Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

• the present invention relates generally to the fields of biology, medicine, and oncology. More particularly, it concerns the use of exosomes to target delivery of therapeutic agents to diseased or disordered cells.
• Exosomes are small extracellular vesicles (EVs) with a lipid bilayer that contain proteins and polynucleotides, including messenger RNAs (mRNAs), non-coding RNAs and double-stranded genomic DNA (Kalluri, 2016; Raposo and Stoorvogel, 2013).
• mRNAs messenger RNAs
• mRNAs messenger RNAs
• non-coding RNAs double-stranded genomic DNA
• Exosomes are part of a larger group of extracellular vesicles, which also include microvesicles and apoptotic bodies (Colombo et al., 2014). Amongst extracellular vesicles, exosomes are typically distinguished through their unique biogenesis via the endocytic pathway. Endocytic vesicles mature into late endosomes, also known as multivesicular bodies, which contain a number of intracellular vesicles (ILVs) generated through invagination of the endosomal membrane.
• ISVs intracellular vesicles
• exosomes are released into the extracellular space and enter circulation (Bastos et al., 2017; Colombo et al., 2014).
• exosomes membranes have a similar polarity to cellular membranes, containing membrane proteins anchored with their intracellular domains facing the lumen and the extracellular domains facing the extracellular space.
• protein content of exosomes varies depending on their cellular origin, several proteins seem to be generally enriched. These include members of the tetraspanin family and components of the endocytic and ILV maturation pathways, such as Rab proteins and members of the ESCRT complex.
• EGFR epithelial growth factor receptor
• EGFR epithelial growth factor receptor
• HER2, HERS and HER4 HER2
• HER4 epitheial growth factor
• the receptor dimerizes, forming either homodimers or heterodimers with other members of the ErbB family (Seshacharyulu et al., 2012).
• This dimerization activates the receptor’s intrinsic kinase activity, leading to the autophosphorylation of different key tyrosine residues on its cytoplasmic domain.
• This authophosphorylation reaction recruits different adaptor proteins containing SH2 and PTB (phosphotyrosine binding) domains, such as She and GRB2, which mediate different downstream signaling activities, including the synthesis of relevant proteins (Normanno et al., 2006; Tomas et al., 2014).
• Phosphorylated EGFR is ultimately ubiquitinated and transported to the endosomal pathway, from which it will either recycle back to the membrane or remain in the late endosomal pathway leading to integration into multivesicular bodies or lysosomal degradation (Tomas et al., 2014). Since multivesicular bodies originate exosomes, it is likely that the post-phosphorylation recycling of EGFR (and other growth factors) contribute to their membrane localization in these extracellular vesicles.
• EGFR signaling has been shown to be important for the progression of different malignancies, such as glioblastoma, lung cancer, and breast cancer (Lim et al., 2016; Liu et al., 2012; Masuda et al., 2012; Morgillo et al., 2016; Westphal et al., 2017; Zhang et al., 2013). Perhaps for this reason, most studies of EGFR in exosomes have been performed in the context of cancer development. EGFR signaling has particularly been implicated in the patterns of cellular uptake and secretion of exosomes from different origins.
• gefitinib an EGFR inhibitor
• incubation with gefitinib has been shown to dramatically decrease the rate of exosomes uptake (Hazan-Halevy et al., 2015).
• Treatment of lung cancer cells with gefitinib leads to an increased secretion of exosomes, which mediate horizontal transfer of cisplatin resistance (Li et al., 2016).
• the transfer of EGFR via cancer cell-derived exosomes has also been known to cause alterations in components of the microenvironment, such as endothelial cells and T cells (Al-Nedawi et al., 2009; Huang et al., 2013).
• exosomes derived from gastric cancer cells containing EGFR were shown to be delivered to stromal cells in the liver, mediating metastasis (Zhang et al., 2017).
• exosomes derived from breast cancer cells were shown to contain functional phosphorylated forms of EGFR, which can be transferred to monocytes mediating their survival through activation of the ERK pathway (Song et al., 2016).
• exosomes that contain DNA, RNA, and proteins, can respond to biological stimuli, and initiate properties such as migration, multiplication, initiation of signaling network/cascade, transcription, and protein translation.
• exosomes with the ability to function like minicells. As discussed further below, these minicell-like exosomes can be employed in numerous therapeutic means to treat various disease and/or disorders.
• kits for treating a disease or disorder in a patient in need thereof comprising (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (c) administering the transfected exosomes to a patient; (d) providing a growth factor gradient at a site of the disease or disorder to attract the exosomes to the site and stimulate production of the therapeutic protein at the site, thereby treating the disease in the patient.
• the method is further defined as a method of administering a therapeutic protein to a diseased cell in a patient.
• the exosomes obtained in step (a) are obtained from a body fluid sample obtained from the patient.
• the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum.
• the nucleic acid is an mRNA, a plasmid, or a cDNA.
• the disease or disorder is cancer, an injuiy, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a urogenital disorder, or a bone disease or disorder.
• the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
• the site of the disease or disorder is a tumor.
• the cancer is metastatic.
• the site of the disease or disorder is a metastatic node.
• the therapeutic protein is a kinase, a phosphatase, or a transcription factor.
• the therapeutic protein corresponds to a wildtype version of a protein that is mutated or inactivated in a cell at the site of the disease or disorder.
• the therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in a cell at the site of the disease or disorder.
• the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor.
• the exosomes comprise CD47 on their surface.
• transfection comprises electroporation.
• the method further comprises administering at least a second therapy to the patient.
• the second therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.
• methods are provided of treating a disease or disorder in a patient in need thereof, the method comprising (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with 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 the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the disease in the patient.
• the method is further defined as a method of administering a therapeutic agent to a diseased cell in a patient.
• the exosomes obtained in step (a) are obtained from a body fluid sample obtained from the patient.
• the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum.
• the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug.
• the antibody binds an intracellular antigen.
• the antibody is a full-length antibody, an scFv, a Fab fragment, a (Fab)2, a diabody, a triabody, or a minibody.
• the inhibitory RNA is a siRNA, shRNA, miRNA, or pre-miRNA.
• the gene editing system is a CRISPR/Cas system.
• the therapeutic protein is a kinase, a phosphatase, or a transcription factor.
• the therapeutic protein corresponds to a wildtype version of a protein that is mutated or inactivated in a cell at the site of the disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in a cell at the site of the disease or disorder.
• the small molecule drug is an imaging agent.
• the disease or disorder is cancer, an injuiy, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a urogenital disorder, or a bone disease or disorder.
• the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
• the site of the disease or disorder is a tumor.
• the cancer is metastatic.
• the site of the disease or disorder is a metastatic node.
• the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor.
• the disease or disorder is cancer, wherein the therapeutic agent is an inhibitory RNA targeting an oncogene.
• the exosomes comprise CD47 on their surface.
• transfection comprises electroporation.
• the method further comprises administering at least a second therapy to the patient.
• the second therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.
• exosomes for use according to the embodiments are comprised in a tissue scaffold matrix.
• a matrix may be a synthetic matrix, such a matrix that degradable or can be absorbed in tissues.
• the matrix may be a living tissue matrix.
• a exosomes of the embodiments are cultured in a matrix.
• essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
• “a” or“an” may mean one or more.
• the words“a” or“an” when used in conjunction with the word“comprising,” the words“a” or“an” may mean one or more than one.
• FIGS. 1A-E EGFR phosphorylation in exosomes.
• FIG. 1A Immunoblot of EGFR expression on exosomes obtained from different human and murine cell lines.
• the exosomes marker CD81 is used as a loading control, and to confirm the exosomal origin of protein extracts.
• FIG. IB Immunoblot showing phosphorylation of EGFR on exosomes derived from MDA-MB-231 cells, but not MCF10A cells, after incubation with 500 ng/ml rhEGF for 15 minutes at 37°C. Phosphorylation levels are detected using an antibody specific for the Tyrl068 residue of EGFR.
• FIG. 1C Immunoblot showing the presence of EGFR adaptor proteins She and GRB2, as well increased levels of phosphorylated-ERK protein, in exosomes derived from MDA-MB-231 cells, with and without rhEGF stimulation for 15 minutes at 37°C.
• the exosomes marker CD81 is used as a loading control, and to confirm the exosomal origin of protein extracts.
• ID - GRB immunecomplexes were obtained from protein extracts of MDA-MB-231 exosomes, with and without incubation with 500 ng/ml rhEGF for 15 minutes at 37°C, using a GRB2 specific antibody. Immunoblot analysis of the immunocomplexes shows association of GRB2 with EGFR only upon rhEGF stimulation. Non-specific isotype control IgG was used as a negative control for the GRB2 pulldown. Equal volumes of the stimulated and unstimulated extracts were probed for b-actin as input control. FIG.
• FIGS. 2A-G EGFR phosphorylation alters the content of exosomes.
• FIG. 2B Immunoblot analysis of exosomes from MDA-MB-231 cells incubated with 500 ng/ml rhEGF at 37°C for 48 h, showing an increase in both pEGFR and GRB2 levels when compared to unstimulated exosomes.
• FIG. 2C Cellular component association analysis of mass spectrometry data obtained for MDA-MB-231 exosomes unstimulated or after incubation with 500 ng/ml EGF at 37°C for 48 h. A list of significant proteins identified for stimulated and unstimulated exosomes was obtained and used as input for the open access FunRich functional enrichment analysis tool in order to identify the subcellular origin of the identified proteins.
• FIG. 2D Venn diagram depicting the overlap in proteins identified in control MDA-MB-231 exosomes and exosomes incubated with 500 ng/ml rhEGF at 37°C for 48 h.
• FIG. 2E Individual EGFR and GRB2 protein scores obtained from mass spectrometry analysis of MDA-MB-231 exosomes with or without incubation with 500 ng/ml rhEGF for 48 h.
• FIGS. 3A-F Exosomes contain functional components for transcription and translation.
• FIG. 3A Ultra-performance liquid chromatography-mass spectrometry (UPLCMS) was used to detect free amino acids in MCF10A-, MDA-MB-231-, HDF, E10-, and NIH-3T3 -derived exosomes. Data are represented as a HeatMap using normalized signal intensities (loglO), with hotter colors corresponding to higher intensity levels, as represented in the color legend.
• FIG. 3A Ultra-performance liquid chromatography-mass spectrometry
• FIG. 3B Immunoblot of eIF4Al, eIF3A, and elFlA 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 the pEMT7-GFP cDNA expression plasmid. Protein lysates obtained from cells were used as controls.
• FIG. 3D Immunoblot analysis of RNA Polymerase II in exosomes protein extracts, with CD9 shown as a loading control.
• FIG. 3E Autoradiography of exosomes derived from MDA-MB-231 and E10 cells cultured in the presence of 35 S-methionine. Exosomes only, as well as exosomes cultured in the presence of 35 S-methionine and cycloheximide, were used as controls.
• FIGS. 4A-J Exosomes synthesize new proteins through DNA transcription and cap-dependent mRNA translation.
• FIG. 4A qPCR analysis of GFP mRNA levels in exosomes isolated from MDA-MB-231 cells and either non-electroporated, mock electroporated, or electroporated with a pCMV-GFP plasmid with or without the presence of a-amanitin. Expression levels were normalized to GAPDH.
• FIG. 4B Transmission electron microscopy images of immunogold labeling, using anti-GFP antibody, of exosomes electroporated with GFP plasmid and incubated in cell-free conditions for 48 h (bottom row).
• FIG. 4C Immunoblot of GFP protein expression in exosomes electroporated with a pCMV-GFP plasmid and incubated for 12 hours, 2 days, or 1 week at 37°C. Exosomes only and mock-electroporated exosomes were used as negative controls. The exosomes marker TSG101 was used as a loading control for the presence of exosomes.
• FIG. 4D Immunoblot of GFP protein expression in exosomes electroporated with GFP plasmid and incubated for several periods of time up to one month.
• Non- electroporated exosomes were used as negative controls.
• the exosomes marker CD63 was used as a loading control for the presence of exosomes.
• FIG. 4E Immunoblot of GFP protein expression in exosomes electroporated with a GFP plasmid immediately after isolation (0 h) or after incubation in cell-free conditions (24 h) and cultured as previously described. Mock electroporated exosomes were used as negative controls.
• the exosomes maricer TSG101 was used as a loading control for the presence of exosomes.
• FIG. 4F Immunoblot of GFP protein expression in exosomes electroporated with a pCMV-GFP plasmid and cultured with the translation inhibitor cycloheximide. Exosomes only and exosomes mock electroporated were used as negative controls. TSG101 was used as a loading control for the presence of exosomes. Band densitometry was performed using Image! software.
• FIG. 4G Immunoblot of GFP protein expression in exosomes electroporated with a GFP plasmid and cultured with the transcription inhibitor a-amanitin. Exosomes only and exosomes mock electroporated were used as negative controls. TSG101 was used as a loading control for the presence of exosomes.
• FIG. 4H Schematic representation of the bicistronic plasmid used as a reporter for cap-dependent or cap-independent translation (pCDNA3-rLuc- polIRESfLuc).
• FIG. 41 Activities of renilla (r-Luc) and firefly luciferase (f-Luc) measured by bioluminescence after 48 h incubation of exosomes electroporated with the bicistronic plasmid. Non-electroporated exosomes were used as negative controls.
• FIG. 4! - Luminescence counts measured from exosomes incubated for 48 h after electroporation with or without a plasmid with firefly luciferase expressed under a CMV promoter.
• FIGS. 5A-E Protein translation in exosomes generates functional proteins and can be increased by growth factor stimulation.
• FIG. 5A Confocal microscopy showing the presence of GFP in MCF10A electroporated cells as well as in MCF10A cells, previously treated with cycloheximide, incubated with MDA-MB-231 -derived exosomes electroporated with a pCMV-GFP plasmid and pre-incubated for 48 h. MCF10A cells treated with non- electroporated MDA-MB-231 -derived exosomes were used as a negative control.
• FIG. 5A Confocal microscopy showing the presence of GFP in MCF10A electroporated cells as well as in MCF10A cells, previously treated with cycloheximide, incubated with MDA-MB-231 -derived exosomes electroporated with a pCMV-GFP plasmid and pre-incubated for 48 h. MCF10
• FIG.5B Immunoblot analysis of GFP expression on protein lysates from MDA-MB-231 -derived exosomes electroporated with a p53-GFP expression plasmid. Non-electroporated exosomes were used as negative controls. TSG101 was used as a loading control for the presence of exosomes.
• FIG.5C p21 mRNA expression in MDA-MB-231 cells treated with mock electroporated MDA-MB-231 -derived exosomes or exosomes electroporated with the p53- GFP plasmid with and without the presence of cycloheximide. Expression levels were normalized to the housekeeping gene GAPDH.
• 5D Immunoblot of exosomes isolated from MDA-MB-231 cells, incubated with 100 pg/ml of the translation inhibitor cycloheximide. Exosomes lysates were probed for b-actin and GAPDH. Band densitometry quantification was performed using Image! software.
• FIG. 5E Immunoblot of GFP protein expression in exosomes electroporated with a pCMV-GFP plasmid, and then incubated in the presence of different concentrations of rhEGF at 37°C for 48 h. Exosomes mock electroporated and exosomes without rhEGF incubation are shown as negative controls. The exosomes marker CD81 is used as a loading control. Band densitometry quantification was performed using Image! software.
• FIG. 6A-C Exosomes derived from MDA-MB-231 cells demonstrate chemotaxis towards a gradient of growth factors.
• 10 c 10 9 exosomes isolated from MDA-MB-231 cells were placed in the bottom well of a Coming Transwell® system.
• An HTS Transwell® insert with 400 nm pores was placed on top of the exosomes suspension containing either PBS, 20% FBS, or 10,000 ng/ml rhEGF and incubated at 37°C. The number of exosomes on the top insert was measured by Nanosight NTA after different time points to assess exosomes motility.
• FIGS. 7A-D Tumor-bearing mice show increased protein synthesis in delivered exosomes.
• FIG. 7A - Schematic depicting the experimental plan for the in vivo translation experiment.
• female Balb/C mice were injected with 4T1 tumor orthotopically and the tumor was allowed to grow to 500 mm 3 , after which mice were injected with 30 billion MDA-MB-231 exosomes electroporated with a pCMV-mCherry plasmid. The mice were euthanized 12 h after exosomes injection and serum was collected for exosomes extraction.
• FIG. 7B Graphic showing the tumor growth of mice injected with 4T1 tumors and electroporated exosomes, or 4T1 tumors alone, showing comparable growth kinetics.
• FIG. 7C Nanosight NTA analysis of exosomes extracted from the serum of healthy mice injected with electroporated exosomes, as well as 4T1 tumor-bearing mice injected with electroporated exosomes and 4T1 tumor bearing mice with no exosomes injection. All exosomes show similar size peaks, around 100 nm.
• FIG. 7D Nanosight NTA quantification of serum exosomes shown in FIG.
• FIGS. 8A-G Exosomes characterization.
• FIG. 8A Nanoparticle tracking analysis of exosomes collected from MDA-MB-231 cells, obtained using the Nanosight NTA 2.1 Analytical Software. Left graph represents the size distribution of particles in solution showing a mean size of 104 nm and also showing no peaks at larger sizes. Right graph represents the distribution by size and concentration of particles in solution.
• FIG. 8B Atomic Force Microscopy image of exosomes (left image).
• FIG. 8C Transmission electron micrograph of MDA-MB-231 exosomes. Scale bar - 100 nm.
• FIG. 8D Transmission electron micrograph of immunogold labeled MDA-MB-231 exosomes using anti-CD9 antibody. Gold particles are depicted as black dots. Scale bar - 100 nm.
• FIG. 8E Immunoblot analysis of exosomes markers CD9, CD63, and TSG101 in protein extracts of exosomes obtained from different cell lines.
• FIG. 8F Imaging Flow Cytometry analysis of exosomes from MDA-MB-231 cells coupled to 0.4 pm beads, using antibodies for markers CD9, CD81, CD82, and CD63.
• FIGS. 9A-B EGFR phosphorylation and downstream biological activity in exosomes from MDA-MB-231 cells.
• FIG. 9 A Immunoblot of protein extracts obtained from MDA-MB-231 cells and probed for p-EGFR and GRB2. b-actin is used as a loading control.
• FIG. 9B Immunoblot of immunocomplexes obtained with an anti-EGFR antibody pull down of protein lysates from MDA-MB-231 exosomes with or without incubation with 500 ng/ml at 37°C for 15 minutes. Immuncomplexes were probed for GRB2.
• Non-specific isotype control IgG was used as a negative control for the GRB2 pulldown. Equal volumes of the stimulated and unstimulated extracts were probed for b-actin as input control.
• FIG. 10 Proteomics analysis of exosomes derived from various cells.
• Heatmap representing the binary identification of all individual proteins included in the Protein Translation pathway in the Reactome (Croft et al., 2014) database, in mass spectrometry data obtained from mouse liver cells (Valadi et al., 2007), mouse fibroblasts (Luga et al., 2012), human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et al., 2013), human thymic tissue (Skogberg et al., 2013), and human urine (Gonzales et al., 2009). Black represents the presence and white represents the absence of each protein in each dataset.
• the summary column represents how ubiquitous each protein is in all analyzed datasets, with warmer colors representing a more widespread distribution among different types of exosomes.
• FIGS. 11A-B Proteomics analysis of exosomes from various origins.
• FIG. 11 A Heatmap representing the number of proteins identified in mass spectrometry data obtained from mouse liver cells (Valadi et al., 2007), mouse fibroblasts (Luga et al., 2012), human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et al., 2013), human thymic tissue (Skogberg et al., 2013), and human urine (Gonzales et al., 2009) that associate with different pathways related to protein translation in the Reactome (Croft et al., 2014) database.
• FIGS. 12A-C Exosomes contain 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 were used to quantify
• FIG. 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.
• FIGS. 13A-E DNA transcription and mRNA translation in exosomes derived from MCF10A and MDA-MB-231 cells.
• FIG. 13A Immunoblot of GFP protein expression in exosomes isolated from MCF10A cells, electroporated with a pCMV-GFP plasmid and incubated at 37°C for different periods of time. Exosomes only and mock electroporated exosomes were used as negative controls. CD63 was used as a loading control and to confirm the presence of exosomes.
• FIG. 13B Plot depicting the amount of green exosomes detected by NanoSight in MCFlOA-derived exosomes electroporated with a GFP plasmid.
• FIG. 13C Flow cytometry analysis of beads attached to exosomes after electroporation with a GFP plasmid using increasing voltages, showing the percentages of beads with green fluorescent signal.
• FIG. 13D Immunoblot of ovalbumin protein levels in MDA-MB-231 exosomes electroporated with a pCMV-Ova plasmid and incubated at 37°C for 48 h. b-actin was used as a loading control.
• Extracellular vesicles including exosomes, are nano-sized intercellular communication vehicles having a lipid bilayer that encloses cytosol-like material. Exosomes participate in several physiological processes and contain DNA, RNA, and proteins. It is generally assumed that all contents in exosomes are derived from cells and they remain as such in the exosomes until they enter other cells and deposit their contents. Exosomes are released by all cells in large numbers and are considered as garbage bags that carry cellular constituents into the extracellular space as a payload without any biological significance for exosomes themselves per se.
• exosomes that behave like minicells and exhibit the ability to biologically respond to stimuli and multiply and migrate just as cells do but without a defined nucleus. These exosomes exhibit chemotaxis towards serum factors and upon stimulation with growth factors such as EGF, will phosphorylate the EGFR receptor on their surface and initiate a signaling cascade that leads to transcription and translation of new proteins. When these exosomes are injected into tumor bearing mice, they preferentially accumulate in the tumors. Collectively, the capacity for protein translation and growth factor response by these exosomes provides them a functional role in tissue homeostasis and modulation of disease. L Aspects of the Present Invention
• exosomes are not just used for passive transfer to recipient cells but can respond to external stimuli to phosphorylate growth factor receptors, such as EGFR, and generate new proteins via active transcription and translation. External stimulation of exosomes can initiate de novo biological activities such as retrograde migration. It is conceivable that exosomes may function like mini cells, albeit primitive with respect to their fine-tuned operations in response to external stimuli. In fact, recently it has been suggested that exosomes could potentially constitute extant representations of protocellular ribosomes, for which they would need to contain rRNAs, which is confirm in this study (Sinkovics, 2015). Exosomes are biologically responsive and migrate towards growth factor gradients in an active manner. Actin remodeling might be involved. The patterns of actin polymerization could therefore constitute an interesting target in the modulation of exosomes biodistribution.
• Vesicles such as prostasomes obtained from different species have been shown to contain different components of the glycolytic pathway, which allow them to produce ATP in cell-free conditions (Ronquist et al., 2013a; Ronquist et al., 2013b). While direct transcription in exosomes has not been previously reported, a 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 was also recently demonstrated that independent production of mature miRNAs in exosomes isolated from cancer cells is possible (Melo et al., 2014).
• exosomes were demonstrated to possess an intrinsic capacity for de novo synthesis of functional proteins via DNA transcription coupled with mRNA translation.
• Platelets can translate proteins from mRNA molecules remaining within them after megakaryocyte differentiation (Weyrich et al., 2004). Nevertheless, DNA transcription resulting in new mRNA molecules is not reported in platelets. Additionally, foci of mRNA translation activity, associated with polyribosomes and mRNA binding proteins, is observed in dendritic spines even when severed from the major body of the cell (Aakalu et al., 2001; Smith et al., 2001; Steward and Levy, 1982).
• exosomes are capable of DNA transcription via RNA polymerase P. It is well established that basic transcription of naked DNA in nucleosome-free regions is possible with minimal components of transcriptional machinery, namely only RNA Pol II and a cocktail of six general transcription factors (GTFs) (Lorch et al., 2014; Nagai et al., 2017). It has also been shown that exosomes contain a plethora of transcription factors that can be delivered to cells in order to alter their patterns of protein expression (Ung et al., 2014). It is therefore conceivable that exosomes contain naked DNA residues unbound by chromatin, which could undergo transcription in the presence of these minimal transcriptional components.
• GTFs general transcription factors
• the ribosome itself has an important role in co-translational protein folding, for instance, it can promote the formation of secondary structures in newly formed proteins.
• the ribosome also acts as a platform for the association of chaperones that can assist with the appropriate folding of nascent proteins (Kramer et al., 2009). It is conceivable that these exosomes components could contribute to the stabilization of newly formed proteins. Physical confinement, as would be the case in the lumen of exosomes, can also have a stabilizing effect on the folding of proteins (Rao and Cruz, 2013). The possibility, however, cannot be ruled out that many proteins might exhibit inappropriate conformation or mis-folding. These could still have important biological implications, as demonstrated with the recent unraveling of unexpected features of the“dark proteome” (Perdigao et al., 2015), which suggested that proteins with unknown structure or intrinsically disordered regions may have important physiological functions.
• a lipid-based nanoparticle is a liposomes, an exosomes, lipid preparations, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., a DOT AP : cholesterol vesicle).
• a lipid-based vesicle e.g., a DOT AP : cholesterol vesicle.
• Lipid-based nanoparticles may be positively charged, negatively charged or neutral.
• Lipid-based nanoparticles may comprise the necessary components to allow for transcription and translation, signal transduction, chemotaxis, or other cellular functions.
• A“liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge.
• a multilamellar liposome has multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution The lipid components undergo self- rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.
• a polypeptide, a nucleic acid, or a small molecule drug may be, for example, encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide/nucleic acid, entrapped in a liposome, complexed with a liposome, or the like.
• a liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art.
• a phospholipid such as for example the neutral phospholipid dioleoylphosphatidylcholine (DOPC)
• DOPC neutral phospholipid dioleoylphosphatidylcholine
• the lipid(s) is then mixed with a polypeptide, nucleic acid, and/or other component(s).
• Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition's weight.
• Excess tert-butanol is added to this mixture such that the volume of tert-butanol is at least 95%.
• the mixture is vortexed, frozen in a dry ice/acetone bath and lyophilized overnight.
• the lyophilized preparation is stored at -20°C and can be used up to three months. When required the lyophilized liposomes are reconstituted in 0.9% saline.
• a liposome can be prepared by mixing lipids in a solvent in a container, e.g., a glass, pear-shaped flask.
• a container e.g., a glass, pear-shaped flask.
• the container should have a volume ten-times greater than the volume of the expected suspension of liposomes.
• the solvent is removed at approximately 40°C under negative pressure.
• the solvent normally is removed within about 5 min to 2 h, depending on the desired volume of the liposomes.
• the composition can be dried further in a desiccator under vacuum. The dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.
• Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended.
• the aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum.
• the dried lipids or lyophilized liposomes prepared as described above may be dehydrated and reconstituted in a solution of a protein or peptide and diluted to an appropriate concentration with a suitable solvent, e.g., DPBS.
• a suitable solvent e.g., DPBS.
• the washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g, about 50-200 mM.
• the amount of additional material or active agent encapsulated can be determined in accordance with standard methods. After determination of the amount of additional material or active agent encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4°C until use.
• a pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.
• Additional liposomes which may be useful with the present embodiments include cationic liposomes, for example, as described in W002/100435A1, U.S Patent 5,962,016, U.S. Application 2004/0208921, W003/015757A1, WO04029213A2, U.S. Patent 5,030,453, and U.S. Patent 6,680,068, all of which are hereby incorporated by reference in their entirety without disclaimer. [0057] In preparing such liposomes, any protocol described herein, or as would be known to one of ordinary skill in the art may be used. Additional non-limiting examples of preparing liposomes are described in U.S.
• the lipid based nanoparticle is a neutral liposome (e.g., a DOPC liposome).
• neutral liposomes or“non-charged liposomes”, as used herein, are defined as liposomes having one or more lipid components that yield an essentially- neutral, net charge (substantially non-charged).
• neutral liposomes may include mostly lipids and/or phospholipids that are themselves neutral under physiological conditions (i.e. , at about pH 7).
• Liposomes and/or lipid-based nanoparticles of the present embodiments may comprise a phospholipid.
• a single kind of phospholipid may be used in the creation of liposomes (e.g., a neutral phospholipid, such as DOPC, may be used to generate neutral liposomes).
• a neutral phospholipid such as DOPC
• more than one kind of phospholipid may be used to create liposomes.
• Phospholipids may be from natural or synthetic sources.
• Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating neutral liposomes.
• the phospholipid DOPC is used to produce non-charged liposomes.
• a lipid that is not a phospholipid e.g., a cholesterol
• Phospholipids include glycerophospholipids and certain sphingolipids.
• Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine (“EPC”), dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine (“DSPC”), 1 -myristoyl-2-palmitoyl phosphatidylcholine
• MPPC 1 -palmitoyl-2-myristoyl phosphatidylcholine
• PMPC 1 -palmitoyl-2-stearoyl phosphatidylcholine
• SPPC l-stearoyl-2-palmitoyl phosphatidylcholine
• DLPG dimyristoylphosphatidylglycerol
• DPPG dipalmitoylphosphatidylglycerol
• DSPG distearoylphosphatidylglycerol
• DSSP distearoyl sphingomyelin
• DSPE dioleoylphosphatidylglycerol
• DOPG dimyristoyl phosphatidic acid
• DPP A dipalmitoyl phosphatidic acid
• microvesicle and “exosomes,” as used herein, refer to a membranous particle having a diameter (or largest dimension where the particles is not spheroid) of between about 10 nm to about 5000 nm, more typically between 30 nm and 1000 nm, and most typically between about 50 nm and 750 nm, wherein at least part of the membrane of the exosomes is directly obtained from a cell.
• exosomes will have a size (average diameter) that is up to 5% of the size of the donor cell. Therefore, especially contemplated exosomes include those that are shed from a cell.
• Exosomes may be detected in or isolated from any suitable sample type, such as, for example, body fluids.
• the term“isolated” refers to separation out of its natural environment and is meant to include at least partial purification and may include substantial purification.
• the term“sample” refers to any sample suitable for the methods provided by the present invention. The sample may be any sample that includes exosomes suitable for detection or isolation.
• Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, broncho-alveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid, and bronchial washes.
• the sample is a blood sample, including, for example, whole blood or any fraction or component thereof.
• a blood sample suitable for use with the present invention may be extracted from any source known that includes blood cells or components thereof, such as venous, arterial, peripheral, tissue, cord, and the like.
• a sample may be obtained and processed using well-known and routine clinical methods (e.g., procedures for drawing and processing whole blood).
• an exemplary sample may be peripheral blood drawn from a subject with cancer.
• Exosomes may also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from tissue sources it may be necessary to homogenize the tissue in order to obtain a single cell suspension followed by lysis of the cells to release the exosomes. When isolating exosomes from tissue samples it is important to select homogenization and lysis procedures that do not result in disruption of the exosomes. Exosomes contemplated herein are preferably isolated from body fluid in a physiologically acceptable solution, for example, buffered saline, growth medium, various aqueous medium, etc.
• a physiologically acceptable solution for example, buffered saline, growth medium, various aqueous medium, etc.
• Exosomes may be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. In some embodiments, exosomes may be isolated from cell culture medium. Although not necessary, higher purity exosomes may be obtained if fluid samples are clarified before precipitation with a volume-excluding polymer, to remove any debris from the sample. Methods of clarification include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by numerous methods well-known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatants.
• exosomes Exemplary methods for isolation of exosomes are described in (Losche et ah, 2004; Mesri and Altieri, 1998; Morel et ah, 2004). Alternatively, exosomes may also be isolated via flow cytometry as described in (Combes et ah, 1997).
• a significant problem is that both blood and cell culture media contain large numbers of nanoparticles (some non-vesicular) in the same size range as exosomes.
• some miRNAs may be contained within extracellular protein complexes rather than exosomes; however, treatment with protease (e.g., proteinase K) can be performed to eliminate any possible contamination with “extraexosomal” protein.
• protease e.g., proteinase K
• cancer cell-derived exosomes may be captured by techniques commonly used to enrich a sample for exosomes, such as those involving immunospecific interactions (e.g., immunomagnetic capture).
• Immunomagnetic capture also known as immunomagnetic cell separation, typically involves attaching antibodies directed to proteins found on a particular cell type to small paramagnetic beads. When the antibody- coated beads are mixed with a sample, such as blood, they attach to and surround the particular cell. The sample is then placed in a strong magnetic field, causing the beads to pellet to one side. After removing the blood, captured cells are retained with the beads.
• a sample such as blood
• the exosomes may be attached to magnetic beads (e.g., aldehyde/sulphate beads) and then an antibody is added to the mixture to recognize an epitope on the surface of the exosomes that are attached to the beads.
• Exemplary proteins that are known to be found on cancer cell-derived exosomes include ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta- 18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and glypican-1 (GPC1).
• Cancer cell-derived exosomes may be isolated using, for example, antibodies or aptamers to one or more of these proteins.
• analysis includes any method that allows direct or indirect visualization of exosomes and may be in vivo or ex vivo.
• analysis may include, but not limited to, ex vivo microscopic or cytometric detection and visualization of exosomes bound to a solid substrate, flow cytometry, fluorescent imaging, and the like.
• cancer cell-derived exosomes are detected using antibodies directed to one or more of ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta- 18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), glypican-1 (GPC1), Histone H2A type 2-A (HIST1H2AA), Histone H2A type 1-A (HIST1H1AA), Histone H3.3 (H3F3A), Histone H3.1 (HIST1H3A), Zinc finger protein 37 homolog (ZFP37), Laminin subunit beta-1 (LAMB1), Tubulointerstitial nephritis antigen-like (TINAGL1), Peroxiredeoxin-4 (PRDX4), Collagen alpha-2(IV) chain (COL4A2), Putative protein C3P1 (C3P1)
• [0074] Mix 1 x 10 8 exosomes (measured by NanoSight analysis) or 100 nm liposomes ⁇ e.g., purchased from Encapsula Nano Sciences) and 1 pg of siRNA (Qiagen) or shRNA in 400 pL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Electroporate the exosomes or liposomes using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012).
• A“therapeutic agent” as used herein is an atom, molecule, or compound that is useful in the treatment of cancer or other conditions.
• 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, photoactive agents, and dyes.
• A“diagnostic agent” as used herein is an atom, molecule, or compound that is useful in diagnosing, detecting or visualizing a disease.
• 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 enhancing agents ⁇ e.g., paramagnetic ions).
• radioactive substances e.g., radioisotopes, radionuclides, radiolabels or radiotracers
• dyes e.g., dyes, contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes and enhancing agents ⁇ e.g., paramagnetic ions).
• a therapeutic recombinant protein may be a protein having an activity that has been lost in a cell of the patient, a protein having a desired enzymatic activity, a protein having a desired inhibitory activity, etc.
• the protein may be a transcription factor, an enzyme, a proteinaceous toxin, an antibody, a monoclonal antibody, etc.
• the monoclonal antibody may specifically or selectively bind to an intracellular antigen.
• the monoclonal antibody may inhibit the function of the intracellular antigen and/or disrupt a protein-protein interaction.
• Other aspects of the present invention provide for diagnosing a disease based on the presence of cancer cell-derived exosomes in a patient sample.
• exosomes are known to comprise 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 a therapeutic protein may be transfected into exosomes. Alternatively, the therapeutic protein itself may be electroporated into the exosomes or incorporated directly into a liposome.
• Exemplary therapeutic proteins include, but are not limited to, a tumor suppressor protein, peptides, a wild type protein counterparts of a mutant protein, a DNA repair protein, a proteolytic enzyme, proteinaceous toxin, a protein that can inhibit the activity of an intracellular protein, a protein that can activate the activity of an intracellular protein, or any protein whose loss of function needs to be reconstituted.
• exemplary therapeutic proteins include 123F2, Abcb4, Abccl, Abcg2, Actb, Ada, Ahr, Akt, Aktl, Akt2, Akt3, Amhr2, Anxa7, Ape, Ar, Atm, Axin2, B2m, Bardl, Bcl211, Becnl, BhlhalS, Binl, Blm, Braf, Brcal, Brca2, Brca3, Braf, Brcata, Brinp3, Bripl, Bublb, Bwscrla, Cadm3, Cascl, Casp3, Casp7, Casp8, Cavl, Ccam, Ccndl, Ccr4, Ccsl, Cd28, Cdc25a, Cd95, Cdhl, Cdknla, Cdknlb, Cdkn2a, Cdkn2b, Cdkn2c, Cftr, Chekl, Chek2, Crcsl, CrcslO, Cr, Cr
• One specific type of protein that it may be desirable to introduce into the intracellular space of a diseased cell is an antibody (e.g, a monoclonal antibody).
• an antibody e.g, a monoclonal antibody
• Such an antibody may disrupt the function of an intracellular protein and/or disrupt an intracellular protein-protein interaction.
• exemplary targets of such monoclonal antibodies include, but are not limited to, proteins involved in the RNAi pathway, telomerase, transcription factors that control disease processes, kinases, phosphatases, proteins required for DNA synthesis, protein required for protein translation.
• exemplary therapeutic antibody targets include proteins encoded by the following genes: Dicer, Agol, Ago2, Trbp, Ras, raf, wnt, btk, Bcl-2, Akt, Sis, src, Notch, Stathmin, mdm2, abl, hTERT, c-fos, c-jun, c-myc, erbB, HER2/Neu, HER3,VEGFR, PDGFR, c-kit, c-met, c-ret, flt3, API, AML1, axl, alk, fins, fps, gip, lck, Stat, Hox, MLM, PRAD-I, and trk.
• any antigen binding fragment there of such as a scFv, a Fab fragment, a Fab’, a F(ab’)2, a Fv, a peptibody, a diabody, a triabody, or a minibody, is also contemplated. Any such antibodies or antibody fragment may be either glycosylated or aglycosylated.
• exosomes are known to comprise DICER and active RNA processing RISC complex (see PCT Publn. WO 2014/152622, which is incorporated herein by reference in its entirety)
• shRNA transfected into exosomes can mature into RISC-complex bound siRNA with the exosomes themselves.
• mature siRNA can itself be transfected into exosomes or liposomes.
• target genes that may be used in the methods of the present invention to modulate or attenuate target gene expression: wild-lype or mutant versions of developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth or differentiation factors and their receptors, neurotransmitters and their receptors), tumor suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2b, pl6, pl9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CF
• developmental genes e.g., adhe
• pro-apoptotic genes e.g, CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PARP, bad, bcl-2, MST1, bbc3, Sax, BEK, and BID
• cytokines e.g ., GM-CSF, G-CSF, IL-lo, IL-Ib, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, EL-10, EL-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
• sh/siRNA may be designed to specifically target a mutant version of a gene expressed in a cancer cell while not affecting the expression of the corresponding wild-type version.
• RNAi there are several factors that need to be considered, such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system.
• the siRNA that is introduced into the organism will typically contain exonic sequences.
• the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences.
• the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective.
• Exosomes may also be engineered to comprise a gene editing system, such as a CRISPR/Cas system.
• CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
• tracrRNA or an active partial tracrRNA a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
• a Cas nuclease and gRNA are introduced into the cell.
• target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing.
• the target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG.
• PAM protospacer adjacent motif
• the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence.
• a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
• target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex.
• the CRISPR system in exosomes engineered to comprise such a system may function to edit the genomic DNA inside a target cell, or the system may edit the DNA inside the exosomes itself.
• exosomes may be used to deliver small molecule drugs, either alone or in combination with any protein- or nucleic acid-based therapeutic.
• exemplary small molecule drugs that are contemplated for use in the present embodiments include, but are not limited to, toxins, chemotherapeutic agents, agents that inhibit the activity of an intracellular protein, agents that activate the activity of intracellular proteins, agents for the prevention of restenosis, agents for treating renal disease, agents used for intermittent claudication, agents used in the treatment of hypotension and shock, angiotensin converting enzyme inhibitors, antianginal agents, anti- arrhythmics, anti-hypertensive agents, antiotensin ii receptor antagonists, antiplatelet drugs, b-blockers bl selective, beta blocking agents, botanical product for cardiovascular indication, calcium channel blockers, cardiovascular/diagnostics, central alpha-2 agonists, coronary vasodilators, diuretics and renal tubule inhibitors, neutral endopeptid
• Exosomes may also be used to deliver diagnostic agents.
• diagnostic agents include, but are not limited to, magnetic resonance image enhancement agents, positron emission tomography products, radioactive diagnostic agents, radioactive therapeutic agents, radio-opaque contrast agents, radiopharmaceuticals, ultrasound imaging agents, and angiographic diagnostic agents.
• subject refers to any individual or patient to which the subject methods are performed.
• the subject is human, although as will be appreciated by those in the art, the subject may be an animal.
• mammals including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.
• rodents including mice, rats, hamsters, and guinea pigs
• farm animals including cows, horses, goats, sheep, pigs, etc.
• primates including monkeys, chimpanzees, orangutans, and gorillas
• “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
• a treatment may include administration of chemotherapy, immunotherapy, or radiotherapy, performance of surgery, or any combination thereof.
• therapeutic benefit refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
• treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
• cancer may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer.
• the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
• the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
• the terms“contacted” and“exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
• a therapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
• one or more agents are delivered to a cell in an amount effective to kill the cell or prevent it from dividing.
• An effective response of a patient or a patient's“responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder.
• Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse.
• an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.
• neoplastic condition treatment involves one or a combination of the following therapies: surgery to remove the neoplastic tissue, radiation therapy, and chemotherapy.
• Other therapeutic regimens may be combined with the administration of the anticancer agents, e.g., therapeutic compositions and chemotherapeutic agents.
• the patient to be treated with such anti-cancer agents may also receive radiation therapy and/or may undergo surgeiy.
• the appropriate dosage of a therapeutic composition will depend on the type of disease to be treated, as defined above, the severity and course of the disease, the patient's clinical history and response to the agent, and the discretion of the attending physician.
• the agent is suitably administered to the patient at one time or over a series of treatments.
• Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect.
• a tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents, or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations.
• a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
• Administration in combination can include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the subject therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, subject therapeutic composition and another therapeutic agent can be simultaneously administered, wherein both the agents are present in separate formulations. In another alternative, the therapeutic agent can be administered just followed by the other therapeutic agent or vice versa. In the separate administration protocol, the subject therapeutic composition and another therapeutic agent may be administered a few minutes apart, or a few hours apart, or a few days apart.
• a first anti-cancer treatment e.g., exosomes that express a recombinant protein or with a recombinant protein isolated from exosomes
• the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
• the first treatment is provided to a patient separately from the second treatment, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
• a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered.
• This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
• a first anticancer therapy is“A” and a second anti-cancer therapy is“B”:
• chemotherapeutic agents may be used in accordance with the present invention.
• the term“chemotherapy” refers to the use of drugs to treat cancer.
• A“chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
• chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particulariy cryptophycin 1 and ciy
• DNA damaging factors include what are commonly known as g-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
• Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
• Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
• Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
• immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
• Rituximab (Rituxan®) is such an 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 serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
• the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
• the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
• Various effector cells include cytotoxic T cells and NK cells.
• the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
• Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, FLAP, laminin receptor, erb B, and pl55.
• An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
• Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
• cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
• chemokines such as MIP-1, MCP-1, IL-8
• growth factors such as FLT3 ligand.
• immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapy, e.g., interferons a, b, and g, IL-1, GM-CSF, and TNF (Bukowski et al, 1998; Davidson et al., 1998; Hellstrand et al, 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S.
• immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds
• Patents 5,830,880 and 5,846,945) ; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
• the immunotherapy may be an immune checkpoint inhibitor.
• Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal.
• Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3 -dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 ( ⁇ M-3) and V-domain Ig suppressor of T cell activation (VISTA).
• the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
• the immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference).
• Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.
• alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
• the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
• the PD-1 ligand binding partners are PDL1 and/or PDL2.
• a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners.
• PDL1 binding partners are PD-1 and/or B7-1.
• the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners.
• a PDL2 binding partner is PD-1.
• the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.
• Other PD-1 axis antagonists for use in the methods provided herein are known in the art such as described in U.S. Patent Publication Nos. 20140294898, 2014022021, and 20110008369, all incorporated herein by reference.
• the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
• the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011.
• the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
• the PD-1 binding antagonist is AMP- 224.
• Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO ® , is an anti- PD-1 antibody described in W02006/121168.
• Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA ® , and SCH-900475, is an anti-PD-1 antibody described in W02009/114335.
• CT-011 also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611.
• AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
• CTLA-4 cytotoxic T-lymphocyte-associated protein 4
• CD 152 cytotoxic T-lymphocyte-associated protein 4
• the complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006.
• CTLA-4 is found 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 an inhibitory signal to T cells.
• CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells.
• CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
• Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
• 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 oligopeptide.
• 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 oligopeptide.
• Anti -human-CTL A-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
• art recognized anti-CTLA-4 antibodies can be used.
• Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
• a humanized CTLA-4 antibody is described in International Patent Application No. W02001014424, W02000037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
• An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424).
• the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab.
• 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.
• the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above- mentioned antibodies.
• the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
• CTLA-4 ligands and receptors such as described in U.S. Patent Nos. 5844905, 5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins such as described in U.S. Patent No. 8329867, incorporated herein by reference.
• the immune therapy could be adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo.
• the T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma. Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010).
• CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule.
• the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully.
• the signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).
• the present application provides for a combination therapy for the treatment of cancer wherein the combination therapy comprises adoptive T- cell therapy and a checkpoint inhibitor.
• the adoptive T-cell therapy comprises autologous and/or allogenic T cells.
• the autologous and/or allogenic T cells are targeted against tumor antigens.
• Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
• Tumor resection refers to physical removal of at least part of a tumor.
• treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
• a cavity may be formed in the body.
• Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. 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 be of varying dosages as well.
• agents may be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of treatment.
• additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
• cytostatic or differentiation agents can be used in combination with certain aspects of the present invention to improve the anti- hyperproliferative efficacy of the treatments.
• Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
• Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present invention to improve the treatment efficacy.
• exosomes that express or comprise a therapeutic protein, inhibitory RNA, and/or small molecule drug can be administered systemically or locally to inhibit tumor cell growth and, most preferably, to kill cancer cells in cancer patients with locally advanced or metastatic cancers. They can be administered intravenously, intrathecally, and/or intraperitoneally. They can be administered alone or in combination with anti-proliferative drugs. In one embodiment, they are administered to reduce the cancer load in the patient prior to surgery or other procedures. Alternatively, they can be administered after surgery to ensure that any remaining cancer (e.g., cancer that the surgery failed to eliminate) does not survive.
• any remaining cancer e.g., cancer that the surgery failed to eliminate
• compositions can be provided in formulations together with physiologically tolerable liquid, gel, solid carriers, diluents, or excipients.
• physiologically tolerable liquid, gel, solid carriers, diluents, or excipients can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents in general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particular requirements of individual subjects.
• compositions comprising recombinant proteins and/or exosomes in a form appropriate for the intended application.
• pharmaceutical compositions 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.
• pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
• compositions comprising a recombinant protein and/or exosomes as disclosed herein, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington’s Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.
• the composition suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent.
• pharmaceutically acceptable carrier includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, ethanol, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., fats, oils, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), vegetable oil, and injectable organic esters, such as ethyloleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, inert gases, parabens
• aqueous solvents e.g., water, alcoholic/aqueous
• the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers.
• the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents.
• auxiliary substances such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents.
• the pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
• a pharmaceutically acceptable carrier is particularly formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but that would not be acceptable (e.g., due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient (e.g., detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein), its use in the therapeutic or pharmaceutical compositions is contemplated.
• the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, and the like. Such procedures are routine for those skilled in the art.
• compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection.
• the compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).
• the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
• parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
• such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the therapeutics may be formulated into a composition in a free base, neutral, or salt form.
• Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine and the like.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as formulated for parenteral administrations, such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations, such as drug release capsules and the like.
• the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
• the mixing can be carried out in any convenient manner, such as grinding.
• Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
• stabilizers for use in a composition include buffers, amino acids, such as glycine and lysine, carbohydrates, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
• the present invention may concern the use of a pharmaceutical lipid vehicle composition comprising one or more lipids and an aqueous solvent.
• lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic ⁇ i.e., designed or produced by man). However, a lipid is usually a biological substance.
• Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof.
• neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof.
• lipids are also encompassed by the compositions and methods.
• the therapeutic agent may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
• the dispersion may or may not result in the formation of liposomes.
• unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
• the quantity to be administered depends on the effect desired.
• the actual dosage amount of a composition of the present invention administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance.
• a dose may also comprise from about 1 pg/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein.
• a derivable range from the numbers listed herein, a range of about 5 pg/kg/body weight to about 100 mg/kg/body weight, about 5 pg/kg/body weight to about 500 mg/kg/body weight, etc., can be administered.
• the practitioner responsible for administration will, in any event, determine the concentration of active ingredients) in a composition and appropriate dose(s) for the individual subj ect.
• the actual dosage amount of a composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredients) in a composition and appropriate dose(s) for the individual subject.
• compositions may comprise, for example, at least about 0.1% of an active compound.
• an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
• the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound.
• Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
• a dose may also comprise from 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.
• a range of about 5 milligram/kg/body weight to about 100 milligram/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
• nucleic acid sequences encoding a therapeutic protein or a fusion protein containing a therapeutic protein may be disclosed.
• nucleic acid sequences can be selected based on conventional methods.
• the respective genes or variants thereof may be codon optimized for expression in a certain system.
• Various vectors may be also used to express the protein of interest. Exemplary vectors include, but are not limited, plasmid vectors, viral vectors, transposon, or liposome-based vectors.
• Some embodiments concern recombinant proteins and polypeptides. Particular embodiments concern a recombinant protein or polypeptide that exhibits at least one therapeutic activity.
• a recombinant protein or polypeptide may be a therapeutic antibody.
• a therapeutic antibody may be an antibody that specifically or selectively binds to an intracellular protein.
• the protein or polypeptide may be modified to increase serum stability.
• the present application refers to the function or activity of“modified protein” or a“modified polypeptide,” one of ordinary skill in the art would understand that this includes, for example, a protein or polypeptide that possesses an additional advantage over the unmodified protein or polypeptide.
• a“modified protein” may be implemented with respect to a“modified polypeptide,” and vice versa.
• Recombinant proteins may possess deletions and/or substitutions of amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments, these proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example.
• A“modified deleted protein” lacks one or more residues of the native protein, but may possess the specificity and/or activity of the native protein.
• A“modified deleted protein” may also have reduced immunogenicity or antigenicity.
• An example of a modified deleted protein is one that has an amino acid residue deleted from at least one antigenic region that is, a region of the protein determined to be antigenic in a particular organism, such as the type of organism that may be administered the modified protein.
• Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and/or bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge.
• Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; 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.
• a modified protein may possess an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
• biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of a control polypeptide are included, provided the biological activity of the protein is maintained.
• a recombinant protein may be biologically functionally equivalent to its native counterpart in certain aspects.
• amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
• the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
• a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids.
• a polypeptide of greater than about 100 amino acids
• a peptide of from about 3 to about 100 amino acids.
• the terms“protein,”“polypeptide,” and “peptide are used interchangeably herein.
• an“amino acid residue” refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art.
• the residues of the protein or peptide are sequential, without any non-amino acids interrupting the sequence of amino acid residues.
• the sequence may comprise one or more non-amino acid moieties.
• the sequence of residues of the protein or peptide may be interrupted by one or more nonamino acid moieties.
• protein or peptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.
• fusion proteins may have a therapeutic protein linked at the N- or C-terminus to a heterologous domain.
• fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
• Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein.
• a protein affinity tag such as a serum albumin affinity tag or six histidine residues
• an immunologically active domain such as an antibody epitope, preferably cleavable
• Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).
• the therapeutic protein may be linked to a peptide that increases the in vivo half-life, such as an XTEN polypeptide (Schellenberger et ah, 2009), IgG Fc domain, albumin, or albumin binding peptide.
• a peptide that increases the in vivo half-life such as an XTEN polypeptide (Schellenberger et ah, 2009), IgG Fc domain, albumin, or albumin binding peptide.
• fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein.
• Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the polypeptides connected in tandem.
• the linker would be of sufficient length to allow proper folding of the resulting fusion protein.
• siNA e.g., siRNA
• siRNA and double-stranded RNA have been described in U.S. Pat. Nos. 6,506,559 and 6,573,099, as well as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.
• a siNA may comprise a nucleotide and a nucleic acid or nucleotide analog.
• siNA form a double-stranded structure; the double- stranded structure may result from two separate nucleic acids that are partially or completely complementary.
• the siNA may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with 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 contiguous nucleobases, including all ranges therein.
• the siNA may comprise 17 to 35 contiguous nucleobases, more preferably 18 to 30 contiguous nucleobases, more preferably 19 to 25 nucleobases, more preferably 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.
• a complementary nucleic acid which may be another part of the same nucleic acid or a separate complementary nucleic acid
• Agents of the present invention useful for practicing the methods of the present invention include, but are not limited to siRNAs.
• 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 phenomena called RNA interference or RNAi.
• RNA interference has been referred to as“cosuppression,”“post- transcriptional gene silencing,”“sense suppression,” and“quelling.”
• RNAi is an attractive biotechnological tool because it provides a means for knocking out the activity of specific genes.
• RNAi there are several factors that need to be considered, such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system.
• the siRNA that is introduced into the organism will typically contain exonic sequences.
• the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene- specific sequences.
• the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater homology between the siRNA and the gene to be inhibited, the less likely expression of unrelated genes will be affected.
• the size of the siRNA is an important consideration.
• the present invention relates to siRNA molecules that include at least about 19-25 nucleotides and are able to modulate gene expression.
• the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in length.
• a target gene generally means a polynucleotide comprising a region that encodes a polypeptide, or a polynucleotide region that regulates replication, transcription, or translation or other processes important to expression of the polypeptide, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
• Any gene being expressed in a cell can be targeted.
• a target gene is one involved in or associated with the progression of cellular activities important to disease or of particular interest as a research object.
• siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art.
• one commercial source of predesigned siRNA is Ambion®, Austin, Tex.
• An inhibitory nucleic acid that can be applied in the compositions and methods of the present invention may be any nucleic acid sequence that has been found by any source to be a validated downregulator of a protein of interest. Without undue experimentation and using the disclosure of this invention, it is understood that additional siRNAs can be designed and used to practice the methods of the invention.
• the siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3 '-hydroxyl group. Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
• the double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain nonnatural intemucleoside linkages. Additional modifications of siRNAs (e.g., 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides,“universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate intemucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Application Publication 2004/0019001 and U.S. Pat. No. 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.
• CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
• a tracr trans-activating CRISPR
• tracr-mate sequence encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
• guide sequence also referred to as a“spacer” in the context of an endogenous CRISPR
• the CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
• a CRISPR system can derive from a type I, type P, or type PI CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
• a Cas nuclease and gRNA are introduced into the cell.
• target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g, the gene, using complementary base pairing.
• the target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG.
• PAM protospacer adjacent motif
• the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence.
• a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
• target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex.
• Full complementarity is not necessarily required, provided there is sufficient complementarity 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 disruptions as discussed herein.
• Cas9 variants deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced.
• 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 DNA or RNA polynucleotides.
• the target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell.
• a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or "editing polynucleotide” or “editing sequence”.
• an exogenous template polynucleotide may be referred to as an editing template.
• the recombination is homologous recombination.
• tracr sequence which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g.
• tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
• One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites.
• Components can also be delivered to cells as proteins and/or RNA.
• a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatoiy elements on separate vectors.
• two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included 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”).
• a restriction endonuclease recognition sequence also referred to as a “cloning site”.
• one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
• a vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein.
• Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, CsxM, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs
• the CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia).
• the CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
• the vector can encode a CRISPR enzyme that is mutated with respect 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 containing a target sequence.
• an aspartate-to-alanine substitution D10 A
• D10 A aspartate-to-alanine substitution
• pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
• a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
• an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
• the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
• codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
• Various species exhibit particular bias for certain codons of a particular amino acid.
• Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
• mRNA messenger RNA
• tRNA transfer RNA
• the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
• a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
• the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
• Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BEAT, Novoalign (Novocraft Technologies, ELAND (Dlumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
• any suitable algorithm for aligning sequences include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BEAT, Novoalign (Novocraft Technologies, ELAND (Dlumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.
• the CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains.
• a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
• protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
• Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
• reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), 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).
• GST glutathione-5- transferase
• HRP horseradish peroxidase
• CAT chloramphenicol acetyltransferase
• beta galactosidase beta- glucuronidase
• a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.
• kits are envisioned containing the necessary components to purify exosomes from a body fluid or tissue culture medium.
• a kit is envisioned containing the necessary components to isolate exosomes and transfect them with a therapeutic nucleic acid, therapeutic protein, or a nucleic acid encoding a therapeutic protein therein.
• the kit may comprise one or more sealed vials containing any of such components.
• the kit may also comprise a suitable container means, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• the container may be made from sterilizable materials such as plastic or glass.
• the kit may further include 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 are known to those of ordinary skill.
• the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of purifying exosomes from a sample and transfecting a therapeutic nucleic acid therein, expressing a recombinant protein therein, or electroporating a recombinant protein therein.
• MCF7, MDA-MB231, E10, HDF, and BJ human cell lines, as well as the NIH 3T3 murine cell line were cultured in DMEM with 10% FBS.
• 4T1 and 67NR murine cell lines were cultured in RPMI with 10% FBS.
• MCF10A human mammary epithelial cell line was cultured in DMEM/F12 media with 5% Horse Serum, 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 100 ng/ml Cholera Toxin, and 10 pg/ml Insulin. All cells originated from the American Type Culture Collection - ATCC.
• Exosomes were purified by differential centrifugation as described previously (Luga et al., 2012; Thery et al., 2006). Supernatant from cells cultured for 48 h were subjected to sequential centrifugation steps of 800g and 2000 g. The resulting supernatant was filtered using a 0.2 pm filter. A pellet was recovered after ultracentrifugation in an SW40Ti swinging bucket rotor at 100,000g for 3 h (Beckman-Coulter). Supernatant was removed and the pellet was re-suspended in PBS, followed by a second ultracentrifugation at 100,000g for 3 h.
• exosomes content was analyzed for exosomes content.
• Exosomes used for RNA extraction were resuspended in 500 pL of Trizol; exosomes used for protein extraction were resuspended in Urea/SDS lysis buffer (8M Urea, 2.5% SDS, 5 pg/mL leupeptin, 1 pg/mL pepstatin, and 1 mM phenylmethylsulphonyl fluoride); and exosomes used for delivery to cells were re-suspended in serum-free DMEM culture medium.
• isolated exosomes were processed as described in the remaining experimental procedures.
• Imaging Flow Cytometry analysis (ImageStream). Exosomes were attached to 4 pm aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) in NaCl 0.9% saline solution (B. Braun Medical Inc, Bethlehem, PA, USA). The reaction was stopped with 100 mM glycine and 2% BSA in saline and blocked with 10% BSA with rotation at room temperature for 30 min.
• the beads were resuspended in saline solution and analyzed on the ImageStream® (Merck Millipore).
• the image acquisition gain (%) was set using a positive sample, in order to avoid pixel saturation.
• Image processing was done using the IDEAS® (Merck Millipore) software. Gates were defined to exclude out-of- focus beads and select single beads. Alexa-488 positive bead gates were defined on the negative control sample. Percentage of positive beads is relative to the number of events analyzed per sample.
• Grids were rinsed with PBS and floated on drops of the appropriate secondary antibody attached with 10 nm gold particles (AURION, Hatfield, PA) for 2 h at room temperature.
• Grids were rinsed with PBS and placed in 2.5% Glutaraldehyde in 0.1 M Phosphate buffer for 15 minutes. After rinsing in PBS and distilled water, the grids were allowed to dry and stained for contrast using uranyl acetate.
• the samples were viewed with a Tecnai Bio Twin transmission electron microscope (FBI, Hillsboro, OR) and images were taken with an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA).
• MB-231 cells as described above. 1-3 * 10 9 exosomes were resuspended in 1 mL PBS and different concentrations of rEGF were added. Exosomes suspensions, with or without EGF, were incubated at 37°C with 5% CO2 for 15 minutes and then placed on ice. Three replicates were pooled and PBS was added to a total volume of 11 mL and stimulated exosomes were collected through ultracentrifugation in an SW40Ti swinging bucket rotor at l00,000g for 3 h, as before.
• Protein extracts were collected from the pelleted stimulated exosomes in Urea/SDS lysis buffer (with 100 mM NaF and 1 mM NaOV4) for immunoblot analysis or a Triton X-100 buffer (150 mM NaCl, 1% (v/v) Triton X-100, 10 mM Na2HP04, 2 mM KH2P04, pH 7.4, 50 mM 6-aminohexanoic acid, 10 mM EDTA, 5 mM N-ethylmaleimide, 5 mM benzamidine, 5 pg/mL leupeptin, 1 pg/mL pepstatin, 1 mM phenylmethylsulphonyl fluoride, 100 mM NaF, and 1 mM NaOV4) for immvmoprecipitation assays.
• Urea/SDS lysis buffer with 100 mM NaF and 1 mM NaOV4
• Exosomes protein extracts were loaded according to a Bicinchoninic Acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific) onto aciylamide gels and transferred onto PVDF membranes (ImmobilonP) by wet electrophoretic transfer.
• BCA Bicinchoninic Acid
• Blots were blocked for 1 h at RT with 5% non-fat dry milk in TBS/0.05% Tween20 and incubated overnight at 4°C with the following primaiy antibodies: 1 :300 anti-CD9 ab92726 (Abeam); 1:300 anti-TSGlOl ab83 (Abeam); 1:1000 anti-EGFR 4267S (CST); 1:300 anti-CD63 sc-365604, (Santa Cruz); 1:200 anti-CD81 cs-166029 (Santa
• HRP-conjugated secondary antibodies (Sigma, 1:2000) were incubated for 1 h at room temperature. Washes after antibody incubations were done on an orbital shaker, four times at 10 min intervals, with lx TBS 0.05% Tween20. Blots were developed with chemiluminescent reagents from Pierce.
• RNA reaction was performed using 3 ng of cDNA, TaqMan® Universal Master mix and QuantStudioTM 3D Digital PCR Master Mix vl (Applied Biosystems) according to manufacture recommendations. Using QuantStudioTM 3D Digital PCR Chip Loader (Applied Biosystems) a total of 14.5 pL of the mix were loaded to a QuantStudioTM 3D Digital PCR 20K Chip Kit vl (Applied Biosystems). PCR reaction was performed on GeneAmp® 9700 (Applied Biosystems) following manufacture protocol. The chips were imaged using QuantStudioTM 3D Digital PCR Instrument (Applied Biosystems). Table 2. Digital PCR Primers.
• Protein extracts were quantified using a BCA protein assay kit, run on acrylamide gels and transferred onto PVDF membranes (ImmobilonP) by wet electrophoretic transfer, after which the membranes were analyzed by autoradiography using the EN3HANCE® autoradiography enhancer according to the manufacturer’s instructions (Perkin-Elmer).
• Threshold cycle (Rothstein et al.), the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined and expression was measured using the 2-ACt formula, as previously reported (Livak and Schmittgen, 2001).
• Lysate preparation for in vitro transcription and translation Exosomes and cell pellets were washed once in ice-cold PBS and resuspended in an equal volume of ice-cold 20 mM HEPES (pH 7.5), 100 mM potassium acetate, 1 mM magnesium acetate, 2 mM dithiothreitol, and 100 pg/mL lysolecithin. After 1 min on ice, they were again pelleted and resuspended in an equal volume of ice-cold hypotonic extraction buffer. After 5 min on ice, the lysates were disrupted by passing 10 times through a 26-gauge needle attached to a 1-mL syringe. The resulting homogenates were centrifuged at lOOOg for 5 min at 4°C. The supernatant was collected, and aliquots were frozen in liquid nitrogen and stored at -80°C for use in the in vitro translation assay.
• HEPES pH
• Exosomes were electroporated using a 4 mm cuvette using a Gene Pulser Xcell Electroporation System (BioRad), as previously described (Alvarez-Erviti et al., 2011). When appropriate, exosomes were electroporated in the presence of cycloheximide (Sigma, 100 pg/mL) or a-amanitin (Sigma, 30 pg/mL), for inhibition of translation and transcription, respectively. Electroporated exosomes were cultured in serum-free DMEM at 37°C for the time points indicated.
• BioRad Gene Pulser Xcell Electroporation System
• Exosomes preparations (5 -10 pg) were incubated with 5 pL of 4 pm diameter aldehyde/sulfate latex beads (Interfacial Dynamics, Portland, OR) and resuspended into 600 pL. Exosomes-coated beads were analyzed on a FACS Calibur flow cytometer (BD Biosciences) and analyzed for green fluorescence.
• Example 1 - EGFR phosphorylation is detected 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 represent a heterogeneous mix, with the same size distribution being consistently observed between preparations.
• NanoSight nanoparticle tracking analysis (NTA) as well as atomic force microscopy (AFM) revealed particles with a size distribution averaging 104 ⁇ 1.5 nm in diameter, and ranging roughly between 30 and 200 nm. This was confirmed by transmission electron microscopy (TEM) showing extracellular vesicles surrounded by a lipid bilayer (FIGS. 8A-C).
• exosomes were further shown by immunogold/TEM imaging, immunoblot analysis and imaging flow cytometry to possess known markers of exosomes (Raposo and Stoorvogel, 2013) (FIGS. 8D-F).
• exosomes samples were inoculated onto solidified LB plates, showing no colony formation when compared to bacterial controls obtained from mouth swabs. This demonstrates the absence of bacterial contamination in the isolated exosomes (FIG. 8G).
• Exosomes obtained from different cell lines were probed by immunoblotting for their EGFR content. While exosomes from all cell lines show low levels of EGFR expression, exosomes derived from the BJ fibroblast cell line and MDA-MB-231 triple negative breast cancer cell line showed strong expression of the receptor.
• the known exosomes 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 1 billion exosomes were incubated with 500 ng/ml of recombinant human EGF (rhEGF) for 15 minutes at 37°C in serum-free culture media.
• Immunoblotting of protein extracts obtained from these exosomes with an antibody specific for the Tyrl068 residue of EGFR revealed an increase in the phosphorylation levels of this receptor in exosomes derived from MDA-MB- 231, but not non-tumori genic MCF10A breast epithelial cells (FIG. IB). Baseline levels of EGFR did not change in any of the samples, confirming the specificity of the observed increase in phosphorylation.
• 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 capacity to generate ATP (Ronquist et al., 2013a).
• an ATP quantification assay 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, albeit in smaller quantities in the latter. Exosomes from MDA-MB-231 cells, but not MCF10A cells, demonstrated a slight decrease in their ATP quantity upon stimulation with EGF (FIG.
• GRB2 was only identified in rhEGF stimulated exosomes (FIG. 2E). It should be stressed however that this does not mean that GRB2 is not present in unstimulated exosomes, but it might be present at a level under the detectable threshold for this type of analysis.
• the Exponentially Modified Protein Abundance Index (emPAI) which allows for label-free quantification of relative changes in protein content based on the observable peptide matches, was employed (Ishihama et al., 2005).
• the top 15 proteins that revealed a stronger increase in rhEGF stimulated exosomes when compared to their unstimulated counterparts included several participants of actin remodeling and membrane dynamics, such as a-actinin, MARCKS, ezrin, moesin, and integrin alpha-2 (Tables 4&5).
• actin remodeling and membrane dynamics such as a-actinin, MARCKS, ezrin, moesin, and integrin alpha-2 (Tables 4&5).
• a gene ontology (GO) analysis was then performed using the PANTHER overrepresentation test.
• Immunoblotting for b-actin also shows an increase in the levels of polymerized actin in exosomes stimulated with different amounts of rhEGF when compared with control unstimulated exosomes (FIG. 2G).
• Example 3 Exosomes derived from different cell types contain the functional constituents required for transcription and translation
• exosomes lysates from both MDA-MB-231 and MCF10A cells allowed for the synthesis of protein from the GFP expression plasmid confirmed that exosomes derived from different cells likely contain all the necessary functional components for both DNA transcription and mRNA translation. Consistent with the potential for DNA transcription, additional immunoblot analysis of protein extracts from exosomes isolated from different cell sources identified the presence of RNA polymerase II subunits, both in its phosphorylated and non-phosphorylated forms (FIG. 3D).
• Example 4 Exosomes are capable of cell-independent protein synthesis
• exosomes have the autonomous capability for de novo mRNA translation
• isolated exosomes obtained from MDAMB-231 cells as well as the murine lung cancer E10 cell line were incubated with 35 S-methionine to enable labeling of newly synthesized proteins by the exosomes.
• the assay was performed at 37°C in order to activate the putative biosynthetic processes and potential autocrine stimulation.
• Autoradiography of protein extracts from exosomes incubated for 72 h in the presence of 35 S-methionine exhibited the incorporation of the radioactive amino acid into several proteins in the range of 40 to 300 kDa.
• NanoSight NTA of electroporated exosomes using a 488 nm laser also detected green fluorescence in exosomes electroporated with the pCMV-GFP plasmid but not in mock- electroporated exosomes or exosomes electroporated with the plasmid and cycloheximide or a-amanitin (FIG. 13B). Additionally, beads based flow cytometry analysis of plasmid- containing exosomes using different electroporation conditions detected the presence of GFP (FIG. 13C). Next, exosomes were incubated for 24 h at 37°C to initiate biological processes, before electroporation with pCMV-GFP plasmid. The GFP production, as detected by immunoblotting, was impaired, suggesting an exhaustion of the required components for transcription and translation in the exosomes that are pre-incubated at 37°C. (FIG. 4G).
• an ovalbumin expression plasmid (pCMV-Ova), a protein that is also not expressed in mammalian cells, was used.
• pCMV-Ova ovalbumin expression plasmid
• immunoblotting analysis of exosomes after electroporation and incubation at 37°C for 48 h showed production of ovalbumin only in exosomes electroporated with the pCMV-Ova plasmid (FIG. 13D).
• Example 5 - mRNA translation in exosomes generates functional proteins and can be stimulated by growth factors
• MCF10A cells pretreated with cycloheximide were incubated with MDA-MB-231 exosomes that were directly electroporated with the pCMV-GFP plasmid.
• Imaging by confocal microscopy detected green fluorescence in the MCF10A cells, likely contributed by the GFP protein (after transcription and translation) delivered by MDAMB- 231 exosomes (FIG. 5 A, top and bottom panels).
• cells directly electroporated with the pCMV-GFP plasmid show a GFP fluorescence pattern distinct from fluorescence pattern observed in cells incubated with pCMV-GFP plasmid containing exosomes (FIG. 5 A, middle and bottom panels).
• MDA-MB-231 cells overexpress an inactive mutant form of the tumor suppressor protein p53, which is therefore unable to activate the p21 promoter (Cartel et al., 2003). Wild-type (wt) p53 typically responds to DNA damage by direct induction of p21, facilitating cell cycle arrest (Zilfou and Lowe, 2009).
• Exosomes isolated from MDA-MB-231 cells were electroporated with a plasmid encoding for wt p53 fused to GFP. The electroporated exosomes were incubated in culture media for 48 h to allow transcription and translation to generate wt p53 protein (FIG. 5B).
• exosomes containing the newly formed wt p53 were incubated with recipient MDA-MB-231 cells under the influence of cycloheximide.
• the recipient MDA-MB-231 cells revealed a substantial increase in expression of p21 (FIG. 5C), confirming the functionality of wt p53 protein that was exclusively synthetized by the exosomes (FIG. 5B).
• MDA-MB-231 derived exosomes were electroporated with the p53-GFP plasmid and either allowed to incubate for 48 h to synthesize the wt p53 protein prior to incubation with the recipient MDA-MB-231 cells (48 h), or delivered immediately to the recipient MDA-MB-231 cells without allowing them to produce the wt p53 protein (0 h, FIG. 13E).
• exosomes from MDA-MB-231 cells exhibit a baseline capacity for intrinsic protein synthesis
• exosomes were incubated at 37°C, with and without the presence of cycloheximide.
• Immunoblotting of protein extracts from these exosomes showed a consistent reduction in the expression levels of small cytoplasmic proteins, b-actin and GAPDH, upon incubation with cycloheximide, again demonstrating the existence of a baseline level of protein synthesis in these exosomes (FIG.
• the protein translation assay was repeated using exosomes derived from MDA-MB-231 cells with electroporated pCMV-GFP plasmid.
• the exosomes were incubated in serum-free culture media at 37°C for 48 h with or without stimulation with rhEGF. While the unstimulated exosomes presented with a baseline level of GFP production, the GFP levels increased upon incubation with different concentrations of rhEGF (FIG. 5E). This again confirmed that, while all exosomes can synthetize proteins, growth factor stimulation can alter their rate of production by leading to increased levels of protein synthesis.
• Example 6 Exosomes actively migrate in response to stimulation by rhEGF and serum factors
• a reverse migration assay based on the Boyden chamber system was designed including rhEGF and serum factors.
• Ten billion exosomes isolated from MDA-MB- 231 cells were placed in the culture wells of a 96-well plate, covered by a polycarbonate surface insert containing 400 nm pores.
• the insert contained either PBS, PBS with 10 mM rhEGF, or PBS with 20% exosomes-depleted FBS (FIG. 6A).
• mice with established 4T1 mammary tumors were injected with 5 billion MDA-MD 231/CD63-mCherry exosomes electroporated with either GFP or ovalbumin expression plasmids.
• Control experimental arms of this study included CD63-mCherry exosomes without the plasmids and CD63-mCherry exosomes electroporated with plasmid and cyclohexamide. Twenty-four hours after the I.P.
• exosomes were FACS isolated using the CD63 mCherry tag and evaluated for GFP or ovalbumin protein. GFP and ovalbumin were predominantly detected in the tumor, lung, bone, brain, and serum of mice with tumors, but were found only at very low levels in the tissues of non-tumor bearing mice and in tumor bearing mice that were injected with cyclohexamide-containing exosomes.
• Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell, 7:469- 483, 2005.
• Kalra et al. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics, 13:3354-3364, 2013.
• Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.
• Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region.
• eIF4E Eukaryotic translation initiation factor 4E
• Poliseno et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature, 465:1033-1038, 2010.
• K-ras oncogene silencing strategy reduces tumor growth and enhances gemcitabine chemotherapy efficacy for pancreatic cancer treatment.
• 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 Immunol., 166:322-329, 2001.
• Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Research, 40:el30, 2012.
• Mutant KRAS is a druggable target for pancreatic cancer. Proc.

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