased levels of phosphorylated ERK, indicating that the observed EGFR phosphorylation triggered downstream signaling events within the exosomes (fig. 1C). Further probing of the protein content of MDA-MB-231 exosomes indicated that they also contained the downstream effectors of EGFR, i.e., GRB2 and Shc (FIG. 1C).

It was then investigated whether EGFR of exosomes could engage (engage) its downstream adaptor after rhEGF stimulation. Exosomes were stimulated with rhEGF for 15 min at 37 ℃. Exosome protein extracts were subjected to pull-down assays using specific antibodies to GRB2 and Shc, and they were detected to show increased co-immunoprecipitation with EGFR following EGF stimulation (fig. 1D, 1E). Isotype IgG was used as a negative control for pull down and EGFR co-immunoprecipitation was not shown. In addition, co-immunoprecipitated GRB2 could also be detected only in EGF stimulated exosomes by reversing the assay and pulling down EGFR (fig. 8B). Taken together, these results indicate that exosomes from MDA-MB-231 cells contain EGFR, which can be phosphorylated by incubation with its ligand under cell-free conditions, leading to putative downstream signaling events in exosomes.

Example 2 EGF stimulation of exosomes alters their protein content

Receptor tyrosine kinases require ATP as a substrate for their kinase activity, and prostate-derived exosomes have been shown to have the ability to produce ATP (Ronquist et al, 2013 a). To further confirm the presence of phosphorylation activity in the absence of cells, ATP quantification was performed on exosomes with or without rhEGF stimulation. Using a luminescence-based kit, ATP was detected in exosomes from both MDA-MB-231 cells and MCF10A cells, although the latter were present in smaller amounts. Exosomes from MDA-MB-231 cells but not MCF10A cells showed a slight decrease in their ATP amount following stimulation with EGF (fig. 2A). To further investigate the effect of EGF stimulation on exosomes, they were incubated under cell-free conditions for a period of 48 hours. GRB2 protein levels were consistently higher in exosomes stimulated with EGF for 48 hours compared to their unstimulated counterparts (fig. 2B). This presents the possibility of arousing interest: the protein content of exosomes may change following growth factor stimulation. To further investigate this possibility, mass spectrometry was performed on protein extracts obtained from unstimulated or stimulated exosomes of rhEGF. The protein extracts were trypsinized and evaluated using an ESI-TRAP mass spectrometer to obtain MS/MS peptide spectra for each sample. The obtained spectra were then evaluated with reference to the SwissProt database for peptide identification to obtain a protein list for each exosome sample. Using the open access FunRich functional enrichment analysis tool (Pathan et al, 2015), it was observed that most of the identified hits (hit) in both unstimulated and stimulated exosomes matched proteins previously identified in exosomes (visiclepidia database) (fig. 2C). In exosomes stimulated with rhEGF, higher amounts of protein were identified compared to unstimulated exosomes (491 versus 371, fig. 2D). Although most of these proteins are common to both stimulated and unstimulated exosomes, 224 of the 491 proteins were detected only after rhEGF stimulation. Although EGFR was identified on both samples, GRB2 was identified only in rhEGF stimulated exosomes (fig. 2E). However, it should be emphasized that this does not mean that GRB2 is not present in unstimulated exosomes, but rather that it may be present at a level below the detectable threshold for this type of assay. An Exponentially Modified Protein Abundance Index (empiai) was used, which allows for unlabeled quantification of the relative changes in Protein content based on the observed peptide matches (Ishihama et al, 2005). The top 15 proteins that showed stronger increases in rhEGF-stimulated exosomes when compared to their unstimulated counterparts include several participants of actin remodeling and membrane dynamics, such as a-actin, MARCKS, ezrin (ezrin), moesin (moesin), and integrin α -2 (tables 4& 5). Gene Ontology (GO) analysis was then performed using the pantech over representation test (over presentation test). Interestingly, several were associated with actin remodeling and migration during the most predominant GO biological processes enriched in rhEGF-stimulated exosomes (5 of the 20 most predominant pathways, table 6).

TABLE 4 exosomes from MDA-MB-231 cells incubated with 500ng/ml EGF for 48 hours at 37 ℃ were identified as the first 15 proteins identified as being up-regulated in exosomes based on protein scores using the empAI method (Ishihama et al, 2005) compared to control exosomes.

TABLE 5 exosomes from MDA-MB-231 cells incubated with 500ng/ml EGF for 48 hours at 37 ℃ were identified as down-regulated first 15 proteins in exosomes based on protein scores using the empAI method (Ishihama et al, 2005) compared to control exosomes.

TABLE 6 top 20 Gene Ontology (GO) pathways determined based on the differential protein score between control exosomes and exosomes incubated with 500ng/ml EGF for 48 hours at 37 ℃. The list of differentially expressed proteins was obtained using the emPAI method and used as input for GO analysis using the PANTHER over-representation test.

Taken together, these mass spectral data indicate that MDA-MB-231 exosomes can change their protein content following rhEGF stimulation. These data also indicate that the same exosomes stimulated with rhEGF may undergo actomyosin remodeling and migration, suggesting a motile phenotype in response to rhEGF stimulation. The bicinchoninic acid (BCA) assay for protein quantification demonstrated an increase in protein content in exosomes stimulated with rhEGF when compared to unstimulated controls (fig. 2F). Immunoblots of β -actin also showed an increase in the level of polymerized actin in exosomes stimulated with varying amounts of rhEGF when compared to control non-stimulated exosomes (fig. 2G). Together, these observations indicate an unexpected degree of exosome bioactivity. Thus, the possibility of de novo protein synthesis by exosomes under permissive conditions was further investigated, and the potential induction of exosome motility following growth factor stimulation was explored.

Example 3 exosomes derived from different cell types contain functional components required for transcription and translation

Analysis of proteomic data from exosomes of different 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) (fig. 10, 11A, 11B). This information, coupled with knowledge that mRNA and its corresponding protein are found in exosomes, further suggests that isolated exosomes may have the ability to translate mRNA into protein.

Using quantitative pcr (qpcr) analysis, the presence of both 18S and 28S rRNA, as well as trnas for methionine, glycine, leucine, serine, and valine were confirmed in all exosomes analyzed (fig. 12A, 12B). In addition, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of exosomes revealed the presence of all free amino acids (fig. 3A). Immunoblot analysis identified the presence of different members of the translation initiation complex in exosomes, including eIF4A, eIF3A, and eIf1A (fig. 3B), confirming the observations made by mass spectrometry. In addition, initiation factors eIF4A and eIF3A were co-immunoprecipitated in protein extracts obtained from exosomes (Morino et al, 2000) (fig. 12C).

To functionally address the correlation of components present in exosomes for protein production, total protein extracts of exosomes isolated from MCF10A and MDA-MB-231 cells were incubated with a cDNA expression plasmid for green fluorescent protein (GFP plasmid) and subjected to coupled in vitro transcription and translation assays. Western blot analysis of the extracts revealed the production of GFP protein after incubation with GFP-encoding plasmids (fig. 3C). The fact that exosome lysates from both MDA-MB-231 and MCF10A cells allowed synthesis of proteins from GFP expression plasmids demonstrates that exosomes derived from different cells may contain all the essential functional components of both DNA transcription and mRNA translation. Consistent with the potential for DNA transcription, additional immunoblot analysis of protein extracts from exosomes isolated from different cellular sources determined the presence of RNA polymerase II subunits in both their phosphorylated and non-phosphorylated forms (fig. 3D).

Example 4 exosomes were able to perform cell-independent protein synthesis

To further validate the discovery that exosomes have autonomous capacity for de novo mRNA translation, isolated exosomes obtained from MDAMB-231 cells and murine lung carcinoma E10 cell line were used³⁵S-methionine was incubated to enable labeling of newly synthesized proteins from exosomes. To activate the putative biosynthetic process and potential autocrine stimulation, the assay was performed at 37 ℃. From the presence of³⁵Autoradiography of protein extracts of exosomes with S-methionine incubated for 72 hours showed incorporation of radioactive amino acids into several proteins in the 40 to 300kDa range. Cycloheximide, a protein translation inhibitor, when used as an exosome and³⁵this was largely inhibited upon S-methionine incubation (fig. 3E). When used in exosomes derived from different cancer cells³⁵Upon S-methionine incubation, a different pattern of labeled proteins was observed. In addition, total protein content was quantified from freshly isolated exosomes incubated in cell-free medium. After 48 hours of incubation, the total exosome protein content increased significantly (fig. 3F).

Next, it was confirmed whether transcription and translation could occur in the intact exosomes and not only in their lysates by establishing an exosome in vitro translation scheme. The pCMV-GFP expression plasmid was electroporated directly into exosomes derived from MDA-MB-231 cells (Borges et al, 2013; El-Andaloussi et al, 2012; Kamerkar et al, 2017) and the electroporated exosomes were incubated in serum-free medium at 37 ℃ for 48 hours. qPCR analysis of isolated exosome RNAs after digestion with dnase showed the presence of GFP mRNA in exosomes electroporated with pCMV-GFP expression plasmid (fig. 4A). Transmission electron microscopy showed that the structure of the exosomes electroporated with the pCMV-GFP plasmid was intact, and immunogold labeling with anti-GFP antibody showed that the protein was only detectable in exosomes containing the GFP plasmid (fig. 4B). Immunoblot analysis of exosome protein extracts using GFP antibodies further confirmed the presence of GFP in exosomes electroporated with the pCMV-GFP plasmid observed as early as 12 hours after electroporation (fig. 4C). After 1 week and up to 1 month, GFP could be observed in exosomes electroporated with expression plasmid (fig. 4C, 4D), although there was no increase relative to the levels observed at 24 hours. The same pattern was also observed in exosomes derived from MCF10A cells, confirming that exosomes derived from different cells (not only tumorigenic) contain all the required components and have the ability to synthesize proteins de novo (fig. 13A).

Immunoblot analysis of exosomes electroporated with GFP plasmid showed that GFP levels decreased by about 80% when incubated in the presence of the protein translation inhibitor cycloheximide (fig. 4E). GFP production was also reduced in the presence of the transcription inhibitor of RNA polymerase II, alpha-amanitine (FIGS. 4E, 4F). NanoSight NTA of electroporated exosomes using 488nm laser also detected green fluorescence in exosomes electroporated with pCMV-GFP plasmid, but not in exosomes mimicking electroporation or electroporated with plasmid and cycloheximide or α -amanitine (fig. 13B). In addition, bead-based flow cytometry analysis of plasmid-containing exosomes using different electroporation conditions detected the presence of GFP (fig. 13C). Next, the exosomes were incubated at 37 ℃ for 24 hours to initiate biological processes prior to electroporation with the pCMV-GFP plasmid. GFP production was impaired as detected by immunoblotting, indicating depletion of components required for transcription and translation in the exosomes preincubated at 37 ℃ (fig. 4G).

To confirm that these results are not specific only for GFP, an ovalbumin expression plasmid (pCMV-Ova), which is a protein that is not expressed in mammalian cells, was used. As with GFP, immunoblot analysis of exosomes after electroporation and incubation at 37 ℃ for 48 hours showed that ovalbumin was produced only in exosomes electroporated with pCMV-Ova plasmid (fig. 13D).

Initiation of protein translation of most mrnas in eukaryotes involves recognition of the 5' cap structure by the eIF4F complex (Merrick, 2004). To determine whether protein translation in exosomes is cap-dependent, a cDNA bicistronic construct consisting of two different luciferase cistrons separated by an internal ribosome entry site was used (fig. 4H) (Poulin et al, 1998). In this system, translation of Renilla luciferase is cap-dependent, whereas translation of firefly luciferase is directed by the poliovirus IRES, and is therefore cap-dependent (FIG. 4H). Electroporation of the plasmid directly into the exosomes resulted in an increase in renilla luciferase activity with no significant change in firefly luciferase activity (fig. 4I) (Poulin et al, 1998), indicating that protein translation in the exosomes occurs in a cap-dependent manner. Since firefly and Renilla luciferases have different activity requirements, the assay is repeated using a plasmid expressing firefly luciferase under the control of the CMV promoter. Luciferase activity was also observed in pCMV-Fluc electroporated exosomes (FIG. 4J).

Example 5 translation of mRNA in exosomes to produce functional protein and can be stimulated by growth factors

MCF10A cells pretreated with cycloheximide were incubated with MDA-MB-231 exosomes electroporated directly with the pCMV-GFP plasmid. Imaging by confocal microscopy detected green fluorescence in MCF10A cells, possibly contributed by GFP protein (after transcription and translation) delivered by MDAMB-231 exosomes (fig. 5A, top and bottom panels). Interestingly, cells electroporated directly with the pCMV-GFP plasmid showed a GFP fluorescence pattern that was different from the fluorescence pattern observed in cells incubated with exosomes containing the pCMV-GFP plasmid (fig. 5A, middle and bottom panels).

MDA-MB-231 cells overexpress an inactive mutant form of the tumor suppressor protein p53, which therefore fails to activate the p21 promoter (Gartel et al, 2003). Wild-type (wt) p53 generally responds to DNA damage by directly inducing p21, thereby promoting cell cycle arrest (Zilfou and Lowe, 2009). Exosomes isolated from MDA-MB-231 cells were electroporated with a plasmid encoding wt p53 fused with GFP. The electroporated exosomes were incubated in culture medium for 48 hours to allow transcription and translation to produce wt p53 protein (fig. 5B). Subsequently, exosomes containing the newly formed wt p53 were incubated with acceptor MDA-MB-231 cells under the influence of cycloheximide. Recipient MDA-MB-231 cells showed a significant increase in p21 expression (fig. 5C), confirming the function of wt p53 protein synthesized only by exosomes (fig. 5B). To additionally confirm that this increase in p21 expression was indeed due to the wt p53 protein being newly translated by the exosome and not due to delivery of the plasmid, MDA-MB-231 derived exosomes were electroporated with the p53-GFP plasmid and either incubated for 48 hours prior to incubation with recipient MDA-MB-231 cells to synthesize wt p53 protein (48 hours) or immediately delivered to recipient MDA-MB-231 cells without letting them produce wt p53 protein (0 hours, fig. 13E). Exosomes that allowed active synthesis of wt p53 protein for 48 hours prior to delivering induced p21 expression in recipient MDA-MB-231 cells as early as 30 minutes after exosome incubation were higher than exosomes in the plasmid-only case, which showed the same baseline of p21 expression observed in control MDA-MB-231 cells (fig. 13E).

To further demonstrate that exosomes from MDA-MB-231 cells exhibited baseline capacity for intrinsic protein synthesis, exosomes were incubated at 37 ℃ in the presence and absence of cycloheximide. Immunoblots of protein extracts from these exosomes showed that expression levels of the small cytoplasmic protein, β -actin and GAPDH consistently decreased after incubation with cycloheximide, again demonstrating the presence of baseline levels of protein synthesis in these exosomes (fig. 5D).

To determine which proteins were produced by MDA-MB-231 exosomes in the absence of external stimuli, stable isotope labeling with amino acids (SILAC) was performed in culture in adapted form. Exosomes derived from MDA-MB-231 cells were supplemented with heavy markers¹³C-lysine and¹⁵incubation in SILAC medium with N-arginine. Culturing MDA-MB-231 exosomes in heavily labeled SILACThe medium was incubated for 5 days and protein extracts were obtained, trypsinized and subjected to mass spectrometry. Although only a few re-labeled peptides matched the MS/MS spectrum obtained, 11 proteins could be identified, each matching 1 or 2 peptides containing re-labeled amino acids (table 7). This confirms that, despite low levels, baseline mRNA translation still occurs in exosomes, resulting in the formation of very small amounts of newly synthesized protein.

TABLE 7 listing of proteins comprising peptides matched to the spectra in case of heavy isotopes, obtained with¹³C-lysine and¹⁵mass spectrometric analysis of protein extracts from MDA-MB-231 exosomes incubated for 5 days in N-arginine SILAC medium. Each of the proteins listed comprises at least 1 and¹³c-lysine or¹⁵N-arginine relabeled spectrally matched peptides.

Next, protein translation assays were performed using exosome repeats derived from MDA-MB-231 cells with the electroporated pCMV-GFP plasmid. Exosomes were incubated in serum-free medium with or without stimulation with rhEGF at 37 ℃ for 48 hours. Although the unstimulated exosomes exhibited baseline levels of GFP production, GFP levels increased after incubation with different concentrations of rhEGF (fig. 5E). This again confirms that although all exosomes can synthesize proteins, growth factor stimulation can alter their production rate by causing increased levels of protein synthesis.

Example 6-active migration of exosomes in response to stimulation by rhEGF and serum factors

To determine whether growth factors can induce a motility phenotype in exosomes, a reverse migration assay based on the Boyden chamber (Boyden chamber) system was designed, which included rhEGF and serum factors. 100 million exosomes isolated from MDA-MB-231 cells were placed in culture wells of a 96-well plate covered with polycarbonate surface inserts containing 400nm wells. Inserts contained PBS, PBS with 10 μ M rhEGF, or PBS with 20% exosome-depleted FBS (fig. 6A). Since the FBS consumed by exosomes still may contain trace amounts of exosomes (data not shown), 20% FBS was placed on the top insert with no exosomes in the bottom well, which served as a control. After incubation at 37 ℃, samples were obtained from the apical insert at different time points and exosomes were quantified using Nanosight NTA.

After 4 hours incubation, exosome levels on the top insert were comparable in all experimental groups (fig. 6B). After 24 hours of incubation, 20% FBS significantly improved efflux migration from bottom to top, indicating a sustained chemotactic effect on MDA-MB-231 exosomes towards higher serum growth factor gradients (fig. 6C). Inserts with 20% FBS had significantly less exosomes after 24 hours relative to wells without exosomes, confirming that the migrating exosomes are from the identity of MDA-MB-231 cells (fig. 6C). PBS resulted in negligible efflux migration of exosomes, but rhEGF alone also induced movement of exosomes, albeit at lower levels compared to the full serum-associated growth factors (fig. 6C). Taken together, these results indicate that exosomes exhibit functional chemotactic capacity that can be induced by growth factors.

Example 7 exosomes specifically show enhanced protein production in tumor-bearing mice

To address whether the ability of exosomes to respond to tumor-induced growth factor gradients is involved in the intrinsic production of new proteins with functional consequences, a reference mouse model was generated. Mice bearing established 4T1 breast tumors were injected with 50 billion MDA-MD 231/CD63-mCherry exosomes electroporated with GFP or ovalbumin expression plasmids. The control panel of this study included CD63-mCherry exosomes without plasmid and CD63-mCherry exosomes electroporated with plasmid and cyclohexamide. Twenty-four hours after i.p. injection of exosomes in tumor-bearing or non-tumor-bearing mice, tumors, sera and several other organs were collected. FACS separation of exosomes was performed using CD63 mCherry tag and GFP or ovalbumin proteins were evaluated. GFP and ovalbumin were detected mainly in tumors, lung, bone, brain and serum of tumor bearing mice, but only at very low levels in tissues of non-tumor bearing mice and tumor bearing mice injected with cyclohexamide-containing exosomes. These results indicate that although exosomes can be detected in the liver, lungs and brain of non-tumor bearing mice, exosomes enter these organs and enter more robustly (presumably through enhanced motility), including tumor tissue, and respond biologically by producing nascent proteins. In addition, serum-derived exosomes from tumor-bearing mice exhibited protein production, suggesting that tumors biologically affect exosomes at a systemic level.

***

All 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 this invention have been described in terms of certain preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes 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.

Reference to the literature

The following references are specifically incorporated by reference herein, to the extent that they provide exemplary procedures or other details supplementary to those set forth herein.

U.S. Pat. No. 4,870,287

U.S. Pat. No.5,739,169

U.S. Pat. No.5,760,395

U.S. Pat. No.5,801,005

U.S. Pat. No.5,824,311

U.S. Pat. No.5,830,880

U.S. Pat. No.5,846,945

Aakalu et al.，Dynamic visualization of local protein synthesis in hippocampal neurons.Neuron，30：489-502，2001.

Almoguera et al.，Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.Cell，53：549-554，1988.

Al-Nedawi et al.，Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR.Proc.Natl.Acad.Sci.U.S.A.，106：3794-3799，2009.

Alvarez-Erviti et al.，Delivery of siRNA to the mouse brain by systemic inj ection of targeted exosomes.Nature Biotechnology，29：341-345，2011.

Austin-Ward&Villaseca，Gene therapy and its applications.Rev.Med.Chil.，126：838-845，1998.

Baietti et al.，Syndecan-syntenin-ALIX regulated the biogenesis of exosomes.Nat.Cell Biol.，14：677-685，2012.

Bastos et al.，Exosomes in eancer：Use them or target themSemin.Cell Dev.Biol.，78：13-21，2018.

Biankin et al.，Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature，491：399-405，2012.

Borges et al.，TGF-beta1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis.J.Amer.Soc.Nephrology，24：385-392，2013.

Bukowski et al.，Signal transduction.abnormalrties in T.lympbocytes from patienis with advanced renal carcinoma.clinical relevance and effects of cvtokine therapy.Clin.Cancer Res.，4-2337-2347，1998.

Chang et al.，Pancreatic cancer genomics.Curreni Ppinion in Genetics&Development，24：74-81，2014.

Choi et al.，The protein interaction network of extracell ular vesicles derived from human colorectal cancer cells.J.Proteome Res.，11：1144-1151，2012.

Christodoulides et al.，Immunization with recombinant elass 1 outer-membrane proiein from Neisseria meningitidis：influence of liposomes and adjuvants on antibody avidity，recognition of native protein and the induction of a baciericidal immime response against meningococci.Microbiology，144：3027-3037，1998.

Clayton et al.，Antigen-presenting cell exosomes are protected from complement-mediated lysis by eptession of C55 and CD59 Eur.J.immunology，33：522-531，2003.

Collins et al.，Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice.J.Climical hivestigafion，122：639-653，2012a.

Collins et al.，Metastatic pancreatic caneer is dependent on oncogeuic Kras in mice.PLoS One，7：e-49707，2012b.

Colombo et al.，Biogenesis，secretion，and intercellular interactions of exoscmes and other extracellular vesicles.Annu.Rev.Cell.Dev.Biol.，30：255-289，2014.

Combes et al.，A new flow cytometry method of platelet-derived microvesicle quantitation in plasma，Thromb.Haemost.，77：220，1997.

Cooper et al.，Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice.Movement Disorders，29：1476-148S，2014.

Croft et al.，The Reactome pathway knowledgebase.Nuc.Acids Res.，42.D472-477，2014.

Davidson et a.，Intralesional cytokine therapy in cancer：a pilot study of GM-CSF infusion in mesothelioma.J.Immnunother.，21：389-398，1998.

Du et al.，A systematie analysis of the sileneing effects of an active siRNA at all single-nucieotide mismatched target sites.Nuc.Acids Reqs.，33：1671-1677，2005.

El-Andaloussi et al.，Extracellular vesicles：biology and emerging therapeutic opportunities.Nature Reviews Drug Discovery，12：347-357，2013.

El-Andaloussi et al.，Exosome-mediated delivery of siRNA in vitro and in vivo.Nature Protocols，7：2112-2126，2012.

Escr et al.，Omcogenic KRAS signalling in pancreatic cancer.British Jomnal of Cancer，111：817-822，2014.

Gartel et al.，A new method for determining the status of p53 in tumor eell lines of different origin.Oncology Research，13：405-408，2003.

Gomes-da-Silva et al.，Lipid-based nanoparticles for siRNA delivery in cancer therapy：paradigms and challenges.Accounts of Chemical Research，45：1163-1171，2012.

Gonzales et al.，Large-scale proteomics and phosphoproteomics of urinary exosomes.J.Amer.Soc.Nephrology，20：363-379，2009.

Gysin et al.，Therapeutic strategies for targeting ras proleins，Genes&Concer，2：359-372，2011.

Hanibuchi et al.，Therapeutic efficacy of mouse-human chimeric anti-ganglioside GM2monoclonal antibody against multiple organ micrometasiases of human lung cancer in NK cell-depleted SCID mice.Int.J.Cancer.78：480-485，1998.

Harding et al.，Endocytosis and intracellular processing of transrerrin and colloidal gold-transferrin in rat reticulocytes：demonstration of a pathway for receptor shedding.European J.Cell Biol.，35：256-263，1984.

Hazan-Halevy et al.，Cell-specific uptake of mantle cell lympboma-derived exosomes by malignant and non-malignant B-lymphocytes.Cancer Lett.，364：59-69，2015.

Hellstrand et al.，Histamine and cytokine therapy.Acta Oncol.，37：347-353，1998.Hingorani et al.，Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in nice.Cancer Cell，7：469-483，2005.

Hollander，Immunotherapy for B-cell lymphoma：current status and prospective advances.Froat Immunol.，3：3，2013.

Howlader et al.，SEER Cancer Statistics Review，1975-2011，National Cancer Institute.Bethesda.，MD.On the World Wide Web at seercancergov/csr/1975_2011/，2013.

Hruban et al.，K-ras oncogene activation in adenocarcinoma of the human pancreas.A study of 82 carcinomas using a combination of mutant-cnriched polymerase chaln reaction analysis and allele-specific oligonucleoti de hybridization.American J.Pathology，143；545-554，1993.

Huang et al.，Epidermal growth factor receptor-containing exosomes induce tumor-specific regulatory T cells.Cancer Invest，31：330-335，2013.

Hui&Hashimoto，Pathways for Poyentiation offmmunogenicity during Adjuvant-Assisted Immunizations with Plasmodium falcipanum Major Merozoite Surface Protein I.Infec.Immun.，66：5329-5336，1998.

Isihama et al.，Exponentially modified protein abundance index(emPAI)for estimation of absolute protein amount In proteomics bythe number of sequenced peprides per protein.Mol.Cell.Proteomics，4：1265-1272，2005.

Ji et al.，Ras activity levels control the development of pancreatic diseases.Gastroenterology，137：1072-1082，82e1-6，2009.

Johnsen et al.，A comprehensive overview of exosomes as drug delivery vehicies-endogenous manocarriers for targeted cancer therapy.Biochimica et Biophysica Acta，1846：75-87，2014.

Kahlert et al.，Identifi cation of Double Stranded Genomic DNA Spanning all Chromosomes with Mutated KEAS and p53 DNA in the Serum Exosomes of Patients with Pancreatic Cancer.J.Biol.Chem.，289：3869-3875，2014.

Kalra et al.，Comparative proteomics evaluation of plasma exosome isolation tecbniques and assessment of the stability of exosomes in normal human blood plasma.Proleomics，13：3354-3364，2013.

Kalluri，Tbe biology and function of exosomes in cancer.J.Clin.Invest.，126：1208-1215，2016.

Kamerkar et al.，Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.Nature，546：498-503，2017.

Kowal et al.，Biogenesis and secretion of exosones.Currenl Opinion in Cell Biology，29：116-125，2014.

Kramer et al.，The ribosome as a platform for co-translational processing，folding and targeting of newly symthesized proteins.Nat.Struct.Mol.Biol.，16：589-597，2009.

Li et al.，Exosomes derived from gefitinib-treated EGFR-mutant lung cancer cells alter cisplatin sensitivity via up-regulating autophagy.Oncoiarget，7：24585-24595,2016.

Lim et al.，EGFR Signaling Enhances Aerobic Glycolysis in Triple-Negative Breasi.Cancer Cells to Promote Tumor Growth and Immune Escape.Cancer Rescarch，76：1284-1296，2016.

Liu et al.，.EGFR expression correlates with decreased disease-free survival in triple-negative breast cancer：a retrospective analy sis based on a tissue microarray.Med.Oncol.，29：401-405，2012.

Livak&Schmittgen，Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method.Methods，25：402-408，2001.

Lorch et al.，Role of DNA sequence in chromatin remodeling and the formation of nucleosome-free regions.Genes Dev.，28：2492-2497，2014.

Luga et al.，Exosomes mediate stromal mobilization of auiocrine Wnt-PCP signaling inbreast cancer cell migration.Cell，151：1542-1556，2012.

Ma et al.，Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain.Nature，429：318-322，2004.

Marcus&Leonard，FedExosomes：Engineering Therapeutic Biological Nanoparticles that Truly Deliver Pharmaceuticals(Basel)，6：659-680，2013.

Masuda et al.，Role of epidermal growth factor receptor in beast cancer.Breast Cancer Res.Treat.，136：331-345，2012.

Melo et al.，Cancer exosomes perform cell-independent microRNA biogenesis and promote rumorigenesis.Cancer Cell，26：707-721，2014.

Melo et al.，Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.Naiure，523：177-182，2015.

Merrick，Cap-dependent and cap-independent translation in eukaryotic systerns.Gene，332：1-11，2004.

Morgillo et al.，Mechanisms of resistanee to EGFR-targeted drugs：lung cancer.ESMO Open，1：e000060，2016.

Morino et al.，Eukaryotic translation initiation factor 4E(elF4E)binding site and the middle one-third of elF4GI constitute the core domain for cap-dependent translation，and the C-terminal one-third functions as a modulatory region.Mol.Cell Biol.，20：468-477，2000.

Nagai et al.，Chromatin potentiates transcription.Proc.Natl.Acad.Scl.U.S.A.，114：1536-1541，2017.

Nakai et al.，A perspective on anti-EGFR therapies targeting tripie-negative breast cancer.Am.J.Cancer Res.，6：1609-1623，2016.

Normanno et al.，Epidermal growth factor recep1or(EGFR)signaling in cancer.Gene，366：2-16，2006.

Ozdernir et al.，Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas caneer with reduced survival.Cancer Cell，25：719-734，2014.

Pardoll，Cancer immunotherapy through checkpoint blockade：the future of cancer treaiment.Medicographia，36：274-284，2014.

Pathan et al.，FunRich：An open access standalone functional enrichment and interaction network analysis tool.Proteomics，15：2597-2601，2015.

Pecot et al.，Therapeutic Silencing of KRAS using Systemically Feiivered siRNAs.Molecular Cancer Therapeutics，13：2876-2885，2014.

Peinado et al.，Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.Nature Medicine，18：883-891，2012.

Perdigao et al.，Unexpected features of the dark proteome.Proc.Natl.Acad.Sci.U.S.A.，112：15898-S903，2015.

Pico de Coana et al.，Checkpoint blockade for cancer therapy：revitalizig.a suppressed immune system.Trends in Molecular Medicine，21：482-492，2015.

Pisitkun et al.，Identification and proteomic profiling of exosomes in human urine.Proc.Natl.Acad.Scl.U.S.A.，101：13368-13373，2004.

Poliseno et al.，A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.Nature，465：1033-1038，2010.

Poulin et al.，4E-BP3，a new member of the eukaryotie initiation factor 4E-binding protein famiiy.J.Biol.Chem.，273：14002-14007，1998.

Qin et al.，Interferon-beta gene therapy inhibits tumor formation and causes regression of established tumors inimmune-deficient mice，Proc.Natl.Acad.Sci.U.S.A.，95：14411-14416，1998.

Rachagani et al.，Activaied KrasG12D is associa1ed with invasion and metastasis of pancreatic cancer cells through inhibition of E-cadherin.Br.J.Cancer，104：1038-1048，2011.

Rao&Cruz，Effects of confinement on the structure and dynamics of anintrinsically disordered peptide：a molecular-dynamics study.J.Phys.Chem.B.，1173707-3719，2013.

Raposo&Sroorvogel，Extracellular vesicles：exosomes，microvesicles，and friends.J.Cell Biol.，200：373-383，2013.

Rejiba et al.，K-ras oncogene silencing strategy reduces tumor growth and enhances gemeitabine chemotherapy efficacy for pancreaic cancer treatment.Cancer Science，98：1128-1136，2007.

Ronquist et al.，prosiasomes from four different species are able to produce extracellular adenosine triphosphate(ATP).Biochim Biophys.Acia，1830：4604-4610，2013a.

Ronquist et al.，Human prostasomes express glycolytic enzymes with capacity for ATP production.Am.J.Physiol.Endocrinol.Metab.，304：E576-582，2013b.

Rothstein et al.，Targeting signal 1 through CD45RB synergizes with CD40 ligand blockade and promotes long term engraftment and tolerance in stringent transplant models.J.Immiinol.，166：322-329，2001.

Seshacharyulu et al.，Targering the EGFR signaling pathway in cancer therapy.Expert Opin.Ther.Targets，16：15-31，2012.

Siegel et al.，Cancer stati stics，2014.CA：A cancer journal for clinicicanss，64：9-29，2014.

Simoes et al.，Cationlc liposomes for gene delivery.Expert Prinion on Drug Delivery，2：237-254，2005.

Sinkovics，The celi survival patliways of the primordial RNA-DNA complex remain conserved in the extant genomes and may function as proto-oncogenes.Eur.J.Microbiol.Immunol.(Bp)，5：25-43，2015.

Skogberg et al.，Characterization of human thymic exosomes.PLoS ONE，8：e67554，2013.

Smakman et al.，Dual effect of Kras(D12)knockdown on tumorigenesis：increased immune-mediated tumor clearance and abrogation of tumor malignancy.Oncogene，24：8338-8342，2005.

Smith et al.，Local protein synthesis in neurons.Curr.Biol.，11：R901-903，2001.

Song et al.，Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.J.Biol.Chem.，291：8453-8464，2016.

Steward&Levy，Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus.J.Neurosci.，2：284-291，1982.

Sun et al.，Characterization ofthe mutations of the K-ras，p53，p16，and SMAD4 genes in 15human pancreatic cancer cell lines.Oncology Reports，8：89-92，2001.

Thery et al.，Exosomes：composition，biogenesis and function.Nature Reviews.Immiimology，2：569-579，2002.

Thery et al.，Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Cttrrent Protocols in Cell.Biology，Cbapter 3，Unit 3 22，2006.

Tomas et al.，EGF receptor trafficking：consequences for signaling and cancer.Trends.Cell Biol.，24：26-34，2014.

Ung et al.，Exosome proteomics reveals transcriptional regulator proreins with potential to mediate downstream pathways.Cancer Sci.，105：1384-1392，2014.

Valadi et al.，Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic cxchangc between cells.Narure Cell Bologv，9：654-659，2007.

van den Boorn et al.，Exosomes as nucleic acid nanocarriers.Advanced Drug Delivery Revlews，65：331-335，2013.

van der Meel et al.，Extracellular vesicles as drug delivery systems：Lessons from the liposome field.J.Conrrolled Release，195：72-85，2014.

Wahlgren et al.，Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes.Nucleic Acids Research，40：e130，2012.

Westphal et al.，EGFR as a Target for Glioblastoma Teatment：An Unfulfilled Promise.CNS Drugs，31：723-735，2017.

Weyrich et al.，Change in protein phenotype withour a nuclens：translational control in platelets.Semin.Thromb.Hemost.，30：491-498，2004.

Willms et al.，Cells release subpopulations of exosomes with distinct moleeular and biological properties.Sci.Rep.，6：22519，2016.

Wykes&Lewin，Immune checkpoint blockade in infectious diseases.Nat.Rev.Immunology，18：91-104，2018.

Xue et al.，Small RNA combination therapy for lung cancer.Proc.Natl.Acad.Sci.U.S.A.，111：E3553-3561，2014.

Yamada et al.，Cell Infectivity in Relation to Bovine Leukemia Virus gpS1 and p24in Bovine Milk Exosomes.PLoS ONE，8：e77359，2013.

Ying et al.，Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.Cell，149：656-670，2012.

Yuan et al.，Development of siRNA payloads to target KRAS-mutant cancer.Concer Discovery，4：1182-1197，2014.

Zhang et al.，Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis.Nat.Commun.，8：15016，2017.

Zhang et al.，A mechanism for the upregulation of EGF receptor ievels in glioblastomas.Cell.Rep.，3：2008-2020，2013.

Zilfou&Lowe，(2009).Tumor suppressive functions of p53.Cold Spring Harb.Perspect.Biol.，1：a001883，2009.

Zorde Khvalevsky et al.，Mutant KRAS is a druggable target for pancreatic cancer.Proc.Natl.Acad.Sci.U.S.A.，110：20723-20728，2013.

Sequence listing

<110> board of system of State university of Texas

<120> use of exosomes for targeted delivery of therapeutic agents

<130> UTFC.P1363WO

<140> unknown

<141> 2019-03-28

<150> US 62/649,057

<151> 2018-03-28

<160> 10

<170> PatentIn 3.5 edition

<210> 1

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 1

agtaggtagc gcgtcagtct cataatctga aggtcgtgag ttcgatcctc acacggggca 60

<210> 2

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 2

aggcgctgga ttaaggctcc agtctcttcg gaggcgtggg ttcgaatccc accgctgcca 60

<210> 3

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 3

agtggttatc acgttcgcct aacacgcgaa aggtccccgg ttcgaaaccg ggcggaaaca 60

<210> 4

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 4

ggcgatggac tagaaatcca ttggggtttc cccgcgcagg ttcgaatcct gccgactacg 60

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 5

tacccttgtg cctcgctcag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 6

gagaagatca gccggcgttt 20

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 7

catgtacgtt gctatccagg c 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 8

ctccttaatg tcacgcacga t 21

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 9

ggctgtattc ccctccatcg 20

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 10

ccagttggta acaatgccat gt 22

Claims (46)

1. A method of treating a disease or disorder in a patient in need thereof, the method comprising:

(a) obtaining an exosome having growth factor receptors on its surface;

(b) transfecting the exosome with a nucleic acid encoding a therapeutic protein;

(c) administering the transfected exosomes to a patient;

(d) providing a growth factor gradient at a site of the disease or disorder to attract exosomes to the site and stimulate production of the therapeutic protein at the site, thereby treating the disease in the patient.

2. The method of claim 1, wherein the method is further defined as a method of administering a therapeutic protein to diseased cells in a patient.

3. The method of claim 1, wherein the exosomes obtained in step (a) are obtained from a bodily fluid sample obtained from the patient.

4. The method of claim 3, wherein the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate/tears, or serum.

5. The method of claim 1, wherein the nucleic acid is mRNA.

6. The method of claim 1, wherein the nucleic acid is a plasmid.

7. The method of claim 1, wherein the nucleic acid is cDNA.

8. The method of claim 1, wherein the disease or disorder is cancer, injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a renal disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a genitourinary disorder, or a bone disease or disorder.

9. The method of claim 8, 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.

10. The method of claim 8, wherein the site of the disease or disorder is a tumor.

11. The method of claim 8, wherein the cancer is metastatic.

12. The method of claim 11, wherein the site of the disease or disorder is a metastatic nodule.

13. The method of claim 1, wherein the therapeutic protein is a kinase, a phosphatase, or a transcription factor.

14. The method of claim 1, wherein the therapeutic protein corresponds to a wild-type form of the protein that is mutated or inactivated in cells at the site of the disease or disorder.

15. The method of claim 1, wherein the therapeutic protein corresponds to a dominant negative form of a protein that is overactive in cells at the site of the disease or disorder.

16. The method of claim 1, wherein the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor.

17. The method of claim 1, wherein the exosome comprises CD47 on its surface.

18. The method of claim 1, wherein transfecting comprises electroporation.

19. The method of claim 1, further comprising administering at least a second treatment to the patient.

20. The method of claim 19, wherein the second therapy comprises surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

21. The method of claim 1, wherein the exosome is comprised in a tissue scaffold matrix.

22. A method of treating a disease or disorder in a patient in need thereof, the method comprising:

(a) obtaining an exosome having growth factor receptors on its surface;

(b) transfecting the exosomes with a therapeutic agent;

(c) administering the transfected exosomes to a patient;

(d) providing a growth factor gradient at a site of the disease or disorder to attract exosomes to the site and deliver the therapeutic agent to the site, thereby treating the disease in the patient.

23. The method of claim 22, wherein the method is further defined as a method of administering a therapeutic agent to diseased cells in a patient.

24. The method of claim 22, wherein the exosomes obtained in step (a) are obtained from a bodily fluid sample obtained from the patient.

25. The method of claim 24, wherein the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate/tears, or serum.

26. The method of claim 22, wherein the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug.

27. The method of claim 26, wherein the antibody binds to an intracellular antigen.

28. The method of claim 26, wherein the antibody is a full length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody.

29. The method of claim 26, wherein the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA.

30. The method of claim 26, wherein the gene editing system is a CRISPR/Cas system.

31. The method of claim 26, wherein the therapeutic protein is a kinase, a phosphatase, or a transcription factor.

32. The method of claim 26, wherein the therapeutic protein corresponds to a wild-type form of the protein that is mutated or inactivated in cells at the site of the disease or disorder.

33. The method of claim 26, wherein the therapeutic protein corresponds to a dominant negative form of a protein that is overactive in cells at the site of the disease or condition.

34. The method of claim 26, wherein the small molecule drug is an imaging agent.

35. The method of claim 22, wherein the disease or disorder is cancer, injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a renal disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a genitourinary disorder, or a bone disease or disorder.

36. The method of claim 35, 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.

37. The method of claim 35, wherein the site of the disease or disorder is a tumor.

38. The method of claim 35, wherein the cancer is metastatic.

39. The method of claim 38, wherein the site of the disease or disorder is a metastatic nodule.

40. The method of claim 22, wherein the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor.

41. The method of claim 22, wherein the disease or disorder is cancer, wherein the therapeutic agent is an inhibitory RNA that targets an oncogene.

42. The method of claim 22, wherein the exosome comprises CD47 on its surface.

43. The method of claim 22, wherein transfecting comprises electroporation.

44. The method of claim 22, further comprising administering at least a second treatment to the patient.

45. The method of claim 44, wherein the second treatment comprises surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

46. The method of claim 22, wherein the exosome is comprised in a tissue scaffold matrix.

Images