WO2025072945A2 - Methods and compositions for production of mrna-loaded exosomes for targeted immunotherapy - Google Patents

Abstract

Provided herein are compositions, systems, and methods for producing small extracellular vesicles (sEVs) that are mRNA-loaded and include CD64 on the sEV surface. Also provided are the immunogenic EVs (imsEVs) that further include a targeting ligand bound to the CD64 on the imsEV surface. The disclosure also provides fusion proteins including a CD64 amino acid sequence fused to an N peptide amino acid sequence. Also provided are nucleic acids that encode the fusion proteins, and vectors and cells that comprise the nucleic acids. Also disclosed are methods for using the imsEVs for immunotherapy, such as for the treatment of cancer in a subject.

Description

METHODS AND COMPOSITIONS FOR PRODUCTION OF MRNA- LOADED EXOSOMES FOR TARGETED IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Serial No. 63/586,666, filed September 29, 2023, the content of which is incorporated herein by this reference as if fully set forth herein.

BACKGROUND

[0002] Messenger RNA (mRNA) has demonstrated a therapeutic benefit in various clinical applications, such as pathologic infections, in recent years. The use of mRNA for treating other human diseases (e.g., cancer) presents challenges as there are physiological barriers with respect to achieving both tissue-targeted and cell-targeted delivery of mRNA with the currently available delivery systems. In addition, in some current mRNA therapeutics, the carrier (e.g., lipid nanoparticle (LNP)) has certain additional limitations, including immunogenicity and storage issues. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for RNA-based therapeutics because of their advantages over other mRNA delivery systems, including their excellent biosafety and biocompatibility, stability against degradation, and ability to cross physiological barriers such as the blood brain barrier (BBB). Small EVs (sEVs) or exosomes have been used successfully to deliver certain full-length transcripts of mRNAs for cancer therapy. However, the efficient production of exosomes harboring a sufficient amount of the mRNAs remains challenging. The present disclosure provides solutions to this problem, providing electroporation systems, methods, and compositions for the efficient production of EVs harboring therapeutic mRNAs for targeted immunotherapy.

BRIEF SUMMARY

[0003] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0004] In one aspect, a process for generating exosomes loaded with mRNAs is provided. In some embodiments, the process includes the following steps: subjecting a population of cells to a nanosecond duration electroporation treatment at a first electroporation station, wherein the first electroporation station is located in a microfluidic channel of a microfluidic device in which the population of cells are flowed past at least one electrical field source to receive the nanosecond duration electroporation treatment; after the nanosecond duration electroporation treatment, transporting the population of cells to a second electroporation station; subjecting the population of cells to a millisecond duration electroporation treatment at the second electroporation station, wherein during at least the millisecond duration electroporation treatment the population of cells are in the presence of extracellular nucleic acids comprising a DNA sequence, and wherein transcription of the DNA sequence produces an mRNA; and after the nanosecond and millisecond duration electroporation treatments, collecting exosomes secreted by the population of cells, wherein the secreted exosomes are loaded with the mRNA.

[0005] In some embodiments, the step of subjecting the population of cells to the nanosecond duration electroporation treatment includes subjecting a solution including the population of cells and the extracellular nucleic acids to the nanosecond duration electroporation treatment. In some embodiments, the step of transporting the population of cells to the second electroporation station comprises transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station. In some embodiments, the step of subjecting the population of cells to the millisecond duration electroporation treatment comprises subjecting the solution including the population of cells and the extracellular nucleic acids to the millisecond duration electroporation treatment.

[0006] In some embodiments, the microfluidic device comprises the first electroporation station and the second electroporation station; wherein transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station comprises flowing the solution from the first electroporation station to the second electroporation station via at least the microfluidic channel. In other embodiments, the second electroporation station comprises a second device, wherein transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station comprises collecting the solution from the microfluidic device and depositing the solution in the second device.

[0007] In some embodiments, the population of cells express a protein or a protein complex, at least a portion of which is located in cell membranes of the population of cells prior to the nanosecond and millisecond duration electroporation treatments; and membranes of the collected exosomes comprise the proteins or protein complexes from the population of cells with the mRNA bound to a portion of the protein or protein complex inside the exosome. In some embodiments, the population of cells express a CD64-N peptide fusion protein. In some embodiments, the cell membranes of the secreted exosomes comprise the CD64-N peptide fusion protein from the population of cells, wherein the CD64 portion of the fusion protein is located in the cell membranes and extends outside the exosome, and wherein the N peptide portion of the fusion protein is located inside the exosome. In some embodiments, the mRNAs are bound to the N-peptide portion of the CD64-N peptide fusion proteins and are located inside the secreted exosomes. In some embodiments, the CD64 portion of the CD64-N peptide fusion proteins of the secreted exosomes binds to an Fc region of an antibody that is outside the secreted exosomes. In some embodiments, the antibody is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response. In certain embodiments, the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

[0008] In some embodiments of the disclosed processes, the electrical field source at the first electroporation station applies an electrical field having a strength of between 5 kV / cm and 125 kV / cm as the population of cells flows past the at least one electrical field source. In some embodiments, the electrical field source at the first electroporation station applies an electrical field having a strength of between 30 kV / cm and 80 kV / cm as the population of cells flows past the at least one electrical field source.

[0009] In some embodiments of the disclosed processes, the nanosecond electroporation treatment has a frequency in the range of 5 Hz to 1 M Hz. In some embodiments, the nanosecond electroporation treatment has a duration in the range of 50 ns to 2000 ns.

[0010] In some embodiments, the millisecond duration electroporation treatment has an amplitude of at least 10 V and a duration of at least 1 millisecond.

[0011] In some embodiments, the microfluidic channel at the first treatment station has a cross-sectional area perpendicular to a fluid flow direction in the range of 10,000 pm² to 50,000 pm². In some embodiments, the microfluidic channel at the first treatment station has a cross-sectional area perpendicular to a fluid flow direction in the range of 1 x 10'⁸ m² to 5 * IO’⁸ m². [0012] In another aspect, small extracellular vesicles (sEVs) are provided. In some embodiments, the sEV includes a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence, wherein the CD64 portion of the fusion protein is located in the cell membrane and extends outside the sEV, and wherein the N peptide is located inside the sEV; and a plurality of mRNAs, wherein the mRNAs are located inside the sEV and may be bound to the N peptide. In some embodiments, the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from c|)21 phage. In certain embodiments, the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the CD64 is a human CD64. In certain embodiments, the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO: 8. In some embodiments, the amino acid sequence of the fusion protein comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NO: 10.

[0013] In some embodiments, the sEV further includes an antibody located outside the sEV that is bound to the CD64. In some embodiments, the antibody is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response. In some embodiments, the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier. In certain embodiments, the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1). In some embodiments, the sEV includes both antibodies to CD71 and antibodies to PDL-1.

[0014] In some embodiments, the sEV includes an mRNA that is translated into a protein that stimulates an immune response. In certain embodiments, the mRNA is an interferon gamma (IFNy) mRNA. In some embodiments, the amino acid sequence of the protein comprises SEQ ID NO: 18 or SEQ ID NO:20, or a sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO:20. In some embodiments, the protein is encoded by a nucleotide sequence that comprises SEQ ID NO: 19 or SEQ ID NO:21, or a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:21.

[0015] In some embodiments, the sEV is produced by subjecting a population of cells to a nanosecond duration electroporation treatment at a first electroporation station, wherein the cells express a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence, and wherein the first electroporation station is located in a microfluidic channel of a microfluidic device in which the population of cells are flowed past at least one electrical field source to receive the nanosecond duration electroporation treatment; after the nanosecond duration electroporation treatment, transporting the population of cells to a second electroporation station; subjecting the population of cells to a millisecond duration electroporation treatment at the second electroporation station, wherein during at least the millisecond duration electroporation treatment the population of cells are in the presence of extracellular nucleic acids comprising a DNA sequence, wherein the DNA sequence encodes a protein that stimulates an immune response, and wherein transcription of the DNA sequence produces an mRNA; after the nanosecond and millisecond duration electroporation treatments, collecting sEVs secreted by the population of cells, wherein the secreted sEVs are loaded with the mRNA, and wherein the mRNA may be bound to the N peptide of the fusion protein; and after collecting the sEVs, binding the CD64 in the membrane of the sEVs to an antibody that is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response. In some embodiments, the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier. In certain embodiments, the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1). In certain embodiments, the mRNA is an interferon gamma (IFNy) mRNA.

[0016] In another aspect, the disclosure provides a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence. In some embodiments, the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from c|)21 phage. In certain embodiments, the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain embodiments, the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO:8. In certain embodiments, the amino acid sequence comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NO: 10.

[0017] In another aspect, nucleic acids that encode the disclosed fusion proteins are provided. In other aspects, the disclosure provides vectors or cells comprising the nucleic acids. [0018] In another aspect, the disclosure provides a system for use in producing small extracellular vesicles (sEVs). In some embodiments, the systems include a cell expressing a fusion protein that comprises a CD64 amino acid sequence fused to an N peptide amino acid sequence; and a vector sequence comprising a DNA sequence encoding a therapeutic protein. In some embodiments, the system further includes an electroporation system including a microfluidic device having a microfluidic channel in which the cell is flowed past at least one electrical field source to receive a nanosecond duration electroporation treatment and a second electroporation station to receive a millisecond duration electroporation treatment.

[0019] In some embodiments of the disclosed systems, the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from c|)21 phage. In certain embodiments, the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the CD64 is a human CD64. In certain embodiments, the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO: 8. In certain embodiments, the amino acid sequence of the fusion protein comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the system also includes an antibody that is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response. In some embodiments, the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier. In certain embodiments, the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1). In certain embodiments, the system includes both an antibody specific for CD71 and an antibody specific for PDL-1. In some embodiments, the DNA sequence encodes a protein that stimulates an immune response. In certain embodiments, the protein is interferon y (IFNy).

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions, systems, and methods, and to supplement any description(s) of the compositions, systems, and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case. [0021] FIG. 1 shows one example of a system for generating exosomes loaded with mRNAs.

[0022] FIGS. 2-8 show an example of a process for generating exosomes loaded with mRNAs using the system of FIG. 1.

[0023] FIG. 9 shows another example of a system for generating exosomes loaded with mRNAs.

[0024] FIG. 10 is a schematic drawing illustrating an exemplary large-scale production of immunologic small extracellular vesicles (imsEVs) based on a nanosecond-electroporation (nsEP) system with mouse embryonic fibroblasts (MEFs) harboring a plasmid for expression of IFN-y. In this example, the imsEVs are loaded simultaneously with anti-CD71 and anti- PD-L1 antibodies on the imsEV surface and with IFN-y mRNA inside the imsEV. These imsEVs are used for glioblastoma (GBM) treatment.

[0025] FIGS. 11A-11F demonstrate the large-scale generation of sEVs by a nsEP system. FIG. 11A is a schematic representation of the nsEP system for sEV generation. FIG. 1 IB is a graph showing the sEV number per cell produced by mouse embryonic fibroblasts (MEFs) in untreated control, microsecond electroporation pulses (msEP), nsEP, and nsEP/PBS treatment groups. The total amount of sEVs = concentration of sEVs x volume. The number of viable cells was calculated by trypan blue staining and cell counting. The number of sEVs produced per cell = total number of sEVs/number of living cells. FIG. 11C is a graph showing the number of sEVs produced per MEF after nsEP system at voltage amplitudes from 0 to 250 V. FIG. HD is a graph showing the viability of MEFs after nsEP system at voltage amplitudes from 0 to 250 V. FIG. HE is a graph showing the size measurements and size distribution of sEVs produced by both the nsEP system (solid line) and the untreated control (dashed line) cells. FIG. HF RT-qPCR of IFN-y mRNA revealed that sEVs produced by nsEP treatment contained much larger quantities of transcribed mRNAs than sEVs produced by other methods. Data in FIGS. 11B-11D, and HF are presented as means ± standard deviation (SD), n = 3 biologically independent samples; statistical significance was calculated by oneway analysis of variance with Tukey’s multiple comparisons test.

[0026] FIGS. 12A-12F illustrate the proteomic profiling of nsEP treated MEF cells. FIG. 12A is a graphical representation of the Gene Ontology annotation of proteins expressed at different levels before and after nsEP treatment. FIG. 12B is a graphical representation of the Gene Ontology enrichment of cellular components. FIG. 12C is a schematic showing the STRING-based protein-protein interaction (PPI) network analysis of identified proteins with higher expression confidence of 0.7. Proteins of interest are displayed as a single diamondshaped node, and differential proteins that interacted with nodes were analyzed by Kyoto Encyclopedia of Genes and Genomes pathway. FIG. 12D is a graphical representation of a Proteomics proteins volcano plot analysis for sEVs derived from nsEP-treated MEFs. Triangles and diamonds represent proteins associated with induction of sEVs, among which diamonds indicate proteins that were more highly expressed than others. A heatmap of the top 95 proteins differentially expressed after nsEP treatment. NA indicates UPF0600 protein C5orf51 homolog (a protein whose gene name is unknown) (data not shown). Proteins associated with sEV secretion are highlighted. FIG. 12E shows photographs of representative Western blots of mixed lineage kinase domain-like protein (MLKL) identified in the indicated treatment conditions (top panels). GAPDH was used as a control (lower panels). FIG. 12F is a graph showing the number of sEVs per cell produced by MLKL knockdown or MLKL-overexpressing MEFs. Equal amounts of protein were used for the proteomic analysis, and data are from three independent biological replicates. Data in FIG. 12F represent means ± SD n = 3 biologically independent samples; statistical significance was analyzed with unpaired two-tailed Student’s t tests.

[0027] FIGS. 13A-13I illustrate the characterization of immunogenic sEVs (imsEVs). Confocal images of MEFs simultaneously transfected with CD64-DsRed and CD63-GFP indicate extensive colocalization of these two surface markers (data not shown). FIG. 13A shows images of the Western blot assessment of CD64 expression in natural sEV and sEV produced by CD64⁺ cells. FIG. 13B shows images of Western blots of an sEV pull-down assay showing that Flag beads could pull down the N-terminal Flag of 3XFlag-CD64, suggesting that the N terminus of CD64 is on the outside of the sEVs. FIG. 13C is a schematic representation of the attachment of IgG to the surface of sEVs through CD64 and the active RNA packaging strategy via the N peptide-box B affinity. The N peptide fused to the C-terminus of CD64 binds specifically to box B to recruit RNA cargo molecules into the sEVs. The inward budding of endosomal membranes leaves the N-terminus of the CD64 protein outside of an sEV when it forms, and the C-terminus conjugated with the N peptide is within the sEV. The former (N-terminus) helps gain the specific surface targeting function while the latter (C-terminus) promotes the enrichment of target mRNA for the imsEVs. FIG. 13D is a graph showing the fold change of IFN-y expression after microsecond electroporation (msEP) or nanosecond electroporation (nsEP) as indicated. CD64-N peptides were co-transfected with box B-IFN-y or control IFN-y plasmids in MEFs, and the resulting imsEVs were pelleted via ultracentrifugation. RT-qPCR was used to detect IFN-y in imsEVs prepared by the various methods, and U6 was used as the internal standard. Representative total internal reflection fluorescence (TIRF) images from a tethered lipoplex nanoparticle (TLN) assay showed IFN-y mRNA colocalized in sEVs (PKH26-sEV) after transfection with IFN-y or IFN-y-box B plasmids (data not shown). The IFN-y-box B group had better IFN y mRNA-loading efficiency compared with sEVs having IFN-y only. FIG. 13E is a graph of IFN-y mRNA fluorescence intensity within sEVs, as measured by the TLN assay in the different treatment groups; n = 5, total 25 images were used for statistical analysis. FIG. 13F is a graph showing colocalization percentage of IFN-y mRNA with sEVs after transfecting with IFN-y or IFN-y-box B plasmid; n = 5, total 25 images were used for statistical analysis. FIG. 13G shows images of a Western blot assessment of CD71 expression in glioblastoma cell lines and MEFs. FIG. 13H shows images of a Western blot assay in which CD64-sEV were incubated with anti-PD-Ll antibody or anti-CD71 antibody for 4 hours and then subjected to immunoprecipitation and Western blot assay. Representative TIRF images of the TLN assay showed that CD64-sEV could simultaneously adsorb two different IgGs in a single sEV (data not shown). FIG. 131 is a graph showing sEV size distribution measured by NS300 after incubating CD64-sEV with IgG; a slight increase in size was observed relative to CD64-sEV only. Red, CD64-sEV; Green, imsEV. Cryo-EM images of sEV, CD64-sEV, and imsEV showed a typical vesicle shape and size (data not shown). Data in FIG. 13D represent means ± SD n = 3 biologically independent samples; analyzed by one-way analysis of variance with Tukey's multiple comparisons test.

[0028] FIGS. 14A-14H show the results of an in vitro study of imsEV for cancer therapy. FIG. 14A shows increased uptake of imsEV conjugated with anti-CD71 antibody by SB28 glioma cells, in photographs (data not shown) and graphically (FIG. 14A). FIGS. 14B-14C show fluorescence intensity of PKH26-labeled sEV taken up by SB28 cells, measured by flow cytometry (shown as flow cytometry plots (FIG. 14B) or graphically (FIG. 14C)), confirming the effective uptake of imsEV by SB28 cells. Representative immunostains showed colocalization of imsEV labeled with PKH26 and imsEV labeled with other endocytosis markers (data not shown). Most imsEV were colocalized with transferrin-Alexa Fluor 647 (A647-Tf), suggesting that imsEV are mainly taken up through clathrin-dependent endocytosis. A647-Tf is a marker of clathrin-dependent endocytosis; cholera toxin subunit B- Alexa Fluor 647 (A647-CT-B) is a marker of caveolae-dependent endocytosis; and A647- dextran is a marker of macropinocytosis. Before being incubated with PKH26-labeled sEVs, tumor cells were incubated for 1 hour with various endocytosis inhibitors (sucrose, 0.4 pM, a clathrin-dependent endocytosis inhibitor; nystatin, 50 pM, a caveolae-dependent endocytosis inhibitor; or cytochalasin D, 5 pM, a micropinocytosis inhibitor) or CD71 monoclonal antibody (10 pg mL'¹), after which the cells were thoroughly washed three times with PBS before being exposed to PKH26-labeled sEVs (data not shown). FIG. 14D is a graph showing the fluorescence intensity of PKH26-labeled imsEV taken up by SB28 cells treated with different endocytosis inhibitors, assessed by flow cytometry, further confirming that imsEVs are primarily taken up through clathrin-dependent endocytosis. Sucrose, clathrin- dependent endocytosis inhibitor; Nystatin, caveolae-dependent endocytosis inhibitor; and Cytochalasin D, macropinocytosis inhibitor. FIG. 14E is a graph showing amounts of IFN-y in the supernatant of SB28 cell culture medium after treatment with PBS, antibody combo (anti-PD-Ll & anti-CD71), CD64-sEV, or imsEV for 48 hours and then measured by ELISA. FIGS. 14F-14G are flow cytometry plots and a graphical representation, showing the expression of IFN-y and MHC-I in SB28 cells by flow cytometry after the indicated treatments. FIG. 14H provides images of a Western blot assessment of MHC-I expression in SB28 cells after treatment with PBS, antibody combo, CD64-sEV, or imsEV. Data in FIGS. 14A, 14C, 14D, 14E, and 14F represent means ± SD n = 3 biologically independent samples; analyzed by one-way analysis of variance with Tukey's multiple comparisons test (FIGS. 14C, 14D, and 14E ) or by unpaired two-tailed Student’s t tests (FIG. 14A).

[0029] FIGS. 15A-15E illustrate the in vivo therapeutic efficacy of imsEV in an orthotopic GL261 glioma model. In vivo imaging by IVIS showed preferential accumulation of DiR- labeled imsEV within orthotopically implanted GL261 tumors in mice (data not shown). FIG. 15A provides a graphical representation of tissue distribution analyses which indicated that imsEV showed increased brain targeting with low hepatic accumulation (n = 3, biologically independent samples). FIG. 15B provides a graphical representation of tumor growth inhibition by tail-vein injection of PBS, empty sEVs (sEVs), antibody combo (anti- PD-Ll & anti-CD71), CD64-sEV, and IFN-y-mRNA containing sEVs plus antibodies (imsEV). (n = 5, biologically independent samples). FIG. 15C is a graph showing that imsEV treatment extended the survival of mice with GL261 glioma, (n = 10, biologically independent samples). Images showing IFN-y, MHC-I, and CD8 staining of residual GBM tumor tissue in the indicated treatment groups which showed that imsEV increased the expression of IFN-y and MHC-I and increased the proportion of CD8⁺ cells in tumor tissues (data not shown). FIG. 15D is a graph reflecting the flow cytometry assessment of the proportions of CD8⁺ cells in tumor tissues of mice in the indicated treatment groups (n = 5, biologically independent samples). FIG. 15E is a graph showing the quantitative analysis of macrophages (gated on F4/80 cells) in the indicated treatment groups (n = 5, biologically independent samples). Data represent means ± SD (FIGS. 15A, 15B, 15D, and 15E); analyzed by one-way analysis of variance with Tukey's multiple comparisons test or by logrank (Mantel-Cox) tests (FIG. 15C).

[0030] FIGS. 16A-16H demonstrate the in vivo therapeutic efficacy of imsEV in an orthotopic SB28 glioma model. In vivo imaging showed preferential accumulation of DiR- labeled imsEV within orthotopically implanted SB28 tumors in mice (data not shown). FIG. 16A provides a graphical representation of tissue distribution analyses which indicated that imsEV showed increased brain targeting and low hepatic accumulation. FIGS. 16B provides a graphical representation of tumor growth inhibition after tail-vein injection of PBS, empty sEV, antibody combo (anti-PD-Ll & anti-CD71), CD64-sEV, and IFN-y mRNA-containing sEV with antibodies (imsEV). (n = 5, biologically independent samples). FIG. 16C is a graph showing imsEV extended the survival of mice with SB28 glioma, (n = 10, biologically independent samples). FIG. 16D provides a graphical representation of tumor size assessed by magnetic resonance imaging after the final treatment. FIGS. 16E-16F are graphs showing IFN-y and MHC-I staining in residual GBM tumor tissue from the indicated treatment groups which showed that imsEV increased the expression of IFN-y and MHC-I. FIGS. 16G-16H are graphs showing the quantitative analysis of T cells and Ml type macrophages in SB28 tumors from the indicated treatment groups analyzed by flow cytometry which showed that imsEV led to increased proportions of CD8⁺ T cells and Ml type macrophages, (n = 5, biologically independent samples). The H&E stain of residual SB28 tumor tissue after the indicated treatments showed that imsEV inhibited cell proliferation in tumor tissue (data not shown). Data represent means ± SD; analyzed by one-way analysis of variance with Tukey's multiple comparisons test (FIGS. 16A, 16B, and 16D-16H), or by log-rank (Mantel-Cox) tests (FIG. 16C)

[0031] FIG. 17 is a schematic representation of the nsEP system for sEV generation.

[0032] FIGS. 18A and 18B are graphs showing results of plasmid loading and mRNA transcription analysis. The copy number of DNA (FIG. 18A) or RNA in cells (FIG. 18B) was determined by correlating the Ct (cycle threshold) values of RT-qPCR. The copy number in each sample was then divided by the cell number (calculated based on the cell viability data) from which DNA was extracted to receive the copies of plasmid (or RNA) per cell. In detail, around 5600 copies of target plasmids were averagely loaded in each cell 3 hours after the transfection, and around 16000 copies of the target mRNA were transcribed after another three hours. In contrast, millisecond electroporation pulse (msEP) supplied only around 1200 copies of DNA and 650 copies of RNA, respectively. Data are presented as means ± standard deviation (SD) for n = 5 biologically independent samples; statistical significance was calculated by one-way analysis of variance with Tukey’s multiple comparisons test.

[0033] FIGS. 19A-19D show the optimization of nsEP conditions for MEFs. FIGS. 19A- 19B are graphs showing the numbers of sEVs per cell produced by the mouse embryonic fibroblasts (MEFs) after nsEP followed by msEP at frequencies from 10 to 400 Hz (FIG. 19A), or the quantity of sEVs per cell produced by MEFs by nsEP with pulses lasting from 300 to 1500 ns (FIG. 19BC). FIGS. 19C-19D are graphs showing the viability of MEFs after nsEP at voltage amplitudes from 10 to 400 Hz (FIG. 19C), or the viability of MEFs after nsEP at pulse durations of 300 to 1500 ns (FIG. 19D). Data for FIGS. 19A-19D are presented as means ± SD, n = 3 biologically independent samples.

[0034] FIGS. 20A-20F show the optimization of nsEP conditions for HEK293T cells. FIG. 20A is a graph showing the sEV numbers per cell produced by HEK293T cells by nsEP system at amplitudes from 0 to 250 V. FIG. 20B is a graph showing the quantity of sEV per cell produced by HEK293T cells after nsEP system at frequencies from 10 to 400 Hz. FIG. 20C is a graph showing the sEV numbers per cell produced by HEK293T cells by nsEP system at durations from 300 to 1500 ns. FIG. 20D is a graph showing the viability of HEK293T cells after nsEP at voltage amplitudes from 0 to 250 V. FIG. 20E is a graph showing the viability of HEK293T cells after nsEP at frequencies from 10 to 400 Hz. FIG. 20F is a graph showing the viability of HEK293T cells after nsEP at durations from 300 to 1500 ns. Data in FIGS. 20A-20F are presented as means ± SD, n = 3 biologically independent samples.

[0035] FIGS. 21A-21D show results of a biocompatibility evaluation. FIGS. 21A and 21B are graphs showing the viability of SB28 cells treated with PBS, sEV, Antibody combo, CD64-sEV-ctrl, CD64-sEV, and imsEV after 24 hours or 48 hours, respectively. FIGS. 21C and 21D are graphs showing the viability of GL261 cells at 24 or 48 hours, respectively, after treatment with PBS, sEV, Antibody combo, CD64-sEV-ctrl, CD64-sEV, and imsEV. These findings suggest that imsEV has good biocompatibility. Data in FIGS. 21A-21D are presented as means ± SD, n = 5 biologically independent samples.

[0036] FIGS. 22A-22C are graphs demonstrating the detection of INF-y concentration in cell culture supernatant. FIG. 22A shows INF-y levels in the supernatant of SB28 cell culture medium after incubation with the indicated sEV preparations for 24 hours, detected by ELISA. FIGS. 22B and 22C show INF-y levels in the supernatant of GL261 cell culture medium after incubation with the indicated sEV preparations for 24 hours or 48 hours, respectively, detected by ELISA. Data in FIGS. 22A-22C are presented as means ± SD, n = 3 biologically independent samples; statistical significance was calculated by one-way analysis of variance with Tukey’s multiple comparisons test.

[0037] FIGS. 23A-23D are graphs illustrating the biosafety evaluation. Levels of blood urea nitrogen (BUN) (FIG. 23A), aspartate transaminase (AST)(FIG. 23B), alanine transaminase (ALT) (FIG. 23C), and creatinine (FIG. 23D) (measured by ELISA) were determined in mice given different doses of imsEV. All data are presented as mean ± SD, n = 3 biologically independent samples.

[0038] FIGS. 24A-24I are graphs showing blood cell counts after a single injection of the indicated preparations. FIGS. 24A-24C show the cell counts after a single injection of the indicated preparations. The cell types being counted were red blood cells (FIG. 24A), white blood cells (FIG. 24B), or lymphocyte counts (FIG. 24C). FIGS. 24D-24F show the cell counts counts in mice after a single injection of different doses of imsEVs. The cell types being counted were red blood cells (FIG. 24D), white blood cells (FIG. 24E), and lymphocytes (FIG. 24F). FIGS. 24G-24I show the cell counts after a single injection of the indicated preparations. The cell type being counted were red blood cells (FIG. 24G), white blood cells (FIG. 24H), and lymphocytes (FIG. 241). All data are presented as mean ± SD, n = 5 biologically independent samples, analyzed by one-way analysis of variance with Tukey's multiple comparisons test.

DETAILED DESCRIPTION

[0039] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

I. INTRODUCTION

[0040] The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. The disclosed methods and compositions provide an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). A microfluidic electroporation approach using a combination of nanosecond and millisecond pulses was shown to efficiently produce large amounts of mRNA-loaded sEVs.

[0041] As discussed in further detail below and in the examples, the present disclosure provides an nsEP system with microfluidic configuration that generates large quantities of sEVs that encapsulate mRNA molecules. Applying millisecond and nanosecond pulses separately shifted the main impact of electroporation from the cell membrane to the membrane structure of cellular organelles. These effects have been confirmed in work involving irreversible electroporation for cancer treatment in vivo and in previous studies of the effective delivery of exogeneous probes into cells. In irreversible electroporation, pulses of 10-300 ns were used to damage only the membrane structure of intracellular organelles, ultimately triggering pulse-induced cell apoptosis. To use electroporation to effectively deliver exogenous cargos without compromising cell viability, it was determined that longer pulses (600-800 ns) led to more transient and reversible disruption of the membrane structures of both cell and cell nuclei. In the present disclosure, the potential of this new stimulation strategy was investigated to leverage sEV secretion for its ability to modify organelle membranes in cells. Impressive enhancement of sEV yield was achieved — more than 40 times the yield from natural secretion — under optimized stimulation conditions. Integration of a microfluidic platform into this nsEP technology not only allows parallel processing for high yield of imsEVs with greater throughput but also suppresses gas bubble formation during electrical stimulation, which can interfere with the electric pulses and damage the treated cells. With the microfluidic setup, the flow quickly sweeps any gas bubbles away from the electrode surface before they grow to undesirable size, to ensure that passing cells receive effective stimulation. This strategy improves the viability of source cells after electroporation, which is crucial for maintaining high sEV yield. [0042] As an example of the disclosed methods, IFN-y mRNAs were loaded in sEVs having CD64 overexpressed on the sEV surface. The CD64 molecule serves as an adaptor to dock targeting ligands (e.g., anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies). The resulting immunogenic sEVs (imsEVs) were shown to preferentially target glioblastoma cells and generate potent antitumor activities in vivo, including against tumors intrinsically resistant to immunotherapy. Therefore, this disclosure provides an improved approach to engineering mRNA-loaded sEVs with targeting functionality and paves the way for their use in cancer immunotherapy applications.

II. TERMINOLOGY

[0043] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0044] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0045] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of those certain elements.” As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0046] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, e.g., In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0047] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0048] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20%; preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0049] The term “nucleic acid” or “nucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. It is understood that when an RNA is described, its corresponding DNA is also described, wherein uridine is represented as thymidine. Similarly, when a DNA is described, its corresponding RNA is also described wherein thymidine is represented by uridine. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et aL, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et aL, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0050] The term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain through translation of an mRNA. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, guide RNA, or micro RNA.

[0051] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass full-length proteins, truncated proteins, and fragments thereof, and amino acid chains, wherein the amino acid residues are linked by covalent peptide bonds. As used throughout, the term “fusion polypeptide” or “fusion protein” is a polypeptide comprising two or more proteins or fragments thereof. In some embodiments, a linker comprising about 3 to 10 amino acids can be positioned between any two proteins or fragments thereof to help facilitate proper folding of the proteins upon expression.

[0052] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein (e.g., a CD64-N peptide fusion protein), refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. It is understood that sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any nucleotide or polypeptide sequence set forth herein, for example, any one of SEQ ID NOs: 1-21, can be used in the compositions and methods provided herein. It is understood that a nucleic acid sequence can comprise, consist of, or consist essentially of any nucleic acid sequence described herein. Similarly, a polypeptide can comprise, consist of, or consist essentially of, any polypeptide sequence described herein. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0053] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, about 20 to 50, about 20 to 100, about 50 to about 200 or about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0054] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et aU). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands.

[0055] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'L Acad. Set. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10'⁵, and most preferably less than about IO'²⁰.

[0056] As used throughout, by “subject” is meant an individual. For example, the subject may be a mammal, such as a primate, and, more specifically, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical uses and formulations are contemplated herein. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder.

[0057] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a vector, such as a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter, followed by a transcription termination signal sequence. An expression cassette may or may not include specific regulatory sequences, such as 5’ or 3’ untranslated regions from human globin genes.

[0058] As used herein the term “extracellular vesicle (EV)” refers to membrane-bound vesicles that are naturally released from eukaryotic cells. As such EVs are cell-derived vesicles, i.e., a lipid bilayer delimited particles, comprising a membrane that encloses an internal space (lumen). EVs are released by cells and found in most biological fluids including urine, plasma, cerebrospinal fluid, saliva etc., as well as in tissue culture conditioned media. Generally, EVs range in diameter from 20 nm to 1000 nm. As used herein, the terms “exosomes,” “small extracellular vesicles,” and “sEVs” are used interchangeably to refer to EVs that are less than 200 nm. In some embodiments, the exosomes are between about 35 nm to about 200 nm. In some embodiments, the exosomes are about 20 to about 200 nm, about 25 to about 190 nm, about 30 to about 180 nm, about 35 to about 170 nm, or about 40 to about 165 nm, and every range located within these ranges. As also used herein, the terms “immunogenic sEVs,” “immune sEVs,” or “imsEVs” are used interchangeably to refer to a sEV that includes a CD64-N peptide fusion protein in its membrane, at least one antibody bound to the extracellular portion of the fusion protein that binds to a protein on a target cell, and a nucleic acid inside the imsEV that stimulates an immune response in the subject upon delivery to the target cell. In some embodiments, the imsEV increases CD8⁺ T cells and Ml type macrophages. In some embodiments, the imsEV inhibits cell proliferation in a tumor tissue.

[0059] “ Treat,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. “Treating” or “treatment” refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the disease condition more tolerable to the patient, slowing in the rate of degeneration or decline, or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the imsEVs of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a disease, condition, or disorder. “Treating” or “treatment” includes the administration of an agent, such as an imsEV, to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. “Treating” or “treatment” using the methods of the present disclosure includes preventing the onset of symptoms in a subject that can be at increased risk of a disease or disorder associated with a disease, condition or disorder as described herein, but does not yet experience or exhibit symptoms, inhibiting the symptoms of a disease or disorder (slowing or arresting its development), providing relief from the symptoms or side effects of a disease (including palliative treatment), and relieving the symptoms of a disease (causing regression). Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition. The term “treatment,” as used herein, includes preventative (e.g., prophylactic), curative, or palliative treatment.

[0060] The term “administer,” as used herein, refers to a method of delivering agents, compounds, or compositions (e.g., imsEVs) to the desired site of biological action. The agents, compounds, or compositions are prepared for administration in a number of ways, including but not limited to injection, ingestion, transfusion, implantation, or transplantation, depending on whether local or systemic treatment is desired, and on the area to be treated. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0061] All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for all purposes. None is admitted to be prior art. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

III. Electroporation Methods and Systems

[0062] FIG. 1 schematically illustrates an example of a system for generating exosomes loaded with mRNAs using a multi-step electroporation process.

[0063] The example system of FIG. 1 includes a first electroporation station 102 and a second electroporation station 104. The first electroporation station 102 is configured to subject a population of cells to a nanosecond duration electroporation treatment. The second electroporation station 104 is configured to subject the population of cells to a millisecond duration electroporation treatment. [0064] The first electroporation station 102 in this example is located in a microfluidic channel 106 of a microfluidic device 108. In use, the population of cells are flowed past an electrical field source 110 adjacent the microfluidic channel to receive the nanosecond duration electroporation treatment. The electrical field source 110 may be a pair of electrodes embedded in the microfluidic device 108 on either side of the microfluidic channel 106. In one implementation the electrodes may be formed by a 50 pm platinum wire that has been embedded by hot embossing in a polymethyl methacrylate (PMMA) block that forms the base substrate of the microfluidic device 108, with the electrodes spaced apart by 300 pm. In some implementations the electrodes may each have a surface area in the range of 10,000 pm² to 50,000 pm² facing the microfluidic channel and be spaced apart by 100 pm to 500 pm. These are just exemplary implementations and other electrode types, geometries, and configurations and other electrical field sources are also possible. In the system of FIG. 1, the electrical field source 110 is shown in electrical communication with a nanosecond pulse generator 112 configured to generate nanosecond pulses at desired amplitude, frequency, and duration parameters.

[0065] In the example of FIG. 1 the microfluidic channel 106 is a channel milled in the base substrate of the microfluidic device 108 perpendicular to the electrical field source 110. In one implementation the microfluidic channel may have a width of 300 pm and a depth of 100 pm where it passes between the electrodes of the electrical field source 110. In some implementations the microfluidic channel may have a cross-sectional area perpendicular to a fluid flow direction in the microfluidic channel 106 at the electrical field source 110 in the range of 10,000 pm² to 50,000 pm². In some implementations the cross-sectional area of the microfluidic channel may be selected to avoid clogging by the cells flowing through the microfluidic channel while still allowing for relatively closely spaced electrodes to facilitate application of uniform and high strength electrical fields across the microfluidic channel. The top of the microfluidic channel may be closed by a thin film or other substrate applied over the base substrate (not shown). The microfluidic channel 106 depicted in FIG. 1 is just an example and other channel geometries and configurations are also possible.

[0066] The microfluidic channel 106 of the microfluidic device 108 of FIG. 1 is fluidically coupled to a fluid inlet 114 and a fluid outlet 116. The fluid inlet 114 is fluidically coupled to a syringe 118. A syringe pump 120 drives fluid from the syringe 118, into fluid inlet 114, through microfluidic channel 106 past electrical field source 110, and to the fluid outlet 116. The syringe pump 120 or another pumping device may be configured to flow fluid through the microfluidic channel 106 at a controlled rate. In some example implementations the syringe pump 120 or other system component may be configured to generate a flow rate in the range of 1 ml / hour to 30 ml / hour past the first electroporation station 102.

[0067] In the example of FIG. 1 the first electroporation station 102 is configured to subject a population of cells to a nanosecond duration electroporation treatment. In some implementations the electrical field source 110 may apply an electrical field having a strength between 5 kV / cm and 125 kV / cm as the population of cells flows past. In some implementations the electrical field source 110 may apply an electrical field having a strength between 30 kV / cm and 80 kV / cm to the population of cells as they flow between the electrodes of electrical field source 110. In some implementations the nanosecond electroporation treatment may have a frequency in the range of 5 Hz to 1 M Hz and may be applied for a duration in the range of 50 ns to 2000 ns.

[0068] In this particular example the microfluidic device 108 is configured to provide suitable conditions for performing a nanosecond electroporation to achieve temporary organelle membrane poration (e.g. nuclear membrane poration) in a population of cells without significantly impacting on cell viability. The closely spaced electrodes of the electrical field source 110 provide for uniform application of the nanosecond duration electrical field to the population of cells and also allows relatively low applied voltage or energy dose and long pulse duration of the nanosecond treatment, helping to collapse the nuclear membrane more effectively without much cell toxicity. The microfluidic configuration of the nanosecond treatment (i.e. the application of this treatment as the population of cells flows in suspension past the electrical field source 110) mitigates electro- hydrolysis-induced side effects such as gas bubble evolution and Joule heating, which could undesirably lead to permanent damage to cells and/or consequent cell apoptosis. Gas bubble formation is common in electroporation, contributed by water electro-hydrolysis. With the presence of gas bubbles, they disturb the instantaneous local electric field, making the followed pulse disruption on neighbor cells much less effective. Moreover, the accompanying Joule heating further elicits the bubble’s growing, rising, and bursting. Since many cells are trapped around gas bubbles, such bubble evolution dynamics cause further detrimental damage to cells, varying from the impact on cell physiological response to complete cell lysis. With a microfluidic nanosecond electroporation treatment, any generated gas bubbles will be taken downstream by flow, away from the Joule heating source (i.e. the electrical field source 110) before growing too big, which mitigates the impact of those air bubbles on the electroporation treatment.

[0069] In the example of FIG. 1 the second electroporation station 104 in this example is located in a separate device 122 from the microfluidic device 108 that houses the first electroporation station 102. In this particular example the separate device 122 is a cuvette with an electrical field source 124 including a pair of spaced apart electrodes. In one implementation the electrodes of the electrical field source 124 may be separated by 4 mm. In some implementations the electrodes of the electrical field source 124 may be separated by a spacing in the range of 1 mm to 5 mm. In the example of FIG. 1 the electrodes of the electrical field source 124 are in electrical communication with a millisecond pulse generator 126 configured to generate electrical pulses at a desired amplitude, frequency, and duration to apply a millisecond duration electroporation treatment to the population of cells held in the device 122. In some implementations, the millisecond duration electroporation may apply an electrical field having a strength between about 400 V/cm and 1,000 V/cm to the population of cells as they flow between the electrodes of electrical field source 124. In some implementations, the millisecond duration electroporation may apply an electrical field having a strength of 1,000 V/cm and may be applied for a duration in the range of 1 ms to 5 ms.

[0070] FIGS. 2-8 illustrate an example process for generating exosomes loaded with mRNA’s using the example system of FIG. 1.

[0071] FIG. 2 illustrates a step of using syringe pump 120 to pump a solution including a population of cells 128 from syringe 118 into microfluidic channel 106 via fluid inlet 114. Syringe pump 120 may operate to flow the solution 128 through microfluidic channel 106 at a flow rate in the range of 5 ml / hour to 20 ml / hour (e.g. at 10 ml / hour).

[0072] In this example the solution pumped through the microfluidic channel 106 includes both the population of cells 128 as well as extracellular nucleic acids 130. In other implementations the extracellular nucleic acids 130 may be added to the solution at a later step after the nanosecond electroporation treatment.

[0073] In this example the cells 128 of the population of cells each include a cellular membrane 132 and a nucleus including a nuclear membrane 134. The cells 128 also are expressing a protein or protein complex 136, a portion of which is located in the cellular membrane 132 of the cells 128. The population of cells 128 may be suspended in a medium suitable for a transfection process such as but not limited to serum free Opti-Mem medium. In some implementations the cells may be present in the solution at a density in the range of 1 x 10⁵ cells per ml ¹ to 1 x 10⁹ cells per mL'¹ (e.g. at 6 x 10⁷ cells per ml/¹). In some embodiments, the process is used for cells that are about 5 to about 200 pm.

[0074] In this example the extracellular nucleic acids 130 may consist of or include a DNA sequence such that transfection of the extracellular nucleic acids 130 into the population of cells 128 will subsequently result in transcription of the DNA sequence to produce mRNA. The extracellular nucleic acids 130 may be present in the solution with the population of cells 128 at a density in the range of 1 pg mL’¹ to 1,000 pg mL’¹ (e.g., 10 pg mL’¹). In some embodiments, the DNA sequence is between about 1 kb and about 100 kb.

[0075] FIG. 3 illustrates a step of subjecting the population of cells 128 to a nanosecond duration electroporation treatment at the first electroporation station 102. In this example the nanosecond pulse generator 112 generates an electrical signal resulting in an electrical field between the electrodes of the electrical field source 110. The population of cells 128 are flowed past the electrodes of the electrical field source 110 to receive the nanosecond duration electroporation treatment. As shown in FIG. 3, the nanosecond duration electroporation treatment results in temporary pore formation in the nuclear membrane 134 of the population of cells 128.

[0076] FIGS. 4-5 illustrate a step of transporting the population of cells 128 to the second electroporation station 104 after receiving the nanosecond duration electroporation treatment. In this particular example, the solution including the population of cells 128 and extracellular nucleic acids 130 is withdrawn from the microfluidic device 108 at the fluid outlet 116 downstream of the first electroporation station 102. In some implementations, the fluid outlet 116 may be a well and the solution may be collected using a pipette device from the well and deposited into the fluid receiving cavity of the second electroporation device 122.

[0077] FIG. 6 illustrates a step of subjecting the population of cells 128 to a millisecond duration electroporation treatment at the second electroporation station 104. In this example the millisecond pulse generator 126 generates an electrical signal resulting in an electrical field between the electrodes of the electrical field source 124. As shown in FIG. 6, the millisecond duration electroporation treatment results in temporary pore formation in the cellular membrane 132 of the population of cells 128. [0078] As shown in FIG. 7, the multi-step nanosecond and millisecond duration electroporation treatments result in transfection of the extracellular nucleic acids 130 into the nuclei of the population of cells 128. The inventors have discovered that performing the nanosecond electroporation treatment (for temporary poration of the nuclear membrane) before the millisecond electroporation treatment (for temporary poration of the cellular membrane) unexpectedly improves cell viability. The inventors have discovered that, when subjected to identical electroporation conditions, cell viability decreased by over 10% when millisecond duration electroporation was administered before nanosecond duration electroporation. This outcome implies that the preceding disruption of the cell membrane diminishes the subsequent polarization of the cell nuclear membrane. The subsequent application of high-frequency nanosecond pulses leads to a greater expulsion of ions or molecules from the treated cells than the effects observed in a millisecond electroporation treatment alone. The severity of the cellular damage inflicted is such that the enhancement of nuclear DNA delivery is largely counterbalanced by the sluggish recovery of the cellular membrane and certain metabolic functions. As a result, the survival rate of these treated cells is substantially lowered. This phenomenon likely constitutes a significant factor contributing to the observed undesirable outcomes of low transfection efficiency and/or diminished cell viability in other prior art treatments.

[0079] After the nanosecond and millisecond duration electroporation treatments, exosomes 138 secreted by the population of cells 128 may be collected. FIG. 8 illustrates the population of cells 128 after transfer to a culture dish. As shown in FIG. 8, the secreted exosomes 138 are loaded with mRNA 142. As also shown in FIG. 8, the membranes of the secreted exosomes 138 include the protein or protein complexes 136 that were expressed by the population of cells 128, with the mRNA 142 bound to a portion of the protein or protein complex 136 inside the exosome 138.

[0080] In the examples illustrated in FIGS. 1-8 the first and second electroporation stations 102, 104 are located in separate devices. In other implementations, both the first and second electroporation stations 102, 104 may be incorporated into a single microfluidic device. In some embodiments, the electroporation stations 102 and 104 are at a sufficient distance apart from one another to reduce or eliminate the influence of the electrical field of either station on the electrical field of the other station. In some embodiments, shielding may be incorporated into the microfluidic device to reduce or eliminate the influence of the electrical field of either station on the electrical field of the other station. [0081] FIG. 9 schematically illustrates an example of a system for generating exosomes loaded with mRNAs using a multi-step electroporation process in which a single microfluidic device 200 includes both a first electroporation station 202 and a second electroporation station 204. The first electroporation station 202 is configured to subject a population of cells to a nanosecond duration electroporation treatment. The second electroporation station 204 is configured to subject the population of cells to a millisecond duration electroporation treatment. A microfluidic channel 206 extends from a fluid inlet 208, to the first electroporation station 202, to the second electroporation station 204, and to a fluid outlet 210. Fluid inlet 208 connects to syringe 212, with syringe pump 214 controlling flow of fluid out of the syringe 212 and into the fluid inlet 208 and through the microfluidic device 200 at a controlled flow rate.

[0082] The first and second electroporation stations 202, 204 each include electrical field sources (e.g. electrodes) 216, 218 and pulse generators 220, 222.

[0083] The first and second electroporation stations 202, 204 are spaced apart on the microfluidic device 200 and are separated by shielding 224 to reduce electromagnetic interference between the two stations 202, 204.

[0084] In some implementations, the example processes and systems described above for FIGS. 1-9 may be used to generate exosomes loaded with mRNA that can be used for therapeutic purposes.

IV. Compositions and Systems for Targeted Immunotherapy

A. Small Extracellular Vesicles (sEVs)

[0085] In one aspect, small extracellular vesicles (sEVs) or exosomes are provided. In some embodiments, the sEVs express a fusion protein in the membrane of the sEV, wherein the fusion protein includes a CD64 amino acid sequence fused to an N peptide sequence, and the sEVs contain a plurality of mRNAs inside the sEV membrane.

[0086] In the disclosed sEVs expressing the CD64-N peptide fusion protein, the CD64 portion of the fusion protein is located in the cell membrane and extends outside the sEV, and the N peptide portion of the fusion protein is located inside the sEV. As used herein, “CD64” refers to an integral membrane glycoprotein. The portion of CD64 that is located on the outside surface of the cell membrane functions as an Fc receptor that binds the Fc portion of an antibody with high affinity. CD64 also may be referred to as “Fc-gamma receptor 1” or “FcyRI.” In certain embodiments, the CD64 amino acid sequence is a human CD64 sequence. In some embodiments, the CD64 is a human FcyRIA (CD64A), FcyRIB (CD64B), or FcyRIC (CD64C) sequence. In certain embodiments, the CD64 sequence comprises the sequence of SEQ ID NO:8 or SEQ ID NO:9 or a sequence that is at least 90% identical to SEQ ID NO:8 or SEQ ID NO:9. In certain embodiments, the fusion protein has an amino acid sequence comprising SEQ ID NO: 10.

[0087] As used herein, “N peptide” refers to a peptide from a bacteriophage that binds to a specific stem loop RNA sequence element called a box B sequence. In some embodiments, the bacteriophage is a lambda (X), P22, or c|)21 bacteriophage. In certain embodiments, the N peptide sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:42, and SEQ ID NO:3, and a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

[0088] In some embodiments, the amino acid sequence of the CD64 portion of the fusion protein and the amino acid sequence of the N peptide portion of the fusion protein are connected by a linker sequence. As used herein, the term “linker” refers to a linkage between two elements, e.g., amino acid sequences. In some embodiments, the linker is a peptide linker. The term “peptide linker” refers to an amino acid or polypeptide that links two amino acid sequences to provide space and/or flexibility between the two amino acid sequences. A peptide linker can include between 1 and 50 amino acids (e.g., between 2 and 50, between 5 and 50, between 10 and 50, between 15 and 50, between 20 and 50, between 25 and 50, between 30 and 50, between 35 and 50, between 40 and 50, between 45 and 50, between 2 and 45, between 2 and 40, between 2 and 35, between 2 and 30, between 2 and 25, between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5 amino acids). Glycine and glycine-serine polymers are relatively unstructured, and therefore may serve as a neutral tether between components. Examples of glycine polymers include (G)n, glycine-alanine polymers (GA)_n, alanine-serine polymers (AS)n, glycine-serine polymers (for example, (GS)n, (GSGGS)n (SEQ ID NO:22), (GmSoGm)n (SEQ ID NO:23), (GmSoGmSoGm)n (SEQ ID NO:24), (GSGGSm)n (SEQ ID NO:25), (GSGS_mG)_n (SEQ ID NO:26), (GGGSm)n (SEQ ID NO:27), and combinations thereof, where m, n, and o are each independently selected from an integer of at least 1 to 20, e.g., 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and other flexible linkers. [0089] In some embodiments, the plurality of mRNAs inside the sEV membrane include a box B sequence. In some embodiments, the box B sequence is at the 3’ end of the mRNA sequence. In certain embodiments, the box B sequence comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and a sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In other embodiments, the box B sequence comprises a sequence that is the reverse sequence of a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and a sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, wherein the box B sequence forms a secondary structure that is bound by the N peptide. For example, a reverse sequence of the sequence agt would be the sequence tga. In some embodiments, at least some of the plurality of mRNAs in the sEV are bound to the N peptide portion of the fusion protein. In some embodiments, the mRNAs are translated into a protein that stimulates an immune response when delivered to a target cell. In certain embodiments, the mRNA is an interferon gamma (IFNy) mRNA. In some embodiments, the amino acid sequence of the IFNy protein comprises SEQ ID NO: 18 or SEQ ID NO:20, or a sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO:20. In some embodiments, the IFNy protein is encoded by a nucleotide sequence that comprises SEQ ID NO: 19 or SEQ ID NO:21, or a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:21.

[0090] In other embodiments, the sEVs further include at least one antibody bound to the CD64 portion of the fusion protein on the external surface of the sEVs. In some embodiments, the antibody is specific for a protein that is expressed on a target cell (e.g., a cancer cell) or is specific for a protein that inhibits an immune response. In certain embodiments, the sEVs include an antibody that is specific for a protein expressed on an epithelial cell of the blood brain barrier. In certain embodiments, the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

[0091] In one embodiment, the mRNA is an interferon gamma (IFNy) mRNA, and the presence of CD64 in the imsEV membrane provides docking sites for both anti-CD71 and anti-PD-Ll antibodies to facilitate sEV targeting to glioblastoma (GBM) tumors. These immunogenic sEVs can successfully target GBM and induce immunotherapy effects in vivo. Moreover, these imsEVs, upon reaching the tumor microenvironment, lead to upregulation of MHC-I expression, which is often downregulated in solid tumors including GBM to promote immune escape, thereby enhancing the antitumor effects of immunotherapy (see, e.g., FIG. 10). These novel imsEVs represent a new strategy for achieving mRNA loading, tumor targeting, and microenvironmental regulation to enhance the effectiveness of cancer immunotherapies.

[0092] In some embodiments, the sEVs can be prepared by any method or process disclosed herein. For example, in some embodiments, the sEVs are produced by (a) subjecting a population of cells to a nanosecond duration electroporation treatment at a first electroporation station, wherein the cells express a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence, and wherein the first electroporation station is located in a microfluidic channel of a microfluidic device in which the population of cells are flowed past at least one electrical field source to receive the nanosecond duration electroporation treatment; (b) after the nanosecond duration electroporation treatment, transporting the population of cells to a second electroporation station; (c) subjecting the population of cells to a millisecond duration electroporation treatment at the second electroporation station, wherein during at least the millisecond duration electroporation treatment the population of cells are in the presence of extracellular nucleic acids comprising a DNA sequence, wherein the DNA sequence encodes a protein that stimulates an immune response, and wherein transcription of the DNA sequence produces an mRNA; (d) after the nanosecond and millisecond duration electroporation treatments, collecting sEVs secreted by the population of cells, wherein the secreted sEVs are loaded with the mRNA, and wherein the mRNA may be bound to the N peptide of the fusion protein; and (e) after collecting the sEVs, binding the CD64 in the membrane of the sEVs to an antibody that is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response.

B. Proteins and Nucleic Acids for Use in imsEVs

[0093] In another aspect, proteins and nucleic acids for use in immunogenic small extracellular vesicles or exosomes are provided. In some embodiments, the disclosure provides a fusion protein that includes a CD64 amino acid sequence fused to an N peptide sequence. In certain embodiments, the fusion protein has an amino acid sequence that comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NOTO. In some embodiments, the CD64 amino acid sequence is a human CD64 sequence. In some embodiments, the CD64 is a human FcyRIA (CD64A), FcyRIB (CD64B), or FcyRIC (CD64C) sequence. In certain embodiments, the CD64 sequence comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9, or a sequence that is at least 90% identical to SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, N peptide is from a bacteriophage selected from the group consisting of a lambda (X), P22, and c|)21 bacteriophage. In certain embodiments, the N peptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, and a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

[0094] In another aspect, the disclosure provides nucleic acids encoding the fusion proteins. For example, in some embodiments, the fusion protein is encoded by a nucleic acid sequence comprising SEQ ID NO: 11 or a sequence that is at least 90% identical to SEQ ID NO:11. Also provided are vectors and cells comprising a nucleic acid encoding the fusion protein.

[0095] In another aspect, the disclosure provides mRNAs that include a box B sequence. In certain embodiments, the box B sequence comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and a sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the mRNAs are translated into a protein that stimulates an immune response. In certain embodiments, the mRNA is an interferon gamma (IFNy) mRNA.

C. Systems or Kits for Production of imsEVs

[0096] In another aspect, systems or kits for the production of imsEVs are provided. In some embodiments, the systems or kits include a cell expressing a fusion protein that comprises a CD64 amino acid sequence fused to an N peptide amino acid sequence; and a vector sequence comprising a DNA sequence encoding a therapeutic protein. In some embodiments, the cell is a mammalian cell, for example a human cell. In some embodiments, the cell is a cell capable of rapid, unlimited proliferation. In some embodiments, the cell is a HEK293T cell. In certain embodiments, the fusion protein has an amino acid sequence that comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the CD64 amino acid sequence is a human CD64 sequence. In some embodiments, the CD64 is a human FcyRIA (CD64A), FcyRIB (CD64B), or FcyRIC (CD64C) sequence. In certain embodiments, the CD64 sequence comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9 or a sequence that is at least 90% identical to SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the N peptide is from a bacteriophage selected from the group consisting of a lambda (X), P22, and c|)21 bacteriophage. In certain embodiments, the N peptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, and a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the DNA sequence encoding a therapeutic protein produces an mRNA that includes a box B sequence. In certain embodiments, the box B sequence comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and a sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the mRNAs are translated into a protein that stimulates an immune response. In certain embodiments, the mRNA is an interferon gamma (IFNy) mRNA.

V. Methods for Targeted Immunotherapy

[0097] In another aspect, methods for targeted immunotherapy are provided. Any of the sEV compositions described herein can be used to treat a disease (e.g., cancer) in a subject. In some embodiments, the methods and compositions are used to treat cancer. The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non- metastatic cancer. In some methods, the cancer to be treated is a brain cancer. In certain embodiments, the cancer to be treated is selected from a glioma, astrocytoma, oligodendroglioma, glioblastoma, subendyoma, myxopapillary ependymoma, and hemangiopericytoma.

[0098] In some methods, any of the imsEVs described herein are administered to the subject. See, for example, Murphy el al.. Experimental and Molecular Medicine 51 : 1-12 (2019)). In some embodiments, the imsEVs can be targeted to a cell or tissue by modification of the EVs expressing the CD64-N peptide fusion protein to include a binding moiety that binds to a target, for example, an antibody that binds a tumor antigen on a tumor cell. As used throughout, the phrase “tumor antigen” or “tumor-specific antigen” means an antigen that is unique to cancer cells or is expressed more abundantly in cancer cells than in in non-cancerous cells. In some embodiments, an effective amount of any of the imsEVs described herein are administered to the subject. As used herein, an “effective amount” is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. In some embodiments, the imsEVs are delivered in a pharmaceutical composition. Typically, such pharmaceutical compositions are formulated for in use in vivo, ex vivo, or in vitro using pharmaceutically acceptable excipients known in the art.

[0099] The dosage of imsEVs administered to a subject will depend on the disease or the symptoms to be treated or alleviated, the administration route, as well as various other parameters of relevance known to a skilled person. The amount of imsEVs to be administered to the subject can be determined by quantitating an imsEV protein using methods well known in the art. The imsEV concentration in any of the compositions described herein may be expressed in many different ways, for instance amount of imsEV protein per unit (often volume) or per dose, number of imsEVs or particles per unit (often volume, per subject, per kg of body weight, etc.). For example, and not to be limiting, a composition comprising from about 10⁶ to about 10²⁵ imsEVs can be administered to a subject in one or more doses. For example, a composition comprising 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, IO²⁰, 10²¹, 10²², 10²³, 10²⁴, 10²⁵ or any other amount of imsEVs, in between these amounts, can be administered to the subject in one or more administrations. In some embodiments, a composition comprising 10¹¹, 10¹², 10¹³, or any other amount of imsEVs, in between these amounts, can be administered to the subject in one or more administrations.

[0100] In some embodiments, the imsEVs are combined with a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier is selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and/or adjuvant to be used with the methods disclosed herein. Such compositions can be used, for example, in a subject with a cancer that would benefit from any of the imsEVs described herein.

[0101] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 2P¹ Edition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and/or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceutically- acceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, 22^nd Edition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release, and/or rate of in vivo clearance of the imsEV.

[0102] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery (e.g., through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intra-ocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration can be in the form of a pyrogen -free, parenterally acceptable aqueous solution (z.e., water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media) comprising an imsEV in a pharmaceutically acceptable vehicle. Preparations for parenteral administration can also include non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0103] In some embodiments, the provided methods may include administering an imsEV and a second form of cancer therapy to the subject. In some embodiments, the second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody).

[0104] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an imsEV and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., antibody or antibody fragment or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an imsEV, 2) an anti-cancer agent, or 3) both an imsEV and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, immunotherapy, or radioimmunotherapy.

[0105] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0106] An imsEV may be administered before, during, after, or in various combinations relative to another anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the imsEV is provided to a patient separately from another anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such embodiments, it is contemplated that one may provide a patient with the imsEV therapy and the anti-cancer therapy within about 6 to 72 hours, about 6 to 48 hours, or about 6 to 24 hours of each other and, more particularly, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0107] In certain embodiments, a course of treatment will last 1-90 days or more (including intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (including intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (including intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more, or any time period within these ranges(including intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. EXAMPLES

[0108] The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1. Materials and Methods

[0109] Cell culture. The SB28 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Thermo Fisher Scientific). SB28 cells were cultured in Dulbecco’s modified essential medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, 26140095; Exosome-depleted FBS, Thermo Fisher Scientific, A2720801) supplemented with 1% penicillin/ streptomycin at 37°C in a humidified condition equilibrated with 10% CO2. The HEK293T, MEF, and GL261 cell lines were purchased from the American Type Culture Collection and cultured in DMEM with 10% FBS supplemented with 1% penicillin-streptomycin at 37°C and 5% CO2.

[0110] Plasmid preparation. Mouse CD64, mouse IFN-y, mouse MLKL, mouse MLKL shRNA, human CD64, and human IFN-y plasmids were purchased from Origene (MC208752, SC300109, MC206757, TR513478, RC207487, RC209993). Primers designed to encode N pep (MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO:2) were used to introduce the ligands into the C terminus of CD64. In the same way, box B (CGGGAAAAAGUCCCG (SEQ ID NO:7)) was introduced into the 3’ end of IFN-y.

[0111] Microfluidic nanopulse channel device fabrication. A microfluidic device for nanopulse electroporation was fabricated by using a computer numeric control machine. One platinum wire of 50 pm in diameter was first embedded in a polymethyl methacrylate (PMMA) block by hot embossing. The platinum wire was then cut into two pieces when a microchannel (width x depth x length = 300 pm x 100 pm x 5 cm) was micro-milled perpendicular to the direction that the wire is positioned on the PMMA block. These two platinum wires serve as the electrodes and connected to the nanopulse circuit during cell stimulation. The flow rate of cell solution in the microfluidic device was regulated by a syringe pump (KDS 100 Legacy Syringe Pump).

[0112] Nanosecond pulse electroporation. A home-made electroporation circuit was designed to generate electrical pulses with both high-voltage and tunable duration of nanosecond pulses. To avoid signal entanglement and pulse profile distortion, the nanosecond pulse generation circuit is separated from the high-voltage supply while connected with a radio frequency metal-oxide-semiconductor field-effect transistor (MOSFET). During the operation, the rectangular signal from the pulse waveform generator (Agilent 33220A) periodically triggers the closure of the electroporation circuit through the MOSFET switch when overcoming its threshold gate voltage. A power supply (KIKUSUI PMC250-0.25A) is used to provide the desired level of energy output by pre-charging a capacitor that stands by until the electroporation circuit is closed by the MOSFET switch to allocate high-voltage pulses on cells with nanosecond pulses while the pulse width, frequency, and number decided by the pulse generator. An oscilloscope was connected to monitor the actual profile of the nanosecond pulses.

[0113] Cell transfection. For nanosecond electroporation (nsEP), MEFs or HEK293T cells were digested, centrifuged at 1000 x g for 10 min, and re-suspended in fresh serum-free OPTI-MEM medium at a density of 6 * 10⁷ cells mL’¹. DNA plasmids were then mixed with the electroporated sample (100 pg mL'¹), which was passed through the microfluidic device and its integrated platinum electrode at a speed of 10 mL h'¹. The treated cell solutions were collected in a traditional electroporation cuvette downstream (with the parallel electrodes separated by 4 mm) and received immediately standard millisecond electroporation, according to the manufacturer’s instructions (BTX Harvard Apparatus ECM630 Electro Cell Manipulator Generator). After electroporation, cells were transferred and further cultured in a fresh exosome-free medium prior to sEV harvesting or further analysis.

[0114] Plasmid loading and mRNA transcription analysis. Copies of plasmids loaded in cells and subsequently transcribed mRNA in the transfected cells were estimated (data not shown). Briefly, 2 * 10⁵ cells were first transfected with 2 pg plasmids by electroporation and divided into two separate groups for further culturing. After cells were re-attached on the culturing surface (approximately 3 hours later), half of the transfected cells were washed with fresh medium to ensure that all plasmids extracted later were those already inside cells. INF-y plasmids were extracted from cells by using DNeasy Blood & Tissue Kits (QIAGEN) in accordance with the manufacturer's instructions. Copies of plasmids in the collected cells of this group were then determined by qPCR. The average plasmid copies per cell were then calculated by dividing the number of cells in each group (estimated by cell viability testing). The average copies of mRNA transcribed from the loaded plasmids were estimated similarly from the other half of the transfected cells (that had been collected 6 hours after plasmid delivery). [0115] Collection and purification of sEVs. The sEVs were collected and purified by ultracentrifugation (See Yang, Z. et cd.. Large-scale generation of functional mRNA- encapsulating exosomes via cellular nanoporation. Nature biomedical engineering 4:69-83 (2020)). Briefly, before cells were transfected, serum-containing cell culture medium was removed. After nsEP, the cells were cultured in exosome-free culture medium for 48 hours. Then, the cell-culture supernatants were centrifuged at 2,000 * for 10 minutes to remove debris, and large vesicles and apoptotic bodies were removed by centrifugation at 10,000 x g for 30 minutes. The final sEV fraction was then purified after ultracentrifugation at 100,000 x g for 2 hours. To prepare imsEVs, CD64-sEVs were incubated with anti-CD71 mAb (Bio X Cell) and anti-PD-Ll mAb (Bio X Cell) (1/1/3, w/w/w, CD64-sEV by protein mass) for 2 hours at 37°C. Subsequently, free antibodies were removed by ultracentrifugation at 100,000 x g for 2 hours.

[0116] sEV number and size measurements. Absolute numbers and size distributions of sEVs were determined with a NanoSight NS300 device (Malvern, PA, USA).

[0117] Cryogenic transmission electron microscopy (cryo-EM). Cryo-EM was used to characterize purified sEVs from MEFs. A concentration of 10¹¹ sEVs mL’¹ was necessary for this experiment. Sample preparation and data acquisition were performed by the Cryo-EM Core Facility at UTHealth Houston. A small aliquot (3 pL) of sample was applied to the Quantifoil R2/1 Cu 200 specimen grid (Electron Microscopy Sciences). Glow discharge of the grid was operated with PELCO easiGlow (Ted Pella). Acquisitions were obtained with a Titan Krios microscope and data were acquired with EPU software (Thermo Fisher Scientific). Images were recorded on a K2 Summit direct electron detector (Gatan) operated in super-resolution counting mode.

[0118] RT-qPCR of exosomal RNA expression levels. The expression of IFN-y mRNA in sEVs was detected by RT-qPCR according to the manufacturer's instructions. Briefly, total RNA was isolated from sEVs by using TRIzol (Invitrogen) and was reverse-transcribed into cDNA with a Reverse Transcription Kit (Thermo Fisher Scientific). Gene expression was measured by using the SYBR Green qPCR kit (BioRad). Expression values were normalized to that of U6. Gene-specific primers included U6 forward (5’-CTCGCTTCGGCAGCACA-3' (SEQ ID NO: 12)), U6 reverse (5’-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO: 13)), IFNG (human) forward (5¹- ACAGCAAGGCGAAAAAGGATG-3' (SEQ ID NO: 14)), IFNG (human) reverse (5’-TGGTGGACCACTCGGATGA-3' (SEQ ID NO: 15)), Ifiig (mouse) forward (5’-CAGCAACAGCAAGGCGAAAAAGG-3' (SEQ ID NO: 16)), and I fug (mouse) reverse (5'-TTTCCGCTTCCTGAGGCTGGAT-3' (SEQ ID NO: 17)).

[0119] The absolute copy number of target mRNA in sEVs was also determined by qPCR results. The average number of target mRNAs per sEV were calculated by dividing by the sEV number measured using NS300. Briefly, the isolated RNA was first reverse-transcribed into complementary DNA (cDNA) by using the TaqMan™ reverse transcription kit (Life Technologies, Carlsbad, CA), following the manufacturer's protocol. The subsequent quantitative polymerase chain reaction (qPCR) analysis was done in triplicate with 100 ng of DNA in a 20 pL reaction volume. Each 20 pL reaction contained 10 pL of TaqManTM Fast Advanced Master Mix, 1 pL of the Gene copy number assay (TaqMan™ If rig Gene copy number assay Mm00734344_cn), and 9 pL of the DNA template. The qPCR conditions included an initial denaturation step at 50°C for 2 minutes, followed by a 10 minute step at 95°C. Subsequently, a total of 40 cycles were performed, consisting of denaturation at 95°C for 15 seconds, followed by annealing and extension at 60°C for 1 minute.

[0120] Proteomics analysis. MEFs were treated with nsEP, harvested, digested overnight with trypsin at 37°C, and incubated with DTT and iodoacetamide to reduce and alkylate proteins⁶¹. Samples were then subjected to solid-phase extraction cleanup with an Oasis HLB plate (Waters), and the resulting samples were loaded onto an EasySpray column (75 pm particles, 750 mm length) to analyze with an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. The gradient consisted of an increase from 1% to 28% solvent B (80% acetonitrile, 10% trifluoroethanol, and 0.1% formic acid in water) over 90 min; solvent A contained 2% acetonitrile and 0.1% formic acid in water. MS scans were acquired at 120,000x resolution in the Orbitrap, and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired by higher- energy collisional dissociation for ions with charges. Dynamic exclusion was set for 25 seconds after an ion was selected for fragmentation. For enrichment analysis of proteins involved in sEV secretion and protein-protein interaction network analysis, the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING databases were applied, and the protein network data were visualized with Cytoscape software (v.3.7.2).

[0121] Preparation of tethered lipoplex nanoparticles (TLN) biochips. The TLN biochips for the exosomal IFN-y mRNA detection were fabricated as follows. An Au layer (~15 nm thick) was coated onto glass coverslips by using a Denton e-beam evaporator (DV- 502A, Moorsetown, NJ), and the freshly coated coverslips were incubated overnight in an ethanol solution containing the lipidic anchor molecule WC14 (20-tetradecyloxy- 3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol), the lateral spacer P-mercapto-ethanol, and biotin-SH at a molar ratio of 30 : 70 : 1. After incubation, the coverslips were washed carefully with 100% ethanol three times and air-dried. A poly dimethylsiloxane chip (24 wells; 3 mm diameter, 4 mm thick) was anchored to the glass side with the Au coating, 20 pL neutravidin ethanol solution (100 pg pL'¹) was added to each well, and the chip was incubated for 15 min at room temperature. During incubation, the lipoplex nanoparticles containing molecular beacons against IFN-y mRNA were freshly prepared as described below, for immediate use thereafter. First 9.75 pL of IFN-y molecular beacon stock solution (at a concentration of 100 pM) was mixed with lipid in ethanol solution (29.5 pL, DOTMA : Cholesterol : Biotin-PEG6-SH = molar ratio of 49 : 49 : 2) and then quickly injected into 675 pL PBS and vortexed for 10 seconds. After the untethered neutravidin was removed from the wells with cold PBS, the freshly prepared lipoplex nanoparticles were added to each well and incubated for 15 minutes at room temperature. After the removal of untethered nanoparticles with cold PBS, the collected imsEVs were added to each well and incubated at 37°C for 2 hours.

[0122] sEV imaging with total internal refractory microscopy (TIRE) and flow cytometry. Purified engineered-sEVs from MEF cells were stained with PKH26, following a tangential flow filtration method to extensively remove the dye residual⁶². Later, anti-CD71, anti-PD-Ll, and IFN-y molecular beacon were added for different staining needs as described in our previously published protocol (Zhang, J. et al, Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids. Scientific reports 11, 1-17 (2021); Zhang, J. et al., Engineering a Single Extracellular Vesicle Protein and RNA Assay (siEVPRA) via In Situ Fluorescence Microscopy in a UV micropattemed Array. bioRxiv, 2022.2008. 2005.502995 (2022); Nguyen, L. T. H. et al., An immunogold single extracellular vesicular RNA and protein ((Au) SERP) biochip to predict responses to immunotherapy in non-small cell lung cancer patients. J Extracell Vesicles 11, el2258, doi: 10.1002/jev2.12258 (2022)). For TLN biochips, sEVs were imaged with a TIRF. Images were recorded by an Andor iXon EMCCD camera with a 100X oil lens, and the exposure time was set at 200 ms. The same staining protocol was used for flow cytometry. sEVs were imaged using Cytek@Aurora (CYTEK). [0123] Image analysis and colocalization. An automatic algorithm was used to quantify detected bright spots present on the TIRF microscopy images. The grey value is the sum of the intensities of all the pixels within the calculated spot area. The open-source plugin EzColocalization was applied with ImageJ to calculate the colocalization efficiency of sEVs stained with different biomarkers acquired from the TIRF microscopy images (Zhang et cd.. Engineering a Single Extracellular Vesicle Protein and RNA Assay (siEVPRA) via In Situ Fluorescence Microscopy in a UV micropattemed Array. bioRxiv, 2022.2008. 2005.502995 (2022); Stauffer, W., Sheng, H. & Lim, H. N. EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms. Scientific reports 8: 1-13 (2018).

[0124] sEV pull-down assay. Protein A magnetic beads (BioRad) were incubated with 5% (w/w) bovine serum albumin in PBS overnight at 4°C, after which the beads were washed three times with cold PBS. Flag antibody (Sigma- Aldrich) was then added to the magnetic beads and the mixture was incubated overnight at 4°C and washed three times with PBS. The purified sEVs were incubated with the magnetic beads overnight at 4°C and washed. The beads were then eluted in 0.1% sodium dodecyl sulfate (SDS), and 20 pL of the supernatant was loaded onto SDS gels for SDS-polyacrylamide gel electrophoresis (PAGE) analysis.

[0125] Confocal microscopy. Transferrin-Alexa Fluor 647 (0.1 mg mL’¹), cholera toxin subunit B-Alexa Fluor 647 conjugates (0.005 mg mL'¹), and dextran-Alexa Fluor 680 (1 mg mL'¹) (Invitrogen) were each incubated with SB28 cells or GL261 cells for 1 hour to label different endonucleases and then washed away. Cells were then incubated with sEVs stained with PKH26 (2 pM) (Sigma-Aldrich) for 4 h, washed twice with PBS, and fixed with 4% formaldehyde in PBS for 15 minutes. Nuclei were stained with 4’,6-diamidino-2- phenylindole in the gold coating solution, and fluorescence was observed and recorded on a laser scanning confocal microscope (LSM880, Carl Zeiss).

[0126] MTS assay. The cytotoxic potential of sEVs was assessed with an MTS assay. SB28 and GL261 cells were plated in 96-well plates (5,000 cells per well) and incubated overnight. Cells were then incubated with sEVs for 24 hours or 48 hours, followed by the addition of MTS reagent (Promega) according to the manufacturer's instructions, and absorbance was measured at 490 nm wavelength after an additional 4 hours of incubation.

[0127] Western blots and antibodies. Protein samples were homogenized in RIPA lysis buffer (Thermo Fisher Scientific) with 1% proteinase inhibitor cocktail (Thermo Fisher Scientific, no. 78429), and the protein lysates were normalized with a BCA protein assay kit (Thermo Fisher Scientific). Protein lysates were separated on 8% / 10% / 12% acrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) under denaturing conditions. The membranes were then blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution at room temperature and incubated overnight at 4°C with primary antibodies (anti-MLKL, catalogue no. ab243142; anti-CD71, catalogue no. ab84036; anti-CD63, catalogue no. ab217345; anti-CD9, catalogue no. ab92726; anti-MHC-I, catalogue no. ab281902; anti-GAPDH, catalogue no. ab8245), after which the membranes were washed three times and then incubated with a secondary antibody (Cell Signaling Technology) conjugated to horseradish peroxidase for 45 minutes at room temperature. The PVDF membranes were developed by using a chemiluminescence detection system.

[0128] Flow cytometry analysis. Cellular uptake of sEVs and assessment of cell surface and internal antigens were analyzed by flow cytometry. To investigate sEV uptake by tumor cells, PKH26-labeled sEVs were incubated with tumor cells for 4 h, after which cells were washed three times with cold PBS and fixed in 4% paraformaldehyde. To assess cell surface and internal antigens, tumor tissues obtained at Day 15 were isolated after transcardial perfusion from each treatment group were collected and digested at 37°C for 60 minutes in 10 mmol L'¹ HEPES buffer with 300 U mL’¹ collagenase D, dispase, and 15 U mL’¹ DNase I to obtain cell suspensions. After dissociation, the cells were filtered through a 70 pm nylon cell strainer and collected. For flow cytometry, cells were fixed and permeated to allow the entry of fluorescence probes. To avoid nonspecific binding to the Fc receptor, cells were first blocked with anti-CD16/CD32 antibody (Bio X Cell, catalog no. BE0307, dilution of 1 :200) for 15 minutes. Then, cells were incubated with various labeled antibodies (anti-IFN-y-PE at a 1 :200 dilution; anti-MHC-I-APC at 1 :300; anti-CD8a-PE at 1 :200; anti-CD86-PE at 1 :300; anti-CD45-PerCP at 1 :200; anti-CD3-APC at 1 :300; or anti-F4/80-APC at 1 :300) according to the manufacturer's instructions. Cell fluorescence intensity was analyzed with a flow cytometer (Gallios 561, Beckman). At least 10,000 events were collected per cell sample. Representative gating strategies for all flow cytometry data were determined (data not shown).

[0129] Enzyme-linked immunosorbent assay. Cytokines in cell culture media were measured by ELISA as follows. Tumor cells were incubated with sEVs for 24 hours or 48 hours, and INF-y (BioLegend) levels were measured in the culture medium. [0130] Levels of aspartate aminotransferase (Abeam), alanine aminotransferase (Abeam), blood urea nitrogen (Abeam), and creatinine (Thermo Fisher Scientific) in serum after 4 hours for the systemic administration of sEVs were also tested using ELISA follow manufacturer’s protocol for the biosafety measurements.

[0131] Blood Safety Assessment. Prior to in vivo application, the sEV safety assessment was conducted. The hemolysis assay and complete blood counts (CBCs) were investigated. For the hemolysis assay, the whole blood from healthy female mice was collected in an anticoagulant solution tube and centrifuged at RT for 15 minutes (900 x g) to get the RBCs. The harvested RBCs were then mildly rinsed with PBS. The RBCs (1 x io⁹ cells) were treated with PBS, imsEV at different concentrations, or 0.5% Triton X-100 (v/v in PBS) at 37 °C for 2 hours. All samples were centrifuged for 15 minutes (900 x g) and photographed. The CBC was obtained using AD VIA 2120i (Siemens, Erlangen, Germany).

[0132] Animals. Six- to eight-week-old C57BL/6J female mice were purchased from Jackson Laboratory or Weitong Lihua Experimental Animal Technology Co. and maintained at the animal facility of The University of Texas MD Anderson Cancer Center or Jilin University in isolator cages in a pathogen-free facility. All experimental procedures were performed in compliance with the institutional policies and approved protocols of Jilin University (no. SY202110005) or MD Anderson Cancer Center (no. 00002163).

[0133] Animal surgery and tumor implantation. GL261-Luc or SB28 cells (1 ^x io⁵) were engrafted into the caudate nucleus of the mice with guide screws as follows. Lal, S. et al. An implantable guide-screw system for brain tumor studies in small animals. Journal of neurosurgery 92, 326-333, doi:10.3171/jns.2000.92.2.0326 (2000). Briefly, anesthetized mice were restrained on the operating table and given preemptive analgesia. A 2- to 3-mm-long incision was then made just to the right of the midline and anterior to the interaural line, and the coronal and sagittal sutures were identified and the bregma marked. Then, a 2-mm diameter twist drill was used to drill a small hole at a point 2.5 mm lateral and 2.5 mm anterior to the bregma, corresponding to a point above the caudate nucleus; a sterilized guide screw was then placed in the hole and gently screwed in until it was flush with the skull. Seven days after placement of the guide screw, the mice were reanesthetized and the tumor cell suspension was infused slowly (0.2 pL min ¹ ) into the brain. The mice were kept warm until recovery from anesthesia and were allowed to move around freely thereafter. [0134] In vivo biodistribution of sEVs. For the sEV biodistribution experiments, at 14 days after implantation of the GBM cells, a luciferase substrate was injected and the presence of tumor was confirmed with an IVIS 200 imaging system (Xenogen). Next, sEVs labeled with the lipophilic dye DiR plus 8 pg protein, or an equal amount of DiR diluted in PBS, were injected into the tail vein of each mouse. The IVIS 200 imaging system was used to assess fluorescence distribution in the intact mice at 1 hour, 2 hours, and 4 hours after injection. Finally, at 4 hours, the mice were euthanized, the heart, liver, spleen, lung, kidney, and brain were removed, and the fluorescence distribution in these organs was assessed with an IVIS 200.

[0135] In vivo tumor-treatment assays. Seven days after implantation, the establishment of intracranial tumors was confirmed using bioluminescence imaging. The mice were randomly divided into five groups and treated with PBS, sEV, Antibody combo, CD64-sEV, or imsEV. The treatment was administered every 3 days through tail vein injections at a dose of 5 x io¹¹ sEVs per injection. It was observed that the fluorescence signals of luciferase were captured and analyzed at 0, 3, 6, 9, 12, and 15 days. Survival curves were constructed using Kaplan-Meier methods for each group.

[0136] Magnetic resonance imaging. Mice were subjected to imaging at the MD Anderson Small Animal Imaging Facility with a 7 Tesla (T) 30-cm horizontal bore magnet (Bruker Biospin MRI, Billerica, MA). Acquisition and image analysis followed the Facility’s protocol and other published procedures. (See Melo, S. A. et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177-182 (2015); Schmid, A. et al., Non-invasive monitoring of pancreatic tumor progression in the RIPl-Tag2 mouse by magnetic resonance imaging. Molecular imaging and biology 15, 186-193 (2013)). Briefly, during the imaging procedure, each mouse was placed under anesthesia with 2% isoflurane. Tumor detection involved acquiring T2-weighted coronal and axial images using specific parameters: T2-weighted coronal slices with a thickness of 0.75 mm were captured in a field of view (FOV) of 30 x 40 and a matrix size of 256 x 192 pixels, resulting in an in-plane resolution of 0.156 pm. Similarly, T2-weighted axial slices with a thickness of 0.75 mm were obtained in a FOV of 30 x 22.5, using a matrix size of 256 x 192 pixels, yielding an in-plane resolution of 0.117 pm. These images were acquired with a RARE (rapid acquisition with relaxation enhancement) sequence, with a repetition time (TR) of 3000 ms and an echo time (TE) of 57 ms. The regions suspected of containing lesions were delineated on each slice in a blinded manner using Image! The volume was calculated by summing the delineated regions of interest in mm² x 1 mm slice intervals.

[0137] Histologic and immunofluorescence assays. Mice from each treatment group were euthanized on the indicated number of days after tumor inoculation. After transcardial perfusion with saline and paraformaldehyde, the brain was extracted and placed in a solution of 30% sucrose until the brain tissue sank to the bottom and was collected afterward. Next, the brain tissues were embedded in Tissue-Tek optimum cutting temperature (OCT) compound, frozen in liquid nitrogen, and maintained at -80°C. Frozen tissue blocks were cut into 10 pm slices using a freezer and attached to adhesive glass slides. The tissue slices were stored in a -80°C refrigerator for later use. For analysis of IFN-y, CD8, or MHC-I, tissue samples were thawed and then incubated for 30 minutes in 0.1% Triton X-100, blocked with 10% goat serum for 1 hour, and incubated with the primary antibodies IFN-y- AF647 (SouthemBiotech), CD8-AF647 (Thermo Fisher Scientific), or MHC-I-AF647 (BioLegend). Images were acquired with an LSM880 microscope (Carl Zeiss) and processed with Zeiss Zen software.

[0138] For histologic evaluation of paraffin sections, after mice were euthanized (Day 15), hearts, lungs, livers, spleens, kidneys and brains were extracted and fixed with paraformaldehyde. The fixed tissue was dehydrated in ethanol solution and xylene successively until the tissue was transparent. Dehydrated tissue was embedded in paraffin and cut into 4 pm wax sections and placed on glass slides. Then, slides were deparaffinized three times in xylene, followed by graded ethanol rehydration. Antigen retrieval, immunofluorescence staining, and immunohistochemical staining were then performed per the manufacturers’ instructions². The primary antibody against Ki67 (Abeam) was used at 1 : 1,000 dilution. Hematoxylin and eosin staining was used to analyze normal organs, including the liver, lung, heart, spleen, and kidney. Image acquisition was done with a BX3 microscope (OLYMPUS).

[0139] Statistical analysis. Statistical analyses were done with GraphPad Prism 9 and presented as means ± SD. Statistical significance was evaluated by one-way analysis of variance with Tukey's multiple comparisons test and Student's t tests. For survival studies, log-rank (Mantel-Cox) tests were used. P values of < 0.05 were considered to indicate statistically significant differences.

Data availability [0140] The sequencing data was deposited in the Center for Computational Mass Spectrometry (CCMS) database (accession number MSV000090923).

Example 2. High-throughput generation of sEVs by nanosecond pulse electroporation (nsEP).

[0141] To design an electroporation system that produces highly efficient mRNA-loaded sEVs, source cells are subjected to a two-step electroporation process. First, nanosecond electropulses are used to transiently permeabilize the membrane structure of organelles inside source cells, which are then exposed to millisecond pulses that permeabilize the cell plasma membrane (FIGS. 11A and 17). This nanosecond pulse electroporation approach (nsEP) allows high cell transfection performance (FIG. 18) and large-scale generation of sEVs, leading to a 46-fold increase in sEV production (relative to control) by mouse embryonic fibroblasts (MEFs) (FIG. 1 IB) and a 40-fold increase by the human embryonic kidney 293 T (HEK293T) cell lines (data not shown). Non-significant differences in sEV release were observed with or without the presence of plasmids in host cells during nsEP treatment (FIG. 1 IB and data not shown). Adjusting the pulse parameters of nsEP allows further optimization of the quantity of sEV secretion as follows: (1) when the pulse voltage is raised from 50 V to 200 V, more sEVs are secreted from host cells, reaching a plateau at 180 V (FIG. 11C). A slight decrease in cell viability is observed when the voltage exceeds 180 V (FIG. 11D and data not shown). (2) The frequency and duration of nanopulses also have an influence on this nsEP -triggered sEV secretion, with cells treated at 100 kHz and 600 ns releasing the most sEVs (FIGS. 19A-19B and data not shown) whilst maintain high cell viability (FIGS. 19C- 19D). The size distribution of sEVs secreted by microsecond electroporation pulses (msEP) or nsEP stimulated cells and natural secretion groups (“untreated control”) were similar and shared the same morphological features, all showing a dominant size of about 120 nm (FIG. HE and data not shown). The sEVs generated by nsEP were further characterized after purification by using western blotting, which demonstrated the sole presence of sEVs without apoptotic bodies (data not shown).

[0142] With the microfluidic setup and multiple units operated in parallel, large production capacity of EVs can be achieved with high throughput (3.0 x 10⁷ cells in 5 minutes), which would be sufficient for many clinical applications with regard to both sEV quantity and processing time. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) confirmed that levels of mRNAs complementary to the IFN-y plasmid DNAs that had been encapsulated within the sEVs were 10³-fold higher in the nsEP system -treated groups than in the control samples (FIG. 1 IF). Similar levels of sEV secretion induced by nsEP were found in HEK293T cells (FIGS. 20A-20F and data not shown).

Example 3. Mechanism of nsEP-induced sEV secretion.

[0143] To reveal the cellular mechanisms underlying the nsEP -triggered release of sEVs, proteomic profiling was used to identify the relevant proteins involved in this process. A total of 4423 quantifiable proteins were evaluated, among which 1344 were expressed at statistically different levels before and after the nsEP stimulation when the fold change threshold was set at 1.5. These 1344 proteins were further classified according to their functions by using Gene Ontology annotations. The results reveal that the nsEP treatment induces multiple cellular and metabolic processes, biological regulation, and responses to stimuli (FIG. 12A). Proteins that differed in the cellular-process component were Gene Ontology-enriched to obtain three sEV-associated clusters of extracellular sEVs, extracellular space, and extracellular vesicles involving 104 proteins (FIG. 12B and data not shown). Upon identifying proteins involved in the regulation of sEV secretion, we used protein-protein interaction network analysis to recapitulate proteins associated with classic sEV-generation pathways, including intraluminal vesicle formation (MLKL, Sdcbp, Cdc2), protein ubiquitination (Ndfipl), endosomal sorting complex required for transport (ESCRT)- dependent cargo sorting (Vps36, Vtal, Chmp4b, Mvbl2a, Chmp3, Chmp5), small GTP- binding proteins leading to exosomal budding from the plasma membranes (Rab8a, Rab27b, Raia), lysosomal degradation of multivesicular bodies by ISGylation (ISG15, Uspl8) or autophagy (Pmp), and SNARE interactions in vesicular transport (Stxl7, Vamp7, Ykt6, Vamp8) (FIGS. 12C-12D and data not shown). A heatmap was generated for the top 95 proteins showing differences of 6-fold or more (data not shown). Among them, MLKL was two orders of magnitude higher than other regulatory proteins involved in sEV generation. This suggests that the increased sEV secretion during nsEP stimulation depends strongly on MLKL (FIG. 12D and data not shown). To confirm the involvement of MLKL in sEV trafficking, MLKL expression was silenced in MEFs, an MLKL-knockdown cell line was generated, and the effects on sEV generation after nsEP treatment were analyzed (FIG. 12E). Downregulating MLKL was found to lead to significant inhibition of sEV production from cells exposed to nsEP. A slight increase in MLKL protein expression was observed after nsEP treatment, leading to limited recovery of sEV generation (FIGS. 12E-12F). Conversely, overexpressing MLKL in MEFs promotes the nsEP-stimulated sEV production, further implicating MLKL in the regulation of sEV secretion during this process (FIGS. 12E-12F).

Example 4. Preparation and characterization of imsEVs.

[0144] A strategy was developed to actively incorporate target mRNAs into these secreted sEVs, with a goal of restoring immunogenicity in solid tumors (Miller, K. D. et aL, Brain and other central nervous system tumor statistics, 2021. CA Cancer J. Clin. 71 :381-406, doi: 10.3322/caac.21693 (2021); Wang, Y. et al., Nanoparticle-mediated convection-enhanced delivery of a DNA intercalator to gliomas circumvents temozolomide resistance. Nat. Biomed. Eng. 5, 1048-1058, doi: 10.1038/s41551-021-00728-7 (2021); Stupp, R. et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England journal of medicine 352, 987-996 (2005)). GBM was selected as a preclinical model system, as it is an aggressive tumor with no effective treatment currently available and does not respond to immunotherapy (Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350-1355 (2018); Sampson, J. H. et al.,. Brain immunology and immunotherapy in brain tumours. Nat. Rev. Cancer 20, 12-25, doi: 10.1038/s41568-019-0224-7 (2020); Simonds, E. F. et al., Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma. Journal for immunotherapy of cancer 9 (2021); Han, R. T. et al., Astrocyte- immune cell interactions in physiology and pathology. Immunity 54, 211-224 (2021)).

[0145] Another major obstacle for effective immunotherapy in GBM is the downregulation of major histocompatibility complex class I (MHC-I) proteins on the tumor surface, which leads to poor antigen presentation (Genoud, V. et al., Treating ICB-resistant glioma with anti- CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin). JCI insight 6 (2021); Castro, M. et al., (American Society of Clinical Oncology, 2020); Yang, W ., Li, Y ., Gao, R., Xiu, Z. & Sun, T. MHC class I dysfunction of glioma stem cells escapes from CTL- mediated immune response via activation of Wnt/p-catenin signaling pathway. Oncogene 39, 1098-1111 (2020)). The low mutational load, with correspondingly few infiltrating T cells and Ml macrophages, plus the downregulation of MHC-I, results in a highly immunosuppressive tumor microenvironment (Lim, M. et al., Current state of immunotherapy for glioblastoma. Nature reviews Clinical oncology 15, 422-442 (2018); Quail, D. F. & Joyce, J. A. The microenvironmental landscape of brain tumors. Cancer cell 31, 326-341 (2017); Arora, S. et aL, Macrophages: their role, activation and polarization in pulmonary diseases. Immunobiology 223:383-396 (2018)).

[0146] To prepare imsEVs for immunotherapy of GBM, both GBM-targeting (anti-CD71) and immunotherapy (anti-PD-Ll) antibodies were attached to the surfaces of imsEVs to enable their specific targeting of GBM for immunotherapeutic effects. The method took advantage of CD64, an Fey receptor that can bind to the constant region of IgG heavy chain (Mancardi, D. A. et al. The high-affinity human IgG receptor FcyRI (CD64) promotes IgG- mediated inflammation, anaphylaxis, and antitumor immunotherapy. Blood, The Journal of the American Society of Hematology 121, 1563-1573 (2013); Bruhns, P. & Jonsson, F. Mouse and human FcR effector functions. Immunological reviews 268, 25-51 (2015)).

[0147] First, an MEF line was constructed that stably expressed CD64-DsRed (CD64- DsRed⁺) (data not shown). In CD64-DsRed⁺ cells, co-localization of CD64-DsRed with CD63-GFP, a classic surface marker protein of sEVs, was associated with a significant increase in CD64 content in sEVs generated from CD64⁺ cells, suggested the presence of CD64 expression on the surface of sEVs generated by this method (FIGS. 13A and data not shown). To further evaluate the topology of CD64 on sEVs, a 3XFLAG epitope was inserted into the N-terminus of CD64, and a Myc epitope was inserted into the C-terminus of CD64. A pulldown assay was performed with anti-FLAG beads to confirm that the N-terminus of CD64 was localized to the external surface of these sEVs (FIG. 13B and data not shown). To achieve active loading of target mRNA into sEVs, the method took advantage of the N peptide, which specifically binds to the box B sequence in the RNA, as follows (Cai, Z. et al., Solution structure of P22 transcriptional antitermination N peptide-box B RNA complex. Nature structural biology 5, 203-212 (1998); Cilley, C. D. & Williamson, J. R. Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE). Rna 3, 57-67 (1997)). The N peptide was cloned into the C- terminus of the CD64 protein (which is located inside sEVs when they are formed by the inward budding of endosomal membranes, as shown in FIG. 13C), and a box B sequence to the 3’ end of IFN-y mRNA by engineering the IFN-y plasmid. It was reasoned that the highly specific binding affinity between N peptide and box B sequence would enrich box B-fused IFN-y mRNA within sEVs during their formation (FIG. 13C). The box B-IFN-y or control IFN-y was transfected into MEFs that stably express CD64-N peptide, and the secreted sEVs were harvested for mRNA analysis by RT-qPCR. As shown in FIG. 13D, IFN-y mRNAs fused with box B-sequence were greatly enriched in sEVs produced by the CD64-N peptide- overexpressing cells. This was further verified by using a tethered lipoplex nanoparticle (TLN) biochip that contains molecular beacons against IFN-y mRNA. The mean fluorescence intensity in a single sEV from the box B-IFN-y group was 3.5 times higher than that from the control IFN-y plasmid group (FIGS. 13E and data not shown). Similarly, the percentage of sEVs containing IFN-y mRNA was 20% higher in the box B-IFN-y group than in the control IFN-y plasmid group (FIG. 13F and data not shown). Flow cytometry -based detection of single sEV yielded similar conclusions (data not shown). These findings suggest that the disclsoed active loading strategy indeed enhances the loading of specific mRNA (IFN-y mRNA in this study) into imsEV, thus potentially improve their potency.

[0148] Since CD71 is overexpressed on GBM cell lines, but not on MEF or HEK293T cells (FIG. 13G), CD71 was chosen as the active GBM-targeting marker. To check the binding affinity of these CD64⁺ sEVs with antibodies to CD71 or PD-L1, the sEVs were incubated with either anti-CD71 (Mouse IgG2a, K) or anti-PD-Ll (Rat IgG2b) at different sEV/antibody (w/w) ratios. As shown in FIG. 13H, binding was noted between total sEV protein and anti- CD71, starting at a ratio of 1 :0.5 as evident by the detection of IgG heavy and light chains on western blotting; additional binding was observed as the IgG concentration was increased before reaching a plateau, in which the ratio of total sEV protein to IgG was 1 :4 (w/w). Similar results were also observed in the anti-PD-Ll group (FIG. 13H). To achieve both targeting and immunotherapy effects in these mRNA-containing imsEV, the sEVs were incubated with anti-CD71 and anti-PD-Ll antibodies, and their co-existence was confirmed by Western blotting (data not shown). Aiming to improve the targeting and immunotherapy efficacy, anti-CD71 and anti-PD-Ll at different antibody ratios were optimized to the optimal co-localization rate. It was found that most (>70%) of the imsEV were conjugated with both anti-CD71 and anti-PD-Ll antibodies when the ratio of anti-CD71 to anti-PD-Ll was 1 :3 (FIG. 13H and data not shown). Therefore, in the following experiments, the ratio of CD64- sEV /anti-CD71 /anti-PD-Ll was set at 1 /I /3 (w/w/w). The binding capability was further confirmed by flow cytometry (data not shown). The binding of antibodies on sEVs makes the particle size of the imsEVs derived from MEFs slightly larger than the regular ones (-about 10 nm), as shown in FIG. 131 and data not shown. Cryo-electron microscopy analysis demonstrated that CD64-sEV and imsEV derived from MEF cells treated with the nsEP system exhibited electron-dense cargo in the lumen, whereas sEV from untreated MEFs were devoid of such content. The surface characteristics of imsEV, relative to CD64-sEV, showed increased depth, thereby confirming the presence of IgG attached to the surface of the imsEVs. Similar results were also obtained in HEK293T cells (data not shown).

Example 5. In vitro study of imsEV for GBM therapy.

[0149] To evaluate the potential therapeutic utility of imsEV, their cytotoxicity was first studied in vitro and no significant cytotoxicity was found in the two GBM cell lines tested (SB28 and GL261) at 24 hours or 48 hours (FIGS. 21A-21D), indicating good biocompatibility. Linking the CD71 antibody to imsEV significantly increased uptake of the imsEVs by both SB28 and GL261 cells in vitro (FIGS. 14A-14C and data not shown). The studies of endocytosis showed strong co-localization of imsEV with transferrin and partial co-localization of imsEV with other endocytosis markers, indicating that entry of imsEV into target cells was regulated mainly by clathrin-mediated endocytosis (data not shown). Indeed, inhibition of clathrin-mediated endocytosis significantly reduced the cellular uptake of imsEVs, confirming the importance of this pathway in the regulation of imsEV uptake (FIG. 14D). After imsEVs had been incubated with GBM cells for 48 hours, much higher concentrations of IFN-y protein were noted in both the culture medium and cytosol as measured by ELISA (FIG. 14E and FIGS. 22A-22C). Because IFN-y can upregulate the expression of MHC-I on GBM cells, thereby affecting their immunogenicity, MHC-I expression was further investigated after imsEV treatment by flow cytometry. It was noted that the proportion of MHC-I-positive cells increased significantly at 48 hours after imsEV treatment (FIGS. 14F and 14G). Western blotting results further verified the increased MHC- I expression in the imsEV-treated condition (FIG. 14H).

[0150] To further investigate the therapeutic potential of imsEV for in vivo applications, the biosafety of the imsEV was evaluated through co-incubation with blood samples. No hemolytic toxicity was observed at the studied concentrations of imsEV (data not shown). The results of mouse biosafety and biocompatibility experiments showed that at 24 hours after administration of imsEV, serum markers including ALT, AST, BUN, and creatinine in the blood of healthy mice were all within the normal range, with values that are similar to the control groups (FIGS. 23A-23D). Most sEVs were found to accumulate in the livers of healthy mice, and their fluorescence in the brain was weak for mice injected with sEV and CD64-sEV, but slightly stronger for those injected with imsEV, possibly because of TfRl expression by the brain capillary endothelial cells forming the BBB (data not shown). In addition, a comprehensive analysis of total blood cell counts revealed no statistically significant changes in red blood cells, white blood cells, or lymphocytes across various preparations and the PBS-negative control in Naive mice (FIGS. 24A-24I). Moreover, no differences were observed in the quantities of distinct T cell subsets (i.e., CD4⁺, CD8⁺) in the blood and spleen samples (data not shown). Collectively, these findings indicate that these imsEV have a favorable safety profile for in vivo administration.

Example 6. Therapeutic efficacy of imsEV in preclinical models.

[0151] To investigate the immunotherapeutic potential of imsEV in vivo, imsEV were injected intravenously at a dose of 5 * 10¹¹ sEVs into immune-competent mice implanted with GL261 tumors, which are moderately immunogenic. Results from IVIS in vivo imaging showed that the imsEV had significantly improved tumor targeting capability than nontargeted sEVs at 2 hours and 4 hours after injection (data not shown). Ex vivo evaluation of systemic biodistribution indicated a significantly higher accumulation of imsEV within tumors as compared with non-targeted sEVs, with a corresponding drastic reduction in hepatic accumulation (FIG. 15A and data not shown). Administering imsEV to the GL261 tumor-bearing mice every 3 days led to significant inhibition of tumor growth at 7 days after tumor implantation (FIGS. 15B and data not shown) and extension of survival, as evidenced by a median survival time of 53.5 days for the imsEV-treated mice versus 35 days for the antibody combo-treated mice and 30 days for the control groups (FIG. 15C). Immunoassays of residual tumor tissue revealed that IFN-y protein expression was increased after imsEV treatment (data not shown). MHC-I levels were also upregulated after imsEV treatment relative to the other treatment conditions (data not shown). Upon restoration of MHC-I expression in GBM, the proportion of CD8⁺ cells in tumor tissues from imsEV-treated mice also increased (data not shown), and the upregulation of IFN-y was associated with increased proportions of Ml -type macrophages at the tumor site (FIG. 15D and data not shown). Immunohistochemical staining further showed that imsEV treatment greatly reduced tumor cell proliferation in the GBM tissue (data not shown). Toxicity effects were not observed in major organs including heart, spleen, liver, lung, and kidney (data not shown).

[0152] Finally, after validating the therapeutic potential of imsEV in the moderately immunogenic GL261 mouse model, its antitumor effects were investigated in the orthotopic SB28 murine GBM model, which is poorly immunogenic owing to its low intrinsic MHC-I expression and is phenotypically similar to human GBM. Similar to the observation with the GL261 model, imsEV accumulated in SB28 tumors to a greater extent than the non-targeted sEVs did, as evidenced by strong DiR fluorescence at 2 hours and 4 hours in the imsEV- treated group (data not shown). Ex vivo data further confirmed that more imsEV accumulated at the tumor and less at the liver relative to the non-targeting sEVs (FIG. 16A). Tumor growth was also drastically reduced after imsEV treatment (FIGS. 16B), and survival time was extended (median survival time of 50 days in the imsEV-treated group versus 27 days in the PBS-treated group) (FIG. 16C). Magnetic resonance imaging further confirmed that the tumors were the smallest in animals from imsEV-treated group as compared with the other treatment groups (FIG. 16D). Again, an increase in IFN-y and MHC-I expression was noted in tumors after imsEV treatment (FIGS. 16E and 16F and data not shown). The proportions of CD8⁺ T cells and CD86⁺ macrophages that penetrated the GBM tumor sites after imsEV treatment were also greatly increased (FIG. 16G and data not shown). Furthermore, increased expression of Ibal was detected in tumors of imsEV-treated mice (data not shown), which is considered evidence of CD8⁺ T cell-mediated adaptive immunity. Additionally, by blood cell counts and T cell immunity in both blood and spleen of mice across different treatment groups, it was observed that imsEV partially mitigated the systemic immune suppression associated with GBM. This finding underscores the potential of imsEV as a therapeutic intervention for alleviating the immune suppression observed in GBM (data not shown). Finally, immunohistochemical staining results confirmed that imsEV treatment reduced the proliferation of SB28 tumor cells, but did not affect proliferation in the heart, liver, spleen, lung, and kidney (data not shown).

Example 7. The imsEVs are efficiently produced and useful for targeted imunotherapy

[0153] In addition to the large-scale production of sEVs, the disclosed method also facilitates enrichment of the doses of target RNA probes in the sEVs, in two ways. First, combining electric pulses of different duration (i.e., nsEP and msEP) enhances the loading efficiency of plasmids as well as their expression kinetics. In detail, the nanosecond pulses help to increase the permeability of the nuclear membrane of the treated cells and accelerate transportation of plasmids to the nucleus and the overall transcription process. The second means of enriching target-mRNA doses in sEVs is by promoting the recruitment of the target mRNA (e.g., IFN-y mRNA) by engineering a small box B sequence in the 3’ end of the target mRNA and the N peptide on CD64, which are overexpressed on the membrane of host cells. The specific binding affinity between the box B and the N peptide on its amino-terminal arginine-rich domain was determined to selectively enrich the target RNA probes in sEVs during their formation and leveraged the average mRNA number in individual sEVs. Considering that the sEV population is similar in both cases (nsEP with and without N- peptide introduction), the increase in mRNA probes in sEVs produced by the nsEP-plus-N peptide approach is mainly attributable to having more than one mRNA per individual sEV.

[0154] For a deeper understanding of the biological mechanisms underlying this nsEP- triggered sEV release, proteomics analysis was used, which implicated three proteins in the sEV secretion process: MLKL, ISG15, and Uspl8. MLKL is known to be required for the effective generation of intraluminal and extracellular vesicles. (Yoon, S. et al., MLKL, the protein that mediates necroptosis, also regulates endosomal trafficking and extracellular vesicle generation. Immunity 47, 51-65. e57 (2017)). MLKL was pivotal in controlling sEV production after nsEP treatment, as MLKL deficiency led to reduced levels of sEV secretion, below the basal level of untreated cells. Others have found that an ISGylation modification of the multivesicular body protein TSG101 by ISG15 can facilitate its co-localization with lysosomes and promote their aggregation, thereby impairing sEV secretion, and that this effect could be reversed by the Ub-specific protease USP18. (Villarroya-Beltri, C. et al. ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins. Nature communications 7, 1-11 (2016)). The ISGylation targets of functional proteins in the secretion of sEVs are TSG101 and heat-shock proteins (HSPs). (See Giannakopoulos, N. V. et al. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochemical and biophysical research communications 336, 496-506 (2005); Sanyal, S. et al. Type I interferon imposes a TSG101/ISG15 checkpoint at the Golgi for glycoprotein trafficking during influenza virus infection. Cell host & microbe 14, 510-521 (2013)). Interestingly, although the proteomics profiling revealed ISG15 and USP18 as top candidates in the sEV secretion process (nsEP led to a 189-fold increase in ISG15 and an 81- fold increase in USP18), most downstream functional proteins of ISG15/USP18 signaling, including TSG101 and HSP90, were not significantly changed. Therefore, ISG15/USP18 were excluded as being the main factors for promoting sEV trafficking during nsEP. This differs from previous findings on sEV secretion after cellular nanoporation (CNP), in which HSP90 and HSP70 were found to be critical for electroporation-stimulated sEV production: inhibiting both greatly reduced the numbers of sEVs produced after CNP. (Yang, Z. et al. Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation. Nature biomedical engineering 4, 69-83 (2020)). One possible explanation for this difference is the formation of a transient, localized heat shock to the cell membrane close to the nanopore during CNP, but not during nsEP. Hence even though an electroporation step is involved in both techniques, the major mechanisms underlying the enhancement of sEV secretion are different, although they may share some similarities.

[0155] Because a natural receptor for the Fc domain on IgG is anchored on the external surface (on the N-terminal of CD64), the sEVs produced by the disclosed methods could be used to selectively target other cell types simply by changing the antibodies. In this work, the potential of these imsEVs for immunotherapy in GBM was investigated. Although the success of checkpoint blockade has generated considerable enthusiasm for immunotherapy in general, immunotherapy for GBM has not been successful clinically. (Jackson, C. M. et al., Mechanisms of immunotherapy resistance: lessons from glioblastoma. Nature immunology 20, 1100-1109 (2019)). GBM effectively evades immune surveillance, in part through downregulating MHC-I is usually downregulated in GBM cells. (Wu, A. et al. Expression of MHC I and NK ligands on human CD133+ glioma cells: possible targets of immunotherapy. Journal of neuro-oncology 83, 121-131 (2007)). Exposing GBM cells to IFN-y is thought to restore MHC-I expression on their surfaces. (Tanaka, K. et al., Expression of major histocompatibility complex class I antigens as a strategy for the potentiation of immune recognition of tumor cells. Proceedings of the National Academy of Sciences 83, 8723-8727 (1986)). IFN-y has antitumor effects by modulating the functions of tumor cells, immune cells, and other cells in the TME, and effective immunotherapy seems to require abundant and constant secretion of IFN-y into the TME. (See Mendoza, J. L. et al. Structure of the IFNy receptor complex guides design of biased agonists. Nature 567, 56-60 (2019); Ivashkiv, L. B. IFNy: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nature Reviews Immunology 18, 545-558 (2018); Tau, G. Z., Cowan, S. N., Weisburg, J., Braunstein, N. S. & Rothman, P. B. Regulation of IFN-y signaling is essential for the cytotoxic activity of CD8+ T cells. The Journal of Immunology 167, 5574-5582 (2001); Jorgovanovic, D., Song, M., Wang, L. & Zhang, Y. Roles of IFN-y in tumor progression and regression: a review. Biomarker research 8, 1-16 (2020)). However, delivery of soluble IFN-y has a wide range of side effects that depend on dose, route of administration, and frequency. (See Yuba, E. et al. pH-sensitive polymer-liposome-based antigen delivery systems potentiated with interferon-y gene lipoplex for efficient cancer immunotherapy. Biomaterials 67, 214-224 (2015); Wu, J. et al. Dynamic distribution and expression in vivo of the human interferon gamma gene delivered by adenoviral vector. BMC cancer 9, 1-7 (2009); Gocher, A. M., Workman, C. J. & Vignali, D. A. Interferon-y: teammate or opponent in the tumor microenvironment? Nature Reviews Immunology 22, 158-172 (2022)). The US FDA has approved the use of the recombinant protein IFN-ylb, given as subcutaneous injections, to reduce the risk of sEV side effects. (Todd, P. A. & Goa, K. L. Interferon gamma- lb: a review of its pharmacology and therapeutic potential in chronic granulomatous disease. Drugs 43, 111-122 (1992)). Moreover, IFN-y is known to have a short half-life, which necessitates frequent dosing or continuous infusion to sustain therapeutic efficacy. Thus far IFN-ylb has shown disappointing results in the clinic because of the short half-life of the IFN-y protein and the toxicity associated with frequent dosing. (Razaghi, A. et cd.. Review of the recombinant human interferon gamma as an immunotherapeutic: Impacts of production platforms and glycosylation. J Biotechnol 240, 48-60, doi: 10.1016/j.jbiotec.2016.10.022 (2016); Gleave, M. E. et al. Interferon gamma- lb compared with placebo in metastatic renal-cell carcinoma. New England journal of medicine 338, 1265-1271 (1998)). Limited tumor targeting is another significant clinical challenge for the clinical use of cytokine immune checkpoint blockade, which in the case of IFN-y is limited because of the widespread expression of IFNGR. The nonspecific distribution of IFN- y can also result in off-target effects and potentially limit its therapeutic efficacy. For these reasons, ways of introducing the IFN-y gene into the targeted tumor or immune cells were explored by encapsulating the mRNA for IFN-y in carriers to result in localized and constant production of IFN-y.

[0156] Various carriers such as adenovirus, oncolytic viruses, and liposomes have been used to load the gene that encodes IFN-y and allow cytokine release in the TME; some of these carriers have had beneficial antitumor effects in vitro. (See Bourgeois-Daigneault, M. C. et al. Oncolytic vesicular stomatitis virus expressing interferon-y has enhanced therapeutic activity. Mol Ther Oncolytics 3, 16001, doi: 10.1038/mto.2016.1 (2016); Oh, E. et al., Oncolytic adenovirus coexpressing interleukin- 12 and decorin overcomes Treg-mediated immunosuppression inducing potent antitumor effects in a weakly immunogenic tumor model. Oncotarget 8, 4730 (2017)). However, those studies were not designed specifically for GBM therapy. To date, adequate and constant IFN-y expression in the TME within the brain has not been confirmed in trials of oncolytic virotherapy. (See Foreman, P. M. et al., Oncolytic virotherapy for the treatment of malignant glioma. Neurotherapeutics 14, 333-344 (2017)). One potential challenge for such studies is that, unlike sEVs, only specific groups of viruses can cross the BBB. Second, encapsulating large molecules (e.g., mRNA) into viruses that can cross the BBB is difficult because of their limited capacity (e.g., 4.5 kb for AAV rhlO, parvovirus). (Hoshino, Y. et al. The adeno-associated virus rhlO vector is an effective gene transfer system for chronic spinal cord injury. Scientific reports 9, 9844 (2019)). Moreover, although one study found that an inserted peptide could increase the infectivity of glioma cells, most virus carriers result in untargeted viral replication, whereas sEVs demonstrate flexible surface functionalization capability to target specific cells. (Salunkhe, S. et al., Surface functionalization of exosomes for target-specific delivery and in vivo imaging & tracking: Strategies and significance. Journal of Controlled Release 326, 599-614 (2020); Das, C. K. et al. Exosome as a novel shuttle for delivery of therapeutics across biological barriers. Molecular pharmaceutics 16, 24-40 (2018)). Hence, sEVs present satisfying gene encapsulation capacity, with easy surface modification for targeting, and excellent biocompatibility as an IFN-y carrier for GBM immunotherapy. Unlike DNA-based drugs, mRNA does not carry a risk of accidental infection or opportunistic insertional mutagenesis, as it does not need to enter the nucleus to be functional. (Corny, R. M. et al. Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res 55, 1397-1400 (1995); Pardi, N. et al., mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov 17, 261- 279, doi: 10.1038/nrd.2017.243 (2018)). An intrinsic advantage of mRNA-based immunotherapy lies in the fact that small amounts of loading are adequate to provide vigorous efficacy signals. (Pastor, F. et al. An RNA toolbox for cancer immunotherapy. Nature Reviews Drug Discovery 17, 751-767 (2018)). Also, the abundance of positive safety and efficacy data obtained from the SARS-CoV-2 mRNA vaccines, together with approval and regulation of such vaccines by the US FDA, underscores the broad therapeutic potential of mRNA therapy, including cancer immunotherapy. (Qin, S. et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduction and Targeted Therapy 7, 166 (2022); Beck, J. D. et al. mRNA therapeutics in cancer immunotherapy. Molecular cancer 20, 1-24 (2021); Shi, J. et al., Cancer nanomedicine: progress, challenges and opportunities. Nature reviews cancer 17, 20-37 (2017)). For all of these reasons, mRNA was selected for encapsulation, rather than other IFN-y encoding drugs for effective immunotherapy.

[0157] In the present disclosure, the imsEVs were found to successfully bind both anti- CD71 and anti-PD-Ll. The GBM cell-targeted imsEV, delivering IFN-y mRNA and PD-L1 antibody, could reprogram the immune microenvironment of the tumor from an immunosuppressive to an immune-stimulating phenotype. Evidence of this reprogramming included the increased infiltration of effector immune cells, upregulation of MHC-I on cancer cells, and polarization of suppressive myeloid cells to an activating phenotype. These changes inhibited tumor growth and extended survival in preclinical GBM models, including models that are intrinsically immune-resistant. Correspondingly, the surface-functionalized, nontoxic, low-immunogenic sEVs allowed specific interactions with targeted cells, protected IFN-y from endonucleases, and prevented its detection by the immune system, leading to targeted delivery to cells of interest, efficient entry into those cells, and potency with few severe side effects. Collectively, the findings demonstrate that an adaptive design strategy that efficiently produces mRNA-loaded sEVs with targeting functionalities could pave the way for their adoption in cancer immunotherapy applications, offering a new avenue for improving the responsiveness of immune-resistant tumors.

SEQUENCE LISTING

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Claims

WHAT IS CLAIMED IS:

1. A process for generating exosomes loaded with mRNAs, the process comprising:

(a) subjecting a population of cells to a nanosecond duration electroporation treatment at a first electroporation station, wherein the first electroporation station is located in a microfluidic channel of a microfluidic device in which the population of cells are flowed past at least one electrical field source to receive the nanosecond duration electroporation treatment;

(b) after the nanosecond duration electroporation treatment, transporting the population of cells to a second electroporation station;

(c) subjecting the population of cells to a millisecond duration electroporation treatment at the second electroporation station, wherein during at least the millisecond duration electroporation treatment the population of cells are in the presence of extracellular nucleic acids comprising a DNA sequence, and wherein transcription of the DNA sequence produces an mRNA; and

(d) after the nanosecond and millisecond duration electroporation treatments, collecting exosomes secreted by the population of cells, wherein the secreted exosomes are loaded with the mRNA.

2. The process of claim 1, wherein:

(i) subjecting the population of cells to the nanosecond duration electroporation treatment comprises subjecting a solution including the population of cells and the extracellular nucleic acids to the nanosecond duration electroporation treatment;

(ii) transporting the population of cells to the second electroporation station comprises transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station; and

(iii) subjecting the population of cells to the millisecond duration electroporation treatment comprises subjecting the solution including the population of cells and the extracellular nucleic acids to the millisecond duration electroporation treatment.

3. The process of claim 2, wherein the microfluidic device comprises the first electroporation station and the second electroporation station; wherein transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station comprises flowing the solution from the first electroporation station to the second electroporation station via at least the microfluidic channel.

4. The process of claim 2, wherein the second electroporation station comprises a second device; wherein transporting the solution including the population of cells and the extracellular nucleic acids to the second electroporation station comprises collecting the solution from the microfluidic device and depositing the solution in the second device.

5. The process of claim 1, wherein the population of cells express a protein or a protein complex, at least a portion of which is located in cell membranes of the population of cells prior to the nanosecond and millisecond duration electroporation treatments; and wherein membranes of the collected exosomes comprise the proteins or protein complexes from the population of cells with the mRNA bound to a portion of the protein or protein complex inside the exosome.

6. The process of claim 1, wherein the population of cells express a CD64- Npeptide fusion protein.

7. The process of claim 6, wherein the cell membranes of the secreted exosomes comprise the CD64-N peptide fusion protein from the population of cells, wherein the CD64 portion of the fusion protein is located in the cell membranes and extends outside the exosome, and wherein the N peptide portion of the fusion protein is located inside the exosome.

8. The process of claim 7, wherein the mRNAs are bound to the N-peptide portion of the CD64-N peptide fusion proteins and are located inside the secreted exosomes.

9. The process of claim 8, wherein the CD64 portion of the CD64-N peptide fusion proteins of the secreted exosomes binds to an Fc region of an antibody that is outside the secreted exosomes.

10. The process of claim 9, wherein the antibody is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response.

11. The process of claim 10, wherein the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

12. The process of claim 1, wherein the electrical field source at the first electroporation station applies an electrical field having a strength of between 5 kV / cm and 125 kV / cm as the population of cells flows past the at least one electrical field source.

13. The process of claim 1, wherein the electrical field source at the first electroporation station applies an electrical field having a strength of between 30 kV / cm and 80 kV / cm as the population of cells flows past the at least one electrical field source.

14. The process of claim 12, wherein the nanosecond electroporation treatment has a frequency in the range of 5 Hz to 1 M Hz.

15. The process of claim 14 wherein the nanosecond electroporation treatment has a duration in the range of 50 ns to 2000 ns.

16. The process of claim 15 wherein the millisecond duration electroporation treatment has an amplitude of at least 10 V and a duration of at least 1 millisecond.

17. The process of claim 13, wherein the microfluidic channel at the first treatment station has a cross-sectional area perpendicular to a fluid flow direction in the range of 10,000 pm² to 50,000 pm².

18. The process of claim 13, wherein the microfluidic channel at the first treatment station has a cross-sectional area perpendicular to a fluid flow direction in the range of 1 ^x 10-8 m² to 5 x 10-8 m².

19. A small extracellular vesicle (sEV) comprising: a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence, wherein the CD64 is located in the cell membrane and extends outside the sEV; and wherein the N peptide is located inside the sEV; and a plurality of mRNAs, wherein the mRNAs are located inside the sEV and may be bound to the N peptide.

20. The sEV of claim 19, wherein the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from (|)21 phage.

21. The sEV of claim 20, wherein the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

22. The sEV of claim 19, wherein the CD64 is a human CD64.

23. The sEV of claim 22, wherein the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO:8.

23. The sEV of claim 19, wherein the amino acid sequence of the fusion protein comprises SEQ ID NOTO or a sequence that is at least 90% identical to SEQ ID NOTO

24. The sEV of claim 19, further comprising an antibody located outside the sEV that is bound to the CD64.

25. The sEV of claim 24, wherein the antibody is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response.

26. The sEV of claim 25, wherein the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier.

27. The sEV of claim 25, wherein the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

28. The sEV of claim 19, wherein the mRNA is translated into a protein that stimulates an immune response.

29. The sEV of claim 28, wherein the mRNA is an interferon gamma (IFNy) mRNA.

30. The sEV of claim 19, wherein the sEV is produced by (a) subjecting a population of cells to a nanosecond duration electroporation treatment at a first electroporation station, wherein the cells express a fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence, and wherein the first electroporation station is located in a microfluidic channel of a microfluidic device in which the population of cells are flowed past at least one electrical field source to receive the nanosecond duration electroporation treatment;

(b) after the nanosecond duration electroporation treatment, transporting the population of cells to a second electroporation station;

(c) subjecting the population of cells to a millisecond duration electroporation treatment at the second electroporation station, wherein during at least the millisecond duration electroporation treatment the population of cells are in the presence of extracellular nucleic acids comprising a DNA sequence, wherein the DNA sequence encodes a protein that stimulates an immune response, and wherein transcription of the DNA sequence produces an mRNA;

(d) after the nanosecond and millisecond duration electroporation treatments, collecting sEVs secreted by the population of cells, wherein the secreted sEVs are loaded with the mRNA, and wherein the mRNA may be bound to the N peptide of the fusion protein; and

(e) after collecting the sEVs, binding the CD64 in the membrane of the sEVs to an antibody that is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response.

31. The sEV of claim 30, wherein the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier.

32. The sEV of claim 30, wherein the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

33. The sEV of claim 30, wherein the mRNA is an interferon gamma (fFNy) mRNA.

34. A fusion protein comprising a CD64 amino acid sequence fused to an N peptide amino acid sequence.

35. The fusion protein of claim 34, wherein the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from (|)21 phage.

36. The fusion protein of claim 35, wherein the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NOT.

37. The fusion protein of claim 34, wherein the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO:8.

38. The fusion protein of claim 34, wherein the amino acid sequence comprises SEQ ID NO: 10 or a sequence that is at least 90% identical to SEQ ID NOTO.

39. A nucleic acid sequence that encodes the fusion protein of claim 34.

40. A vector comprising the nucleic acid sequence of claim 39.

41. A cell comprising the nucleic acid sequence of claim 39 or a vector comprising the nucleic acid sequence.

42. A system, comprising: a cell expressing a fusion protein that comprises a CD64 amino acid sequence fused to an N peptide amino acid sequence; and a vector sequence comprising a DNA sequence encoding a therapeutic protein.

43. The system of claim 42, wherein the system further comprises an electroporation system comprising a microfluidic device having a microfluidic channel in which the cell is flowed past at least one electrical field source to receive a nanosecond duration electroporation treatment and a second electroporation station to receive a millisecond duration electroporation treatment.

43. The system of claim 42, wherein the N peptide is selected from a group consisting of an N peptide from P22 phage, an N peptide from lambda phage, or an N peptide from (|)21 phage.

44. The system of claim 43, wherein the N peptide amino acid sequence comprises a sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NOT.

45. The system of claim 42, wherein the CD64 is a human CD64.

46. The system of claim 45, wherein the CD64 amino acid sequence comprises SEQ ID NO:8 or a sequence that is at least 90% identical to SEQ ID NO:8.

47. The system of claim 42, wherein the amino acid sequence of the fusion protein comprises SEQ ID NOTO or a sequence that is at least 90% identical to SEQ ID NOTO

48. The system of claim 42, further comprising an antibody that is specific for a protein expressed on a target cell or is specific for a protein that inhibits an immune response.

49. The system of claim 48, wherein the antibody is specific for a protein expressed on an epithelial cell of the blood brain barrier.

50. The system of claim 48, wherein the antibody is specific for CD71 or programmed cell death ligand 1 (PDL-1).

51. The system of claim 42, wherein the DNA sequence encodes a protein that stimulates an immune response.

52. The system of claim 51, wherein the protein is interferon y (IFNy).