Therapeutic extracellular vesicles

Abstract

Described herein are therapeutic extracellular vesicle compositions, as well as methods and systems for producing therapeutic extracellular vesicles. Also described herein are methods for treating diseases using therapeutic extracellular vesicles.

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Application No. 62 / 883,319, filed on 6 August 2019, and U.S. Provisional Application No. 62 / 947,228, filed on 12 December 2019, which are incorporated herein by reference in their entirety.

[0002] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were explicitly and individually indicated. [Background technology]

[0003] Extracellular vesicles are secreted by a wide variety of cell types. Generally, extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies are membrane-bound and can carry therapeutic cargo. Exosomes are one type of extracellular vesicle secreted by the vast majority of eukaryotic cells. Exosome biosynthesis may begin when the invagination of an endosome is pinched away to form a multivesicle, creating an intermembryonic vesicle. If the multivesicle fuses with the cell's plasma membrane, the intermembryonic vesicle may be released as an exosome. Microvesicles are another type of extracellular vesicle that budding outward from the cell surface membrane. Apoptotic bodies, on the other hand, are extracellular vesicles formed from dead cell fragments. Exosomes, microvesicles, and apoptotic bodies can be released in vivo or in vitro, such as in cell culture medium.

[0004] The delivery of therapeutic genetic material may be useful in treating diseases. Extracellular vesicles have been considered as carriers for therapeutic nucleic acids. However, most current methods for producing extracellular vesicles and encapsulating therapeutic nucleic acids within them have several drawbacks. First, the production of extracellular vesicles containing therapeutic nucleic acids is generally low, which often results in a small number of vesicles being produced or a small number of copies of therapeutic nucleic acids being encapsulated within the vesicles. For example, with certain transfection methods, messenger RNA (mRNA) is generally too large to be effectively encapsulated by extracellular vesicles. Other problems arising from currently available methods include fragmentation and degradation of nucleic acids encapsulated by extracellular vesicles. Finally, since the majority of extracellular vesicles in circulation accumulate and are metabolized in the liver, spleen, and kidneys, the challenge remains of directing extracellular vesicles to in vivo targets.

[0005] Therefore, there is a need for pharmaceutical compositions containing extracellular vesicles capable of effectively delivering a sufficient amount of therapeutic nucleic acid to target cells, tissues, or organs. Furthermore, there is a need for methods and systems for producing pharmaceutical compositions containing extracellular vesicles to deliver a sufficient amount of high-quality therapeutic nucleic acid to targets and treat target diseases. [Overview of the Initiative]

[0006] This specification describes, in several embodiments, a method for producing extracellular vesicles, the method comprising nanoelectroporating at least one polynucleotide into extracellular vesicle donor cells, the at least one polynucleotide encoding a targeting polypeptide comprising (i) an adapter polypeptide comprising a transmembrane domain and an extracellular domain, and (ii) a heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide; incubating the extracellular vesicle donor cells under conditions such that (i) the targeting polypeptide is expressed in the extracellular vesicle donor cells and (ii) the targeting polypeptide is incorporated into extracellular vesicles released from the extracellular vesicle donor cells; and collecting the at least one extracellular vesicle released from the extracellular vesicle donor cells, the at least one extracellular vesicle comprising the targeting polypeptide. In some embodiments, the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the heterologous targeting domain is covalently linked to the C-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the transmembrane domain of the adapter polypeptide is at least 70% identical to the transmembrane domain of the CD47 polypeptide, or the extracellular domain of the adapter polypeptide is at least 70% identical to the extracellular domain of the CD47 polypeptide. In some embodiments, the transmembrane domain of the adapter polypeptide is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the transmembrane domain of the CD47 polypeptide, or the extracellular domain of the adapter polypeptide is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the extracellular domain of the CD47 polypeptide.In some embodiments, the adapter polypeptide is selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to a polypeptide selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to CD47. In some embodiments, the adapter polypeptide is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to CD47. In some embodiments, the heterologous targeting domain includes a tumor targeting domain. In some embodiments, the tumor targeting domain is a CDX peptide.In some embodiments, the tumor targeting domain is a CREKA peptide. In some embodiments, the method further comprises nanoelectroporating a polynucleotide into the extracellular vesicle donor cell, wherein the polynucleotide encodes a ribonucleic acid (RNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is incorporated into an extracellular vesicle released from the extracellular vesicle donor cell, and the method further comprises collecting the extracellular vesicle released from the extracellular vesicle donor cell. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is a messenger RNA (mRNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof. In some embodiments, the RNA therapeutic agent is a cancer therapeutic agent. In some embodiments, the extracellular vesicle contains the RNA therapeutic agent in a fully intact or substantially intact form. In some embodiments, the RNA therapeutic agent is a fully intact or substantially intact messenger RNA. In some embodiments, the RNA therapeutic agent comprises at least five copies of a fully intact messenger RNA. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one copy of the RNA therapeutic agent. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one fully intact or substantially intact copy of the RNA therapeutic agent. In some embodiments, prior to nanoelectroporation, the extracellular vesicle donor cells are primary cells or unmodified cells. In some embodiments, the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells.In some embodiments, the extracellular vesicle donor cells are not neutrophils. In some embodiments, the extracellular vesicles are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles are exosomes. In some embodiments, the polynucleotides are nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. In some embodiments, the nanochannels consist of a diameter of 1 nanometer to 1000 nanometers. In some embodiments, the nanochannels consist of a diameter of 200 nanometers to 800 nanometers. In some embodiments, the nanochannels consist of a diameter of approximately 500 nanometers. In some embodiments, the biochip includes a nanochannel array comprising nanochannels with spacings between them of 1 micrometer to 100 micrometers. In some embodiments, the nanoelectroporation constitutes an electric field. In some embodiments, the electric field has an electric field intensity of 1 volt / mm to 1000 volts / mm. In some embodiments, the electric field consists of multiple pulses having pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse. In some embodiments, the tumor targeting domain is located at the N-terminus of the tumor targeting polypeptide. In some embodiments, the tumor targeting domain is located at the C-terminus of the tumor targeting polypeptide.

[0007] This specification describes, in several embodiments, a method for producing extracellular vesicles, the method comprising: nano-electroporating primary cells with at least one heterologous deoxyribonucleic acid (DNA) polynucleotide to obtain primary cells containing the heterologous DNA polynucleotide, the heterologous DNA polynucleotide encoding a therapeutic ribonucleic acid (RNA) polynucleotide; incubating the primary cells containing the heterologous DNA polynucleotide under conditions that enable transcription of the heterologous DNA polynucleotide to produce the therapeutic ribonucleic acid (RNA) polynucleotide, the therapeutic ribonucleic acid (RNA) polynucleotide being incorporated into extracellular vesicles released from the primary cells; and collecting the extracellular vesicles released from the primary cells, the extracellular vesicles released from the primary cells containing, on average, at least one copy of the therapeutic ribonucleic acid (RNA) polynucleotide. In some cases, the extracellular vesicles released from the primary cells contain, on average, at least one copy of the therapeutic ribonucleic acid (RNA) polynucleotide for every 5, 10, 20, 50, 100, 500, or 1000 extracellular vesicles released from the primary cells. In some cases, the extracellular vesicles released from the primary cells contain, on average, at least 2, 5, 10, 25, or 50 copies of the therapeutic ribonucleic acid (RNA). In some embodiments, prior to nanoelectroporation, the primary cells are unmodified primary cells. In some embodiments, the primary cells are selected from the group consisting of mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells. In some embodiments, the primary cells are not neutrophils. In some embodiments, the extracellular vesicles are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles are exosomes.

[0008] This specification describes, in several embodiments, compositions comprising extracellular vesicles, wherein the extracellular vesicles are: adapter polypeptides comprising an extracellular domain, and the adapter polypeptides comprising the following polypeptides: CD47 extracellular domain, CD47 transmembrane domain, CD63, CD81, CD82, CD47, CD315, heterotrimeric G protein, MHC class I, integrin, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycan, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidase, CD73, complement-binding proteins CD55 and CD59, and sonic hedgehog (SHH). The present invention comprises an adapter polypeptide comprising a polypeptide sequence that is at least 70% identical to one of the following: TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-associated proteins; and a heterologous targeting polypeptide covalently attached to the extracellular domain of the adapter polypeptide, wherein the targeting polypeptide specifically binds to target cells. In some embodiments, the adapter polypeptide comprises a transmembrane domain that is at least 70% identical to the transmembrane domain of the CD47 polypeptide, or an extracellular domain that is at least 70% identical to the extracellular domain of the CD47 polypeptide. In some embodiments, the heterologous targeting polypeptide is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the heterologous targeting polypeptide is covalently linked to the C-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the adapter polypeptide contains CD47. In some embodiments, the heterologous targeting polypeptide contains a targeting domain that binds to a cell surface marker associated with disease cells.In some embodiments, the targeting domain is a tumor targeting domain. In some embodiments, the tumor targeting domain is a CDX peptide. In some embodiments, the tumor targeting domain is a CREKA peptide. In some embodiments, the extracellular vesicle contains at least one copy of a ribonucleic acid (RNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is a messenger RNA (mRNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof. In some embodiments, the RNA therapeutic agent is a cancer therapeutic agent. In some embodiments, the RNA therapeutic agent is in a fully intact or substantially intact form. In some embodiments, the RNA therapeutic agent is a fully intact or substantially intact messenger RNA. In some embodiments, the RNA therapeutic agent comprises at least five copies of a fully intact or substantially intact messenger RNA. In some embodiments, the extracellular vesicle is an exosome, microvesicle, or apoptotic body. In some embodiments, the extracellular vesicle is an exosome. In some embodiments, the tumor targeting domain is located at the N-terminus of the tumor targeting polypeptide. In some embodiments, the tumor targeting domain is located at the C-terminus of the tumor targeting polypeptide.

[0009] This specification describes, in several embodiments, methods for treating a target tumor, the method comprising systemically administering to at least one extracellular vesicle containing a therapeutic agent, wherein the at least one extracellular vesicle containing the therapeutic agent is: nanoelectroporating at least one polynucleotide into an extracellular vesicle donor cell, the at least one polynucleotide encoding a targeting polypeptide comprising (i) an adapter polypeptide comprising a transmembrane domain and an extracellular domain, and (ii) a heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide, and the at least one polynucleotide encoding a ribonucleic acid (RNA) therapeutic agent. The systemic administration obtained by: (i) performing nanoelectroporation; (ii) incubating the extracellular vesicle donor cells under conditions such that the targeting polypeptide is expressed in the extracellular vesicle donor cells and (ii) the targeting polypeptide is incorporated into the extracellular vesicles released from the extracellular vesicle donor cells; and collecting the at least one extracellular vesicle released from the extracellular vesicle donor cells, wherein the at least one extracellular vesicle contains the targeting polypeptide, wherein the accumulation rate of the at least one extracellular vesicle in the tumor is higher than the accumulation rate of extracellular vesicles lacking the heterologous targeting domain. In some embodiments, the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the heterologous targeting domain is covalently linked to the C-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the transmembrane domain of the adapter polypeptide is at least 70% identical to the transmembrane domain of the CD47 polypeptide, or the extracellular domain of the adapter polypeptide is at least 70% identical to the extracellular domain of the CD47 polypeptide.In some embodiments, the adapter polypeptide is selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to a polypeptide selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to CD47. In some embodiments, the heterologous targeting domain includes a tumor targeting domain. In some embodiments, the tumor targeting domain is a CDX peptide. In some embodiments, the tumor targeting domain is a CREKA peptide.In some embodiments, the ribonucleic acid (RNA) therapeutic agent is incorporated into extracellular vesicles released from the extracellular vesicle donor cells, and the method further includes collecting the extracellular vesicles released from the extracellular vesicle donor cells. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is a messenger RNA (mRNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof. In some embodiments, the RNA therapeutic agent is a cancer therapeutic agent. In some embodiments, the extracellular vesicles contain the RNA therapeutic agent in a fully intact or substantially intact form. In some embodiments, the RNA therapeutic agent is a fully intact or substantially intact messenger RNA. In some embodiments, the RNA therapeutic agent constitutes at least five copies of a fully intact messenger RNA. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one copy of the RNA therapeutic agent. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one fully intact or substantially intact copy of the RNA therapeutic agent. In some embodiments, prior to nanoelectroporation, the extracellular vesicle donor cells are primary cells or unmodified cells. In some embodiments, the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells. In some embodiments, the extracellular vesicle donor cells are not neutrophils. In some embodiments, the extracellular vesicles are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles are exosomes.In some embodiments, the polynucleotide is nanoelectroporated into the extracellular vesicle donor cell via nanochannels located on the biochip. In some embodiments, the nanochannels consist of a diameter of 1 nanometer to 1000 nanometers. In some embodiments, the nanochannels consist of a diameter of 200 nanometers to 800 nanometers. In some embodiments, the nanochannels consist of a diameter of approximately 500 nanometers. In some embodiments, the biochip includes a nanochannel array comprising nanochannels with spacings between them of 1 micrometer to 100 micrometers. In some embodiments, the nanoelectroporation constitutes an electric field. In some embodiments, the electric field has an electric field intensity of 1 volt / mm to 1000 volts / mm. In some embodiments, the electric field consists of multiple pulses with pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse. In some embodiments, the tumor targeting domain is located on the N-terminus of the tumor targeting polypeptide. In some embodiments, the tumor targeting domain is located on the C-terminus of the tumor targeting polypeptide. In some embodiments, the tumor is cancer. In some embodiments, the cancer is a glioma.

[0010] This specification describes, in several embodiments, methods for treating a target muscular dystrophy, the method comprising systemically administering to at least one extracellular vesicle containing a therapeutic agent, wherein the at least one extracellular vesicle containing the therapeutic agent is: nanoelectroporating at least one polynucleotide into an extracellular vesicle donor cell, wherein the at least one polynucleotide encodes a targeting polypeptide comprising (i) an adapter polypeptide comprising a transmembrane domain and an extracellular domain, and (ii) a heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide, and the at least one polynucleotide is a ribonucleic acid (RNA) therapeutic agent The systemic administration comprising: (i) nanoelectroporation; (ii) incubation of extracellular vesicle donor cells under conditions such that the targeting polypeptide is expressed in the extracellular vesicle donor cells and (ii) the targeting polypeptide is incorporated into extracellular vesicles released from the extracellular vesicle donor cells; and collecting the at least one extracellular vesicle released from the extracellular vesicle donor cells, wherein the at least one extracellular vesicle contains the targeting polypeptide, wherein the accumulation rate of the at least one extracellular vesicle in the tumor is higher than the accumulation rate of extracellular vesicles lacking the heterologous targeting domain. In some embodiments, the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the heterologous targeting domain is covalently linked to the C-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the transmembrane domain of the adapter polypeptide is at least 70% identical to the transmembrane domain of the CD47 polypeptide, or the extracellular domain of the adapter polypeptide is at least 70% identical to the extracellular domain of the CD47 polypeptide.In some embodiments, the adapter polypeptide is selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to a polypeptide selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to CD47. In some embodiments, the heterologous targeting domain includes a tumor targeting domain. In some embodiments, the tumor targeting domain is a CDX peptide. In some embodiments, the tumor targeting domain is a CREKA peptide.In some embodiments, the ribonucleic acid (RNA) therapeutic agent is incorporated into extracellular vesicles released from the extracellular vesicle donor cells, and the method further includes collecting the extracellular vesicles released from the extracellular vesicle donor cells. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is a messenger RNA (mRNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof. In some embodiments, the extracellular vesicles contain the RNA therapeutic agent in a fully intact or substantially intact form. In some embodiments, the RNA therapeutic agent is a fully intact or substantially intact messenger RNA. In some embodiments, the RNA therapeutic agent constitutes at least five copies of a fully intact messenger RNA. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one copy of the RNA therapeutic agent. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one fully intact or substantially intact copy of the RNA therapeutic agent. In some embodiments, prior to nanoelectroporation, the extracellular vesicle donor cells are primary cells or unmodified cells. In some embodiments, the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells. In some embodiments, the extracellular vesicle donor cells are not neutrophils. In some embodiments, the extracellular vesicles are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles are exosomes. In some embodiments, the polynucleotide is nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip.In some embodiments, the nanochannels are configured with a diameter of 1 nanometer to 1000 nanometers. In some embodiments, the biochip includes a nanochannel array comprising nanochannels with spacings between nanochannels of 1 micrometer to 100 micrometers. In some embodiments, the nanoelectroporation constitutes an electric field. In some embodiments, the electric field has an electric field intensity of 1 volt / mm to 1000 volts / mm. In some embodiments, the electric field comprises multiple pulses having pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse. In some embodiments, the tumor targeting domain is located on the N-terminus of the tumor targeting polypeptide. In some embodiments, the tumor targeting domain is located on the C-terminus of the tumor targeting polypeptide. In some embodiments, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0011] This specification describes, in several embodiments, a method for treating a target retinal disease, the method comprising systemically administering to at least one extracellular vesicle containing a therapeutic agent, wherein the at least one extracellular vesicle containing the therapeutic agent is: nanoelectroporating at least one polynucleotide into an extracellular vesicle donor cell, the at least one polynucleotide encoding a targeting polypeptide comprising (i) an adapter polypeptide comprising a transmembrane domain and an extracellular domain, and (ii) a heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide, and the at least one polynucleotide encoding a ribonucleic acid (RNA) therapeutic agent. The systemic administration obtained by: (i) nano-electroporation; (ii) incubation of extracellular vesicle donor cells under conditions such that the targeting polypeptide is expressed in the extracellular vesicle donor cells and (ii) the targeting polypeptide is incorporated into extracellular vesicles released from the extracellular vesicle donor cells; and collecting the at least one extracellular vesicle released from the extracellular vesicle donor cells, wherein the at least one extracellular vesicle contains the targeting polypeptide, wherein the accumulation rate of the at least one extracellular vesicle in the tumor is higher than the accumulation rate of extracellular vesicles lacking the heterologous targeting domain. In some embodiments, the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the heterologous targeting domain is covalently linked to the C-terminus of the extracellular domain of the adapter polypeptide. In some embodiments, the transmembrane domain of the adapter polypeptide is at least 70% identical to the transmembrane domain of the CD47 polypeptide, or the extracellular domain of the adapter polypeptide is at least 70% identical to the extracellular domain of the CD47 polypeptide.In some embodiments, the adapter polypeptide is selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to a polypeptide selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, Sonic Hedgehog (SHH), TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, the adapter polypeptide is at least 70% identical to CD47. In some embodiments, the heterologous targeting domain includes a tumor targeting domain. In some embodiments, the tumor targeting domain is a CDX peptide. In some embodiments, the tumor targeting domain is a CREKA peptide.In some embodiments, the ribonucleic acid (RNA) therapeutic agent is incorporated into extracellular vesicles released from the extracellular vesicle donor cells, and the method further includes collecting the extracellular vesicles released from the extracellular vesicle donor cells. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is a messenger RNA (mRNA) therapeutic agent. In some embodiments, the ribonucleic acid (RNA) therapeutic agent is uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof. In some embodiments, the extracellular vesicles contain the RNA therapeutic agent in a fully intact or substantially intact form. In some embodiments, the RNA therapeutic agent is a fully intact or substantially intact messenger RNA. In some embodiments, the RNA therapeutic agent constitutes at least five copies of a fully intact messenger RNA. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one copy of the RNA therapeutic agent. In some embodiments, on average, after nanoelectroporation, each extracellular vesicle released by the extracellular vesicle donor cells contains at least one fully intact or substantially intact copy of the RNA therapeutic agent. In some embodiments, prior to nanoelectroporation, the extracellular vesicle donor cells are primary cells or unmodified cells. In some embodiments, the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells. In some embodiments, the extracellular vesicle donor cells are not neutrophils. In some embodiments, the extracellular vesicles are exosomes, microvesicles, or apoptotic bodies.

[0012] In some embodiments, the extracellular vesicles are exosomes. In some embodiments, the polynucleotides are nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. In some embodiments, the nanochannels consist of a diameter of 1 nanometer to 1000 nanometers. In some embodiments, the biochip includes a nanochannel array comprising nanochannels with spacings between them of 1 micrometer to 100 micrometers. In some embodiments, the nanoelectroporation constitutes an electric field. In some embodiments, the electric field has an electric field intensity of 1 volt / mm to 1000 volts / mm. In some embodiments, the electric field consists of multiple pulses with pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse. In some embodiments, the tumor targeting domain is located on the N-terminus of the tumor targeting polypeptide. In some embodiments, the tumor targeting domain is located on the C-terminus of the tumor targeting polypeptide. In some embodiments, the retinal disease is retinitis pigmentosa. In some embodiments, the retinal disease is Leber congenital amaurosis.

[0013] This specification describes, in several embodiments, methods for treating a target tumor, the method comprising systemically administering to an extracellular vesicle containing a therapeutic polynucleotide, wherein the extracellular vesicle containing the therapeutic polynucleotide is: nanoelectroporating an extracellular vesicle donor cell with at least a first vector (e.g., plasmid) and at least a second vector (e.g., plasmid), wherein the first vector (e.g., plasmid) encodes a tumor or tissue targeting polypeptide comprising an extracellular vesicle surface protein covalently bound to a tumor or tissue targeting domain, and the second vector The systemic administration comprises: nano-electroporation encoding a therapeutic polynucleotide; expressing a first vector (e.g., a plasmid) in an extracellular vesicle donor cell to obtain a tumor or tissue-targeting polypeptide; transcribing a second vector (e.g., a plasmid) in an extracellular vesicle donor cell to obtain a therapeutic polynucleotide; and collecting at least one extracellular vesicle released from the extracellular vesicle donor cell, wherein the accumulation rate of at least one extracellular vesicle in the target tumor or tissue is higher than the accumulation rate of extracellular vesicles lacking the tumor or tissue-targeting polypeptide. In some embodiments, the extracellular vesicle is an exosome. In some embodiments, the accumulation rate of at least one extracellular vesicle containing the tumor or tissue-targeting polypeptide in the tumor or tissue is at least 100 times higher than the accumulation rate of extracellular vesicles lacking the tumor-targeting polypeptide. In some embodiments, the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells. In some embodiments, multiple first and second plasmids are nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. In some embodiments, the nanochannels consist of a diameter of 1 nanometer to 1000 nanometers.In some embodiments, the biochip includes a nanochannel array comprising nanochannels with spacings of 1 micrometer to 100 micrometers between nanochannels. In some embodiments, nanoelectroporation constitutes an electric field. In some embodiments, the electric field has an electric field intensity of 1 volt / mm to 1000 volts / mm. In some embodiments, the electric field comprises multiple pulses with pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse. In some embodiments, the tumor or tissue targeting domain of the extracellular vesicle domain is located at the N-terminus of the tumor or tissue targeting polypeptide. In some embodiments, the tumor or tissue targeting domain is located at the C-terminus of the tumor targeting polypeptide. In some embodiments, the tumor or tissue targeting domain comprises a CDX peptide. In some embodiments, the tumor or tissue targeting domain comprises a CREKA peptide. In some embodiments, the extracellular vesicle surface protein comprises a peptide sequence that is at least 70% identical to the peptide sequence of a naturally occurring extracellular vesicle surface protein. In some embodiments, naturally occurring extracellular vesicle surface proteins are selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptor (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, and sonic hedgehog (SHH). In some embodiments, naturally occurring extracellular vesicle surface proteins are selected from the group consisting of TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins. In some embodiments, naturally occurring extracellular vesicle surface proteins include CD47.In some embodiments, at least one extracellular vesicle comprises a copy of at least one therapeutic polynucleotide. In some embodiments, at least one extracellular vesicle comprises at least 2 copies, at least 5 copies, at least 10 copies, or at least 50 copies of a copy of a therapeutic polynucleotide. In some embodiments, at least one extracellular vesicle comprises at least 100 copies of a copy of a therapeutic polynucleotide. In some embodiments, at least one extracellular vesicle comprises at least 1000 copies of a copy of a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is selected from the group consisting of mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some embodiments, the therapeutic polynucleotide comprises mRNA. In some embodiments, the mRNA comprises at least 100 RNA nucleotides. In some embodiments, the therapeutic polynucleotide comprises at least one modified nucleotide. In some embodiments, the therapeutic polynucleotide comprises a modified oligonucleotide. In some embodiments, the described method comprises treating a tumor with extracellular vesicles. In some embodiments, the tumor is cancer. In some embodiments, the cancer is glioma.

[0014] This specification describes, in several embodiments, methods for treating a target muscular dystrophy, the method comprising systemic administration of at least one extracellular vesicle containing a therapeutic polynucleotide, the at least one extracellular vesicle containing the therapeutic polynucleotide being: nanoelectroporating at least a first vector (e.g., a plasmid) and at least a second vector (e.g., a plasmid) into an extracellular vesicle donor cell, the first vector (e.g., a plasmid) encoding a myocyte-targeting polypeptide comprising an extracellular vesicle surface protein covalently bound to a myocyte-targeting domain, and the second vector encoding the therapeutic polynucleotide, the systemic administration comprising: nanoelectroporation; expressing the first vector in the extracellular vesicle donor cell to obtain the myocyte-targeting polypeptide; transcribing the second vector in the extracellular vesicle donor cell to obtain the therapeutic polynucleotide; and collecting at least one extracellular vesicle released from the extracellular vesicle donor cell. In some embodiments, the extracellular vesicles for treating muscular dystrophy are exosomes. In some embodiments, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments, the therapeutic polynucleotide for treating muscular dystrophy constitutes mRNA. In some embodiments, the therapeutic polynucleotide for treating muscular dystrophy comprises at least one modified nucleotide. In some embodiments, the therapeutic polynucleotide for treating muscular dystrophy comprises a modified oligonucleotide.

[0015] This specification describes, in several embodiments, methods for treating a target retinal disease, the method comprising systemic administration of at least one extracellular vesicle containing a therapeutic polynucleotide, the at least one extracellular vesicle containing the therapeutic polynucleotide being: nanoelectroporating at least a first vector and at least a second vector into an extracellular vesicle donor cell, the first vector encoding a retinal cell targeting polypeptide comprising an extracellular vesicle surface protein covalently bound to a retinal cell targeting domain, and the second vector encoding the therapeutic polynucleotide, the systemic administration being obtained by: nanoelectroporating; expressing the first vector in the extracellular vesicle donor cell to obtain the retinal cell targeting polypeptide; transcribing the second vector in the extracellular vesicle donor cell to obtain the therapeutic polynucleotide; and collecting at least one extracellular vesicle released from the extracellular vesicle donor cell. In some embodiments, the extracellular vesicle for treating the retinal disease is an exosome. In some embodiments, the retinal disease is retinitis pigmentosa. In some embodiments, the retinal disease is Leber congenital amaurosis.

[0016] In some embodiments, the present specification describes a pharmaceutical composition comprising at least one extracellular vesicle, wherein the at least one extracellular vesicle comprises: at least one targeting polypeptide comprising an extracellular vesicle surface protein covalently bound to a targeting domain; and at least one therapeutic polynucleotide. In some embodiments, the extracellular vesicles of the pharmaceutical composition are exosomes. In some embodiments, the extracellular vesicle surface protein comprises an extracellular vesicle transmembrane domain. In some embodiments, the extracellular vesicle transmembrane domain is at least 70% identical to a CD47 peptide sequence. In some embodiments, the extracellular vesicles of the pharmaceutical composition comprise at least two targeting domains. In some embodiments, the at least two targeting domains are different. In some embodiments, the therapeutic polynucleotide of the pharmaceutical composition is selected from the group consisting of mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some embodiments, the therapeutic polynucleotide comprises mRNA. In some embodiments, the pharmaceutical composition is administered to a subject by intrathecal, intraorbital, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraparenchymal, subcutaneous, or a combination thereof.

[0017] This patent application contains at least one drawing created in color. Copies of this patent or patent application with color drawings(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees.

[0018] The novelty of the present disclosure is described in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description of the invention, which illustrates exemplary embodiments.

Brief Description of the Drawings

[0019] [Figure 1A]This demonstrates the large-scale generation of extracellular vesicles (EVs) carrying transcribed mRNA by cellular nanoporation (CNP). A schematic diagram of CNP generating EVs for targeted nucleic acid delivery is shown. Left: A model CNP system consists of a nanochannel array with each channel having a diameter of approximately 500 nm (top of inset). A DNA vector added to a buffer solution enters attached cells through nanochannels under transient electrical pulses. The attached cells then release large quantities of exosomes containing transcribed mRNA, which can be collected for tumor-targeted delivery via the blood-brain barrier (BBB) ​​and the blood-brain tumor barrier (BBTB) (right). [Figure 1B] This shows that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). The number of EVs per cell is shown for untreated MEF in PBS buffer (PBS), MEF treated with lipofectamine 2000 (Lipo), bulk electroporation (BEP), and MEF after transfection with the Ascl1 / Brn2 / Myt1l (A / B / M) vector via cellular nanoelectroporation (CNP), as well as CNP using only PBS buffer (CNP / PBS). All data are from three independent experiments and are presented as mean ± SEM. *P<0.05 (vs BEP), Student's t-test (Figures 1B, 1C, 1D, and 1H). [Figure 1C] This study demonstrates that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). The study compares CNP-mediated EV release with other stress-induced EV release methods, including starvation, hypoxia, and heat treatment. Starvation: MEF cells were cultured in DMEM without FBS; Hypoxia: MEF cells were cultured in a hypoxic chamber with 1% O2 and 5% CO2 at a humidified environment of 37°C; Heat: MEF cells were cultured at 42°C for 2 hours, then transferred to normal cell culture conditions at 37°C. All data are from three independent experiments and are presented as mean ± SEM. *P<0.05 (vs BEP), Student's t-test (Figures 1B, 1C, 1D, and 1H). [Figure 1D] This shows that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). The number of EVs per cell produced by mouse bone marrow-derived dendritic cells (BMDCs) in different treatment groups, including PBS, Lipo, BEP, CNP, and the CNP / PBS group. All data are from three independent experiments and are presented as mean ± SEM. *P<0.05 (vs BEP), Student's t-test (Figures 1B, 1C, 1D, and 1H). [Figure 1E] This demonstrates that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). Exosome release from MEFs transfected by CNP peaks approximately 8 hours after CNP. [Figure 1F] This demonstrates that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). Dynamic light scattering (DLS) measurements of MEF exosome concentration by CNP at various voltages were performed. The results revealed that increasing the voltage from 200V to 220V did not increase the number of exosomes. **P<0.01 (vs voltage 0V), #P<0.05 (vs voltage 150V), Student's t-test. [Figure 1G] This shows that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). Agarose gel analysis of EV-mRNA collected from EVs after CNP. CNP / PBS: Total RNA recovered from 10⁷MEF after CNP using PBS buffer only; PTEN mRNA: 200 ng of synthetic PTEN mRNA; CNP / PTEN: Total RNA (approximately 1.0 μg) recovered from 10⁷MEF after CNP using a PTEN vector. [Figure 1H]This demonstrates that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). qPCR of mRNAs A, B, and M revealed that exosomes produced by CNP contained significantly higher amounts of transcribed mRNA compared to those produced by other methods. All data are from three independent experiments and are presented as mean ± SEM values. *P<0.05 (vs BEP), Student's t-test (Figures 1B, 1C, 1D, and 1H). [Figure 1I] This demonstrates that extracellular vesicles (EVs) carrying transcribed mRNA are generated in large quantities by cellular nanoporation (CNP). When mRNA from EVs A, B, and M derived from MEFs transfected with CNP was subjected to qPCR (with culture medium changed every 4 hours for 24 hours), it was shown that the largest transcript took the longest time to reach peak concentration. [Figure 2A] This shows the characteristics of exosomes generated from CNP. DLS measurement of the particle size distribution of vesicles produced by CNP. A peak was observed around 70-110 nm in the CNP group, indicating that a large amount of exosomes are produced by CNP. Top panel: PBS group, Bottom panel: CNP group. [Figure 2B] This shows the characteristics of exosomes generated from CNP. DLS measurements of the number of exosomes per MEF cell using a gene gun at various pressures. The results revealed that the number of exosomes slightly increased with increasing pressure used in the gene gun. Data are from three independent experiments and are mean ± SEM. *P<0.05 (vs PBS), Student's t-test. [Figure 2C] This shows the characteristics of exosomes generated from CNP. It also shows the number of extracellular assets (EVs) per cell produced by mouse mesenchymal stem cells (MSCs) in different treatment groups, including PBS, Lipo, BEP, CNP, and the CNP / PBS group. [Figure 2D]This shows the characteristics of exosomes generated from CNP. It also shows the number of EVs per cell produced by human embryonic kidney 293T (HEK293T) in different treatment groups, including PBS, Lipo, BEP, CNP, and the CNP / PBS group. [Figure 2E] This shows the characteristics of exosomes generated from CNPs. It also shows the number of EVs per cell produced by MEF in CNP groups at different CNP temperatures. [Figure 2F] This shows the characteristics of exosomes generated from CNP. When PTEN mRNA in EVs produced by various transfection methods using PTEN vectors was measured by qPCR, it was shown that EVs produced by CNP in MEF contained a much larger amount of transcribed PTEN mRNA than those produced by other methods. [Figure 2G] This shows the characteristics of exosomes generated from CNP. When PTEN mRNA in EVs produced by various transfection methods using PTEN vectors was measured by qPCR, it was shown that EVs produced by CNP in BMDC contained a much larger amount of transcribed PTEN mRNA than those produced by other methods. [Figure 2H] This shows the characteristics of exosomes generated from CNP. When the miR-128 levels in EVs produced by various transfection methods using the miR-128 vector were measured by qPCR, it was shown that in MEFs, EVs produced by CNP contained significantly more transcribed miR-128 than those produced by other methods. [Figure 2I] This shows the characteristics of exosomes generated from CNP. Western blots of in vitro protein translations in whole vesicles secreted from MEF using different transfection methods indicate that the whole vesicles contained transcribed mRNA that could be translated into functional proteins. [Figure 3A]This paper compares the efficiency of miRNA loading into exosomes using CNP and BEP. DLS measurements were performed on the vesicle particle size distribution produced by CNP in exosome fractions collected by ultracentrifugation. [Figure 3B] This paper compares the efficiency of miRNA loading into exosomes using CNP and BEP. DLS measurements were performed on the vesicle size distribution produced by CNP in microvesicle (MV) fractions collected by ultracentrifugation. [Figure 3C] This paper compares the efficiency of miRNA loading into exosomes using CNP and BEP. Representative TIRF images obtained from TLN assays of miR-128 coexisting in exosomes (CD63-GFP) after CNP and BEP showed that CNP resulted in superior miRNA-128 loading efficiency into exosomes compared to BEP. [Figure 3D] This report compares the efficiency of miRNA loading into exosomes using CNP and BEP. It shows the percentage of miR-128 coexistence in exosomes after CNP and BEP. 100 images were used for statistical analysis. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM values. *P<0.05 (vs CNP), Student's t-test (Figures 3D, 3E, and 3F). [Figure 3E] This report compares the efficiency of miRNA loading into exosomes using CNP and BEP. The miR-128 fluorescence intensity within exosomes, measured by TLN, is shown for the CNP and BEP groups. 100 images were used for statistical analysis. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM values. *P<0.05 (vs CNP), Student's t-test (Figures 3D, 3E, and 3F). [Figure 3F]This paper compares the efficiency of miRNA loading into exosomes using CNP and BEP. DLS measurements of relative exosome numbers before and after BEP showed that BEP disrupted approximately 50% of exosomes. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM values. *P<0.05 (vs CNP), Student's t-test (Figures 3D, 3E, and 3F). [Figure 4A] This study demonstrates that exosomes, in addition to microvesicles (MVs), contain functionally transcribed mRNA after CNP (continuous necrosis). Western blotting detected exosome markers (CD9, CD63, and Tsg101) and MV marker (Arf6) in equal amounts (20 μg protein) of exosomes and MVs. [Figure 4B] This indicates that, in addition to microvesicles (MVs), exosomes contain functional transcribed mRNA after CNP. Nanodrop measurement of RNA levels in exosomes versus MVs derived from CNP-transfected 108-MEFs showed that the majority of RNA was found in exosomes rather than MVs. **P<0.01 (vs exosomes), Student's t-test (Figures 4B and 4D). [Figure 4C] This indicates that, in addition to microvesicles (MVs), exosomes contain functional transcribed mRNA after CNP. Cryo-TEM images of exosomes from the PBS group (PBS) and the CNP group (CNP) showed no difference in appearance, but the CNP group contained more RNA internally. [Figure 4D] This indicates that, in addition to microvesicles (MVs), exosomes also contain functionally transcribed mRNA after CNP. qPCR of Ascl1 (A), Brn2 (B), and Myt1l (M) mRNAs from exosomes and MVs showed that the majority of transcribed mRNA was found in exosomes. **P<0.01 (vs exosomes), Student's t-test (Figures 4B and 4D). [Figure 4E]This demonstrates that, in addition to microvesicles (MVs), exosomes contain functional transcribed mRNA after CNP. The results show in vitro protein translations derived from mRNA extracted from exosomes and MVs secreted by MEFs transfected with CNP. [Figure 4F] This shows that, in addition to microvesicles (MVs), exosomes contain functionally transcribed mRNA after CNP (concentrated necrosis). A schematic diagram illustrates the tethered dripoplex nanoparticle (TLN) assay method. Nanoparticles containing specific molecular beacons (MBs) were immobilized on coverslips, and exosomes were captured by the nanoparticles. Hybridization between mRNA within exosomes and MBs within the nanoparticles generated fluorescence, which was detected using total internal reflection fluorescence (TIRF) microscopy. [Figure 4G] This shows that, in addition to microvesicles (MVs), exosomes contain functional transcription mRNA after CNP. Representative TIRF images from TLN assays in the CNP group and S-CNP group show that S-CNP optimized the loading of different mRNAs into individual exosomes. Medium gray (green) dots: Ascl1 mRNA, dark gray (red) dots: Brn2 mRNA, light gray (purple) dots: Myt1l mRNA, dark gray (pink) arrows: exosomes with one mRNA, light gray (turquoise) arrows: exosomes with two mRNAs, medium gray (yellow) arrows: exosomes with three mRNAs. [Figure 4H] This shows that, in addition to microvesicles (MVs), exosomes contain functionally transcribed mRNA after CNP. The percentage of exosomes with different RNAs in the CNP group and S-CNP group is shown. 100 images were selected for statistical analysis in each group. [Figure 5A]This section compares the mRNA loading efficiency of CNP and BEP in exosomes. Representative TIRF images obtained from TLN assays of Brn2 mRNA coexisting in exosomes (CD63-GFP) after CNP and BEP showed that CNP resulted in significantly higher mRNA loading efficiency in exosomes than BEP. [Figure 5B] This report compares the mRNA loading efficiency of exosomes using CNP and BEP. It shows the percentage of Brn2 mRNA coexistence in exosomes after CNP and BEP. 100 images were used for statistical analysis. All data are from three independent experiments and are presented as mean ± SEM values. **P<0.01 (vs CNP), ##P<0.01 (vs BEP without RNase), Student's t-test (Figures 5B, 5C, and 5D). [Figure 5C] This report compares the mRNA loading efficiency of CNP and BEP in exosomes. Brn2 mRNA fluorescence intensity within EV measured by TLN in the CNP and BEP groups. 100 images were used for statistical analysis. All data are from three independent experiments and are presented as mean ± SEM values. **P<0.01 (vs CNP), ##P<0.01 (vs BEP without RNase), Student's t-test (Figures 5B, 5C, and 5D). [Figure 5D]This paper compares the mRNA loading efficiency of exosomes using CNP and BEP. miR-128 and Brn2 mRNA expression levels are measured by qPCR (CT values) for exosomes secreted from 107MEF transfected with CNP (CNP), exosomes derived from 107MEF transfected with CNP / PBS mixed with free RNA from 107MEF transfected with CNP (mixture), exosomes from the mixture after RNA insertion by bulk electroporation (RNase-free BEP), and exosomes from the mixture after BEP treatment with RNase to remove RNA molecules attached to the outer surface of the exosomes (RNase-enabled BEP). All data are from three independent experiments and are presented as mean ± SEM values. **P<0.01 (vs CNP), ##P<0.01 (vs RNase-free BEP), Student's t-test (Figures 5B, 5C, and 5D). [Figure 6A] This shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. The epifluorescence image shows increased intracellular vesicle formation in MEFs induced by CNP / PBS stimulation, measured using a red fluorescence spot of PKH26 dye. [Figure 6B] This study shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. In MEFs (CNPs) perforated with CNP / PBS, CD63-GFP-containing polyvesicular bodies (MVBs) were increased compared to BEPs. Inset image: 3D intensity profile where peaks in the image represent bright spots, indicating active MVB formation. [Figure 6C] This shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Transmission electron microscopy (TEM) images of MEFs containing different amounts of MVBs and intermembryonic vesicles (ILVs) with and without CNP / PBS stimulation. [Figure 6D]This study demonstrates that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Quantification of MVB in CNP / PBS-stimulated or unstimulated MEFs. TEM images of n=20. [Figure 6E] This study shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Quantification of ILV in CNP / PBS-stimulated or unstimulated MEFs. TEM images of n=20. [Figure 6F] This study shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Western blotting revealed an increase in proteins involved in exosome biosynthesis after CNP. [Figure 6G] This study demonstrates that CNP-induced exosome secretion is associated with post-CNP Ca2+ ion influx. Longitudinal fluorescence intensity measurements of propidium iodide (PI) diffusing across membrane pores were performed in MEFs perforated with BEP and CNP in PBS buffer. A sharp increase in PI intensity at the cell attachment surface (upper insertion point) indicates the formation of a series of large pores, while the increase in PI on the opposing cell surface (lower insertion point) indicates the formation of smaller pores. A moderate increase in PI intensity was observed in MEFs perforated with BEP. [Figure 6H] This study demonstrates that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Fluorescence imaging of cells after CNP indicated that membrane pores formed during CNP closed within 1–2 minutes post-transfection. PI was applied to cells at the required time after CNP. [Figure 6I] This study demonstrates that CNP-induced exosome secretion is associated with Ca2+ ion influx after CNP. Furthermore, cell fluorescence intensity measurements confirmed that membrane pores closed within 2 minutes after CNP. Each group consisted of n=20 cells. [Figure 6J]This indicates that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. The number of exosomes per cell produced by MEF at various calcium ion concentrations after CNP. [Figure 6K] This shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Post-CNP intracellular calcium ion concentrations at various calcium ion concentrations in buffer. [Figure 6L] This indicates that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Correlation between post-CNP intracellular calcium ion concentration and exosome release. [Figure 6M] This indicates that CNP-induced exosome secretion was associated with the influx of Ca2+ ions after CNP. The number of exosomes per cell produced by MEF at various calcium ion concentrations after CNP in the presence of the calcium chelating agent EGTA. [Figure 6N] This shows that CNP-induced exosome secretion was associated with post-CNP Ca2+ ion influx. Post-CNP intracellular calcium ion concentrations at various calcium ion concentrations in the buffer in the presence of EGTA. [Figure 6O] This study shows that CNP-induced exosome secretion is associated with post-CNP Ca2+ ion influx. It also shows a correlation between post-CNP intracellular calcium ion concentration and exosome release in the presence of EGTA. [Figure 7A] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. A schematic diagram of the temperature rise simulation in a single nanochannel is shown. Data are from three independent experiments and are presented as mean ± SEM values. [Figure 7B] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Five different locations within / near the nanochannel were selected. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7C] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Temperature changes were simulated at five selected locations. A 200V and 10ms pulse created a localized "hot spot" at the nanochannel exit, resulting in peak temperatures ranging from ambient temperature to a maximum of 60°C. The "hot spot" rapidly disappeared as soon as the pulse was removed. Data are from three independent experiments and are presented as mean ± SEM values. [Figure 7D] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Top-down image of MEF attached to the CNP device surface. Before CNP (0s), dots indicate the location of nanochannels and room temperature. CNP electrical pulse (CNP) caused a rapid increase in temperature at the nanochannel / cell surface interface. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7E] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. The nanochannel cross-sections show the temperature changes within the nanochannel before (0 s), during (1 s), and after (0 s) the CNP pulse. Data are from three independent experiments and are presented as mean ± SEM values. [Figure 7F] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Temperatures measured at the cell-nanochannel interface temporarily rose to approximately 60°C (<1 s). Data are from three independent experiments and are presented as mean ± SEM values. [Figure 7G] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Western blots of HSP90 and HSP70 from untreated (PBS) and CNP / PBS-stimulated (CNP) MEFs are shown. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7H]The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. DLS measurements of exosome concentrations in CNP-stimulated 108-MEF cells with and without HSP inhibitors demonstrated that HSP70 and HSP90 have significant implications for exosome production. NVP-HSP990: HSP90 inhibitor, VER155008: HSP70 inhibitor. **P<0.01 (vs CNP), ##P<0.01 (vs single inhibitor group), Student's t-test. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7I] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. Western blotting results showed that CNP increased the expression of P53 and TSAP6 proteins in P53-WT MEFs, but did not affect the expression of P53 and TSAP6 proteins in p53- / - MEFs. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7J] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. DLS measurements of exosome concentration indicated that P53 knockdown could block exosome release after CNP to some extent. ##P<0.01 (vs CNP-P53+ / + group), Student's t-test. Data are from three independent experiments and are presented as mean ± SEM. [Figure 7K] The thermal effect of CNP increased exosome release via the HSP-P53-TASP6 signaling pathway. A schematic diagram illustrating the proposed mechanism by which CNP induces exosome release in CNP-transfected cells is shown. Data are from three independent experiments and are presented as mean ± SEM values. [Figure 8A]This shows in vitro testing and immunogenicity evaluation in mice of CNP-generating exosomes for gene therapy. A schematic diagram of a glioblastoma (GBM) targeting peptide cloned at the N-terminus of the CD47 transmembrane protein is also shown. [Figure 8B] This report describes in vitro testing and immunogenicity evaluation in mice of CNP-generating exosomes for gene therapy. Western blotting of the exosome pull-down assay showed that the FLAG beads were able to pull down FLAG-CD47, which was cloned at the N-terminus, indicating that the N-terminus of CD47 was located outside the exosome. [Figure 8C] This document presents in vitro studies and immunogenicity evaluations in mice of CNP-generating exosomes for gene therapy. It shows increased uptake by glioma (GL261) cells of CNP-generating exosomes coated with a CD47-linked brain tumor-targeting peptide. Exosomes: Uncoated exosomes. Exo-T: Exosomes generated from CNP-stimulated BMDCs transfected with the CREKA-CD47 vector. [Figure 8D] This report shows in vitro testing of CNP-generating exosomes for gene therapy and immunogenicity evaluation in mice. The fluorescence intensity of PKH26-labeled Exo-T incorporated into GL261 cells was evaluated by flow cytometry, indicating better uptake of Exo-T by GL261 cells. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosomes). Student's t-test (Figures 8D, 8F, and 8H). [Figure 8E] This shows in vitro testing of CNP-generating exosomes for gene therapy and immunogenicity evaluation in mice. Representative confocal microscopy images of GL261 cells stained with PTEN 24 hours after PBS, exosome, or Exo-T treatment. [Figure 8F]This report presents in vitro testing of CNP-generating exosomes for gene therapy and immunogenicity evaluation in mice. Flow cytometry of PTEN staining fluorescence intensity 24 hours after incubation of GL261 with exosomes showed stronger PTEN protein expression in the Exo-T group. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosomes). Student's t-test (Figures 8D, 8F, and 8H). [Figure 8G] This shows in vitro testing of CNP-generating exosomes for gene therapy and immunogenicity evaluation in mice. Representative immunostaining images show co-localization of PKH26-labeled Exo-T vesicles (red) with different endocytosis markers (green). The results indicate that most Exo-T vesicles co-localize with A488-Tf, suggesting that Exo-T vesicles are primarily taken up via clathrin-dependent endocytosis. A488-Tf: clathrin-dependent endocytosis marker, A488-CT-B: caveolae-dependent endocytosis marker, and FITC-dextran: macropinocytosis marker. [Figure 8H] This report describes in vitro testing of CNP-generating exosomes for gene therapy and immunogenicity evaluation in mice. Flow cytometry fluorescence intensity analysis of PKH26-labeled Exo-T uptake by GL261 under different inhibitory conditions further indicated that Exo-T was primarily taken up via clathrin-dependent endocytosis. Sucrose: clathrin-dependent endocytosis inhibitor; Filipinopropyl alcohol: caveolae-dependent endocytosis inhibitor; and Wortinin: macropinocytosis inhibitor. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosomes). Student's t-test (Figures 8D, 8F, and 8H). [Figure 8I]This report describes in vitro testing of CNP-generating exosomes for gene therapy and their immunogenicity evaluation in mice. The viability of GL261 cells treated with empty lipofectamine (E-Lipo), exosomes, and Exo-T indicates the good biocompatibility of Exo-T. [Figure 8J] This document presents in vitro studies and immunogenicity evaluations in mice of CNP-generating exosomes for gene therapy. It also shows the viability of GL261 cells treated with lipofectamine containing PTEN mRNA, exosomes, and Exo-T. [Figure 8K] This document shows in vitro testing and immunogenicity evaluation in mice of CNP-generating exosomes for gene therapy. The circulating half-life of PKH26-labeled exosomes administered systemically to mice is shown. Overexpression of CD47 protein significantly extended the circulating half-life of exosomes, and insertion of the CREKA peptide did not affect this. Exo-C: Exosomes derived from BMDC transfected with the CNP / CD47 vector. Exo-T: Exosomes derived from BMDC transfected with the CNP / CREKA-CD47 vector. Inserted image: Confirmation of CD47 protein expression in BMDC-derived exosomes transfected with the CREKA-CD47 vector. [Figure 8L] This document shows in vitro studies and immunogenicity evaluations in mice of CNP-generating exosomes for gene therapy. AST, ALT, creatinine, BUN, IL-6, and TNF levels are measured by ELISA after administration of different doses of CREKA-CD47-targeted exosomes (Exo-T). [Figure 9] This document presents an exemplary gating strategy for flow cytometry analysis targeting exosomes. [Figure 10A]This shows an in vitro test of CNP-generating exosomes for gene therapy in U87 cells. Increased uptake of CNP-generating exosomes coated with CD47-linked brain tumor targeting peptide (CDX) by glioma (U87) cells. Exosome: Uncoated exosome. Exo-T: Exosomes generated from CNP-stimulated MEFs transfected with the CDX-CD47 vector. [Figure 10B] This shows an in vitro test of CNP-generating exosomes for gene therapy in U87 cells. Flow cytometry fluorescence intensity of PKH26-labeled Exo-T taken up by U87 cells further confirmed that Exo-T was better taken up by U87 cells. [Figure 10C] This shows in vitro testing of CNP-producing exosomes for gene therapy in U87 cells. Representative confocal microscopy images of PTEN staining in U87 cells 24 hours after PBS, exosome, or Exo-T treatment. [Figure 10D] This shows an in vitro test of CNP-generating exosomes for gene therapy in U87 cells. Flow cytometry fluorescence intensity of PTEN staining 24 hours after incubation of U87 cells with exosomes showed that PTEN protein expression was stronger in the Exo-T group. [Figure 10E] This image shows an in vitro test of CNP-producing exosomes for gene therapy in U87 cells. Representative immunohistochemical images show the co-localization of PKH26-labeled Exo-T vesicles (red) with different endocytosis markers (green). The results indicate that most Exo-T vesicles co-localize with A488-Tf, suggesting that Exo-T vesicles are primarily taken up via clathrin-dependent endocytosis. A488-Tf: clathrin-dependent endocytosis marker, A488-CT-B: caveolae-dependent endocytosis marker, and FITC-dextran: macropinocytosis marker. [Figure 10F]This shows in vitro testing of CNP-producing exosomes for gene therapy in U87 cells. Flow cytometry fluorescence intensity analysis of PKH26-labeled Exo-T uptake by U87 under different inhibitory conditions further confirmed that Exo-T was primarily taken up via clathrin-dependent endocytosis. Sucrose: clathrin-dependent endocytosis inhibitor; Filipinolite: caveolae-dependent endocytosis inhibitor; and waltinin: macropinocytosis inhibitor. [Figure 10G] This shows in vitro testing of CNP-generating exosomes for gene therapy in U87 cells. The viability of U87 cells treated with empty lipofectamine (E-Lipo), exosomes, and Exo-T indicates the good biocompatibility of Exo-T. [Figure 10H] This shows in vitro testing of CNP-producing exosomes for gene therapy in U87 cells. The viability of U87 cells treated with lipofectamine containing PTEN mRNA, exosomes, and Exo-T is also shown. [Figure 10I] This shows in vitro testing of CNP-producing exosomes for gene therapy in U87 cells. AST, ALT, creatinine, BUN, IL-6, and TNF levels measured by ELISA at different time points in mice using different types of exosomes are also shown. The results demonstrated that Exo-T did not exhibit significant in vivo toxicity or immunogenicity in mice. [Figure 11] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11A]This demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in an orthotopic glioma model of U87. In vivo imaging shows selective accumulation of PKH-26-labeled Exo-T in U87 tumors orthotopically transplanted into nude mice. [Figure 11B] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in an orthotopic glioma model of U87. In vivo imaging shows the selective accumulation of PKH-26-labeled Exo-T in U87 tumors orthotopically transplanted into nude mice. Targeted delivery of Exo-T to brain tumors was also confirmed by biofluorescence microscopy, which showed a significant increase in Exo-T accumulation in the tumor stroma compared to uncoated exosomes (exosomes) or TurboFect nanoparticles (Turbo). [Figure 11C] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Exosome intensity at tumor sites was quantified at various time points. Three mice were used per group, with 10 images per mouse. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11D] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Tissue distribution analysis showed that Exo-T accumulated less in the liver and spleen, and improved targeting to the brain. [Figure 11E]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Tissue distribution analysis showed that Exo-T accumulated less in the liver and spleen, and improved targeting to the brain. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11F] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Tumor growth inhibition was observed via tail vein injection with PBS, PTEN mRNA-containing exosomes (exosomes), Exo-T, empty Exo-T (E-Exo-T), or TurboFect nanoparticles (Turbo). n=3 mice per group. [Figure 11G] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Tumor growth inhibition was mediated by tail vein injection of PBS, PTEN mRNA-containing exosomes (exosomes), Exo-T, empty Exo-T (E-Exo-T), or TurboFect nanoparticles (Turbo). n=3 mice per group. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11H]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. PTEN mRNA Exo-T extended the survival time of mice with U87 glioma (p<0.001, Bonferroni-corrected log-rank test). n=8 mice per group. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11I] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Western blotting of PTEN protein levels in GBM tumors indicated restoration of PTEN protein expression in PTEN-deficient U87 GBM tumors. n=3 mice per group. [Figure 11J] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. qPCR for mRNA levels in GBM tumors indicated restoration of mRNA expression in PTEN-deficient U87 GBM tumors. n=3 mice per group. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11K] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. PTEN, Ki67, and H&E staining of residual GBM tumor tissue treated with different methods showed that Exo-T restored PTEN expression and inhibited tumor cell proliferation. [Figure 11L]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. Ki67 intensity measurements were taken from IHC images using ImageJ software. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 11M] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a U87 orthotopic glioma model. PTEN intensity was measured using IHC images with ImageJ software. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 11C, 11E, 11G, 11H, 11J, 11L, and 11M). [Figure 12A] This shows the in vivo biodistribution of Exo-T within the tumor stroma. Representative biofluorescence images of mouse GBM tumor stroma at various time points after administration of different nanocarriers labeled with PKH26 showed that Exo-T accumulation in GBM tumors was superior compared to other treatments. Each group consisted of n=3 mice. [Figure 12B] This image shows the in vivo distribution of Exo-T within the tumor stroma. PKH26-bound exosomes are segmented from the overall image. [Figure 13] This shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. [Figure 13A] This shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN, Ki67, and H&E staining of normal brain tissue treated differently did not have a direct effect on normal brain tissue. [Figure 13B]This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN and H&E staining of differently treated cardiac tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 13C] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN and H&E staining of liver tissue treated differently demonstrated that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 13D] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN and H&E staining of spleen tissue treated differently demonstrated that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 13E] This image shows immunohistochemical staining of different tissues in a U87 orthotopic glioma model. PTEN and H&E staining of differently treated lung tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 13F] This shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN and H&E staining of differently treated kidney tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 13G] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. Ki67 intensity measurement of IHC images using ImageJ software. [Figure 13H] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 13I] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 13J]This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 13K] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 13L] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 13M] This image shows immunohistochemical staining of different tissues in the U87 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 14A] This demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in an orthotopic glioma model of GL261. In vivo imaging shows selective accumulation of PKH-26-labeled Exo-T in GL261 tumors orthotopically transplanted into C57BL / 6 mice. [Figure 14B] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. In vivo imaging shows the selective accumulation of PKH-26-labeled Exo-T in GL261 tumors orthotopically transplanted into C57BL / 6 mice. Targeted delivery of Exo-T to brain tumors was also confirmed by biofluorescence microscopy, which showed a significant increase in Exo-T accumulation in the tumor stroma compared to uncoated exosomes (exosomes) or PEG-liposome nanoparticles (liposomes). [Figure 14C]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Exosome intensity at tumor sites was quantified at various time points. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM values. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14D] This shows the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Distribution of PBS (top panel) and PKH26-bound Exo-T (bottom panel) in normal tissue and tumor regions; scale bar: 500 μm. [Figure 14E] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Tissue distribution analysis showed that Exo-T accumulated less in the liver and spleen, and improved targeting to the brain. [Figure 14F] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Tissue distribution analysis showed that Exo-T accumulated less in the liver and spleen, and improved targeting to the brain. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14G] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Tumor growth inhibition was achieved by treatment with PBS, PTEN mRNA-containing exosomes (exosomes), Exo-T, empty Exo-T (E-Exo-T), or PEG-liposome nanoparticles (liposomes) via tail vein injection. n=3 mice per group. [Figure 14H]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Tumor growth inhibition was induced by treatment with PBS, PTEN mRNA-containing exosomes (exosomes), Exo-T, empty Exo-T (E-Exo-T), or PEG-liposome nanoparticles (liposomes) via tail vein injection. n=3 mice per group. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14I] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. PTEN mRNA Exo-T extended the survival time of mice with GL261 gliomas (p<0.001, Bonferroni-corrected log-rank test). Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14J] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Western blotting of PTEN protein levels in GBM tumors showed that PTEN protein expression was restored in PTEN-deficient GL261 GBM tumors. (n=3 mice per group.) [Figure 14K]This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. qPCR for mRNA levels in GBM tumors showed restored mRNA expression in PTEN-deficient GL261 GBM tumors. Each group consisted of 3 mice. Unless otherwise noted, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14L] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. PTEN, Ki67, and H&E staining of residual GBM tumor tissue treated with different methods showed that Exo-T restored PTEN expression and inhibited tumor cell proliferation. [Figure 14M] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. Ki67 intensity measurements were taken from IHC images using ImageJ software. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 14N] This study demonstrates the in vivo therapeutic efficacy of CNP-producing exosomes in a GL261 orthotopic glioma model. PTEN intensity was measured using IHC images with ImageJ software. Unless otherwise specified, data are from three independent experiments and are presented as mean ± SEM. *P<0.05, **P<0.01 (vs PBS), ##P<0.01 (vs exosome group), Student's t-test (Figures 14C, 14F, 14H, 14I, 14K, 14M, and 14N). [Figure 15] This shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. [Figure 15A] This shows immunohistochemical staining of different tissues in the GL261 orthotopic glioma model. PTEN, Ki67, and H&E staining of normal brain tissue treated differently did not have a direct effect on normal brain tissue. [Figure 15B] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN and H&E staining of differently treated cardiac tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 15C] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN and H&E staining of liver tissue treated differently demonstrated that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 15D] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN and H&E staining of spleen tissue treated differently demonstrated that Exo-T had no effect on the tissues examined. Magnification: ×100. [Figure 15E] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN and H&E staining of differently treated lung tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 15F] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN and H&E staining of differently treated kidney tissues showed that Exo-T had no effect on the tissues examined. Magnification: ×400. [Figure 15G] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. Ki67 intensity measurements of IHC images were performed using ImageJ software. [Figure 15H]This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 15I] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 15J] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 15K] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 15L] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Figure 15M] This image shows immunohistochemical staining of different tissues in a GL261 orthotopic glioma model. PTEN intensity measurement of IHC images using ImageJ software. [Modes for carrying out the invention]

[0020] While preferred embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions will be conceived by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in the practice of the present disclosure. The claims define the scope of the present disclosure, and the methods and structures within these claims, as well as their equivalents, are intended to be covered thereby.

[0021] The use of absolute or chronological terms, such as “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” is exemplary and not intended to limit the scope of the embodiments disclosed herein.

[0022] As used herein, the singular forms "a," "an," and "the" are intended to similarly include the plural forms unless the context explicitly indicates otherwise. Furthermore, the terms "including," "include," "having," "has," "with," or variations thereof are intended to be comprehensive in a similar manner to the term "comprising," to the extent that they are used in any form for carrying out the invention and / or in the claims.

[0023] As used herein, the phrases “at least one,” “one or more,” and “and / or” are non-restrictive expressions that are both conjunctive and disjunctive in use. For example, each of the expressions “at least one of A, B, and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “one or more of A, B, or C,” and “A, B, and / or C” means A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together.

[0024] As used herein, "or" may refer to "and," "or," or "and / or," and may be used exclusively and inclusively. For example, the term "A or B" may refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the specific meaning may be determined by the context.

[0025] Any systems, methods, software, and platforms described herein are modular and not limited to sequential processes. Therefore, terms such as "First" and "Second" do not necessarily imply priority, order of importance, or order of actions.

[0026] As used herein, the term "about" refers to a number or range of numbers, meaning that the number or range referred to is an approximation within the range of experimental variation (or statistical experimental error), and that the number or range may vary by, for example, 1% to 15% of the stated number or range. Unless otherwise indicated by the context, the term "about" refers to ±10% of the stated number or value.

[0027] The term "approximately" means within an acceptable margin of error for a particular value, as determined by those skilled in the art, and this depends in part on how the value is measured or determined, for example, on the limitations of the measuring system. For example, "approximately" may mean, in a given value, a standard deviation of 1 or greater than 1, according to convention. Where a particular value is described in this application and claims, unless otherwise indicated, the term "approximately" shall be assumed to mean within an acceptable margin of error for that particular value.

[0028] In this specification, the terms “increased,” “growth,” or “increase” are used to generally mean an increase of a statistically significant amount. In some cases, the terms “increased” or “increase” mean an increase of at least 10% compared to a baseline level, for example, an increase of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or the maximum increase including 100%, or any increase between 10% and 100%. Other examples of “increase” include increases of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 1000, or more compared to a baseline level.

[0029] In this specification, the terms “decreased,” “decrease,” or “decrease” are used to generally mean a decrease of a statistically significant amount. In some cases, the terms “decreased” or “decrease” mean a decrease of at least 10% compared to a reference level, for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or the greatest decrease including 100% (e.g., the level is absent or undetectable compared to a reference level), or any decrease between 10% and 100%. With respect to markers or symptoms, these terms mean a statistically significant decrease to such a level. For example, a decrease could be at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably a decrease to a level that would be accepted as within the normal range for an individual without a given disease.

[0030] The terms “patient” or “subject” are used interchangeably herein and include mammals. Non-exclusive examples of mammals include any member of the mammal class: humans, non-human primates such as chimpanzees, and other apes and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs.

[0031] As used herein, “cell” generally refers to a biological cell. A cell is the basic structural, functional, and / or biological unit of a living organism. A cell may originate from any living organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, plant-derived cells, fungal cells (e.g., yeast cells, mushroom-derived cells), animal cells, cells of invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells of vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), or cells of mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, a cell does not originate from a natural living organism (e.g., a cell is synthesized and is sometimes called an artificial cell). In some cases, a cell is a primary cell. In some cases, a cell is a cell derived from a cell line.

[0032] As used herein, the term “nucleotide” generally refers to a combination of base-sugar-phosphate. Nucleotides include synthetic nucleotides. Nucleotides include synthetic nucleotide analogs. Nucleotides are monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Examples of such derivatives include [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

[0033] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides, or analogs thereof, and which are single-stranded, double-stranded, or multi-stranded. In some cases, polynucleotides are exogenous (e.g., heterologous polynucleotides). In some cases, polynucleotides are intracellular. In some cases, polynucleotides can exist in a cell-free environment. In some cases, polynucleotides are genes or fragments thereof. In some cases, polynucleotides are DNA. In some cases, polynucleotides are RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. In some cases, polynucleotides contain one or more analogs (e.g., modified backbone, sugar, or nucleic acid base). Modification of the nucleotide structure may be performed before or after polymer construction. Some non-exclusive examples of analogs include 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholinos, immobilized nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin.Non-limiting examples of polynucleotides include the coding or uncoding regions of genes or gene fragments, loci(s) revealed by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), uncoding RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In some cases, nucleotide sequences are interrupted by non-nucleotide components.

[0034] "Completely intact" and "substantially intact" refer to nucleic acids described herein that have a nucleic acid sequence that can be transcribed and / or translated into any of the therapeutic polypeptides described herein. A completely intact nucleic acid refers to a full-length nucleic acid sequence that is not partially degraded or fragmented. For example, a completely intact nucleic acid may be a messenger RNA that can be translated into a full-length protein, such as one of the therapeutic polypeptides described herein. In general, completely intact or substantially intact messenger RNA can be translated into polypeptides. Generally, messenger RNA includes a 5' cap that can assist in binding to ribosomes and a poly(A) chain that may be useful for translation. The term "substantially intact" refers to a nucleic acid sequence that may be partially degraded or fragmented but is still transcribed and / or translated into any of the therapeutic polypeptides described herein. For example, a substantially intact nucleic acid may be a partially degraded or fragmented messenger RNA that can be translated into any of the therapeutic polypeptides described herein.

[0035] As used herein, the terms “polypeptide,” “peptide,” and “protein” may be used interchangeably with respect to polymers of amino acid residues. A polypeptide may refer to a full-length polypeptide, such as one translated from a coding region open reading frame or one obtained by processing into its mature form. A polypeptide may also refer to a protein degradation or processing fragment that is still uniquely or identifiable within a particular protein. A polypeptide can be a single linear polymer of amino acids, linked to one another by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrates or phosphorylation. Polypeptides can be heterologous polypeptides.

[0036] As used herein, the term “fragment” or its synonyms may refer to a protein locus that is shorter than that of a full-length protein and, if desired, maintains protein function. “Percentage of identity” and “identity %” refer to the degree to which two sequences (nucleotides or amino acids) have identical residues at the same positions in alignment. For example, “the amino acid sequence is X% identical to sequence number Y” refers to the % identity of the amino acid sequence with respect to sequence number Y, and further details that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0037] As used herein, the term "in vivo" is used to describe events occurring within the body of a subject.

[0038] As used herein, the term “ex vivo” is used to describe events occurring outside the body of a subject. “Ex vivo” assays cannot be performed directly on the subject; rather, they are performed on samples isolated from the subject, such as biological samples obtained from the subject. “Ex vivo” is used to describe events occurring inside intact cells or other types of biological samples outside the body of the subject.

[0039] As used herein, the term "in vitro" is used to describe the events that occur within a laboratory reagent container when a substance derived from a living organism is placed in a container to separate it from that organism. In vitro assays may include cell-based assays that use living or dead cells or other biological substances. In vitro assays may also include cell-free assays that do not use intact cells.

[0040] "To treat" or "treatment" refers to both therapeutic procedures and preventive or protective measures, the purpose of which is to prevent or slow (alleviate) the targeted pathological condition or disorder. Those requiring treatment include those who are already ill, those who are susceptible to the ill, or those whose illness should be prevented. Therapeutic effect may refer to the eradication or remission of the treated symptom or underlying disorder. Therapeutic effect is achieved by the eradication or remission of one or more physiological symptoms associated with the underlying disorder, so that the subject shows improvement, even though the subject may still suffer from the underlying disorder. Preventive effects may include delaying, preventing, or eliminating the onset of the disease or condition, delaying or eliminating the onset of the disease or condition, slowing, stopping, or reversing the progression of the disease or condition, or any combination thereof. With regard to preventive effects, subjects at risk of progression of a particular disease, or those reporting one or more physiological symptoms of the disease, may receive treatment even if they have not been diagnosed with the disease.

[0041] As used interchangeably herein, the terms “effective dose” and “therapeutic dose” generally refer to an amount sufficient to produce the desired activity when administered to a target subject, such as a pharmaceutical composition containing the composition described herein. In the context of this disclosure, the term “therapeutic” refers to an amount of a pharmaceutical composition that may be sufficient to delay the onset, slow the progression, alleviate or reduce at least one symptom of a disorder treated by the methods of this disclosure.

[0042] The terms “medically acceptable carrier,” “medically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refer to medicinally acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. A component is “medically acceptable” in the sense that it is compatible with other components of a pharmaceutical formulation. Furthermore, the component may be suitable for use in contact with human and non-human mammalian tissues or organs, corresponding to a reasonable benefit-risk ratio, without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications.

[0043] The term "pharmaceutical composition" refers to a system or a mixture of systems, or a composition containing each component of a system disclosed herein, having other chemical components such as diluents or carriers. Pharmaceutical compositions can facilitate the administration of a system or components of a system to a subject. There are several techniques in the art for administering compounds, including, but not limited to, oral, injectable, spray, parenteral, and topical administration.

[0044] The term “transfection” or “transfected” generally refers to the introduction of nucleic acid constructs into cells by non-viral or virus-based methods. In some cases, the nucleic acid molecule is a gene sequence encoding a complete protein or a functional portion thereof. In other cases, the nucleic acid molecule is an untranslated sequence. In some cases, transfection methods are used to introduce nucleic acid molecules into cells in order to produce genetically modified animals. Such techniques include pronuclear microinjection, retroviral-mediated gene transfer into germline cells, gene targeting into embryonic stem cells, embryonic electroporation, sperm-mediated gene transfer, and pre-nuclear transfer in vitro transformation of somatic cells such as cumulus oophorus or mammary gland cells, or adult, embryonic, or embryonic stem cells.

[0045] "Nanoelectroporation" or "nanochannel electroporation" refers to the transfection of cells with at least one heterologous polynucleotide, such as a vector, by generating an electric field to load at least one heterologous polynucleotide into a nanochannel, thereby promoting the entry of at least one heterologous polynucleotide into the cell. The cell to be transfected is positioned at the opening of the nanochannel, where the electric field of nanoelectroporation creates a pore in the cell membrane, allowing at least one heterologous polynucleotide to be introduced into the cell.

[0046] As used herein, “plasmid” generally refers to a non-viral expression vector, such as a nucleic acid molecule encoding a gene and / or a regulatory element necessary for gene expression. As used herein, the term “vector” generally refers to a nucleic acid molecule capable of transporting or carrying a payload nucleic acid molecule. The payload nucleic acid molecule can generally be ligated (e.g., inserted) into the vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication within a cell, or sequences sufficient to be integrated into a host cell gene (e.g., host cell DNA). Examples of vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. As used herein, “viral vector” generally refers to a viral nucleic acid capable of carrying another nucleic acid into a cell. A viral vector, when present in a suitable environment, can direct the expression of one or more proteins encoded by the genes transported by the vector. Examples of viral vectors include, but are not limited to, gamma retroviruses, alpha retroviruses, formy viruses, lentiviruses, adenoviruses, or adeno-associated virus vectors. Vectors in any aspect of this disclosure may include exogenous, endogenous, or heterogeneous regulatory sequences such as promoters and / or enhancers.

[0047] Overview This disclosure relates to the design and production of one or more extracellular vesicles (e.g., exosomes) expressing at least one targeting polypeptide and / or transporting a therapeutic cargo (e.g., mRNA). In some examples, the targeting polypeptide may enhance the targeting and accumulation of the extracellular vesicle to target cells such as diseased cells, cancer cells, tumor cells, non-cancerous lesion cells, cells of damaged tissue, or cells of healthy tissue. In some cases, the targeting polypeptide is a tumor-targeting polypeptide. The targeting polypeptide may include an adapter polypeptide comprising a transmembrane domain and an extracellular domain. The targeting polypeptide may also include a heterologous targeting domain ligated to the extracellular domain of the adapter polypeptide. The extracellular vesicle may be designed to transport a payload, such as a therapeutic agent, to be delivered to a target cell. In some cases, therapeutic agents delivered by extracellular vesicles may include therapeutic compounds (e.g., therapeutic polynucleotides, therapeutic DNA, therapeutic RNA, therapeutic mRNA, therapeutic miRNA, therapeutic tRNA, therapeutic rRNA, therapeutic siRNA, therapeutic shRNA, therapeutic SRPRNA, therapeutic tmRNA, therapeutic gRNA, or therapeutic crRNA). In some cases, therapeutic agents delivered by extracellular vesicles may include therapeutic untranslated polynucleotides (e.g., untranslated RNA, lncRNA, piRNA, snoRNA, snRNA, exRNA, or scaRNA), therapeutic polypeptides, therapeutic compounds, or cancer drugs. In some cases, extracellular vesicles may transport non-therapeutic compounds (e.g., non-therapeutic polynucleotides).

[0048] This disclosure provides a method for producing a large number of exosomes containing large amounts of mRNA transcripts, even from cells with low basal exosome secretion. One approach provided herein involves nanoelectroporating at least one heterologous polynucleotide, such as a vector (e.g., plasmid), into extracellular vesicle donor cells, where at least one heterologous polynucleotide encodes a targeting polypeptide. The targeting polypeptide can enhance the targeting and accumulation of extracellular vesicles into targeted cancer cells, tumors, non-cancerous lesion cells, damaged tissue, or healthy tissue. In some cases, the extracellular vesicle donor cells are primary cells (e.g., primary adherent cells). In some cases, the extracellular vesicle donor cells are cell lines. In some cases, the extracellular vesicle donor cells are not genetically modified prior to nanoelectroporation.

[0049] This specification describes a method for treating a disease or disorder such as cancer or tumor (e.g., malignant tumor, benign tumor) in a subject, comprising systemically administering at least one extracellular vesicle to the subject. In some cases, the extracellular vesicle contains at least one therapeutic polynucleotide (e.g., therapeutic mRNA, miRNA, etc.). In some cases, the extracellular vesicle containing the therapeutic polynucleotide can be obtained by nanoelectroporating at least one extracellular vesicle donor cell with at least a first vector and at least a second vector (e.g., a plasmid), wherein the first vector encodes a tumor-targeting polypeptide comprising an extracellular vesicle surface protein covalently linked to a tumor-targeting domain, and the second vector encodes the therapeutic polynucleotide. In some embodiments, the extracellular vesicle surface protein is CD47. In some cases, the extracellular surface protein (e.g., CD47) is covalently linked to the tumor-targeting domain. In some cases, the tumor-targeting polypeptide can be obtained by expressing the first vector in the extracellular vesicle donor cell. In some cases, a second vector can be expressed in extracellular vesicle donor cells to obtain therapeutic polynucleotides. In some embodiments, the extracellular vesicles released from the extracellular vesicle donor cells contain both tumor-targeting polypeptides and therapeutic polynucleotides. In some cases, the extracellular vesicles are collected and administered systemically to the subject. In some cases, the accumulation rate of extracellular vesicles containing tumor-targeting polypeptides in the target tumor is higher than the accumulation rate of extracellular vesicles lacking tumor-targeting polypeptides in the target tumor.

[0050] Extracellular vesicle donor cells In some cases, the extracellular vesicle donor cells described herein produce the extracellular vesicles described herein. Extracellular vesicle donor cells can be any cells that can be genetically modified or manipulated to secrete extracellular vesicles at levels higher than the basal secretion level of extracellular vesicles in that cell. Therefore, even cells with low or very low basal secretion levels of extracellular vesicles can be extracellular vesicle donor cells. In some cases, extracellular vesicle donor cells can be nucleated cells. In some cases, extracellular vesicle donor cells can be autologous cells. In such cases, extracellular vesicle donor cells can be obtained from a subject, which is then modified (e.g., by introducing a vector), the secreted extracellular vesicles are collected, and then administered to the same subject. In some cases, extracellular vesicle donor cells are allogeneic cells. In such cases, extracellular vesicle donor cells are cells obtained from a genetically different source from the subject that will later receive the extracellular vesicles secreted by the extracellular vesicle donor cells. In most cases of allogeneic extracellular vesicle donor cells, the extracellular vesicle donor cells are of the same species, but they are genetically different from the recipient cells that later receive the extracellular vesicles produced and secreted by the extracellular vesicle donor cells.

[0051] Extracellular vesicle donor cells can be of any cell type. In some cases, extracellular vesicle donor cells are eukaryotic cells (e.g., mammalian cells, human cells, non-human mammalian cells, rodent cells, mouse cells, etc.). In some examples, extracellular vesicle donor cells are cells from cell lines, stem cells, primary cells, or differentiated cells. In some embodiments, extracellular vesicle donor cells are primary cells. In some examples, extracellular vesicle donor cells are mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, enucleated cells, neural stem cells, immature dendritic cells, or immune cells. Extracellular vesicle donor cells may also be adherent cells. In some cases, extracellular vesicle donor cells are adherent cells. In some cases, extracellular vesicle donor cells are suspension cells.

[0052] In some cases, extracellular vesicle donor cells contain at least one heterologous polynucleotide. In some cases, at least one heterologous polynucleotide is introduced into the extracellular vesicle donor cell by transfection. At least one heterologous polynucleotide may be transfected into the extracellular vesicle donor cell by any of the biological, chemical, or physical methods described herein, or by any other biological, chemical, or physical method. In some examples, at least one heterologous polynucleotide is transfected into the extracellular vesicle donor cell by electroporation (e.g., nanoelectroporation). In some cases, the electroporation is microchannel electroporation or nanochannel electroporation. In some examples, at least one heterologous polynucleotide is transfected into the extracellular vesicle donor cell by nanochannel electroporation. In some cases, extracellular vesicle donor cells include genetically modified cells. Examples of genetically modified cells include induced pluripotent stem cells or cells genetically modified by nucleic acid-induced nucleases (e.g., CRISPR-Cas). In some cases, extracellular vesicle donor cells are not genetically modified. For example, in some cases, extracellular vesicle donor cells are not genetically modified prior to electroporation (e.g., nanoporation).

[0053] In some cases, heterologous polynucleotides transfected into extracellular vesicle donor cells are integrated into the chromosomes of the extracellular vesicle donor cells. In some cases, heterologous polynucleotides transfected into extracellular vesicle donor cells are not integrated into the chromosomes of the extracellular vesicle donor cells. In some cases, extracellular vesicle donor cells are stably transfected with heterologous polynucleotides. In some cases, extracellular vesicle donor cells are transiently transfected with heterologous polynucleotides. In some cases, the extracellular vesicle donor cells being transfected are cells derived from a cell line. In some cases, at least one heterologous polynucleotide is a vector (e.g., a plasmid).

[0054] In some cases, extracellular vesicle donor cells may be electroporated by multiple vectors to produce and secrete extracellular vesicles. In some cases, extracellular vesicle donor cells may be nanoelectroporated by multiple vectors to produce and secrete extracellular vesicles. In some cases, the multiple vectors include at least a first vector, at least a second vector, or any additional vectors. In some cases, the first and second vectors may be nanoelectroporated simultaneously within the extracellular vesicle donor cell. In some cases, the first and second vectors may be nanoelectroporated within the extracellular vesicle donor cell at different times. In some cases, the time difference between nanoelectroporating the first and second vectors may be at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 5 hours, 12 hours, 1 day, 2 days, 5 days, 10 days, 30 days, or longer.

[0055] In some cases, the first vector may encode a tumor-targeting polypeptide. In some examples, when the first vector is nanoelectroporated into extracellular vesicle donor cells, the first vector is translated to obtain a tumor-targeting polypeptide. In some cases, the extracellular vesicle donor cells may produce extracellular vesicles or exosomes containing the tumor-targeting polypeptide. In some cases, the extracellular vesicle donor cells may secrete the extracellular vesicles or exosomes containing the produced tumor-targeting polypeptide.

[0056] In some cases, the second vector may encode at least one therapeutic polynucleotide. In some cases, when the second vector is nanoelectroporated into extracellular vesicle donor cells, the second vector is transcribed to obtain the therapeutic polynucleotide. In some cases, the extracellular vesicle donor cells produce and secrete extracellular vesicles or exosomes encapsulating the therapeutic polynucleotide encoded by the second vector. In some cases, the extracellular vesicle donor cells may produce and secrete extracellular vesicles or exosomes containing tumor-targeting peptides and the therapeutic polynucleotide encoded by the second vector.

[0057] In some cases, when a second vector is nanoelectroporated into extracellular vesicle donor cells, the second vector is transcribed or translated to obtain a therapeutic polynucleotide or therapeutic polypeptide. In some cases, the extracellular vesicle donor cells may produce and secrete extracellular vesicles or exosomes containing the therapeutic polynucleotide or therapeutic polypeptide encoded by the second vector. In some cases, the extracellular vesicle donor cells may produce and secrete extracellular vesicles containing a tumor-targeting peptide and the therapeutic polynucleotide or therapeutic polypeptide encoded by the second vector. In some examples, the therapeutic polynucleotide and therapeutic polypeptide may be encapsulated in the same extracellular vesicle or exosome. In some examples, the therapeutic polynucleotide and therapeutic polypeptide may be encapsulated in different extracellular vesicles or exosomes.

[0058] In some cases, extracellular vesicle donor cells continuously produce and secrete extracellular vesicles at a steady or basal rate. Extracellular vesicle donor cells can be any cell type, including cells with a low or very low basal rate of extracellular vesicle production and secretion. For example, extracellular vesicle donor cells may be primary or non-cancerous cells that generally do not secrete extracellular vesicles or secrete only a small number.

[0059] In some cases, extracellular vesicle donor cells produce and secrete extracellular vesicles at a basal rate. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. For example, by subjecting extracellular vesicle donor cells to heat shock treatment or by stimulating extracellular vesicle donor cells with Ca 2+By contact with an extracellular vesicle donor cell, the extracellular vesicle donor cell may be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. In some cases, by activating stress response signaling pathways such as the p53-TSAP6 signaling pathway, the extracellular vesicle donor cell may be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. In some cases, by electroporating at least one heterologous polynucleotide into the extracellular vesicle donor cell, the extracellular vesicle donor cell may be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. In some cases, by microchannel electroporation or nanochannel electroporation into the extracellular vesicle donor cell, the extracellular vesicle donor cell may be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. In some cases, by nanochannel electroporation of at least one heterologous polynucleotide into extracellular vesicle donor cells, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate greater than the basal rate. In some examples, extracellular vesicle donor cells stimulated by nanochannel electroporation may produce and secrete extracellular vesicles at a rate at least 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, 50,000 times, 100,000 times, or more than the basal rate at which the extracellular vesicle donor cells produce and secrete extracellular vesicles. In some cases, extracellular vesicle donor cells stimulated by nanochannel electroporation may produce and secrete extracellular vesicles at a rate at least 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, 50,000 times, 100,000 times, or more than the rate at which extracellular vesicle donor cells stimulated by methods other than nanochannel electroporation produce and secrete extracellular vesicles.

[0060] In some cases, the heterologous polynucleotide transfected into an extracellular vesicle donor cell encodes at least one of the targeting polypeptides described herein. In some cases, the heterologous polynucleotide transfected into an extracellular vesicle donor cell encodes a targeting polypeptide comprising at least one of the adapter polypeptides described herein. In some examples, the adapter polypeptide comprises an extracellular domain. In some examples, the adapter polypeptide comprises a transmembrane domain. In some cases, at least one targeting polypeptide comprises a peptide sequence of a heterologous targeting domain that is complexed with the extracellular domain of the adapter polypeptide. In some cases, the heterologous targeting domain is covalently complexed (e.g., fused) with the extracellular domain of the adapter polypeptide.

[0061] In some cases, the heterologous polynucleotide transfected into an extracellular vesicle donor cell encodes at least one therapeutic agent described herein. In some cases, the therapeutic agent is a therapeutic polynucleotide. In some examples, the therapeutic agent is a therapeutic polypeptide. In some examples, an extracellular vesicle donor cell transfected with at least one heterologous polynucleotide produces and secretes an extracellular vesicle containing at least one targeting polypeptide. In some examples, an extracellular vesicle donor cell transfected with at least one heterologous polynucleotide produces and secretes an extracellular vesicle containing at least one therapeutic agent. In some examples, an extracellular vesicle donor cell transfected with at least one heterologous polynucleotide produces and secretes an extracellular vesicle containing at least one targeting polypeptide comprising an adapter polypeptide (e.g., CD47 or genetically modified CD47) and a heterologous targeting domain linked to the adapter polypeptide. In some cases, extracellular vesicle donor cells transfected with at least one heterologous polynucleotide produce and secrete extracellular vesicles comprising at least one targeting polypeptide, which includes an adapter polypeptide (e.g., CD47 or genetically modified CD47) and a heterologous targeting domain linked to the adapter polypeptide, as well as at least one therapeutic agent (e.g., mRNA).

[0062] Extracellular vesicles In some cases, this specification provides compositions comprising extracellular vesicles and methods for producing extracellular vesicles. In some cases, the extracellular vesicles are any membrane-bound particles (e.g., vesicles having a lipid bilayer). In many cases, the extracellular vesicles provided herein are secreted by cells. In some examples, the extracellular vesicles are membrane-bound particles produced in vitro. In some cases, the extracellular vesicles are produced and secreted by extracellular vesicle donor cells transfected with at least one heterologous polynucleotide. In some examples, the extracellular vesicles are other very large extracellular vesicles such as exosomes, microvesicles, retrovirus-like particles, apoptosomes, oncosomes, exotheras, enveloped viruses, exomers, or large oncosomes. In some cases, the extracellular vesicle is an exosome.

[0063] In some cases, extracellular vesicles can have diameters ranging from approximately 10 nm to approximately 50,000 nm. ,000nm, approx. 20nm ~ approx. 2,000nm, approx. 20nm ~ approx. 5,000nm, approx. 20nm ~ approx. 10,000nm, approx. 20nm ~ Approximately 50,000nm, approximately 30nm to approximately 50nm, approximately 30nm to approximately 100nm, approximately 30nm to approximately 200nm, approximately 30nm to approximately 500nm , about 30nm to about 1,000nm, about 30nm to about 2,000nm, about 30nm to about 5,000nm, about 30nm to about 10,000nm nm, approximately 30 nm to approximately 50,000 nm, approximately 50 nm to approximately 100 nm, approximately 50 nm to approximately 200 nm, approximately 50 nm to approximately 500 nm, approximately 5 0nm to approx. 1,000nm, approx. 50nm to approx. 2,000nm, approx. 50nm to approx. 5,000nm, approx. 50nm to approx. 10,000nm, Approximately 50nm to approximately 50,000nm, approximately 100nm to approximately 200nm, approximately 100nm to approximately 500nm, approximately 100nm to approximately 1,000nm, Approx. 100nm to approx. 2,000nm, approx. 100nm to approx. 5,000nm, approx. 100nm to approx. 10,000nm, approx. 100nm to approx. 50 ,000nm, approx. 200nm ~ approx. 500nm, approx. 200nm ~ approx. 1,000nm, approx. 200nm ~ approx. 2,000nm, approx. 200nm ~ Approx. 5,000nm, approx. 200nm ~ approx. 10,000nm, approx. 200nm ~ approx. 50,000nm, approx. 500nm ~ approx. 1,000nm, Approximately 500nm to approximately 2,000nm, approximately 500nm to approximately 5,000nm, approximately 500nm to approximately 10,000nm, approximately 500nm to approximately 50, 000nm, approx. 1,000nm ~ approx. 2,000nm, approx. 1,000nm ~ approx. 5,000nm, approx. 1,000nm ~ approx. 10,000nm , about 1,000nm to about 50,000nm, about 2,000nm to about 5,000nm, about 2,000nm to about 10,000nm, about 2,Extracellular vesicles may have diameters of approximately 10 nm to 50,000 nm, approximately 5,000 nm to 10,000 nm, approximately 5,000 nm to 50,000 nm, or approximately 10,000 nm to 50,000 nm. In some cases, extracellular vesicles may have diameters of approximately 10 nm, approximately 20 nm, approximately 30 nm, approximately 50 nm, approximately 100 nm, approximately 200 nm, approximately 500 nm, approximately 1,000 nm, approximately 2,000 nm, approximately 5,000 nm, approximately 10,000 nm, or approximately 50,000 nm. In some cases, extracellular vesicles may have diameters of at least approximately 10 nm, 20 nm, 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm, 5,000 nm, or 10,000 nm. In some cases, extracellular vesicles may have diameters of up to approximately 20 nm, 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm, 5,000 nm, 10,000 nm, or 50,000 nm.

[0064] In some cases, the extracellular vesicle contains at least one targeting polypeptide. In some cases, the extracellular vesicle contains at least one targeting polypeptide and at least one therapeutic agent. In some cases, at least one targeting polypeptide contains an adapter polypeptide comprising a transmembrane domain and an extracellular domain. In some cases, the targeting polypeptide contains a heterologous targeting domain linked to the extracellular domain of the adapter polypeptide. In some cases, the adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the extracellular vesicle surface protein. In some cases, the adapter polypeptide contains a transmembrane domain of any one of the extracellular vesicle surface proteins or fragments thereof described herein. In some cases, at least one adapter polypeptide contains a transmembrane domain comprising a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the extracellular vesicle surface proteins described herein. In some cases, at least one adapter polypeptide includes an extracellular domain comprising a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the extracellular vesicle surface proteins described herein. In some cases, the targeting polypeptide is a tumor-targeting polypeptide comprising a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the extracellular vesicle surface protein. In some cases, the targeting polypeptide or tumor-targeting polypeptide may be covalently bound to at least one of the targeting domains or tumor-targeting domains described herein.

[0065] Extracellular vesicle surface proteins are generally proteins associated with extracellular vesicles. In some cases, extracellular vesicle surface proteins may be expressed by extracellular vesicle donor cells and integrated and secreted as part of extracellular vesicles produced and secreted by extracellular vesicle donor cells. In some examples, extracellular vesicle surface proteins include at least one extracellular domain, which may include the N-terminus, C-terminus, or both the N-terminus and C-terminus. In some cases, extracellular vesicle surface proteins may be encoded by at least one heterologous polynucleotide or vector as described herein. In some cases, extracellular vesicle surface proteins may be members of the immunoglobulin superfamily. Members of the immunoglobulin superfamily include antigen receptors, antigen-presenting molecules, co-receptors, antigen-receptor accessory molecules, co-stimulatory or inhibitory molecules, receptors on natural killer cells, receptors on leukocytes, immunoglobulin-like cell adhesion molecules, cytokine receptors, growth factor receptors, receptor tyrosine kinases, receptor tyrosine phosphatases, immunoglobulin-binding receptors, cytoskeleton, or other members. In some cases, extracellular vesicle surface proteins containing members of the immunoglobulin superfamily contain either an immunoglobulin variable domain (IgV) or an immunoglobulin constant domain (IgC). In some cases, extracellular vesicle surface proteins containing members of the immunoglobulin superfamily contain an IgV domain. Examples of members of the immunoglobulin superfamily containing IgV include differentiation antigen group proteins (e.g., CD2, CD4, CD47, CD80, or CD86), myelin membrane adhesion molecules, JAMs (junction adhesion molecules), tyrosine-protein kinase receptors, programmed cell death 1 protein (PD1), or T cell antigen receptors.

[0066] In some cases, extracellular vesicle surface proteins may be modified at the N-terminus, C-terminus, or both the N-terminus and C-terminus to include the targeting domain described herein. Generally, extracellular vesicle proteins are transmembrane proteins (e.g., proteins that span the membrane of an extracellular vesicle) having (a) an extracellular domain, (b) a transmembrane domain (e.g., a transmembrane domain), and / or (c) an intracellular domain. A model extracellular vesicle surface protein is the 14-3-3 epsilon protein, 78 kDa Glucose-regulating proteins, acetylcholinesterase (AChE-S), actin, ADAM10, alkaline phosphatase, alpha-enolase, alpha-synuclein, aminopeptidase N, amyloid A4 (APP), annexin 5A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, β-actin (ACTB), beta-amyloid 42, caveolin-1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24, CD26 / DPP4, CD29 / ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73, CD81, CD82, CD9, CD90, CD315, PTGFRN, claudin, cofilin-1, complement-binding proteins CD55 and CD59, cytoplasmic protein 1 (ACTA), cytoplasmic heat shock protein 90 alpha, cytoplasmic heat shock protein 90 beta, EBV LMP1, EBV LMP2A, EF-1 alpha-1, EF2, EFGR EGFR VIII, EMMPRIN, emmprin / CD147, Enolase 1 alpha (ENO1), EPCAM, ERBB2, Fatty acid synthase, Fetuin-A, Flotilin-1, Flotilin-2, Fructose diphosphate aldolase A, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Glycophorin A, GPC1, GPI-anchored 5' nucleotidase, GTPase, Heat shock protein 8 (HSPA8), Heat shock proteins (HSP70 and HSP90), Heparan sulfate proteoglycan, Heparinase, Heterotrimeric G protein, HIV Gag, HIVNef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax, Huntintin, ICAM1, Integrin, LAMP1 / 2, Leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, Lysosomal membrane glycoprotein 1, Lysosomal membrane glycoprotein 2, MHC class I, MHC class II, MUC1, Multidrug resistance-associated protein, Myopyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP / Alix, PECAM1, Phosphoglycerate kinase, Placental prion protein, Prostate-specific antigen (PSA), Pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap Examples include 1B, resistin, sonic hedgehog (SHH), residual, syndecan-1, syndecan-4, syntenin-1, tetraspanin (CD9, transferrin receptor (TFR2)), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, TYRP-2, vacuolar localized protein 35, zeta polypeptide (YWHAZ), or 14-3-3 zeta / delta protein. In some cases, naturally occurring extracellular vesicle surface proteins may be non-tissue specific or tissue- or cell-specific. In some cases, naturally occurring extracellular vesicle surface proteins are selected from the group consisting of CD63, CD81, CD82, CD47, CD315, heterotrimeric G proteins, MHC class I, integrins, transferrin receptors (TFR2), LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, GPI-anchored 5' nucleotidases, CD73, complement-binding proteins CD55 and CD59, and sonic hedgehog (SHH). In some cases, naturally occurring extracellular vesicle surface proteins are selected from a group consisting of TSPAN8, CD37, CD53, CD9, PECAM1, ERBB2, EPCAM, CD90, CD45, CD41, CD42a, glycophorin A, CD14, MHC class II, CD3, acetylcholinesterase / AChE-S, AChE-E, amyloid beta A4 / APP, PTGFRN, and multidrug resistance-related proteins.

[0067] In some cases, the adapter polypeptide includes at least one targeting polypeptide containing a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of CD47. In some cases, CD47 contains the sequence or a fragment of SEQ ID NO: 1. In some cases, CD47 contains a transmembrane domain that matches amino acids at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of SEQ ID NO: 1. In some cases, CD47 contains an extracellular domain that matches amino acids at positions 19-141, 198-207, or 257-268. In some cases, CD47 contains an extracellular domain that matches amino acids at positions 19-141. In some cases, CD47 contains an IgV domain that may interact with signal regulatory proteins (SIRPs) expressed by myeloid cells such as macrophages. Such interactions between CD47 and SIRP may inhibit phagocytosis of myeloid cells. In some cases, the IgV domain may be part of the extracellular domain of CD47. In some cases, the adapter polypeptides described herein contain a CD47 peptide sequence that includes the IgV domain as part of the extracellular domain. [Table 1]

[0068] In some cases, the adapter polypeptide includes an extracellular domain containing a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the CD47 peptide sequence. In some examples, the adapter polypeptide includes a transmembrane domain containing a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the CD47 peptide sequence. In some cases, the heterologous targeting domain is ligated to the extracellular domain of the adapter polypeptide containing the CD47 peptide sequence. In some cases, the heterologous targeting domain is ligated to the N-terminus of the extracellular domain of the adapter polypeptide containing the CD47 peptide sequence. In some cases, the heterologous targeting domain is ligated to the C-terminus of the extracellular domain of the adapter polypeptide containing the CD47 peptide sequence. In some cases, the heterologous targeting domain is covalently ligated to the N-terminus of the extracellular domain of the adapter polypeptide containing the CD47 peptide sequence. In some cases, the heterologous targeting domain is covalently linked to the C-terminus of the extracellular domain of an adapter polypeptide containing the CD47 peptide sequence.

[0069] In some examples, the extracellular vesicles described herein comprise multiple targeting polypeptides, each adapter polypeptide, which may comprise a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the extracellular vesicle surface polypeptides described herein. In some cases, the extracellular vesicles comprise multiple targeting polypeptides, where the multiple adapter polypeptides are identical. In some cases, the extracellular vesicles comprise multiple targeting polypeptides, where the multiple adapter polypeptides are different. In some cases, the extracellular vesicles comprise multiple targeting polypeptides, where at least one of the multiple adapter polypeptides comprises CD47.

[0070] In some cases, extracellular vesicles containing at least one targeting polypeptide (or adapter polypeptide) exhibit a longer circulating half-life compared to extracellular vesicles without targeting or adapter polypeptides. In some cases, extracellular vesicles containing at least one targeting polypeptide (or adapter polypeptide) containing CD47 or a fragment thereof exhibit a longer circulating half-life compared to extracellular vesicles without a targeting polypeptide containing CD47 or a fragment thereof. In some cases, the half-life of an extracellular vesicle containing at least one targeting polypeptide containing CD47 or a fragment thereof is at least 0.1 times, 0.2 times, 0.5 times, 1 time, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1000 times, or more, compared to the half-life of an extracellular vesicle without a targeting polypeptide containing CD47 or a fragment thereof. In some cases, the half-life of an extracellular vesicle containing at least one targeting polypeptide containing CD47 or a fragment thereof is at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer compared to an extracellular vesicle not containing a targeting polypeptide containing CD47 or a fragment thereof.

[0071] In some cases, extracellular vesicles containing at least one targeting polypeptide (and / or adapter polypeptide) indicate a circulating half-life in mammals (e.g., humans, rodents, mice) of at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, or at least 10 minutes. In some cases, extracellular vesicles containing at least one targeting polypeptide (and / or adapter polypeptide) containing CD47 or a fragment thereof indicate a circulating half-life in mammals (e.g., humans, rodents, mice) of at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, or at least 10 minutes. In some cases, extracellular vesicles containing targeting and / or adapter polypeptides indicate a circulating half-life in mammals of less than 5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes.

[0072] In some cases, extracellular vesicles contain an adapter polypeptide containing a modified CD47, where a heterologous targeting domain is attached to or complexed with the extracellular domain of the adapter polypeptide. In some cases, extracellular vesicles containing the modified CD47 adapter polypeptide exhibit a longer circulating half-life compared to extracellular vesicles without the CD47 adapter polypeptide. In some cases, the half-life of extracellular vesicles containing the modified CD47 adapter polypeptide is 0.1 times, 0.2 times, 0.5 times, 1 time, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1000 times, or more longer compared to extracellular vesicles without the CD47 adapter polypeptide. In some cases, the half-life of extracellular vesicles containing the modified CD47-containing adapter polypeptide is at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer compared to the half-life of extracellular vesicles without the CD47-containing adapter polypeptide. In some cases, the rate of circulating decrease in the number of extracellular vesicles containing the modified CD47 adapter polypeptide is 0.1 times, 0.2 times, 0.5 times, 1 time, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1000 times or more compared to the rate of circulating decrease in the number of extracellular vesicles without the CD47 adapter polypeptide, and in this case, extracellular vesicles with CD47 and CD47 Comparisons between extracellular vesicles that do not possess extracellular vesicles are made at time intervals of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer.

[0073] In some cases, extracellular vesicles containing the modified CD47 adapter polypeptide do not exhibit a shorter circulating half-life compared to extracellular vesicles containing the unmodified CD47 adapter polypeptide.

[0074] In some cases, the targeting polypeptide contains a heterologous targeting domain. In some cases, the heterologous targeting domain is attached to or complexed with the extracellular domain of the adapter polypeptide. In some cases, the heterologous targeting domain is complexed with the N-terminus, C-terminus, or both the N-terminus and C-terminus of the adapter polypeptide. In some examples, the heterologous targeting domain is covalently fused with the N-terminus, C-terminus, or both the N-terminus and C-terminus of the adapter polypeptide. Figure 8A shows an example in which a heterologous targeting domain containing either CDX or CREKA is fused with the N-terminus of the extracellular domain of an adapter polypeptide containing CD47. In some cases, the heterologous targeting domain is fused with the adapter polypeptide as part of a fusion polypeptide. In some cases, a fusion polypeptide containing a heterologous targeting domain fused with an adapter polypeptide is encoded by at least one heterologous polynucleotide or vector described herein.

[0075] In some cases, heterologous targeting domains are tumor targeting domains, tissue targeting domains, cell membrane-permeable peptides, viral membrane proteins, or combinations thereof. Heterologous targeting domains may target cell surface markers expressed on the surface of target cells. Cell surface markers can be any macromolecules or proteins expressed on the surface of target cells. Non-limiting examples of cell surface markers include vascular receptors, fibronectin receptors, A2B5, CD44, CD24, ESA, SSEA1, CD133, CD34, CD19, CD38, CD26, CD166, or CD90.

[0076] In some cases, the accumulation rate of extracellular vesicles containing at least one targeting polypeptide in target cells expressing a cell surface marker is higher than the accumulation rate of extracellular vesicles without at least one targeting polypeptide in the same target cells expressing the same cell surface marker. In some cases, the accumulation rate of extracellular vesicles containing at least one targeting polypeptide in target cells expressing a cell surface marker is at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times or more higher than the accumulation rate of extracellular vesicles without targeting polypeptide in the same target cells expressing the same cell surface marker.

[0077] In some cases, the liver and spleen accumulation rate of extracellular vesicles containing at least one targeting polypeptide directed toward target cells expressing cell surface markers, for example, the accumulation rate of extracellular vesicles in non-target cells, is reduced compared to the liver and spleen accumulation rate of extracellular vesicles without at least one targeting polypeptide directed toward the same target cells. In some cases, the liver and spleen accumulation rate of extracellular vesicles containing at least one targeting polypeptide is reduced by at least 0.1, 0.2, 0.5, 2, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times compared to the liver and spleen accumulation rate of extracellular vesicles without the targeting polypeptide.

[0078] In some cases, the targeting polypeptide includes at least one heterologous targeting domain attached to or complexed with the extracellular domain of the adapter polypeptide. In some cases, the at least one heterologous targeting domain is a tumor targeting domain, a tissue targeting domain, a cell membrane permeable peptide, a viral membrane protein, or a combination thereof. In some cases, the at least one heterologous targeting domain is a tumor targeting domain, which targets cancer cells. In some cases, the at least one heterologous targeting domain is a tumor targeting domain, which targets non-cancerous lesion cells.

[0079] In some cases, a targeting polypeptide contains at least one, two, three, four, five, or more heterologous targeting domains. In some examples, at least two heterologous targeting domains may be identical. In some cases, at least two heterologous targeting domains may be different. Heterologous targeting domains may be complexed at the N-terminus of the adapter polypeptide. Alternatively, heterologous targeting domains may be complexed at the C-terminus of the adapter polypeptide. In some cases, the complexation between heterologous targeting domains and adapter polypeptides may be covalent. For example, heterologous targeting domains may be covalently fused to the adapter polypeptide. In some examples, heterologous targeting domains may be integrated within the adapter polypeptide. In some cases, heterologous targeting domains are complexed to the adapter polypeptide via a peptide linker. In some cases, the peptide linker contains 5 to 200 amino acids. In other cases, the peptide linker contains 5 to 25 amino acids.

[0080] In some cases, the targeting polypeptide contains at least one tumor targeting domain. In some cases, the targeting polypeptide contains at least two, three, four, five, or more tumor targeting domains. In some examples, at least two tumor targeting domains are identical. In some cases, at least two tumor targeting domains are distinct. In some cases, the tumor targeting domain is fused to the N-terminus of the adapter polypeptide. In some cases, the tumor targeting domain is fused to the C-terminus of the adapter polypeptide. In some cases, the tumor targeting domain can be integrated at any peptide position on the adapter polypeptide. In some examples, the tumor targeting domain contains at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. In some cases, the tumor targeting domain is the CDX(FKESWREARGTRIERG(SEQ ID NO: 2)) peptide. In some cases, the tumor targeting domain is the CREKA(SEQ ID NO: 3) peptide. In some cases, the tumor targeting domain is the CKAAKN (SEQ ID NO: 4) peptide. In other cases, the tumor targeting domain is the ARRPKLD (SEQ ID NO: 5) peptide. Other exemplary tumor targeting domains may be included.

[0081] In some cases, the targeting polypeptide contains at least one tissue-targeting domain, which causes the extracellular vesicle containing the targeting polypeptide to target and direct cells in a specific tissue. In some cases, the targeting polypeptide contains at least two, three, four, five, or more tissue-targeting peptides. In some examples, at least two tissue-targeting peptides are identical. In some cases, at least two tissue-targeting peptides are distinct. In some cases, the tissue-targeting peptide is fused to the N-terminus of the adapter polypeptide. In some cases, the tissue-targeting peptide is fused to the C-terminus of the adapter polypeptide. In some cases, the tissue-targeting peptide can be integrated at the position of any peptide in the adapter polypeptide. In some examples, the tissue-targeting peptide contains at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Exemplary tissue-targeting domains that target endothelial or cardiac tissue include SIGYPLP (SEQ ID NO: 6), LSIPPKA (SEQ ID NO: 7), FQTPPQL (SEQ ID NO: 8), LTPATAI (SEQ ID NO: 9), CNIWGVVLSWIGVFPEC (SEQ ID NO: 10), NTTTH (SEQ ID NO: 11), VHPKQHR (tetramer) (SEQ ID NO: 12), CRKRLDRNCCRTLTVRKC (SEQ ID NO: 13), CLWTVGGGC (SEQ ID NO: 14), QPWLEQAYYSTF (SEQ ID NO: 15), and YPHIDSLGHWRR (SEQ ID NO: 6). 16) Examples include LLADTTHHRPWT (SEQ ID NO: 17), SAHGTSTGVPWP (SEQ ID NO: 18), VPWMEPAYQRFL (SEQ ID NO: 19), TLPWLEESYWRP (SEQ ID NO: 20), HWRR (SEQ ID NO: 21), CSTSMLKAC (SEQ ID NO: 22), DDTRHWG (SEQ ID NO: 23), CARPAR (SEQ ID NO: 24), CKRAVR (SEQ ID NO: 25), CRSTRANPC (SEQ ID NO: 26), CPKTRRVPC (SEQ ID NO: 27), CSGMARTKC (SEQ ID NO: 28), or CRPPR (SEQ ID NO: 29).Exemplary tissue targeting domains that target pancreatic tissue include CRVASVLPC (SEQ ID NO: 30), SWCEPGWCR (SEQ ID NO: 31), LSGTPERSGQAVKVKLKAIP (SEQ ID NO: 32), CHVLWSTRCCVSNPRWKC (SEQ ID NO: 33), or LSALPRT (SEQ ID NO: 34). Exemplary tissue targeting domains that target kidney tissue include CLPVASC (SEQ ID NO: 35), ELRGD(R / M)AX(W / L) (SEQ ID NO: 36), GV(K / R)GX3(T / S)RDXR (SEQ ID NO: 37), HITSLLSHTTHREP (SEQ ID NO: 38), or ANTPCGPYTHDCPVKR (SEQ ID NO: 39). Exemplary tissue-targeting domains that target lung tissue include CGFELETCCGFECVRQCPERC (SEQ ID NO: 40), QPFMQCLCLIYDASCRNVPPIFNDVYWIAF (SEQ ID NO: 41), VNTANST (SEQ ID NO: 42), CTSGTHPRC (SEQ ID NO: 43), or SGEWVIKEARGWKHW-VFYSCCPTTPYLDITYH (SEQ ID NO: 44). Exemplary tissue-targeting domains that target intestinal tissue include YSGKWGW (SEQ ID NO: 45), LETTCASLCYPSYQCSYTMPHPPVVPPHPMTYSCQY (SEQ ID NO: 46), YPRLLTP (SEQ ID NO: 47), CSQSHPRHC (SEQ ID NO: 48), CSKSSDYQC (SEQ ID NO: 49), CKSTHPLSC (SEQ ID NO: 50), CTGKSCLRVG (SEQ ID NO: 51), SFKPSGLPAQSL (SEQ ID NO: 52), or CTANSSAQC (SEQ ID NO: 53).Exemplary tissue-targeting domains that target brain tissue include CLSSRLDAC (SEQ ID NO: 54), GHKAKGPRK (SEQ ID NO: 55), HAIYPRH (SEQ ID NO: 56), THRPPMWSPVWP (SEQ ID NO: 57), HLNILSTLWKYRC (SEQ ID NO: 58), CAGALCY (SEQ ID NO: 59), CLEVSRKNC (SEQ ID NO: 60), RPRTRLHTHRNR(D-aa) (SEQ ID NO: 61), ACTTPHAWLCG (SEQ ID NO: 62), and GLAHSFSDFA. Examples include RDFV (sequence code 63), GYRPVHNIRGHWAPG (sequence code 64), TGNYKALHPHNG (sequence code 65), CRTIGPSVC (sequence code 66), CTSTSAPYC (sequence code 67), CSYTSSTMC (sequence code 68), CMPRLRGC (sequence code 69), TPSYDTYAAELR (sequence code 70), RLSSVDSDLSGC (sequence code 71), CAQK (sequence code 72), or SGVYKVAYDWQH (sequence code 73). Additional exemplary tissue targeting domains that target various tissues include LMLPRAD (SEQ ID NO: 74) (targets the adrenal gland), CSCFRDVCC (SEQ ID NO: 75) (targets the retina), CRDVVSVIC (SEQ ID NO: 76) (targets the retina), CVALCREACGEGC (SEQ ID NO: 77) (targets the vascular system of the skin and subcutaneous tissue), GLSGGRS (SEQ ID NO: 78) (targets the uterus), WYRGRL (SEQ ID NO: 79) (targets cartilage), CPGPEGAGC (SEQ ID NO: 80) (targets the vascular system of the chest), and SMSIARLVSFLEYR (SEQ ID NO: 81) (targets the prostate gland). Examples include (targeting the thymus), GPEDTSRAPENQQKTGC (SEQ ID NO: 82) (targeting the cutaneous Langerhans gland), CKGGRAKDC (SEQ ID NO: 83) (targeting vascular white adipose tissue), CARSKNKDC (SEQ ID NO: 84) (targeting wound or injured tissue), CHAQGSAEC (SEQ ID NO: 85) (targeting the thymus), LEPRWGFGWWLKLSTHTTESRSMV (SEQ ID NO: 86) (targeting ear or cochlear tissue), ACSTEALRHCGGGS (SEQ ID NO: 87) (targeting retinal blood vessels), or ASSLNIA (SEQ ID NO: 88) (targeting muscle tissue).

[0082] In some cases, the targeting polypeptide contains at least two, three, four, five, or more cell membrane-permeable peptides. In some cases, the targeting polypeptide containing cell membrane-permeable peptides increases the rate at which extracellular vesicles are fused or endocytized by target cells. In some examples, at least two cell membrane-permeable peptides are identical. In some cases, at least two cell membrane-permeable peptides are distinct. In some cases, the cell membrane-permeable peptides are fused to the N-terminus of the adapter polypeptide. In some cases, the cell membrane-permeable peptides are fused to the C-terminus of the adapter polypeptide. In some cases, the cell membrane-permeable peptides can be integrated at the position of any peptide in the adapter polypeptide. In some examples, the cell membrane-permeable peptides contain at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Non-limiting examples of cell membrane-permeable peptides include DSLKSYWYLQKFSWR (SEQ ID NO: 89), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 90), KSKTEYYNAWAVWERNAP (SEQ ID NO: 91), GNGEQREMAVSRLRDCLDRQA (SEQ ID NO: 92), HTPGNSNKWKHLQENKKGRPRR (SEQ ID NO: 93), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 94), R9GPLGLAGE8 (SEQ ID NO: 95), and Ac-GA Examples include FSWGSLWSGIKNFGSTVKNYG (sequence number 96), RLRWR (sequence number 97), LGQQQPFPPQQPY (sequence number 98), ILGKLLSTAAGLLSNL (sequence number 99), TFFYGGSRGKRNNFKTEEY (sequence number 100), Ac-LRKLRKRLLRX-Bpg-G (sequence number 101), Ac-LRKLRKRLLR (sequence number 102), or MVRRFLVTLRIRRACGPPRVRV (sequence number 103).

[0083] In some cases, the targeting polypeptide contains at least two, three, four, five, or more viral membrane proteins or fragments thereof. In some cases, the targeting polypeptide containing viral membrane proteins increases the rate at which extracellular vesicles are fused or endocytized by target cells. In some examples, at least two viral membrane proteins are identical. In some cases, at least two viral membrane proteins are distinct. In some cases, the viral membrane proteins are fused to the N-terminus of the adapter polypeptide. In some cases, the viral membrane proteins are fused to the C-terminus of the adapter polypeptide. In some cases, the viral membrane proteins can be integrated at any peptide position in the adapter polypeptide. In some examples, the viral membrane proteins contain at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Non-exclusive examples of viral membrane proteins include hemagglutinins, glycoprotein 41, envelope proteins, VSV G, HSV O1 gB, Ebola virus glycoproteins, or fusion-associated small transmembrane (FAST) proteins.

[0084] In some cases, the extracellular vesicles described herein contain at least one therapeutic agent. In some cases, at least one therapeutic agent is contained within the extracellular vesicle (e.g., encapsulated). In some cases, the therapeutic agent is a therapeutic polynucleotide. In some cases, the therapeutic agent is a therapeutic polypeptide. In some examples, the therapeutic agent is a therapeutic compound. In some cases, the therapeutic agent is a cancer treatment comprising a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, or a combination thereof. In some examples, the extracellular vesicles contain multiple therapeutic agents, which comprise therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, or a combination thereof.

[0085] In some cases, the extracellular vesicles described herein contain at least one targeting polypeptide. In some cases, the targeting polypeptide is a tumor-targeting polypeptide containing a tumor-targeting domain. In some cases, the accumulation rate of extracellular vesicles containing a tumor-targeting polypeptide containing a tumor-targeting domain in tumors is higher than the accumulation rate of extracellular vesicles without a tumor-targeting polypeptide. In some cases, the accumulation rate of extracellular vesicles containing a tumor-targeting polypeptide in tumors is at least 2, 5, 10, 50, 100, 200, 500, 1,000, 5,000, or 10,000 times higher than the accumulation rate of extracellular vesicles lacking a tumor-targeting polypeptide. In some cases, the accumulation rate of extracellular vesicles containing a tumor-targeting polypeptide in tumors is at least 100 times higher than the accumulation rate of extracellular vesicles lacking a tumor-targeting polypeptide.

[0086] In some cases, a tumor-targeting polypeptide contains at least one tumor-targeting domain. In some cases, the tumor-targeting domain may be located at the N-terminus of the tumor-targeting polypeptide. In some cases, the tumor-targeting domain may be located at the C-terminus of the tumor-targeting polypeptide. In some cases, the tumor-targeting domain may be located at any peptide position in the tumor-targeting polypeptide. In some cases, at least two targeting domains may be located on the same tumor-targeting polypeptide. In some cases, at least two targeting domains located on the same tumor-targeting polypeptide may be identical. In some cases, at least two targeting domains located on the same tumor-targeting polypeptide may be different. In some examples, a targeting domain contains at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. In some cases, the tumor-targeting domain may be a CDX peptide. In some cases, the tumor-targeting domain may be a CREKA peptide.

[0087] In some cases, extracellular vesicles containing extracellular vesicle surface proteins have a longer circulating half-life compared to extracellular vesicles without extracellular vesicle surface proteins. In some cases, the half-life of extracellular vesicles extended by extracellular vesicle surface proteins is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the half-life of extracellular vesicles lacking extracellular vesicle surface proteins.

[0088] In some cases, extracellular vesicles containing extracellular vesicle surface proteins exhibit reduced toxicity compared to extracellular vesicles lacking these proteins. In such cases, the extracellular vesicle surface proteins often bind specifically to their targets and exhibit no significant off-target binding. In some cases, toxicity includes toxicity to cells not targeted by tumor-targeting polypeptides. In some cases, extracellular vesicles containing extracellular vesicle surface proteins exhibit reduced toxicity by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or more, compared to extracellular vesicles lacking these proteins. In some cases, the toxicity reduction of extracellular vesicles containing extracellular vesicle surface proteins is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to extracellular vesicles lacking extracellular vesicle surface proteins.

[0089] In some cases, extracellular vesicles (e.g., exosomes) are tolerated by the patient after administration. For example, in some cases, extracellular vesicles do not induce an immune response or are not immunogenic.

[0090] Therapeutic polynucleotides In some cases, extracellular vesicles containing at least one therapeutic polynucleotide are described herein. In some examples, the at least one therapeutic polynucleotide is encoded by at least one heterologous polynucleotide or vector that is transfected into an extracellular vesicle donor cell. In some cases, the at least one therapeutic polynucleotide comprises a peptide sequence that can be targeted by a targeting polypeptide described herein and translated into a therapeutic polypeptide by the bound cell.

[0091] In some cases, the extracellular vesicle contains at least one therapeutic polynucleotide. In some cases, each extracellular vesicle contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000 or more copies of therapeutic polynucleotides. In some cases, each extracellular vesicle contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000 or more copies of therapeutic mRNA as described herein. In some cases, extracellular vesicles contain at least two therapeutic polynucleotides. In some cases, extracellular vesicles contain at least two therapeutic polynucleotides, and these at least two therapeutic polynucleotides are distinct. In some cases, the at least two distinct therapeutic polynucleotides encapsulated by the extracellular vesicle constitute different ratios. For example, the ratio between the first and second of two distinct therapeutic polynucleotides may be 1:1,000,000, 1:500,000, 1:100,000, 1:50,000, 1:10,000, 1:5,000, 1:1,000, 1:50, 1:10, 1:5, 1:4, 1:3, 1:2, or 1:1. In some cases, extracellular vesicles contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic polynucleotides encapsulated in the same extracellular vesicle. In some cases, extracellular vesicles can be exosomes.

[0092] In some cases, therapeutic polynucleotides constitute mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some cases, therapeutic polynucleotides constitute mRNA. In some cases, mRNA is fully intact or substantially intact. In some cases, mRNA codes for part of a protein. In some cases, mRNA contains at least 50, 100, 200, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 RNA nucleotides. In some examples, therapeutic polynucleotides constitute DNA. In some examples, therapeutic polynucleotides constitute DNA such as a therapeutic polypeptide or a vector encoding an RNA therapeutic agent. Therapeutic polynucleotides can encode therapeutic polypeptides (including, but not limited to, tumor suppressor proteins, peptides, wild-type protein counterparts of mutant proteins, DNA repair proteins, proteases, proteinotoxins, proteins that can inhibit the activity of intracellular proteins, proteins that can activate the activity of intracellular proteins, or any protein whose lost function needs to be reconstituted). Examples of therapeutic polypeptides that can be encoded by therapeutic polynucleotides (e.g., messenger RNA therapeutics) include 123F2, Abcb4, Abcc1, Abcg2, Actb, Ada, Ahr, Akt, Akt1, Akt2, Akt3, Amhr2, Anxa7, Apc, Ar, Atm, Axin2, B2m, Bard1, Bc1211, Becn1, Bhlhal5, Bin1, Blm, Braf, Brca1, Brca2, Brca3, Braf, Brcata, Brinp3, Brip1, Bub1b, Bwscr1a, Cadm3, Casc1, Casp3, Casp7, Casp8, Cav1, Ccam, Ccnd1, Ccr4, Ccs1, Cd28, Cdc25a, Cd95, Cdh1, Cdkn1a, Cdkn1b, Cdkn2a, Cdkn2b, Cdkn2c, Cftr, Chek1, Chek2, Crcs1, Crcs10,Crcs11、Crcs2、Crcs3、Crcs4、Crcs5、Crcs6、Crcs7、Crcs8、Crcs9 、Ctnnb1、Cts1、Cypla1、Cyp2a6、Cyp2b2、Cy1d、Dcc、Dkc1、Dicer1 、Dmtf1、Dnmt1、Dpc4、E2f1、Eaf2、Eef1a1、Egfr、Egfr4、Erbb2、Er bb4、Ercc2、Ercc6、Ercc8、Errfi1、Esr1、Etv4、Fas1g、Fbxo10、Fcc 、Fgfr3、Fntb、Foxm1、Foxn1、Fus1、Fzd6、Fzd7、Fzr1、Gadd45a、Ga st、Gnai2、Gpc1、Gpr124、Gpr87、Gprc5a、Gprc5d、Grb2、Gstm1、Gst m5、Gstp1、Gstt1、H19、H2afx、Hck、Lims1、Hdac、Hexa、Hic1、Hin1 、Hmmr、Hnpcc8、Hprt、Hras、Htatip2、I11b、I1110、I12、I16、I18rb Inha, Itgav, Jun, Jak3, Kit, Klf4, Kras, Kras2, Kras2b, Lig1, Lig4, Lkb1, Lmo7, Lncr1, Lncr2, Lncr3, Lncr4, Ltbp4, Luca1, Luca2, Lyz2, Lzts1, Mad111, Mad211, Madr2 / Jv18, Mapk14, Mcc, Mcm4, Men1, Men2, Met, Mgat5, Mif, M1h1, M1h3, Mmac1, Mmp8, Mnt, Mpo, Msh2, Msh3, Msh6, Msmb, Mthfr, Mts1, Mutyh, Myh11, Nat2, Nbn, Ncoa3, Neil1, Nf1 、Nf2、Nfe211、Nhej1、Nkx2-1、Nkx2-9、Nkx3-1、Npr12、Nqo1、Nras、Nudt1、Ogg1、Oxgr 1、p16、p19、p21、p27、p27mt、p57、p14ARF、Pa1b2、Park2、Pggt1b、Pgr、Pi3k、Pik3ca、 Piwil2、P16、Pla2g2a、Plg、P1k3、Pms1、Pms2、Pold1、Pole、Ppard、Pparg、Ppfia2、Pp m1d、Prdm2、Prdx1、Prkar1a、Ptch、PTEN、Prom1、Psca、Ptchl、Ptf1a、Ptger2、Ptpn13、Examples include Ptprj, Rara, Rad51, Rassf1, Rb, Rb1, Rb1cc1, Rb12, Recg14, Ret, Rgs5, Rhoc, Rint1, Robo1, Rp138, S100a4, SCGB1A1, Skp2, Smad2, Smad3, Smad4, Smarcb1, Smo, Snx25, Spata13, Srpx, Ssic1, Sstr2, Sstr5, Stat3, St5, St7, St14, Stk11, Suds3, Tap1, Tbx21, Terc, Tnf, Tp53, Tp73, Trpm5, Tsc2, Tsc1, Vh1, Wrn, Wt1, Wt2, Xrccl, Xrcc5, Xrcc6, or Zac1. In some cases, the therapeutic polynucleotides described herein may encode PTEN. In some cases, the therapeutic polypeptide encoded from the therapeutic polynucleotides described herein may be PTEN.

[0093] In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics, mRNA therapeutics) encapsulated in extracellular vesicles is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more per extracellular vesicle. In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics, mRNA therapeutics) encapsulated in each extracellular vesicle or exosome described herein is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more.

[0094] In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times or more compared to the copy number of therapeutic polynucleotides encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection, etc.). In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells that have been microchannel electroporated or nanochannel electroporated is increased by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times or more compared to the copy number of therapeutic polynucleotides encapsulated in extracellular vesicles produced by directly introducing the therapeutic polynucleotide into the extracellular vesicles (e.g., directly transfecting the therapeutic polynucleotide into the extracellular vesicles).

[0095] In some cases, therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation are completely or substantially intact, with at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more copies of the encapsulated therapeutic polynucleotide being completely or substantially intact. In some cases, the percentage of fully intact or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is higher compared to the percentage of fully intact or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection, etc.). In some cases, the number of fully intact or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times or more compared to the number of fully intact or substantially intact therapeutic polynucleotides encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection, etc.).In some cases, the number of fully intact or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times or more compared to the number of fully intact or substantially intact therapeutic polynucleotides encapsulated in extracellular vesicles produced by direct introduction of therapeutic polynucleotides into extracellular vesicles (e.g., direct transfection of therapeutic polynucleotides into extracellular vesicles).

[0096] In some cases, therapeutic polynucleotides constitute at least one modified nucleic acid or nucleic acid analog. Exemplary modified nucleic acids include uracil-5-yl, hypoxanthin-9-yl(I), 2-aminoadenine-9-yl, 5-methylcytosine(5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and 6-azouracil. Examples include, but are not limited to, ur, cytosine and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain modified nucleic acids include 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine (5-me-C), those that enhance the stability of double-strand formation, universal nucleic acids, hydrophobic nucleic acids, cross-hybrid nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, and adenine. 2-propyl and other alkyl derivatives of guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl(-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothimine, 5-uracil (pseuducil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanine, 5-halo especially 5-bromo,5-Trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazine cytidine (1H-pyrimide [5,4-b][1,4]benzothiazine-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indole-2-one), pyridoindole cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one), purines or These are compounds in which the pyrimidine base is replaced by another heterocycle: 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil These include 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, etc. In the art, modified nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available, and in some cases, nucleic acids contain one or more heterocyclic bases other than the five main base components of naturally occurring nucleic acids. For example, in some cases, heterocyclic bases include uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl,Examples include the 4-aminopyrrolo[2,3-d]pyrimidine-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-3-yl groups. Purines attach to the sugar portion of nucleic acids via position 9, pyrimidines via position 1, pyrrolopyrimidines via position 7, and pyrazolopyrimidines via position 1.

[0097] In some cases, nucleotide analogs are also modified at the phosphate group. Phosphate group modifications include, but are not limited to, modifications at the linkage between two nucleotides, and include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate ester or modified phosphate ester links between two nucleotides are understood to be via 3'-5' or 2'-5' linkages, and the linkages are understood to include inverted orientations such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0098] In some cases, modified nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleoside 5'-substituted DNA and RNA derivatives or 5'-substituted monomers prepared as monophosphates having modified bases.

[0099] In some cases, modified nucleic acids include modifications at the 5' and 2' positions of the sugar ring, such as 5'-CH2-substituted 2'-O-protected nucleosides. In some cases, modified nucleic acids include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleosides (5' to 3') of the dimer include 2'-OCH3 and 5'-(S)-CH3. Modified nucleic acids may include 2'-substituted 5'-CH2 (or O) modified nucleosides. Modified nucleic acids may include 5'-methylenephosphonate DNA and RNA monomers, as well as dimers. Modified nucleic acids may include 5'-phosphonate monomers with 2'-substitutions and other modified 5'-phosphonate monomers. Modified nucleic acids may include 5'-modified methylenephosphonate monomers. Modified nucleic acids may include 5' or 6'-phosphonate ribbonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions. Modified nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group. Modified nucleic acids may include nucleosides having a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogues), a methyleneamino group (CH2NH2) (and its analogues), or a cyano group (CN) (and its analogues).

[0100] In some cases, modified nucleic acids also include sugar modifications. In some cases, nucleic acids contain one or more nucleosides that have been modified with sugar groups. Such sugar-modified nucleosides may confer enhanced nuclease stability, improved binding affinity, or some other beneficial biological properties. In certain cases, nucleic acids contain chemically modified ribofuranose rings. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents); bridging of two ring atoms to form bicyclic nucleic acids (BNAs); S, N(R), or C(R1)(R2)(R=H, C1~C) of the ribosyl ring oxygen atom. 12 Substitution with alkyl groups or protecting groups; and combinations thereof.

[0101] In some examples, modified nucleic acids include modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or sugar "analog" cyclopentyl group. The sugar may be in pyranosyl or furanosyl form. The sugar moiety may also be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar may be attached to the respective heterocyclic base in either an [alpha] or [beta] anomeric configuration. Examples of sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, the sugar modification may include 2'-O-methyluridine or 2'-O-methylcytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs, and the respective "nucleosides" on which such sugars or analogs are attached to heterocyclic bases (nucleic acid bases), is known. Sugar modifications may be made with other modifications or in combination with other modifications.

[0102] Sugar modifications include not only modified modifications but also natural modifications of ribose and deoxyribose. Examples of sugar modifications, though not limited to them, include the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl, or C2-C 10 Alkenyl and alkynyl forms may also be used. The 2' sugar modification is not limited to -O[(CH2) n O] m CH3, -O(CH2) nOCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, and -O(CH2)nON[(CH2)nCH3]2 are also included, where n and m are from 1 to about 10.

[0103] Other modifications at the 2'-position include, but are not limited to, C1-C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3'-position of the sugar in the 3'-terminal nucleotide or 2'-5' linked oligonucleotide, and the 5'-position of the 5'-terminal nucleotide. Modified sugars include those containing modifications to the bridging ring oxygen such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars. There are numerous U.S. patents that teach the preparation of such modified sugar structures and elaborate on and describe a series of base modifications.

[0104] Examples of nucleic acids having sugar moiety modifications include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2'-position may also be selected from allyl, amino, azide, thio, O-allyl, O-(C1-C 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O-N(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), where each R m and R nThese are independently H or substituted or unsubstituted C1~C 10 It is alkyl.

[0105] In certain cases, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In such certain cases, the bicyclic nucleic acid comprises a bridge between a 4'-ribosyl ring atom and a 2'-ribosyl ring atom. In certain cases, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, the bridges forming a 4'-to-2' bicyclic nucleic acid. Examples of such 4'-to-2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2', and their analogues; 4'-C(CH3)(CH3)-O-2' and its analogues.

[0106] In certain cases, nucleic acids include linked nucleic acids. Nucleic acids can be linked to one another using any internucleotide linkage. The two main classes of internucleotide linkage groups are determined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide linkage groups include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleotide linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamic acid (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N * -Dimethylhydrazine (-CH2-N(CH3)-N(CH3)) is an example. In certain embodiments, internucleotide linking groups having chiral atoms, such as alkylphosphonates and phosphorothioates, can be prepared as racemic mixtures or as separate enantiomers. Modified nucleic acids may contain a single modification. Modified nucleic acids may contain multiple modifications within one part or between different parts.

[0107] Phosphate backbone modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (crosslinked or uncrosslinked), phosphotryesters, phosphorodithioates, phosphodithioates, and boranophosphates, and may be used in any combination. Other non-phosphoric linkages may also be used.

[0108] In some cases, backbone modifications (e.g., internucleotide linkages of methylphosphonates, phosphorothioates, phosphoramidates, and phosphorodithioates) can confer immunomodulatory activity to modified nucleic acids and / or enhance their stability in vivo.

[0109] In some cases, the phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and may be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc.

[0110] In some cases, backbone modifications involve replacing phosphodiester bonds with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include anionic nucleoside linkages; N3'→P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral nucleoside linkages such as methylphosphonates; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; sulfonyl group-containing backbones; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179). Modified nucleic acids may include a chimeric or mixed backbone containing one or more modifications, such as a combination of phosphate linkages, including a combination of a phosphodiester bond and a phosphorothioate bond.

[0111] Examples of phosphate substituents include short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These include those having morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 constituent sites. It is also understood that both the sugar and phosphate moieties of a nucleotide can be replaced by, for example, amide-type linkages (aminoethylglycine) (PNA) in nucleotide substituents. U.S. Patents 5,539,082; 5,714,331; and 5,719,262 teach methods for the preparation and use of PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to increase, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs.Examples of such conjugates include, but are not limited to, lipid portions (cholesterol portion, cholic acid, thioether (e.g., hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate), polyamines or polyethylene glycol chains, etc.), or adamantane acetate, palmityl portion, or octadecylamine or hexylamino-carbonyl-oxycholesterin portion.

[0112] In some cases, at least one modified nucleotide or nucleotide analog described herein may be resistant to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonuclease and 3'-5' exonuclease, compared to natural nucleic acid molecules. In some examples, at least one modified nucleotide or nucleotide analog includes 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifiers, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, such as RNase. These nucleic acids are resistant to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonuclease and 3'-5' exonuclease. In some cases, 2'-O-methyl modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-deoxy-modified nucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, T-deoxy-2'-fluoro modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl (2'-O-AP) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA-modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholino molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, PNA-modified nucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified nucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiolphosphonate-modified nucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, nucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0113] In further cases, the modified nucleotides or nucleotide analogs described herein are modified to enhance their stability. In some embodiments, the nucleic acid molecule is RNA (e.g., mRNA). In some examples, mRNA may be modified by one or more modifications to enhance its stability. In some cases, mRNA may be modified by modifications at the 2' hydroxyl position, such as 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA), or by immobilization or cross-linked ribose conformation (e.g., LNA or ENA). In some cases, at least one modified nucleotide or nucleotide analog is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, at least one modified nucleotide or nucleotide analog also includes morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoroN3-P5'-phosphoramidite to enhance its stability. In some examples, at least one modified nucleotide or nucleotide analog is a chiral-pure (or sterically-pure) nucleic acid molecule. In some examples, a chiral-pure (or sterically-pure) nucleic acid molecule is modified to enhance its stability.

[0114] In some cases, the extracellular vesicles described herein comprise at least one of the therapeutic polypeptides described herein. In some cases, at least one therapeutic polypeptide is encoded by at least one heterologous polynucleotide or vector that is transfected into the extracellular vesicle donor cell.

[0115] In some cases, the therapeutic polynucleotide is translated by extracellular vesicle donor cells to obtain at least one therapeutic polypeptide. In some cases, the therapeutic polypeptide encoded by the therapeutic polynucleotide can be encapsulated by extracellular vesicles produced and secreted by extracellular vesicle donor cells. In some cases, the extracellular vesicles can encapsulate both the therapeutic polynucleotide and the therapeutic polypeptide encoded by the nanoelectroporated vector. In some cases, the extracellular vesicles can be exosomes.

[0116] In some cases, the extracellular vesicles described herein may contain at least one therapeutic compound. In some cases, at least one therapeutic compound is complexed or anchored by one of the extracellular vesicle surface proteins described herein. In some cases, at least one therapeutic compound is located within the extracellular vesicle. Exemplary therapeutic compounds for use in the compositions and methods described herein include those for treating breast cancer, ovarian cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), pancreatic cancer, brain cancer (including brain tumors such as glioblastoma multiforme and anaplastic astrocytoma), bladder cancer, Kaposi's sarcoma, lymphoma, acute lymphoblastic leukemia, and cervical cancer. Exemplary therapeutic compounds for use in the compositions and methods described herein include therapeutic compounds that are nucleoside analogs, alkylating agents, inserts, and tubulin-targeting agents. In some embodiments, the therapeutic compounds for use in the compositions and methods described herein are selected from the group consisting of gemcitabine hydrochloride, temozolomide, doxorubicin, and paclitaxel.

[0117] Therapy using extracellular vesicles This specification describes a method for treating a target disease by administering a therapeutically effective dose of a composition or pharmaceutical composition comprising extracellular vesicles as described herein. In some cases, the extracellular vesicles comprise at least one targeting polypeptide and at least one therapeutic agent as described herein. In some cases, the targeting polypeptide comprises a heterologous targeting domain, including a tumor targeting domain, a tissue targeting domain, a cell membrane permeable peptide, a viral membrane protein, or any combination or fragment thereof. In some examples, each targeting domain binds to a cell surface marker associated with diseased cells, in which case the extracellular vesicle binds to the diseased cells while simultaneously delivering at least one therapeutic agent to the diseased cells. In some cases, the diseased cells are cancer cells. In some cases, the diseased cells are non-cancerous lesion cells. In some examples, the diseased cells are tumor cells. In some examples, the at least one therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, a cancer drug, or a combination thereof.

[0118] In some cases, uptake by target cells of therapeutic agents delivered by extracellular vesicles containing at least one targeting polypeptide is increased by at least 0.1, 0.2, 0.5, 2, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, or more compared to uptake by target cells of therapeutic agents delivered by extracellular vesicles without a targeting polypeptide. In some examples, the target cells in which uptake of therapeutic agents delivered by extracellular vesicles containing at least one targeting polypeptide is increased are cancerous cells, non-cancerous lesion cells, cells as part of a tumor, or cells as part of a tissue.

[0119] In some cases, methods for treating diseases using extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides described herein are described herein. In some cases, methods for treating tumors using extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides are described herein. In some cases, methods for treating tumors using extracellular vesicles described herein inhibit tumor growth. For example, in some cases, tumor growth may be inhibited by at least 20%, at least 30%, at least 40%, or more. In some cases, methods for treating tumors using extracellular vesicles described herein suppress tumor growth (e.g., reduction or elimination of tumor size due to tumor cell death). In some cases, methods for treating tumors include delivering therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, cancer drugs, or combinations thereof to tumor cells via extracellular vesicles. Non-limiting examples of tumor cells that may be treated by therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, cancer drugs, or combinations thereof delivered by extracellular vesicles include acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentigo melanoma, hidradenoma acromega, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute differentiated myeloid leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, malignant NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, and hydatidiform soft tissue. Tumor, ameloblastoma, anal cancer, anaplastic large cell lymphoma, histoplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratomatoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brennel tumor, bronchial tumor, bronchioloalveolar carcinoma, pheochromocytoma, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman disease, central nervous system germ cell tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, intrahepatic cholangiocarcinoma, chondroma,Chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papillary tumor, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colorectal cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cystoma, fibrinogenic round cell tumor, diffuse large B-cell lymphoma, germinal dysplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, intestinal disease-associated T-cell lymphoma, ependymoblastoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, nasal neuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer, fetal fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneurotomycosis, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational trophoblastic carcinoma, gestational trophoblastic tumor, giant cell tumor of bone, pleomorphic gliablastoma, glioma, cerebral gliomatosis, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, trichorrhizomatous cell tumor. Leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, periangiocarcinoma, angiosarcoma, hematological malignancies, primary hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, kidney cancer, Kratzkin's tumor, Krukenberg's cancer, laryngeal cancer, lentigo malignant melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteal malformation, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, bone Malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant schwannoma, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medullary epithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, primary occult metastatic cervical squamous cell carcinoma, metastatic urothelial carcinoma, Müllerian mixed tumor, monocytic leukemia, oral cancer, myxoid neoplasm, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic disorder, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder,Myxoma, nasal cavity cancer, nasopharyngeal cancer, neoplasm, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular tumor, oligodendron astrocytoma, oligodendron glioma, pallocyte tumor, optic nerve sheath meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, papillary thyroid carcinoma, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, Pharyngeal cancer, chromaffin cell tumor, intermediate pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polygermoma, progenitor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, embryonic neuroextrinsic germ cell tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancers involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland Adenocarcinoma, sarcoma, schwannoma, sebaceous carcinoma, secondary neoplasm, seminoma, serous neoplasm, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, blue small round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumor, sooty wart, spinal cord tumor, vertebral tumor, perisplenic zone lymphoma, squamous cell carcinoma, gastric cancer, superficial melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell Examples of cancers targeted include large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prelymphocytic leukemia, teratoma, end-stage lymphoma, testicular cancer, ovarian follicular cell tumor, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Werner-Morrison syndrome, wart-like carcinoma, optic tract glioma, vulvar cancer, Waldenström macroglobulinemia, Warsin tumor, Wilms tumor, and combinations thereof. In some cases, cancer cells targeted by extracellular vesicles represent subpopulations within the cancer cell population, such as cancer stem cells.

[0120] In some cases, a method for treating a muscle disease is described herein, which involves administering extracellular vesicles containing the targeting polypeptide and therapeutic polynucleotide described herein to a subject with a muscle disease. In some cases, a method for treating a muscular dystrophy in a subject using extracellular vesicles containing myocyte-targeting polypeptide and therapeutic polynucleotide is described herein. In some cases, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. In some cases, the therapeutic polynucleotide delivered to the muscle cell constitutes mRNA encoding a full-length or truncated protein. In some cases, the therapeutic polynucleotide delivered to the muscle cell constitutes an antisense oligonucleotide that induces protein exon skipping.

[0121] In some cases, a method for treating an eye disease is described herein by administering to a subject extracellular vesicle containing the targeting polypeptide and therapeutic polynucleotide described herein. In some cases, a method for treating an eye disease of a subject using an extracellular vesicle containing the ophthalmic cell targeting polypeptide and therapeutic polynucleotide is described herein. In some examples, the eye disease is a retinal disease. In some cases, the retinal disease is retinitis pigmentosa. In some examples, the retinal disease is Leber congenital amaurosis. In some examples, a method for treating a retinal disease is described herein using therapeutic polynucleotide delivered to retinal cells by the extracellular vesicle described herein.

[0122] In some cases, methods for treating a disease by administering extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides to a subject in need thereof are described herein. In some cases, extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides may be administered daily, every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. Extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides may be administered for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or longer.

[0123] If the patient's condition improves, the dose of extracellular vesicles containing the targeting polypeptide and therapeutic polynucleotide being administered may be temporarily reduced or discontinued for a certain period of time ("drug-free period"). In some cases, the length of the drug-free period varies between 2 days and 1 year, and is only an example, but could be 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. The dose reduction rate during the drug-free period can range from 10% to 100%, and these are just examples: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0124] In some cases, an effective dose of extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides may be administered to subjects in need as once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, once every thirteen weeks, once every fourteen weeks, once every fifteen weeks, once every sixteen weeks, once every seventeen weeks, once every eighteen weeks, once every nineteen weeks, once every twenty weeks, once every twenty-one weeks, once every twenty-two weeks, once every twenty-three weeks, once every twenty-four weeks, once every twenty-five weeks, once every twenty-six weeks, once every twenty-seven weeks, or once every twenty-eight weeks.

[0125] If the target disease or disease-related symptoms improve, a maintenance dose of extracellular vesicles may be administered as needed. Thereafter, the dose, frequency, or both may be reduced, depending on the symptoms, to a level that maintains improvement in the disease, disorder, or symptoms.

[0126] In some cases, the amount of extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides equivalent to such amounts varies depending on factors such as the severity of the disease and the characteristics of the subject or host requiring treatment (e.g., body weight). Nevertheless, it is routinely determined in the manner known in the art, according to the individual circumstances surrounding the case, including, for example, the specific extracellular vesicles being administered, the route of administration, and the subject or host being treated. In some cases, the desired dose is provided as a single dose, or as divided doses simultaneously (or over a short period) or at appropriate intervals, for example, two, three, four or more divided doses per day.

[0127] In some cases, the dosage may be determined at least in part by the occurrence or severity of grade 3 or grade 4 adverse events in the patient. Non-limiting examples of adverse events include hypothermia, shock, bradycardia, ventricular premature contractions, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second-degree atrioventricular (AV) block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disease, stomatitis, nausea and vomiting, abnormal liver function tests, gastrointestinal bleeding, hematemesis, bloody diarrhea, gastrointestinal disorders, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, increased alkaline phosphatase, increased blood urea nitrogen (BUN), hyperuricemia, increased nonprotein nitrogen (NPN), respiratory acidosis, lethargy, agitation, peripheral neuropathy, delusional reactions, convulsions, grand mal seizures, delirium, asthma, pulmonary edema, hyperrespiratory hyperventilation, hypoxia, hemoptysis, respiratory depression, pneumothorax, mydriasis, pupillary disorders, renal dysfunction, renal failure, and acute These include renal tubular necrosis, duodenal ulcer, intestinal necrosis, myocarditis, supraventricular tachycardia, permanent or transient blindness secondary to optic neuritis, transient ischemic attack, meningitis, cerebral edema, pericarditis, allergic interstitial nephritis, tracheoesophageal fistula, malignant hyperthermia, cardiac arrest, myocardial infarction, pulmonary embolism, stroke, hepatic or renal failure, severe depression leading to suicide, pulmonary edema, respiratory arrest, respiratory failure, leukopenia, thrombocytopenia, increased alanine aminotransferase (ALT), eating disorders, arthralgia, back pain, chills, diarrhea, hyperlipidemia, fatigue, fever, flu-like symptoms, hypoalbuminemia, increased lipase, injection site reactions, myalgia, nausea, night sweats, pruritus, rash, erythematous rash, maculopapular rash, hypertransaminasemia, vomiting, and weakness.

[0128] Given the numerous variables involved in individual treatment regimens, and the fact that significant deviations from these recommendations are not uncommon, the above ranges are merely suggestions. Such dosages will vary depending on many variables (but are not limited to, the activity of the compound used, the disease or condition being treated, the method of administration, the requirements of the individual patient, the severity of the disease or condition being treated, and the physician's judgment).

[0129] In some cases, the toxicity and therapeutic effect of such treatment regimens are determined by standard pharmaceutical methods in cell culture or experimental animals (including, but not limited to, the LD50 (the dose at which 50% of the population dies) and ED50 (the dose at which 50% of the population is therapeutically effective)). The dose-to-toxicity ratio is the therapeutic index, expressed as the ratio between the LD50 and the ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate the range of dosages to be used in humans. The dosage of such compounds is preferably within the range of circulating concentrations that include the ED50 with the lowest toxicity. The dosage varies within this range depending on the dosage form used and the route of administration utilized.

[0130] Production of extracellular vesicles In some cases, methods and systems for producing extracellular vesicles containing targeting polypeptides, therapeutic polypeptides, therapeutic compounds, cancer drugs, or combinations thereof are described herein.

[0131] In some cases, the method involves introducing at least one heterologous polynucleotide into an extracellular vesicle donor cell. In some cases, the at least one heterologous polynucleotide is a vector. In some examples, the at least one heterologous polynucleotide introduced into the extracellular vesicle donor cell encodes at least one targeting polypeptide as described herein. In some cases, the at least one heterologous polynucleotide encodes at least one heterologous targeting domain. In some examples, the at least one heterologous polynucleotide constitutes at least one therapeutic polynucleotide as described herein. In some examples, the at least one heterologous polynucleotide encodes at least one therapeutic polynucleotide as described herein. In some examples, the at least one heterologous polynucleotide encodes at least one therapeutic polypeptide as described herein.

[0132] In some cases, at least two heterologous polynucleotides are introduced into an extracellular vesicle donor cell, where the first heterologous polynucleotide constitutes a first vector encoding at least one targeting polypeptide or tumor-targeting polypeptide. In some cases, a second heterologous polynucleotide introduced into the extracellular vesicle donor cell constitutes a second vector encoding at least one therapeutic polynucleotide or at least one therapeutic polypeptide.

[0133] In some cases, heterologous polynucleotides may be introduced into cells via the use of expression vectors. With respect to expression vectors, the vectors may be readily introduced into cells as described herein by any method in the art. For example, expression vectors may be transferred into cells by biological, chemical, or physical methods. In some cases, extracellular vesicle donor cells may be any cell type described herein. In some cases, extracellular vesicle donor cells may be nucleated cells.

[0134] Biological methods for introducing a target heterologous polynucleotide into cells include the use of DNA or RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into non-human mammalian cells. In some cases, other viral vectors are derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. Exemplary viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some examples, retroviral vectors include gamma retroviral vectors such as those derived from Moloney's mouse leukemia virus (MoMLV, MMLV, MuLV, or MLV) or mouse stem cell virus (MSCV) genomes. In some examples, retroviral vectors include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some cases, AAV vectors may include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. In some cases, the viral vector is a chimeric viral vector containing a portion of a virus derived from two or more viruses. In further cases, the viral vector is a recombinant viral vector.

[0135] Chemical methods for introducing heterologous polynucleotides into cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes (e.g., artificial membrane vesicles) are exemplary colloidal systems for use as delivery media in vitro and in vivo. Other state-of-the-art methods for targeted delivery of nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0136] When nonviral delivery systems are used, the exemplary delivery medium is the liposome. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids are associated with lipids. In some cases, nucleic acids associated with lipids are encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that bind to both liposomes and oligonucleotides, enclosed within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, in some cases they exist in a bilayer structure, as micelles, or with a “disintegrated” structure. Alternatively, they may simply be dispersed in solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances, and in some cases, they can be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives (fatty acids, alcohols, amines, amino alcohols, and aldehydes, etc.).

[0137] Suitable lipids for use are obtained from commercial sources. For example, in some cases, dimyristylphosphatidylcholine ("DMPC") is obtained from Sigma (St. Louis, MO); in some cases, dicetyl phosphate ("DCP") is obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is sometimes obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala). Lipid stock solutions in chloroform or chloroform / methanol are often stored at approximately -20°C. Chloroform is used as the optimal solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, encapsulating water and dissolved solute between the lipid bilayers. However, compositions with solutions that differ from the usual vesicle structure are also included. For example, in some cases, the lipids may appear as micelle structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0138] Physical methods for introducing heterologous polynucleotides into cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, gene guns, electroporation, microneedle arrays, nanoneedle arrays, sonication, or chemical permeation. Electroporation methods include microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. In certain cases, extracellular vesicle donor cells are transfected with at least one heterologous polynucleotide by microchannel electroporation or nanochannel electroporation. In some examples, microchannel electroporation or nanochannel electroporation involves the use of microporous patterned silicon wafers, nanoporous patterned silicon wafers, tracked etched membranes, ceramic microporous membranes, ceramic nanoporous membranes, other porous materials, or combinations thereof. In some examples, at least one heterologous polynucleotide or at least one vector is nanoelectroporated into extracellular vesicle donor cells via nanochannels located on a biochip.

[0139] In some cases, extracellular vesicle donor cells may be proliferated and attached to the substrate surface. In some cases, the substrate may contain a biochip. In some cases, the substrate surface may contain a metallic material. In some cases, the substrate may contain a metallic material. Non-limiting examples of metallic materials include aluminum (Al), indium tin oxide (ITO, In2O3:SnO2), chromium (Cr), gallium arsenide (GaAs), gold (Au), molybdenum (Mo), organic residues and photoresists, platinum (Pt), silicon (Si), silicon dioxide (SiO2), SOI (silicon on insulator), silicon nitride (Si3N4), tantalum (Ta), titanium (Ti), titanium nitride (TiN), and tungsten (W). In some cases, the metallic material may be processed or etched to create an array or channel. In some cases, metal surfaces may be treated or etched with phosphoric acid (H3PO4), acetic acid, nitric acid (HNO3), water (H2O), hydrochloric acid (HCl), cerium ammonium nitrate (NH4)2Ce(NO3)6, citric acid (C6H8O7), hydrogen peroxide (H2O2), aqua regia, iodine solution, sulfuric acid (H2SO4), hydrofluoric acid (HF), potassium hydroxide (KOH), ethylenediamine pyrocatechol (EDP), tetramethylammonium hydroxide (TMAH), buffered oxides, ammonium fluoride (NH4F), SCl, Cl2, CCl4, SiCl4, BCl3, CCl2F2, CF4, O2, SF6, NF3, CHF3, or combinations thereof.

[0140] In some cases, metal surfaces may be treated with gas or plasma to increase their hydrophilicity. In other cases, metal surfaces may be treated with gas or plasma to increase their hydrophobicity. Exemplary gases or plasmas for increasing the hydrophilicity or hydrophobicity of metal surfaces include oxygen, nitrogen, ammonia, argon, chlorine, fluorine, bromine, iodine, astatine, hydrogen, or combinations thereof.

[0141] In some cases, extracellular vesicle donor cells may be grown and attached to substrate surfaces made of polymers such as polypropylene, polyethylene, polystyrene, ABS, polyamide, copolymer polyethylene, epoxy, polyester, polyvinyl chloride, phenol, polytetrafluoroethylene, copolymer polyethylene, fluorinated ethylene propylene, polymerized vinylidene, silicone, natural rubber, latex, polyurethane, styrene-butadiene rubber, fluorocarbon elastomer copolymer, polyethylene terephthalate, polycarbonate, polyamide, polyaramid, polyaryl ether ketone, polyacetal, polyphenylene oxide, PBT, polysulfone, polyethersulfone, polyarylsulfone, polyphenylene sulfide, polytetrafluoroethylene, and beryllium oxide. In some cases, the polymer surface may be semipermeable. In some embodiments, the pore size of the semipermeable polymer surface may be between about 0.01 μm and about 10 μm.In some embodiments, the pore sizes of the semipermeable polymer surface are approximately 0.01 μm to 0.03 μm, 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.2 μm, 0.01 μm to 0.3 μm, 0.01 μm to 0.4 μm, 0.01 μm to 0.5 μm, 0.01 μm to 1 μm, 0.01 μm to 3 μm, 0.01 μm to 5 μm, 0.01 μm to 10 μm, 0.03 μm to 0.05 μm, 0.03 μm to 0.1 μm, and 0.03 μm to 0. 2μm, about 0.03μm to about 0.3μm, about 0.03μm to about 0.4μm, about 0.03μm to about 0.5μm, about 0.03μm to about 1μm, about 0.03μm to about 3μm, about 0.03μm to about 5μm, about 0.03μm to about 10μm, about 0.05μm to about 0.1 μm, approximately 0.05 μm to approximately 0.2 μm, approximately 0.05 μm to approximately 0.3 μm, approximately 0.05 μm to approximately 0.4 μm, approximately 0.05 μm to approximately 0.5 μm, approximately 0.05 μm to approximately 1 μm, approximately 0.05 μm to approximately 3 μm, approximately 0.05 μm to approximately 5 μm, approximately 0.05 μm to approximately 10 μm , about 0.1 μm to about 0.2 μm, about 0.1 μm to about 0.3 μm, about 0.1 μm to about 0.4 μm, about 0.1 μm to about 0.5 μm, about 0.1 μm to about 1 μm, about 0.1 μm to about 3 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 10 μm, about 0.2 μm to about 0.3μm, about 0.2μm to about 0.4μm, about 0.2μm to about 0.5μm, about 0.2μm to about 1μm, about 0.2μm to about 3μm, about 0.2μm to about 5μm, about 0.2μm to about 10μm, about 0.3μm to about 0.4μm, about 0.3μm to about 0.5μm, about 0.3 0.3 μm to 3 μm, 0.3 μm to 5 μm, 0.3 μm to 10 μm, 0.4 μm to 0.5 μm, 0.4 μm to 1 μm, 0.4 μm to 3 μm, 0.4 μm to 5 μm, 0.4 μm to 10 μm, 0.5 μm to approx. It can be between about 1 μm, about 0.5 μm to about 3 μm, about 0.5 μm to about 5 μm, about 0.5 μm to about 10 μm, about 1 μm to about 3 μm, about 1 μm to about 5 μm, about 1 μm to about 10 μm, about 3 μm to about 5 μm, about 3 μm to about 10 μm, or about 5 μm to about 10 μm.In some embodiments, the pore size of the semipermeable polymer surface may be any of approximately 0.01 μm, approximately 0.03 μm, approximately 0.05 μm, approximately 0.1 μm, approximately 0.2 μm, approximately 0.3 μm, approximately 0.4 μm, approximately 0.5 μm, approximately 1 μm, approximately 3 μm, approximately 5 μm, or approximately 10 μm. In some embodiments, the pore size of the semipermeable polymer surface may be at least 0.01 μm, approximately 0.03 μm, approximately 0.05 μm, approximately 0.1 μm, approximately 0.2 μm, approximately 0.3 μm, approximately 0.4 μm, approximately 0.5 μm, approximately 1 μm, approximately 3 μm, or approximately 5 μm. In some embodiments, the pore size of the semipermeable polymer surface may be as large as approximately 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1 μm, 3 μm, 5 μm, or 10 μm.

[0142] In some cases, polymer surfaces may be treated with gas or plasma to increase their hydrophilicity. In some cases, polymer surfaces may be treated with gas or plasma to increase their hydrophobicity. Exemplary gases or plasmas for increasing the hydrophilicity or hydrophobicity of metal surfaces include oxygen, nitrogen, ammonia, argon, chlorine, fluorine, bromine, iodine, astatine, hydrogen, or combinations thereof.

[0143] Nanoelectroporation In some cases, extracellular vesicle donor cells grown or attached to a metal or polymer surface may be nanoelectroporated by a nanoelectroporation system as described herein. In some cases, extracellular vesicle donor cells nanoelectroporated by a nanoelectroporation system as described herein may be grown or attached to a metal or polymer surface such as a biochip as described herein. In some cases, extracellular vesicle donor cells nanoelectroporated by a nanoelectroporation system as described herein may be grown or attached in a single layer to a metal or polymer surface such as a biochip as described herein. In some cases, the system includes a fluid chamber having an upper boundary and a lower boundary. The upper and lower chambers are created by placing a substrate containing extracellular vesicle donor cells into the fluid chamber. In some cases, the system further includes at least one nanochannel. In some cases, the nanochannel may be embedded in the substrate.

[0144] In some cases, extracellular vesicle donor cells grown on or attached to a metal or polymer surface and nano-electroporated with heterologous polynucleotides as described herein may produce extracellular vesicles (e.g., exosomes) at high throughput. In some cases, such high-throughput production of exosomes may be involved in the use of multiple (e.g., more than 1, more than 2, more than 3, more than 5, more than 10, or further numbers) biochips (e.g., CNP biochips). In some cases, the CNP biochips may constitute widths of at least 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 100 cm, or more. In some cases, the CNP biochip will be at least 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 15cm, 20cm, 25cm, 30cm, 35cm, 40cm, 45cm, 50cm, 100cm, or longer in length. In some cases, the biochip will be 1cm x 1cm in size. In some cases, the biochip may consist of exemplary dimensions of 1cm x 2cm, 1cm x 3cm, 1cm x 5cm, 1cm x 10cm, 2cm x 1cm, 2cm x 2cm, 2cm x 3cm, 2cm x 5cm, 2cm x 10cm, 3cm x 1cm, 3cm x 2cm, 3cm x 3cm, 3cm x 5cm, 3cm x 10cm, 5cm x 1cm, 5cm x 2cm, 5cm x 3cm, 5cm x 5cm, 5cm x 10cm, 10cm x 1cm, 10cm x 2cm, 10cm x 3cm, 10cm x 5cm, or 10cm x 10cm.

[0145] In some cases, nanoelectroporation of extracellular vesicle donor cells may constitute a cycle that includes collecting extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells for periods of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, or longer, following nanochannel electroporation (CNP). In some cases, extracellular vesicle donor cells may produce and secrete extracellular vesicles over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CNP cycles.

[0146] In some cases, the secreted extracellular vesicles are biocompatible, approximately 40–150 nm in diameter, and may endogenously express transmembrane and membrane tethering proteins. The presence of these proteins may prolong blood circulation, facilitate tissue-directed delivery, and facilitate cellular uptake of encapsulated exosome contents. In some cases, the methods provided herein may involve the use of nanoelectroporation without significant aggregate formation. In some examples, the heterologous polynucleotides or vectors introduced into the extracellular vesicle donor cells do not encode the peptide sequences incorporated into the extracellular vesicles but bind to target mRNA.

[0147] In some cases, nanochannels constitute pores in a semipermeable polymer substrate. In some embodiments, the nanochannels are composed of heights ranging from approximately 0.01 μm to approximately 500 μm. In some embodiments, the nanochannels are approximately 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.2 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, and 0.01 μm to 0.5 μm. , about 0.05 μm to about 2 μm, about 0.05 μm to about 5 μm, about 0.05 μm to about 10 μm, about 0.05 μm to about 20 μm, about 0.05 μm to about 50 μm, about 0.05 μm to about 100 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 0.5 μm, approximately 0.1 μm to approximately 1 μm, approximately 0.1 μm to approximately 2 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 20 μm, approximately 0.1 μm to approximately 50 μm, approximately 0.1 μm to approximately 100 μm, approximately 0.1 μm to approximately 500 μm, approximately 0.5 0.5 μm to 2 μm, 0.5 μm to 5 μm, 0.5 μm to 10 μm, 0.5 μm to 20 μm, 0.5 μm to 50 μm, 0.5 μm to 100 μm, 0.5 μm to 500 μm, 1 μm to 2 μm, approx. 1 μm to about 5 μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 50 μm, about 1 μm to about 100 μm, about 1 μm to about 500 μm, about 2 μm to about 5 μm, about 2 μm to about 10 μm, about 2 μm to about 20 μm, about 2 μm to about 50 μm, about It is composed of heights of approximately 2 μm to 100 μm, approximately 2 μm to 500 μm, approximately 5 μm to 10 μm, approximately 5 μm to 20 μm, approximately 5 μm to 50 μm, approximately 5 μm to 100 μm, approximately 5 μm to 500 μm, approximately 10 μm to 20 μm, approximately 10 μm to 50 μm, approximately 10 μm to 100 μm, approximately 10 μm to 500 μm, approximately 20 μm to 50 μm, approximately 20 μm to 100 μm, approximately 20 μm to 500 μm, approximately 50 μm to 100 μm, approximately 50 μm to 500 μm, or approximately 100 μm to 500 μm.In some embodiments, the nanochannels are configured with heights of approximately 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, or 500 μm. In some embodiments, the nanochannels are configured with heights of at least approximately 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, or 100 μm. In some embodiments, the nanochannels are configured with heights of up to approximately 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, or 500 μm. In some cases, the height of each nanochannel may be the same. In some cases, the height of each nanochannel may be different. In some cases, the height of the nanochannels must be high enough to facilitate the nanoelectroporation of molecules in a high-electric-field region (e.g., inside the nanochannel), while being low enough for larger molecules being nanoelectroporated by short-duration electrical pulses to just barely pass through.

[0148] In some embodiments, the nanochannels are composed of diameters ranging from approximately 0.01 nm to approximately 10,000 nm.In some embodiments, the nanochannels are approximately 0.01nm to 0.1nm, 0.01nm to 0.5nm, 0.01nm to 1nm, 0.01nm to 5nm, 0.01nm to 10nm, 0.01nm to 50nm, 0.01nm to 100nm, 0.01nm to 500nm, 0.01nm to 1,000nm, 0.01nm to 5,000nm, 0.01nm to 10,000nm, 0.1nm to 0.5nm, 0.1nm to 1nm, 0.1nm to 5nm, 0.1nm to 10nm, and 0.1nm to 5nm. Approx. 50nm, approx. 0.1nm ~ approx. 100nm, approx. 0.1nm ~ approx. 500nm, approx. 0.1nm ~ approx. 1,000nm, approx. 0.1nm ~ approx. 5,0 00nm, about 0.1nm to about 10,000nm, about 0.5nm to about 1nm, about 0.5nm to about 5nm, about 0.5nm to about 10nm, about 0 .5nm to approx. 50nm, approx. 0.5nm to approx. 100nm, approx. 0.5nm to approx. 500nm, approx. 0.5nm to approx. 1,000nm, approx. 0.5nm ~about 5,000nm, about 0.5nm to about 10,000nm, about 1nm to about 5nm, about 1nm to about 10nm, about 1nm to about 50nm, about 1n m ~ approx. 100nm, approx. 1nm ~ approx. 500nm, approx. 1nm ~ approx. 1,000nm, approx. 1nm ~ approx. 5,000nm, approx. 1nm ~ approx. 10,000 nm, approximately 5 nm to approximately 10 nm, approximately 5 nm to approximately 50 nm, approximately 5 nm to approximately 100 nm, approximately 5 nm to approximately 500 nm, approximately 5 nm to approximately 1,000 nm m, about 5 nm to about 5,000 nm, about 5 nm to about 10,000 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm ~500nm, approx. 10nm ~ approx. 1,000nm, approx. 10nm ~ approx. 5,000nm, approx. 10nm ~ approx. 10,000nm, approx. 50nm ~ approx. It consists of diameters of 100nm, approximately 50nm to 500nm, approximately 50nm to 1,000nm, approximately 50nm to 5,000nm, approximately 50nm to 10,000nm, approximately 100nm to 500nm, approximately 100nm to 1,000nm, approximately 100nm to 5,000nm, approximately 100nm to 10,000nm, approximately 500nm to 1,000nm, approximately 500nm to 5,000nm, approximately 500nm to 10,000nm, approximately 1,000nm to 5,000nm, approximately 1,000nm to 10,000nm, or approximately 5,000nm to 10,000nm.In some embodiments, the nanochannels are composed of diameters of approximately 0.01 nm, approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, approximately 5,000 nm, or approximately 10,000 nm. In some embodiments, the nanochannels are composed of diameters of at least approximately 0.01 nm, approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, or approximately 5,000 nm. In some embodiments, the nanochannels are composed of diameters of up to approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, approximately 5,000 nm, or approximately 10,000 nm. In some cases, the diameters of each nanochannel may be the same. In other cases, the diameters of each nanochannel may be different.

[0149] In some cases, nanochannels may be arranged within a nanochannel array. In other cases, nanochannels may be arranged within a nanochannel array with spacing between them. In some examples, the spacing between nanochannels can range from about 0.01 μm to about 5,000 μm.In some examples, the spacing between nanochannels is approximately 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 1 μm, 0.01 μm to 5 μm, 0.01 μm to 10 μm, 0.01 μm to 50 μm, 0.01 μm to 100 μm, 0.01 μm to 500 μm, 0.01 μm to 1,000 μm, 0.01 μm to 5,000 μm, 0.05 μm to 0.1 μm, 0.05 μm to 0.5 μm, 0.05 μm to 1 μm, and 0.05 μm to 5 μm. μm, approximately 0.05 μm to approximately 10 μm, approximately 0.05 μm to approximately 50 μm, approximately 0.05 μm to approximately 100 μm, approximately 0.05 μm to approximately 500 μm m, about 0.05μm to about 1,000μm, about 0.05μm to about 5,000μm, about 0.1μm to about 0.5μm, about 0.1μm to about 1μ m, about 0.1 μm to about 5 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 100 μm, about 0.1 μm m ~ approx. 500 μm, approx. 0.1 μm ~ approx. 1,000 μm, approx. 0.1 μm ~ approx. 5,000 μm, approx. 0.5 μm ~ approx. 1 μm, approx. 0.5 μm ~ Approximately 5μm, approximately 0.5μm to approximately 10μm, approximately 0.5μm to approximately 50μm, approximately 0.5μm to approximately 100μm, approximately 0.5μm to approximately 500μm, Approximately 0.5μm to approximately 1,000μm, approximately 0.5μm to approximately 5,000μm, approximately 1μm to approximately 5μm, approximately 1μm to approximately 10μm, approximately 1μm to approximately 5 0μm, about 1μm to about 100μm, about 1μm to about 500μm, about 1μm to about 1,000μm, about 1μm to about 5,000μm, about 5μm m ~ about 10μm, about 5μm - about 50μm, about 5μm - about 100μm, about 5μm - about 500μm, about 5μm - about 1,000μm, about 5μm m can range from approximately 5,000 μm, 10 μm to approximately 50 μm, 10 μm to approximately 100 μm, 10 μm to approximately 500 μm, 10 μm to approximately 1,000 μm, 10 μm to approximately 5,000 μm, 50 μm to approximately 100 μm, 50 μm to approximately 500 μm, 50 μm to approximately 1,000 μm, 50 μm to approximately 5,000 μm, 100 μm to approximately 500 μm, 100 μm to approximately 1,000 μm, 100 μm to approximately 5,000 μm, 500 μm to approximately 1,000 μm, 500 μm to approximately 5,000 μm, or approximately 1,000 μm to approximately 5,000 μm.In some examples, the spacing between nanochannels may be approximately 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1,000 μm, or 5,000 μm. In some examples, the spacing between nanochannels may be at least approximately 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, or 1,000 μm. In some examples, the spacing between nanochannels can be up to approximately 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1,000 μm, or 5,000 μm.

[0150] In some cases, the nanoelectroporation system includes upper and lower electrode layers for generating an electric field within a fluid chamber. In some cases, the electric field for nanoelectroporation generated by the electrodes consists of voltages between approximately 10V and approximately 500V. In some cases, the electric field for nanoelectroporation generated by the electrodes consists of voltages between approximately 10V and 25V, approximately 10V and 50V, approximately 10V and 100V, approximately 10V and 125V, approximately 10V and 150V, approximately 10V and 175V, approximately 10V and 200V, approximately 10V and 225V, approximately 10V and 250V, approximately 10V and 300V, approximately 10V and 500V, approximately 25V and 50V, approximately 25V and 100V, approximately 25V and 125V, approximately 25V and 150V, approximately 2 5V to approximately 175V, approximately 25V to approximately 200V, approximately 25V to approximately 225V, approximately 25V to approximately 250V, approximately 25V to approximately 300V, approximately 25V to approximately 500V, approximately 50V to approximately 100V, approximately 50V to approximately 125V, approximately 50V to approximately 150V, approximately 50V to approximately 175V, approximately 50V to approximately 200V, approximately 50V to approximately 225V, approximately 50V to approximately 250V, approximately 50V to approximately 300V, approximately 50V to approximately 500V, approximately 100V to approximately 125V, approximately 100V to approximately 150V, approximately 100V to approximately 175V, approximately 100 V ~ about 200V, about 100V - about 225V, about 100V - about 250V, about 100V - about 300V, about 100V - about 500V, about 125V - about 150V, about 125V - about 175V, about 125V - about 200V, about 125V - about 22 5V, about 125V to about 250V, about 125V to about 300V, about 125V to about 500V, about 150V to about 175V, about 150V to about 200V, about 150V to about 225V, about 150V to about 250V, about 150V to about 300V, about 15 It consists of voltages between 0V and approximately 500V, approximately 175V and approximately 200V, approximately 175V and approximately 225V, approximately 175V and approximately 250V, approximately 175V and approximately 300V, approximately 175V and approximately 500V, approximately 200V and approximately 225V, approximately 200V and approximately 250V, approximately 200V and approximately 300V, approximately 200V and approximately 500V, approximately 225V and approximately 250V, approximately 225V and approximately 300V, approximately 225V and approximately 500V, approximately 250V and approximately 300V, or approximately 300V and approximately 500V.In some cases, the electric field for nanoelectroporation generated by electrodes consists of one of the following voltages: approximately 10V, 25V, 50V, 100V, 125V, 150V, 175V, 200V, 225V, 250V, 300V, or 500V. In some cases, the electric field for nanoelectroporation generated by electrodes consists of one of the following voltages: at least approximately 10V, 25V, 50V, 100V, 125V, 150V, 175V, 200V, 225V, 250V, or 300V. In some cases, the electric field for nanoelectroporation generated by the electrodes consists of one of the following voltages: up to approximately 25V, 50V, 100V, 125V, 150V, 175V, 200V, 225V, 250V, 300V, or 500V.

[0151] In some cases, the electric field for nanoelectroporation generated by the electrodes constitutes an electric field strength of approximately 0.1 volts / mm to approximately 50,000 volts / mm. In some cases, the electric field for nanoelectroporation generated by the electrodes is approximately 0.1 volts / mm to approximately 0.5 volts / mm, approximately 0.1 volts / mm to approximately 1 volt / mm, approximately 0.1 volts / mm to approximately 5 volts / mm, approximately 0.1 volts / mm to approximately 10 volts / mm, approximately 0.1 volts / mm to approximately 50 volts / mm, approximately 0.1 volts / mm to approximately 100 volts / mm, approximately 0.1 volts / mm to approximately 500 volts / mm, approximately 0.1 volts / mm to approximately 1,000 volts / mm, approximately 0 0.1 volts / mm to approximately 5,000 volts / mm, approximately 0.1 volts / mm to approximately 10,000 volts / mm, approximately 0.1 volts / mm to approximately 50,000 volts / mm, approximately 0.5 volts / mm to approximately 1 volt / mm, approximately 0.5 volts / mm to approximately 5 volts / mm, approximately 0.5 volts / mm to approximately 10 volts / mm, approximately 0.5 volts / mm to approximately 50 volts / mm, approximately 0.5 volts / mm to approximately 100 volts / mm, approximately 0.5 volts / mm to approximately 500 volts / mm, approximately 0.5 volts / mm to approximately 1.0 00 volts / mm, approximately 0.5 volts / mm to approximately 5,000 volts / mm, approximately 0.5 volts / mm to approximately 10,000 volts / mm, approximately 0.5 volts / mm to approximately 50,000 volts / mm, approximately 1 volt / mm to approximately 5 volts / mm, approximately 1 volt / mm to approximately 10 volts / mm, approximately 1 volt / mm to approximately 50 volts / mm, approximately 1 volt / mm to approximately 100 volts / mm, approximately 1 volt / mm to approximately 500 volts / mm, approximately 1 volt / mm to approximately 1,000 volts / mm, approximately 1 volt / mm to approximately 5, 000 volts / mm, approximately 1 volt / mm to approximately 10,000 volts / mm, approximately 1 volt / mm to approximately 50,000 volts / mm, approximately 5 volts / mm to approximately 10 volts / mm, approximately 5 volts / mm to approximately 50 volts / mm, approximately 5 volts / mm to approximately 100 volts / mm, approximately 5 volts / mm to approximately 500 volts / mm, approximately 5 volts / mm to approximately 1,000 volts / mm, approximately 5 volts / mm to approximately 5,000 volts / mm, approximately 5 volts / mm to approximately 10,000 volts / mm, approximately 5 volts / mm to approximately 50,000 volts / mm, approximately 10 volts / mm to approximately 50 volts / mm, approximately 10 volts / mm to approximately 100 volts / mm, approximately 10 volts / mm to approximately 500 volts / mm, approximately 10 volts / mm to approximately 1,000 volts / mm, approximately 10 volts / mm to approximately 5,000 volts / mm, approximately 10 volts / mm to approximately 10,000 volts / mm, approximately 10 volts / mm to approximately 50,000 volts / mm, approximately 50 volts / mm to approximately 100 volts / mm, approximately 50 volts / mm to approximately 500 volts / mm, approximately 50 volts / mm to approximately 1,000 volts / mm, approximately 50 volts / mm to approximately 5,000 volts / mm, approximately 50 volts / mm to approximately 10,000 volts / mm, approximately 50 volts / mm to approximately 50,000 volts / mm, approximately 100 volts / mm to approximately 500 volts / mm, approximately 100 volts / mm to approximately 1,000 volts / mm Approximately 100 volts / mm to approximately 5,000 volts / mm, approximately 100 volts / mm to approximately 10,000 volts / mm, approximately 100 volts / mm to approximately 50,000 volts / mm, approximately 500 volts / mm to approximately 1,000 volts / mm, approximately 500 volts / mm to approximately 5,000 volts / mm, approximately 500 volts / mm to approximately 10,000 volts / mm, approximately 500 volts / mm to approximately 50,000 volts / mm, approximately 1,000 volts / mm to approximately 5,000 volts / mm, approximately 1,000 volts / mm to approximately 10,000 volts / mm, approximately 1,000 volts / mm to approximately 50,000 volts / mm, approximately 5,000 volts / mm to approximately 10,000 volts / mm, approximately 5,000 volts / mm to approximately 50,000 volts / mm, or approximately 10,000 volts / mm to approximately 50,000 volts / mm. In some cases, the electric field for nanoelectroporation generated by the electrodes can be approximately 0.1 volts / mm, approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, approximately 10,000 volts / mm, or approximately 50,This constitutes an electric field strength of 000 volts / mm. In some cases, the electric field for nanoelectroporation generated by the electrodes constitutes an electric field strength of at least approximately 0.1 volts / mm, approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 50 volts / mm, approximately 100 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, or approximately 10,000 volts / mm. In some cases, the electric field for nanoelectroporation generated by the electrodes constitutes an electric field strength of up to approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 50 volts / mm, approximately 100 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, approximately 10,000 volts / mm, or approximately 50,000 volts / mm.

[0152] In some examples, the electric field for nanoelectroporation generated by the electrodes constitutes multiple pulses with pulse durations ranging from approximately 0.01 milliseconds / pulse to approximately 5,000 milliseconds / pulse. In some examples, the electric field for nanoelectroporation generated by the electrodes constitutes multiple pulses with pulse durations ranging from approximately 0.01 milliseconds / pulse to approximately 0.05 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 0.1 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 0.5 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.01 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 50 milliseconds / pulse, and approximately 0.01 milliseconds / pulse. Approximately 100 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 0.1 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 0.5 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.05 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 50 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 100 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 500 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 1,000 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 5,000 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 0.5 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 1 millisecond / pulse, approximately 0.1 milliseconds / pulse ~Approximately 50 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 10 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 50 milliseconds / pulse milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.5 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 50 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 5 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 10 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 50 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 100 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 500 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 1,000 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 5,000 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 50 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 50 milliseconds / pulse / pulse, approximately 10 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 5,000 Multiple pulses are composed of pulse durations of approximately milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 500 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 500 milliseconds / pulse to approximately 5,000 milliseconds / pulse, or approximately 1,000 milliseconds / pulse to approximately 5,000 milliseconds / pulse. In some examples, the electric field for nanoelectroporation generated by the electrodes is approximately 0.01 milliseconds / pulse, approximately 0.05 milliseconds / pulse, approximately 0.1 milliseconds / pulse, approximately 0.Multiple pulses are composed of pulse durations of 5 milliseconds / pulse, approximately 1 millisecond / pulse, approximately 5 milliseconds / pulse, approximately 10 milliseconds / pulse, approximately 50 milliseconds / pulse, approximately 100 milliseconds / pulse, approximately 500 milliseconds / pulse, approximately 1,000 milliseconds / pulse, or approximately 5,000 milliseconds / pulse. In some examples, the electric field for nanoelectroporation generated by the electrodes constitutes multiple pulses with pulse durations of at least approximately 0.01 milliseconds / pulse, approximately 0.05 milliseconds / pulse, approximately 0.1 milliseconds / pulse, approximately 0.5 milliseconds / pulse, approximately 1 millisecond / pulse, approximately 5 milliseconds / pulse, approximately 10 milliseconds / pulse, approximately 50 milliseconds / pulse, approximately 100 milliseconds / pulse, approximately 500 milliseconds / pulse, or approximately 1,000 milliseconds / pulse. In some examples, the electric field for nanoelectroporation generated by electrodes constitutes multiple pulses with pulse durations of up to approximately 0.05 milliseconds / pulse, 0.1 milliseconds / pulse, 0.5 milliseconds / pulse, 1 millisecond / pulse, 5 milliseconds / pulse, 10 milliseconds / pulse, 50 milliseconds / pulse, 100 milliseconds / pulse, 500 milliseconds / pulse, 1,000 milliseconds / pulse, or 5,000 milliseconds / pulse. In some cases, nanoelectroporation constitutes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pulses.

[0153] In some cases, extracellular vesicles produced and secreted by extracellular vesicle donor cells are collected and purified from the cell culture medium by centrifugation or ultracentrifugation, which allows for the purification of extracellular vesicles from other cell fragments or molecules based on the density of extracellular vesicles.

[0154] In some cases, methods and systems for producing extracellular vesicles containing targeting polypeptides and / or therapeutic polynucleotides include loading multiple vectors into nanochannels to be nanoelectroporated into cells. In some cases, molecules other than vectors (e.g., proteins, biomolecules, compounds, etc.) may be loaded into nanochannels and nanoelectroporated into cells. In some cases, electric fields generated by upper and lower electrodes facilitate the entry of vectors into cells. In some cases, the electric field generated for nanoelectroporation creates pores in the cell membrane, enabling the nanoelectroporation of vectors. In some cases, pores in the extracellular vesicle donor cell membrane may be formed locally, for example, at the nanochannel exit where the electric field directly contacts the cell membrane.

[0155] In some cases, nanoelectroporated extracellular vesicle donor cells may produce and secrete at least 10%, 50%, 1x, 5x, 10x, 50x, 100x, 500x, 1000x, 5000x, or more extracellular vesicles than extracellular vesicle donor cells transfected by non-nanoelectroporation (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection). In some cases, nanoelectroporated extracellular vesicle donor cells may produce and secrete at least 10%, 50%, 1x, 5x, 10x, 50x, 100x, 500x, 1000x, 5000x, or more extracellular vesicles compared to the number of extracellular vesicles produced and secreted by extracellular vesicle donor cells stimulated by non-nanoelectroporation (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection, general cellular stress responses, starvation, hypoxia, and heat treatment).

[0156] In some cases, culturing and nano-electroporating extracellular vesicle donor cells at higher temperatures can cause them to produce and secrete more extracellular vesicles. For example, culturing and nano-electroporating extracellular vesicle donor cells at 37°C results in the production and secretion of more extracellular vesicles than extracellular vesicle donor cells cultured and nano-electroporated at 4°C. In some cases, extracellular vesicle donor cells produce and secrete at least 10%, 50%, 1x, 5x, 10x, 50x, 100x, 1000x, or more extracellular vesicles for every 1°C increase above 4°C during culture and nano-electroporation of extracellular vesicle donor cells.

[0157] In some cases, extracellular vesicle donor cells are used with Ca 2+ When cultured in a buffer containing , more extracellular vesicles may be produced and secreted. For example, 500 nM Ca after nanoelectroporation. 2+ Extracellular vesicle donor cells cultured in a buffer containing Ca after nanoelectroporation 2+ Compared to extracellular vesicle donor cells cultured in a buffer that does not contain Ca, extracellular vesicles are produced and secreted. In some cases, extracellular vesicle donor cells produce and secrete more extracellular vesicles after nanoelectroporation. 2+ When cultured in a buffer with increased concentration of Ca, after nanoelectroporation 2+ Compared to extracellular vesicle donor cells cultured in a buffer that does not contain Ca, these cells produce and secrete at least 10%, 50%, 1x, 5x, 10x, 50x, 100x, 1000x, or more extracellular vesicles. 2+ Examples of concentrations include 10nM, 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM, 1100nM, 1200nM, 1300nM, 1400nM, 1500nM, 2000nM, 2500nM, 3000nM, 5000nM, 10000nM, or higher levels of Ca. 2+ Concentration is one example.

[0158] In some cases, transfection of extracellular vesicle donor cells with at least one heterologous polynucleotide, including a vector encoding 6kbp Ascl1 (Achaete-Scute Complex Like-1), 7kbp Pou3f2 (Pou Domain Class 3 Transcription Factor 2) or Brn2, and 9kbp Myt1l (Myelin Transcription Factor 1 Like), may cause the extracellular vesicle donor cells to produce and secrete more extracellular vesicles. Transfection of extracellular vesicle donor cells with 6kbp Ascl1 (Achaete-Scute Complex Like-1), 7kbp Pou3f2 (Pou Domain Class 3 Transcription Factor 2) or Brn2, and 9kbp Myt1l (Myelin Transcription Factor 1 Like) can increase the number of extracellular vesicles produced and secreted by nanoelectroporation-stimulated extracellular vesicle donor cells by at least 10%, 50%, 1-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more compared to the number of extracellular vesicles produced and secreted by nanoelectroporation of extracellular vesicle donor cells not transfected with 6kbp Ascl1 (Achaete-Scute Complex Like-1), 7kbp Pou3f2 (Pou Domain Class 3 Transcription Factor 2) or Brn2, and 9kbp Myt1l (Myelin Transcription Factor 1 Like).

[0159] In some cases, extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells contain at least 50%, 1x, 2x, 5x, 100x, 500x, 1000x, or more therapeutic polynucleotides compared to extracellular vesicles produced and secreted by non-nanoelectroporated extracellular vesicle donor cells. In some cases, therapeutic polynucleotides encapsulated by extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more likely to have intact encoded therapeutic polypeptides than therapeutic polynucleotides encapsulated by extracellular vesicles produced and secreted by non-nanoelectroporated extracellular vesicle donor cells.

[0160] In some cases, the percentage of extracellular vesicles containing at least one copy of therapeutic polynucleotides produced and secreted by extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased compared to the percentage of extracellular vesicles containing at least one copy of therapeutic polynucleotides produced and secreted by extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection, etc.). In some cases, the percentage of extracellular vesicles containing at least one copy of therapeutic polynucleotide produced and secreted by extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1, 0.2, 0.5, 2, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000 or more compared to the percentage of extracellular vesicles containing at least one copy of therapeutic polynucleotide produced and secreted by extracellular vesicle donor cells transfected by other transfection methods. In some cases, the percentage of extracellular vesicles containing at least one copy of a therapeutic polynucleotide can be determined by measuring the number of extracellular vesicles containing at least one copy of a therapeutic polynucleotide produced and secreted by extracellular vesicle donor cells over periods of 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer.In some cases, extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation produce and secrete an increased number of extracellular vesicles containing at least one copy of therapeutic polynucleotide (e.g., therapeutic mRNA, therapeutic miRNA) compared to the number of extracellular vesicles containing at least one copy of therapeutic polynucleotide produced and secreted by extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection, etc.). In some cases, extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation produce and secrete at least one copy of a therapeutic polynucleotide, resulting in an increase of at least 0.1, 0.2, 0.5, 2, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000 or more extracellular vesicles produced and secreted by extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection). In some cases, extracellular vesicle donor cells subjected to microchannel electroporation or nanochannel electroporation produce and secrete an increased number of extracellular vesicles, in which at least one of 500 extracellular vesicles, at least one of 200 extracellular vesicles, at least one of 100 extracellular vesicles, at least one of 50 extracellular vesicles, at least one of 25 extracellular vesicles, or at least one of 10 extracellular vesicles contains at least one copy of a therapeutic polynucleotide (e.g., therapeutic mRNA, therapeutic miRNA).

[0161] Pharmaceutical composition In some cases, extracellular vesicles may be formulated into pharmaceutical compositions. In some cases, pharmaceutical compositions containing extracellular vesicles or exosomes may be administered to a subject by multiple routes of administration (including, but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal routes). In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreous, intraosseous, intraperitoneal, or intrathecal administration. In some examples, pharmaceutical compositions are formulated for topical administration. In other examples, pharmaceutical compositions are formulated for systemic administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intravenous, subcutaneous, and intramuscular administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intravenous administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by subcutaneous administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intramuscular administration.

[0162] Kit / Manufactured product In certain embodiments, kits and products for use in conjunction with one or more methods and compositions described herein are disclosed herein. Systems for producing extracellular vesicles or exosomes are also described herein. In some cases, the system includes a method of stimulating the production of extracellular vesicles or exosomes containing targeting polypeptides and therapeutic polynucleotides by nanoelectroporating extracellular vesicle donor cells.

[0163] In some cases, a kit may include a carrier, package, or container that is divided to accept one or more containers such as vials, tubes, etc., and each container(s) may include one of the individual elements used in the manner described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some cases, containers may be formed from a variety of materials such as glass or plastic. A kit usually includes a label describing the contents and / or instructions for use, as well as an accompanying leaflet with instructions for use. A set of instructions is usually also included.

[0164] In one embodiment, the label is on the container or attached to the container. In one embodiment, if the letters, numbers, or other symbols forming the label are written, molded, or etched onto the container itself, the label is on the container; if the label is located within a container or carrier that also supports the container, the label is attached to the container, for example, as accompanying documentation. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic purpose. The label also indicates how the contents are to be used, such as in the methods described herein.

[0165] In certain cases, extracellular vesicles containing targeting polypeptides and therapeutic polynucleotides may be present in a pack or dispenser device containing one or more dosage forms containing the compounds provided herein. The pack may contain metal foil or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied by a notice relating to the form of container prescribed by a government agency that regulates the manufacture, use, or sale of the drug, the notice reflecting the government agency's approval of the form of the drug for administration to humans or animals. Such notice may be, for example, a label approved for the drug by the U.S. Food and Drug Administration or an approved product insert. In one embodiment, extracellular vesicles containing tumor-targeting polypeptides and therapeutic polynucleotides, formulated in a suitable pharmaceutical carrier and containing the compounds provided herein, are also prepared, placed in a suitable container, and labeled as for the treatment of an indication.

[0166] In some cases, the kit includes a product useful for deploying the adoptive therapies and therapeutic methods described herein. In some cases, the kit includes components for producing at least one extracellular vesicle containing a targeting polypeptide and a therapeutic polynucleotide, or at least one extracellular vesicle containing a tumor-targeting polypeptide and a therapeutic polynucleotide. In some cases, the kit includes at least one exosome containing a targeting polypeptide and a therapeutic polynucleotide, or components for producing at least one exosome containing a targeting polypeptide and a therapeutic polynucleotide. [Examples]

[0167] The following exemplary examples are representative of embodiments of the compositions, systems, and methods described herein and are not intended to limit the means to any of them.

[0168] Example 1. Quantification of extracellular vesicles generated by cell nanoporation (CNP) We have developed and describe herein a cell nanoporation (CNP) biochip, CNP system, and CNP method for stimulating cells to produce and release exosomes containing a target nucleotide sequence, including mRNA, microRNA, and shRNA. This system and method enable the culture of a monolayer of source cells, such as mouse embryonic fibroblasts (MEFs) and dendritic cells (DCs), on the surface of a chip containing a nanochannel array (Figure 1A). The nanochannels (approximately 500 nm in diameter) allow transient electrical pulses to pass through, enabling the round-trip transport of DNA plasmids from the buffer to the attached cells (Figure 1A). When 6kbp Ascl1 (Achaete-Scute Complex Like-1, sometimes referred to as "A" herein), 7kbp Pou3f2 (Pou Domain Class 3 Transcription Factor 2) or Brn2 (sometimes referred to as "B" herein), and 9kbp Myt1l (Myelin Transcription Factor 1 Like, sometimes referred to as "M" herein) plasmids were added to the buffer, CNP increased the yield of secreted extracellular vesicles (EVs) with a similar vesicle size distribution by >50 times compared to other conventional techniques (Figure 1B and Figures 2A-B). Similarly, EV production methods dependent on general cellular stress responses such as starvation, hypoxia, and heat treatment resulted in only moderate EV release (Figure 1C). In contrast, CNP-induced EV secretion was very potent and could be applied to different cell type sources and transfection vectors (Figure 1D and Figures 2C-D). Dynamic analysis further demonstrated that EV release peaked 8 hours after CNP induction and continued for 24 hours (Figure 1E). The degree of EV secretion could be controlled by adjusting the voltage applied to the nanochannels; increasing the voltage from 100V to 150V increased the number of released EVs until a plateau was observed at 200V (Figure 1F). Ambient temperature was another variable that could influence CNP-induced EV secretion, as cells prepared at 37°C released more EVs than those prepared at 4°C (Figure 2E).To evaluate the internal nucleic acid content of released extracellular viable cells (EVs), CNP was performed on source cells using a PTEN plasmid, followed by agarose gel analysis using RNA collected from the EVs. Compared to the CNP / PBS group, a greater number of intact PTEN mRNA molecules were encapsulated within the EVs (Figure 1G), and in EVs induced by the CNP / PTEN plasmid, intact PTEN mRNA constituted 55.5 ± 9.2% by weight of the total large RNA. Quantitative reverse transcription polymerase chain reaction (qtr-PCR) showed that CNP yielded 10% higher levels of mRNA or miRNA within EVs complementary to plasmid DNA compared to the BEP or Lipo method. 3 Further confirmation was obtained of a twofold increase (Figure 1H and Figures 2F-H). In addition, complementary mRNA extracted from EVs generated by CNP maintained the ability of mRNA to encode polypeptides for protein synthesis (Figure 2I). When multiple plasmid DNAs were used in CNP, the levels of complementary mRNA showed a stepwise increase, with Myt1l, the largest transcript, taking the longest time (16 hours) to reach the peak concentration (Figure 1I). This is thought to be because transcription of longer nucleic acid sequences requires more time.

[0169] Example 2. mRNA loading on extracellular vesicles To better understand the distribution of transcribed mRNA in different EV subgroups, exosomes were first separated from microvesicles (MVs) by standard multi-step ultracentrifugation (Figure 3A-B). Exosome markers (CD9, CD63, and Tsg101) and MV marker (Arf6) were detected in exosomes and MVs by Western blotting (Figure 4A). 100% of the cells transfected with CNP were then separated. 8The majority (>75%) of all EV RNA derived from MEF cells was located in exosomes rather than in MVs (Figure 4B), with an average weight ratio of 1 μg / 20 μg of large RNA / protein in exosomes. This contrasted sharply with the absence of large RNA from the same number of exosomes derived from CNP-free MEF cells. Cryo-TEM images in Figure 4C further revealed that exosomes generated by plasmid DNA-based CNP contained a large amount of nucleic acid, while exosomes from untreated MEF cells were empty. Furthermore, qRT-PCR analysis confirmed that the majority of transcribed mRNA was also located in exosomes rather than in MVs (Figure 4D), indicating that the mRNA retained its ability to encode polypeptides for protein synthesis (Figure 4E).

[0170] To further quantify the potential diversity of mRNA packed within individual exosomes, a tethered lipoplex nanoparticle (TLN) assay was used (Figure 4F). Cationic lipoplex nanoparticles containing specific molecular beacons (MBs) were immobilized on coverslips, and negatively charged exosomes were individually captured by the nanoparticles using electrostatic interactions. Hybridization between mRNA within exosomes and MBs within the nanoparticles generated a fluorescence signal, which was captured using total internal reflection fluorescence (TIRF) microscopy to quantify the mRNA content. After CNP using three different DNA plasmids (A, B, and M), it was found that approximately 50% of exosomes contained only one transcript, 25% contained two mRNAs, and the remaining 25% contained all three mRNA sequences (Figures 4G-H). This multiple packing was improved by using sequential CNP (S-CNP) technology, which delivers different plasmids separately according to their peak times (Figure 1I) (Figures 4G-H). Therefore, S-CNP significantly improved the rate of multiple mRNA loading within individual exosomes, exceeding 50%.

[0171] Example 3. Comparison of CNP and BEP for therapeutic exosomes The main difference between CNP and existing BEP technology is that CNP encapsulates endogenously transcribed RNA within exosomes, while BEP delivers exogenous nucleotides into pre-isolated exosomes. To compare the efficiency of the two approaches, we first delivered both miR-128 and CD63-GFP plasmids into MEFs using CNP to generate GFP-labeled exosomes containing miR-128. For BEP insertion, free miR-128 was mixed with pre-isolated empty exosomes. Because BEP can only insert small nucleic acid sequences into exosomes, microRNA was used as the nucleic acid cargo. The inability of BEP to encapsulate anything but small nucleic acid sequences is a major limitation when using BEP in mRNA-based exosome generation. To compare the amount of miR-128 in exosomes prepared by both CNP and BEP, a tethered dripoplex nanoparticle (TLN) biochip containing a Cy5-miR128 molecular beacon was designed to capture negatively charged exosomes and allow the two types of vesicles to fuse. Subsequently, hybridization of the miR-128 molecule and the Cy5-miR128 molecular beacon released red fluorescence detectable by TIRF microscopy (Figure 3C). Approximately 80% of exosomes containing miR-128 were produced by both CNP and BEP, but the concentration of miR-128 in CNP exosomes was significantly higher (Figures 3D-F). Furthermore, unlike BEP, CNP efficiently produced exosomes containing large mRNA (Figures 5A-C), and exosomes secreted by CNP contained >100 times more Brn2 mRNA than exosomes inserted by BEP (Figures 1H and 5D).

[0172] Example 4. Mechanism of CNP-induced exosome secretion To investigate the underlying cellular mechanisms of CNP-induced exosome release, we first examined intracellular structural changes after CNP exposure. CNP significantly increased the formation of multivesicular bodies (MVBs) within mesenchymal follicular cells (MEFs) (Figure 6A). When the CD63-GFP plasmid was delivered to MEFs via CNP, numerous GFP-positive MVBs were formed within 4 hours of induction (Figure 6B). Furthermore, transmission electron microscopy (TEM) revealed that CNP-treated MEFs showed approximately a twofold increase in MVBs and an approximately eightfold increase in intraluminal membrane vesicles (ILVs) (Figures 6C-E). Correspondingly, increased expression of proteins involved in exosome biosynthesis was observed (Figure 6F). Since CNP relies on delivering a local electric field across the plasma membrane of the source cell, damage may occur at the contact point. First, numerous pores were formed in the plasma membrane facing the basal surface, and then fluorescence gradually increased beyond the cell tip surface (slower than in cells subjected to BEP). This suggests that CNP caused small pores to form beyond the contact points with nanochannels (Figure 6G). Interestingly, the nanopores in the cell membrane were found to close within 2 minutes after electroporation, suggesting that a recovery process occurred to repair membrane damage (Figures 6H-I). Since it has been reported that increased intracellular calcium ion levels promote exosome release, we investigated the role of calcium ion influx through these nanopores after the exosome secretion level increased by CNP. In fact, the amount of exosomes released after CNP was increased by Ca in the extracellular space. 2+ It increases with the increase in [something], and consequently the amount of Ca in the cell increases. 2+ It was found that the amount increased (Figure 6J-L). When EGTA, a calcium chelating agent, was added, the increase in intracellular calcium ions was largely blocked, and exosome release caused by CNP was suppressed (Figure 6M-N). This indicates that the amount of Ca in the cell increased. 2+ This suggests that the increase in [value] may be a factor that induces exosome secretion by CNP (Figure 6O).

[0173] Next, we evaluated the stress response within the source cell that contributes to the increased exosome formation after CNP treatment. It has been shown that heat shock stimulates exosome biosynthesis through increased production of heat shock proteins (HSPs). Numerical simulations showed that a temporary (<1s) temperature increase approaching 60°C occurred near the nanochannel exit in CNP (Figures 7A-C). This temperature increase was concentrated on the cell surface near the nanochannel exit (Figures 7D-F). As expected, CNP significantly increased HSP expression in cells, and the addition of an HSP inhibitor severely suppressed exosome secretion (Figures 7G-H). This suggests that the heat shock response plays a significant role in CNP-mediated exosome production. Since HSPs can regulate P53 activity, and P53 subsequently regulates exosome production through TSAP6, we investigated whether the increase in HSP levels via CNP promoted exosome production through the P53-TSAP6 signaling pathway. In line with this, the expression levels of P53 and TSAP6 were upregulated after CNP (Figure 7I), and in MEF cell lines with stable knockdown of P53 (MEF P53- / -), TSAP6 expression levels remained unchanged (Figure 7I). Furthermore, in P53- / - MEF cells, exosome release did not increase after CNP (Figure 7J). These results suggest that, under conditions of CNP-induced localized thermal stress, the heat shock response promotes activation of the P53-TSAP6 signaling pathway, leading to increased exosome production as part of the cellular recovery process (Figure 7K).

[0174] Example 5. Functional and pharmacokinetic evaluation of CNP-generating exosomes The tumor suppressor gene PTEN, which is commonly mutated in PTEN-deficient human U87 and mouse GL261 glioma models, was evaluated for the clinical utility of mRNA-exosomes. To achieve glioma targeting function, glioma-targeting peptides were cloned into the N-terminus of CD47, a transmembrane protein abundant on the exosome surface (Figure 8A). Two peptides, the CDX peptide for U87 targeting (FKESWREARGTRIERG (SEQ ID NO: 104)) and CREKA for GL261 targeting, along with the FLAG epitope, were separately inserted into the N-terminus of CD47. Since the topology of CD47 on exosomes was unknown, a pull-down assay was performed using anti-FLAG beads to confirm that the N-terminus of CD47 was localized to the outer surface of the exosome (Figure 8B). The addition of targeting peptides dramatically increased the uptake of CD47-exosomes (Exo-T) in U87 and GL261 cells, as well as the translation of PTEN protein (Figures 8C-F, 9, and 10A-D). Staining for endocytosis markers revealed that Exo-T strongly co-localized with transferrin (Figures 8G and 10E). Inhibition of clathrin-mediated endocytosis significantly reduced exosome uptake into cells (Figures 8H and 10F), suggesting that Exo-T entry into target cells may be mediated by clathrin-mediated endocytosis. Exo-T was able to inhibit tumor cell proliferation and exhibited minimal cytotoxicity (Figures 8I-J and 10G-H).

[0175] To investigate the potential role of Exo-T for in vivo application, its pharmacokinetic properties were evaluated. After transfection of cells with the CD47 plasmid, CD47 was strongly expressed on the exosome surface (Figure 8K, top panel). Overexpression of CD47 on the exosome surface was found to triple the in vivo circulating half-life of Exo-T, and the addition of a targeting peptide did not significantly affect the function of CD47 (Figure 8K). Immunogenicity results showed that Exo-T did not exhibit significant in vivo toxicity or immunogenicity in mice tested at different doses and time points (2 hours, 8 hours, and 24 hours) (Figures 8L and 10I).

[0176] Example 6. In vivo therapeutic efficacy of Exo-T in a preclinical model of glioma. To evaluate the therapeutic potential of Exo-T in a PTEN-deficient glioma model, Exo-T was intravenously injected into glioma-immunodeficient mice orthotopically transplanted with human U87. Exo-T showed significantly improved accumulation in tumors compared to untargeted exosomes (exosomes) or TurboFect (Turbo) nanoparticles (Figure 11A). To further evaluate the in vivo distribution of Exo-T within the tumor stroma, PKH26-labeled Exo-T, exosomes, and Turbo were systemically administered to tumor-bearing mice and imaged using a biofluorescence microscope. Strong PKH fluorescence was observed in the tumor stroma 4 hours after Exo-T administration, but not with exosomes or TurbFect nanoparticles (Figures 11B-C and 12A-B). Furthermore, evaluation of systemic distribution revealed that the number of labels was reduced in the accumulation of Exo-T in the liver and spleen (Figures 11D-E).

[0177] In U87 mice treated with Exo-T, tumor growth was significantly inhibited (Figure 11F-G), and the median survival time was 49 days, showing an extension of survival time compared to 37 days in untargeted exosomes (Figure 11H). Evaluation of residual tumor tissue in both groups revealed that both PTEN mRNA and protein levels were upregulated after Exo-T treatment (Figure 11I-J). Immunohistochemical staining further confirmed that Exo-T treatment restored PTEN expression, inhibited tumor cell proliferation, and had no direct effect on other tissues examined (Figures 11K-M and 13).

[0178] Next, the therapeutic efficacy of Exo-T was investigated in a PTEN-deficient GL261 glioma model with immune competence. In Exo-T, accumulation in tumors was shown to be significantly improved compared to non-targeted exosomes (exosomes) or PEG-liposomes (liposomes) (Figure 14A). To further evaluate the in vivo biodistribution of Exo-T in the tumor stroma, Exo-T, exosomes, and PEG-liposomes labeled with PKH26 were intravenously administered to tumor-bearing mice and imaged with a live fluorescence microscope. Strong PKH fluorescence was observed in the tumor stroma 4 hours after Exo-T administration, but not in the exosome cohort or the liposome nanoparticle cohort (Figures 14B–C). Ex vivo results showed that most of the exosomes were taken up by glioma cells, but minimal uptake of exosomes was observed in normal brain cells after administration (Figure 14D). Furthermore, upon evaluation of the systemic distribution, it became clear that the number of labels decreased in the accumulation of Exo-T in the liver and spleen (Figures 14E–F). In GL261 mice treated with Exo-T, tumor growth was demonstrated to be significantly inhibited (Figures 14G–H), and the median survival time was 45 days, showing an extension of the survival time compared to 31 days with non-targeted exosomes (Figure 14I). Evaluation of the remaining tumor tissues in both groups revealed that both PTEN mRNA and protein levels were upregulated after Exo-T treatment (Figures 14J–K). From the results of immunohistochemical staining, it was further confirmed that PTEN expression was restored by Exo-T treatment, tumor cell proliferation was inhibited, and there was no direct effect on other tissues investigated (Figures 7L–N and Figure 15).

[0179] Example 7. Methods and Systems for Synthesizing Therapeutic Extracellular Vesicles Exemplary methods and systems for producing extracellular vesicles or exosomes for encapsulating therapeutic polynucleotides are described herein. The extracellular vesicles and exosomes produced by the methods and systems may be suitable for formulation into pharmaceutical compositions for use in therapy.

[0180] Cell Culture Mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% non-essential amino acids (NEAAs). Human glioma U87-MG and HEK293T cell lines were cultured at 37°C under humidified conditions equilibrated with 5% CO2 in DMEM supplemented with 10 mmol / L HEPES, 10% FBS, and 1% penicillin / streptomycin.

[0181] Plasmid preparation Ascl1 (Achaete-Scute Complex Like-1), Pou3f2 (Pou Domain Class 3 Transcription factor 2) or Brn2, and Myt1l (Myelin Transcription Factor 1 Like) were synthesized. PTEN, CD47, CD63-GFP, and miR-128 plasmids were available for purchase. A ligand was introduced to the N-terminus of CD47 using primers designed to encode CDX (FKESWREARGTRIERG (SEQ ID NO: 105)), CREKA, and the FLAG tag. Isolation of monocytes from mouse bone marrow. C57BL / 6 mice at 4 - 12 weeks of age were euthanized by cervical dislocation and sterilized with 70% ethanol for 5 minutes. Femurs and tibias were removed and purified under sterile conditions. Subsequently, intact bones were washed with PBS and both ends were cut with scissors. The bone marrow was flushed with RPMI-1640 medium using a 0.45 mm diameter needle. Cells were collected by centrifugation at 1,000 rpm for 5 minutes. Tris-NH4Cl erythrocyte lysis buffer was added to the cell pellet to remove erythrocytes. The cell suspension was further centrifuged at 1,000 rpm for 5 minutes to collect monocytes. Inductive culture of bone marrow-derived DCs (BMDCs). The isolated monocytes were cultured at 37°C in an incubator containing 5% CO₂ in RPMI-1640 medium supplemented with 10% FBS. 20 ng / ml GM-CSF and 10 ng / ml IL-4 were added to the culture medium. After 12 hours of culture, non-adherent cells were removed and replaced with fresh complete medium containing GM-CSF and IL-4. On the 7th day, gently adherent cells were collected by gently pipetting the medium against the flask. The cells were seeded onto CNP chips and further incubated with lipopolysaccharide for 24 hours.

[0182] Cell transfection For CNP, MEF, MSC, DC, or HEK-293T cells (-200,000 cells) were spread in a monolayer on a 1cm × 1cm 3D CNP silicon chip surface and incubated overnight. Plasmids pre-prepared in PBS buffer were injected into individual cells via nanochannels (-500nm diameter and 5μm spacing) by applying five 10ms / pulse pulses at 0.1-second intervals with a 200V electric field. Various electroporation conditions were tested for best selection. Bulk electroporation (BEP), gene gun, and lipofectamine 2000 transfection were performed. For transfection, Ascl1 / Brn2 / Myt1l plasmids were pre-mixed in a 2 / 1 / 1 weight ratio and at a concentration of 100ng / mL in PBS buffer. Cell transfection with PTEN, miR-128, CD47, CDX-CD47, CREKA-CD47, and CD63-GFP plasmids was performed using the same procedure.

[0183] Collection and purification of extracellular viable cells (EVs) secreted by donor cells Cells were cultured in a serum-containing culture medium. Before transfection, the serum-containing cell culture medium was removed. Cells were washed three times with PBS and cultured in serum-free cell culture medium. For qRT-PCR, extracellular viable (EVs) were collected from the cell culture supernatant by centrifugation at 1500 g for 10 minutes. For dynamic light scattering angle measurement (DLS) and EV particle measurement by NanoSight® in vitro cell transfection and in vivo animal experiments, EVs were collected from the cell culture supernatant by a series of centrifugation and ultracentrifugation steps. 【0184...

Claims

1. A method for producing extracellular vesicles, a. Electroporating at least one polynucleotide into an extracellular vesicle donor cell, wherein the at least one polynucleotide encodes a targeting polypeptide comprising (i) a transmembrane domain and an extracellular domain, wherein the adapter polypeptide comprises a peptide sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and (ii) a heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide, the heterologous targeting domain of the targeting polypeptide being a cell surface marker binding domain; b. Incubating the extracellular vesicle donor cells under conditions such that (i) the targeting polypeptide is expressed within the extracellular vesicle donor cells, and (ii) the targeting polypeptide is incorporated into the extracellular vesicles released from the extracellular vesicle donor cells; and c. Collecting at least one extracellular vesicle released from the extracellular vesicle donor cell, wherein the at least one extracellular vesicle comprises the targeting polypeptide; This includes, here The transmembrane domain of the adapter polypeptide contains an amino acid sequence that is at least 90% identical to the transmembrane domain amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of a human CD47 polypeptide containing the amino acid sequence of SEQ ID NO: 1, and The extracellular domain of the adapter polypeptide is capable of binding to the SIRP protein and contains an amino acid sequence that is at least 90% identical to the extracellular domain amino acid sequence at positions 19-141 of a human CD47 polypeptide containing the amino acid sequence of SEQ ID NO:

1. The aforementioned method.

2. The method according to claim 1, wherein the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide.

3. The method according to claim 1, wherein the transmembrane domain of the adapter polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of SEQ ID NO: 1, and the extracellular domain of the adapter polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

4. The method according to claim 1, wherein the heterogeneous targeting domain includes a tumor targeting domain.

5. The method according to claim 4, wherein the tumor targeting domain is a CDX peptide.

6. The method according to claim 4, wherein the tumor targeting domain is a CREKA peptide.

7. The method according to any one of claims 1 to 6, further comprising electroporating a polynucleotide into the extracellular vesicle donor cell, wherein the polynucleotide encodes a ribonucleic acid (RNA) therapeutic agent.

8. The method according to claim 7, wherein the ribonucleic acid (RNA) therapeutic agent is incorporated into the at least one extracellular vesicle released from the extracellular vesicle donor cell, and the method further comprises collecting the at least one extracellular vesicle released from the extracellular vesicle donor cell.

9. The method according to claim 7 or 8, wherein the ribonucleic acid (RNA) therapeutic agent is messenger RNA (mRNA), uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof.

10. The method according to claim 7 or 8, wherein the RNA therapeutic agent is a cancer therapeutic agent.

11. The method according to claim 7, wherein the RNA therapeutic agent is a fully intact or substantially intact messenger RNA.

12. The method according to claim 11, wherein the RNA therapeutic agent comprises at least five copies of a completely intact messenger RNA.

13. The method according to any one of claims 7 to 12, wherein the RNA therapeutic agent comprises PTEN mRNA.

14. The method according to any one of claims 1 to 13, wherein the extracellular vesicle donor cells are selected from the group consisting of primary cells, mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells.

15. The method according to any one of claims 1 to 13, wherein the extracellular vesicle donor cells are primary cells.

16. The method according to any one of claims 1 to 15, wherein the extracellular vesicle is an exosome.

17. The method according to any one of claims 1 to 16, wherein the electroporation is nanoelectroporation, and the at least one polynucleotide is nanoelectroporated into the extracellular vesicle donor cell via a nanochannel located on the biochip.

18. The method according to any one of claims 1 to 17, wherein the electroporation constitutes an electric field.

19. The method according to claim 18, wherein the electric field has an electric field strength of 1 volt / mm to 1000 volts / mm.

20. The method according to claim 18, wherein the electric field comprises a plurality of pulses having pulse durations of 0.1 milliseconds / pulse to 100 milliseconds / pulse.

21. The method according to any one of claims 1 to 20, wherein the collection comprises collecting the at least one extracellular vesicle released from the extracellular vesicle donor cell at least 6 hours after the electroporation.

22. The electroporation comprises electroporating primary cells with at least one heterologous deoxyribonucleic acid (DNA) polynucleotide, thereby obtaining primary cells containing the heterologous DNA polynucleotide, wherein the heterologous DNA polynucleotide encodes a therapeutic ribonucleic acid (RNA) polynucleotide. The incubation comprises incubating the primary cells containing the heterologous DNA polynucleotide under conditions that enable transcription of the heterologous DNA polynucleotide, thereby producing the therapeutic ribonucleic acid (RNA) polynucleotide, wherein the therapeutic ribonucleic acid (RNA) polynucleotide is incorporated into extracellular vesicles released from the primary cells, and The collection includes collecting the extracellular vesicles released from the primary cells, wherein the extracellular vesicles released from the primary cells, on average, contain at least one copy of the therapeutic ribonucleic acid (RNA) polynucleotide. The method according to claim 1.

23. A composition comprising extracellular vesicles designed to deliver cargo to target cells, wherein the extracellular vesicles are a. Adapter polypeptide, wherein the adapter polypeptide comprises an extracellular domain and a transmembrane domain, and the adapter polypeptide comprises a peptide sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

1. (i) The transmembrane domain of the adapter polypeptide contains an amino acid sequence that is at least 90% identical to the transmembrane domain amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of a human CD47 polypeptide containing the amino acid sequence of SEQ ID NO: 1, and (ii) The extracellular domain of the adapter polypeptide is capable of binding to the SIRP protein and contains an amino acid sequence that is at least 90% identical to the extracellular domain amino acid sequence at positions 19-141 of the human CD47 polypeptide containing the amino acid sequence of SEQ ID NO: 1; and b. A heterologous targeting domain covalently linked to the extracellular domain of the adapter polypeptide, wherein the heterologous targeting domain is a cell surface marker binding domain that specifically binds to the target cell, thereby enabling the extracellular vesicle to deliver the cargo to the target cell. The composition comprising a targeting polypeptide containing the following.

24. The composition according to claim 23, wherein the cell surface marker binding domain binds to a cell surface marker associated with diseased cells.

25. The composition according to claim 24, wherein the diseased cells are cancer cells, non-cancerous lesion cells, muscle cells, or tumor cells.

26. The composition according to claim 23, wherein the transmembrane domain of the adapter polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of SEQ ID NO: 1, and the extracellular domain of the adapter polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

27. The composition according to any one of claims 23 to 26, wherein the heterologous targeting domain is covalently linked to the N-terminus of the extracellular domain of the adapter polypeptide.

28. The composition according to any one of claims 23 to 27, wherein the heterogeneous targeting domain includes a tumor targeting domain.

29. The composition according to claim 28, wherein the tumor targeting domain is a CDX peptide.

30. The composition according to claim 28, wherein the tumor targeting domain is a CREKA peptide.

31. The composition according to any one of claims 23 to 27, wherein the heterogeneous targeting domain includes a muscle cell targeting domain.

32. The composition according to claim 31, wherein the myocyte targeting domain comprises the amino acid sequence of SEQ ID NO: 88 (ASSLNIA).

33. The composition according to any one of claims 23 to 32, wherein the extracellular vesicle comprises a ribonucleic acid (RNA) therapeutic agent.

34. The composition according to claim 33, wherein the ribonucleic acid (RNA) therapeutic agent is messenger RNA (mRNA), uncoding RNA, microRNA, shRNA, siRNA, or a combination thereof.

35. The composition according to claim 33 or 34, wherein the RNA therapeutic agent is a cancer therapeutic agent.

36. The composition according to claim 33, wherein the RNA therapeutic agent is a fully intact or substantially intact messenger RNA.

37. The composition according to claim 33, wherein the RNA therapeutic agent comprises at least five copies of a completely intact messenger RNA.

38. The RNA therapeutic agent is the composition according to any one of claims 33 to 37, comprising PTEN mRNA.

39. The composition according to any one of claims 23 to 38, wherein the extracellular vesicles are secreted from extracellular vesicle donor cells selected from the group consisting of primary cells, mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, and immune cells.

40. The composition according to any one of claims 23 to 39, wherein the extracellular vesicle donor cells are primary cells.

41. The composition according to any one of claims 23 to 40, wherein the extracellular vesicle is an exosome.

42. The method according to any one of claims 1 to 22, wherein the heterogeneous targeting domain includes a muscle cell targeting domain.

43. The method according to claim 42, wherein the myocyte targeting domain comprises the amino acid sequence of SEQ ID NO: 88 (ASSLNIA).

44. The method according to any one of claims 1 to 22, wherein the adapter polypeptide comprises a peptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

1.

45. The method according to any one of claims 1 to 22, wherein the adapter polypeptide comprises a peptide sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

1.

46. The method according to any one of claims 1 to 22, wherein the adapter polypeptide comprises a peptide sequence identical to the amino acid sequence of SEQ ID NO:

1.

47. The composition according to any one of claims 23 to 41, wherein the adapter polypeptide comprises a peptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

1.

48. The composition according to any one of claims 23 to 41, wherein the adapter polypeptide comprises a peptide sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

1.

49. The composition according to any one of claims 23 to 41, wherein the adapter polypeptide comprises a peptide sequence identical to the amino acid sequence of SEQ ID NO:

1.

50. The method according to any one of claims 1 to 22, wherein the transmembrane domain of the adapter polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of SEQ ID NO: 1, and the extracellular domain of the adapter polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

51. The method according to any one of claims 1 to 22, wherein the amino acid sequence of the transmembrane domain of the adapter polypeptide is the same as the amino acid sequences at positions 142-162, 177-197, 208-228, 236-256, and 269-289 of SEQ ID NO: 1, and the amino acid sequence of the extracellular domain of the adapter polypeptide is the same as the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

52. The composition according to any one of claims 23 to 41, wherein the transmembrane domain of the adapter polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence at positions 142-162, 177-197, 208-228, 236-256, or 269-289 of SEQ ID NO: 1, and the extracellular domain of the adapter polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

53. The composition according to any one of claims 23 to 41, wherein the amino acid sequence of the transmembrane domain of the adapter polypeptide is the same as the amino acid sequences at positions 142-162, 177-197, 208-228, 236-256, and 269-289 of SEQ ID NO: 1, and the amino acid sequence of the extracellular domain of the adapter polypeptide is the same as the amino acid sequence at positions 19-141 of SEQ ID NO:

1.

54. The method according to any one of claims 1 to 22, wherein the extracellular domain includes an IgV domain.

55. The composition according to any one of claims 23 to 41, wherein the extracellular domain includes an IgV domain.

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