Method for producing therapeutic extracellular vesicles from nanoelectroporation and other non-endocytosis cell transfection

Abstract

To provide a method for producing extracellular vesicles (EVs) for treatment in large quantities.SOLUTION: Provided is a method for producing extracellular vesicles (EVs) for treatment in large quantities containing high copies of functional nucleic acids and other biomolecules, comprising: laying donor cells on a surface of a chip; adding various plasmids, other transfection vectors, and combinations thereof to a buffer solution on the chip surface having a three-dimensional (3D) nanochannel electroporation (NEP) biochip formed thereon; applying a pulse electric field across the cells laid on top of the chip surface and plasmids / vectors buffer below the chip surface, and as a result, significantly stimulating the cells to non-endocytically deliver the plasmids / vectors into the cells; and collecting the EVs secreted by the transfected cells.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing therapeutic extracellular vesicles (EVs), particularly exosomes, containing functional messenger RNA (mRNA), microRNA (miR), short hairpin RNA (shRNA), proteins, and other biomolecules by non-endocytotic delivery of DNA plasmids and other vectors to donor cells, wherein non-endocytotic delivery of the DNA plasmid / vector results in high-speed transcription of RNA and translation of proteins in the cytoplasm, and a large number of vesicles are produced intracellularly in the donor cells by the strong stimulus caused by the delivery, and their functional biomolecules are endogenously encapsulated into the vesicles before being secreted from the donor cells as EVs.

Background Art

[0002] Extracellular vesicles (EVs), including exosomes, microvesicles, and other vesicles, are secreted by a number of cell types. In the human body, there are >10E12 EVs in 1 mL of blood, and they are also present in various body fluids. Exosomes are nanovesicles (40 - 150 nm), while the size of microvesicles varies from <100 nm to >1 μm. They contain both coding and non-coding RNAs and their fragments, DNA fragments, proteins, and other cell-related biomolecules. EVs and their biomolecule content have been proposed as biomarkers for disease diagnosis. Furthermore, they play a major role in cell-to-cell communication in the tumor microenvironment and circulation.

[0003] EVs loaded with functional RNAs and proteins have also been proposed as drugs and drug carriers for therapeutic use. A method is needed to produce EVs with either endogenous or exogenous therapeutic cargo to deliver specific nucleic acids and / or proteins to target tissues or cell types in vivo or in vitro.

[0004] In recent years, the post-insertion of exogenous small interfering RNA (siRNA) and plasmid of shRNA into existing exosomes by conventional bulk electroporation (BEP) has been developed. Their therapeutic functions have been successfully demonstrated in several mouse models of cancer and non-cancer diseases, but this approach faces many limitations. First, it is inefficient to post-insert large biomolecules such as DNA plasmids, mRNA, and proteins into nano-sized exosomes. Second, the strong electric field generated by BEP will destroy many exosomes and lead to a decrease in the yield of therapeutic exosomes. Furthermore, many large biomolecules such as mRNA and proteins are difficult to synthesize in vitro and are expensive.

[0005] It would be highly desirable to develop a new method that can produce a large number of exosomes or other EVs that intrinsically contain therapeutic RNA and protein targets by transfecting donor cells with DNA plasmids or other vectors.

[0006] In the prior US Patent Application No. 14 / 282630, we developed a nanochannel electroporation (NEP) biochip that can deliver DNA plasmids or other charged particles and molecules to individual cells in a non-endocytotic manner with good dose control. The above patent shows that NEP can generate a large number of therapeutic exosomes containing high copies of functional mRNA and microRNA targets, which cannot be achieved by the aforementioned post-insertion methods. If appropriate cell stimulation and rapid plasmid / vector delivery can be provided, in addition to NEP, other non-endocytotic delivery methods such as gene guns and micro / nano injections may also be able to achieve similar performance.

Summary of the Invention

Means for Solving the Problems

[0007] The present invention relates to the development of a novel concept and method for delivering DNA plasmids and other vectors to donor cells by non - endocytosis through strong cell stimulation so that a large number of vesicles, transcribed RNA, translated proteins, etc. are formed within the transfected cells. The cells will secrete many extracellular vesicles (EVs) containing specific RNA and protein targets with therapeutic functions.

[0008] To demonstrate the above - mentioned design concept, a three - dimensional (3D) NEP biochip is fabricated that can secrete 10 to 100 times or more EVs containing exosomes with high copies of intact mRNA and miR targets, up to thousands of times more than the EVs secreted from non - transfected donor cells, by transfecting many donor cells with a pre - specified DNA plasmid.

[0009] Some aspects of the present invention are achieved by a method of generating a large number of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules. Such a method comprises placing donor cells on a surface of a chip having a three - dimensional (3D) nanochannel electroporation (NEP) biochip formed thereon; adding various plasmids, other transfection vectors, and combinations thereof to a buffer on the chip; applying a pulsed electric field across the entire cell placed on the chip surface and the plasmid / vector buffer under the chip surface, thereby strongly stimulating the cells to deliver the plasmid / vector to the cells by non - endocytosis; and collecting the EVs secreted by the transfected cells.

[0010] In some of these methods, the diameter of the nanochannels is between 50 and 900 nm.

[0011] In some of these methods, plasmids and vectors transcribe mRNA, microRNA, shRNA, and other RNAs, resulting in the translation of proteins and other biomolecules within the transfected cells.

[0012] In some embodiments of the methods of the present invention, the EVs secreted by the transfected cells contain the transcribed mRNA, microRNA, shRNA, and other RNAs, as well as the translated proteins and other biomolecules.

[0013] In some embodiments of the methods of the present invention, means for increasing the expression of heat shock proteins and other proteins are added to the procedure so as to promote vesicle formation and exocytosis within the transfected cells, and the means include heat shock treatment of the cells or addition of heat shock proteins to the cultured cells.

[0014] In some embodiments, means for increasing the expression of proteins that promote exosome formation within the transfected cells are added to the procedure, and the means include co-transfection of CD63, CD9, and other DNA plasmids.

[0015] In some embodiments, a variety of DNA plasmids and other vectors are sequentially delivered to the transfected cells to promote co-localization of RNA / protein targets and EV secretion.

[0016] In some embodiments, exogenous biomolecules such as DNA plasmids, other transfection vectors, RNA, proteins / peptides, small molecule drugs, etc. are encapsulated within intracellular vesicles by sequential transfection of donor cells with NEP and secreted as therapeutic EVs. In some of these forms, in addition to NEP, other cell transfection methods that strongly stimulate donor cells and promote EV secretion and plasmid / vector delivery by non-endocytosis for high-speed RNA transcription and protein translation are used to generate therapeutic EVs with similar effects. Further in these forms, other cell transfection methods include gene guns and micro- or nano-injections.

[0017] In some embodiments, plasmids and / or other vectors are tethered to nano- or micron-sized gold or other solid particles, and these particles are injected into donor cells under air pressure using a gene gun, causing strong cell stimulation and non-endocytic plasmid / vector delivery.

[0018] In some embodiments, plasmids and / or other vectors are tethered to nano-sized or micron-sized chip arrays, and donor cells are aspirated by these chips, causing strong cell stimulation and non-endocytic plasmid / vector delivery into the donor cells.

[0019] Another aspect of the present invention is achieved by a device for producing a large number of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules, the device including a three-dimensional (3D) nanochannel electroporation (NEP) biochip and a receiving buffer formed thereon, the buffer being suitable for receiving plasmids and other transfection vectors.

[0020] Another aspect of the present invention includes cells transfected by any of the methods described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components of the drawings are not necessarily to scale, and emphasis instead is placed upon clearly depicting the principles of the present disclosure.

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Mode for Carrying Out the Invention

[0022] Example 1: Schematic diagram of the 3D NEP biochip and comparison of EV secretion and mRNA content of EVs using different transfection methods. Figure 1 shows a schematic diagram of a 3D NEP biochip with a single layer of donor cells placed on the chip surface. After incubating the cells overnight, a DNA plasmid pre-loaded in PBS buffer was injected into individual donor cells through the nanochannels using an electric field of 220 V across the nanochannels. Various electroporation conditions such as voltage level, number of pulses, pulse length, etc. can be selected.

[0023] Using a DNA plasmid of Achaete-Scute Complex Like-1 (Ascl1) fluorescently labeled with YOYO-1, Figures 2A and 2B show transfected cells 1 hour after transfection by BEP or NEP imaged using a fluorescence microscope at a wavelength of 488 nm. Fluorescence intensity was calculated by NIS software. Comparison of the fluorescence intensities of these two groups is shown in a bar graph. As a result, it shows that BEP under the best conditions recommended by the manufacturer can deliver almost three times more plasmid than NEP at 220 V with 5 times 10 milliseconds pulses to MEF cells. However, most of the plasmid was still near the cell surface 1 hour after BEP transfection, whereas the plasmid injected by NEP at the same time had already diffused uniformly into the cytoplasm. This means that BEP-based cell transfection mainly depends on endocytosis via electroporation, while NEP-based cell transfection is non-endocytotic.

[0024] In Figure 3, the number of EVs secreted from the same number of MEF cells (5E6 cells) transfected with the same DNA plasmids of Ascl1, Brn2, and Myt1l at a weight ratio of 2 / 1 / 1 by either Lipofectamine (Lipo), BEP, or NEP was compared. All EVs were collected from the cell culture medium 24 hours after transfection, and the total number of EVs was measured by NanoSight (registered trademark). In the case of BEP, the transfection voltage was 1250 V with a single 30 - millisecond pulse. In the case of NEP, the transfection voltage was 220 V with five 10 - millisecond pulses. The concentration of the plasmids used was Ascl1 / Brn2 / Myt1l = 200 / 100 / 100 ng / μl. For Lipofectamine transfection, 5 μg of the plasmid mixture (Ascl1 / Brn2 / Myt1l = 2 / 1 / 1) was used according to the manufacturer's instructions. EVs were collected from the cell culture medium by simple centrifugation at 1500 g for 10 minutes. The results show that Lipofectamine (Lipo) - based cell transfection did not change EV secretion. The EV concentration was approximately 2E9 / ml regardless of the presence or absence of transfection. Perhaps the slow endocytosis process of the plasmid by the nanoparticle carrier did not stimulate the transfected cells much, and as a result, there was little change in EV secretion. In contrast, BEP - based cell transfection increased EV secretion to nearly 6E9 / ml. Depending on the transfection of NEP cells, a significant increase in EV secretion exceeding 1.3E11 / ml was observed regardless of the addition of the plasmid. This means that the transfected cells were somewhat stimulated by BEP but very strongly stimulated by NEP, resulting in a very significant increase in EVs in the latter case.

[0025] During electroporation, Joule heating caused by the applied electric field can temporarily increase the cell temperature and cause heat shock to the transfected cells. Heat shock is known to potentially increase the cellular secretion of EVs through chaperone-mediated autophagy induced by an increase in heat shock proteins (HSPs) in the cells (8-10). Indeed, NEP was found to increase the expression of heat shock protein 70 (HSP70) by 13.8-fold and heat shock protein 90 (HSP) by 4.2-fold in transfected MEF cells compared to non-transfected MEF cells (Ctrl). Adding an HSP inhibitor to the cell culture medium after electroporation may suppress EV secretion. Figure 4 shows a 50%, 40%, and 70% decrease in EV secretion of MEF cells transfected with NEP containing an HSP70 inhibitor (VER 155008, 50 μM), an HSP90 inhibitor (NVP-HSP990, 1 μM), and a mixture thereof, respectively. Here, immediately after NEP transfection, the cell culture was replaced with fresh medium containing an HSP70 inhibitor (VER 155008), an HSP90 inhibitor (NVP-HSP990), or a mixture thereof. The medium was collected 24 hours after transfection, and the number of EVs was detected by dynamic light scattering (DLS) goniometry. These results imply that cell stimulation that can increase the expression of heat shock proteins will promote EV secretion.

[0026] Similarly, an increase in the proteins required for the formation of late endosomal multivesicular bodies (MVBs) in cells may also promote exosome secretion. Figure 5 shows the effect of NEP transfection of the DNA plasmid of CD63 on EV secretion from MEF cells. Cells were transfected with NEP regardless of the presence or absence of the DNA plasmid of CD63. The cell culture medium was collected and replaced with fresh medium every 4 hours. The number of EVs was detected by DLS goniometry. The results show similar EV secretion profiles in all cases during the first 16 hours after NEP transfection. However, more EVs were secreted 16 - 44 hours after NEP transfection with the DNA plasmid of CD63. The CD63 protein is essential for the reorganization of endosomal membranes into tetraspanin-enriched microdomains, which are precursors of exosome secretion.

[0027] Figure 6 shows the size distribution of EVs measured by DLS goniometry for MEF cells (ctrl) and NEP-transfected MEF cells. NEP stimulation did not change the distribution of larger EVs (mainly microvesicles) much, but significantly increased the secretion of exosomes in the size range of 40 - 110 nm.

[0028] Figures 8 - 9 show that EVs secreted from NEP cell transfection of the DNA plasmids of Ascl1, Brn2, and Myt1l contain large amounts of the corresponding Ascl1, Brn2, and Myt1l mRNAs or their fragments, measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Similar to the number of EVs, lipofectamine (Lipo)-based cell transfection did not change mRNA expression much, whereas BEP-based cell transfection could increase mRNA expression several-fold. In contrast, NEP-based cell transfection resulted in a several-thousand-fold increase in the target mRNA. Here, after obtaining the same amount of total RNA, reverse transcription was performed by qRT-PCR according to the manufacturer's instructions.

[0029] Figure 10 shows that the mRNAs of several EVs were able to translate Ascl1, Brn2, and Mytl1 proteins, indicating that they were intact and functional. Here, the same amount of total RNA (1 μg) from each transfection method was applied to in vitro protein translation according to the manufacturer's instructions using the rabbit reticulocyte lysate system (Promega Corporation). The samples were separated by SDS-PAGE, and the proteins were detected with various antibodies as shown in the Western blotting plots.

[0030] For all the collected EVs, larger microvesicles were selected by ultracentrifugation at 10,000 g for 30 minutes. The supernatant was further centrifuged at 100,000 g for 2 hours to collect smaller exosomes. Total RNA was collected from these two fractions described above. The total mRNA concentration was measured by Nanodrop (registered trademark), and the ABM expression of the mRNAs of Ascl1, Brn2, and Myt1l was measured by qRT-PCR. Figure 11 shows that there was more than twice as much RNA in exosomes than in microvesicles, but most of the mRNAs of Ascl1, Brn2, and Myt1l were present in exosomes. Figure 12 shows that functional mRNAs of Ascl1, Brn2, and Myt1l were also present in exosomes, and these exosomes had CD9, CD63, and Tsg101, which are typical exosome protein markers. In comparison, larger microvesicles had Arf6, which is a typical protein marker.

[0031] Figure 13 shows the EV secretion amount and content profiles as a function of time after transfection of NEP with DNA plasmids of Ascl1, Brn2, and Mytl1. The Ascl1 plasmid was the smallest among the three (7 kbp), the Myt1l plasmid was the largest (9 kbp), and the Brn2 plasmid was intermediate (8 kbp). EVs in the cell culture medium were collected at the indicated time points, and the culture medium was replaced with fresh medium. The number of EVs was detected by DLS goniometry, and the mRNA expression of EVs was detected by qRT-PCR as described above. As a result, a rapid increase in EV secretion was shown within 4 hours after transfection, reaching a peak at 8 hours, and resulting in continuous EV secretion for more than 24 hours. EVs containing Ascl1 and Brn2 mRNAs also had a profile that well matched the EV secretion profile and appeared within 4 hours after transfection. EVs containing Myt1l mRNA appeared later but within 24 hours. This means that it is necessary to match the EV secretion time and mRNA transcription time intracellularly, which can be achieved by NEP-based cell transfection.

[0032] To demonstrate that NEP-producing EVs containing endogenous mRNA have therapeutic functions, MEF cells were treated with their EVs every other day at a total EV RNA concentration of 1 μg per 100,000 cells. After several days, the treated MEF cells began to show a neuron-like morphology, and on day 24, the treated cells showed electrophysiological activity as indicated by the ability to receive induced action potentials, as shown in Figure 14. In comparison, NEP-transfected MEF cells also showed similar electrophysiological activity on day 21. The cells presented a voltage-dependent current necessary to fire an action potential. In response to a depolarizing voltage simulation, both transient inward currents and sustained outward currents were observed. A typical response to a 20 pA current injection is shown in Figure 14, which indicates that the cells fired an action potential in response to the depolarizing current.

[0033] Excitability was measured using whole-cell patch-clamp recordings. Cells were continuously perfused with a cell bath solution containing 115 mM NaCl, 2 mM KCl, 1.5 mM MgCl2, 3 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). The glass electrode (3 - 4 MΩ) was filled with a pipette solution containing 115 mM potassium gluconate, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 4 mM NaCl, 0.5 mM ethylene glycol tetraacetic acid (EGTA), 1.5 mM MgCl2 (pH 7.3). After accessing the whole cell, the patch resistance of the cell exceeded 100 MOhm and the series resistance was corrected by 40 - 50%. Data were acquired using an Axopatch 200B amplifier, Digidata 1322A digitizer, and Clampex 9 software (Molecular Devices, Sunnyvale, CA). For the analysis of voltage-dependent currents, the basal holding potential was -70 mV and the cells were stepped from -120 mV to 80 mV in 10 mV increments for 400 milliseconds. Transient inward currents due to the activity of voltage-dependent sodium channels were separated from the measured peak amplitudes. The sustained plateau current reflecting the voltage-dependent potassium current was measured as the average of the last 50 milliseconds of the voltage step in the plateau phase of the current. Action potential induction was measured using current clamp. The current was held at 0 pA and then stepped at 20 pA intervals for 1 second.

[0034] Example 2: MicroRNA content of EVs using different transfection methods. To demonstrate broader therapeutic applicability, we transfected MEF cells with DNA plasmids that transcribe intracellular microRNA targets. Figure 15 shows miR-128 expression in EVs collected from cell culture media at 24 hours after transfection with various methods (miR-128 plasmid). Total RNA was obtained according to the manufacturer's instructions. The same amount of total RNA (30 ng) was used for miR-128 detection by qRT-PCR using the aforementioned procedure. Here too, NEF-based transfection was able to generate EVs containing large amounts of miR-128 (more than 4,500-fold increase), which could not be achieved with BEP- or Lipofectamine-based cell transfection.

[0035] Example 3: Comparison of EVs containing endogenous RNA by NEP transfection of DNA plasmids into MEF cells and existing EVs loaded with pre-collected RNA by BEP post-insertion. Here, we compared the production effects of therapeutic EVs using NEP-based cell transfection and a post-insertion approach of BEP used by some researchers. For the former, according to the aforementioned procedure, the miR-128 plasmid was co-transfected with the CD63-GFP plasmid into MEF cells by NEP to generate EVs containing miR-128. For the latter, blank EVs were first collected from MEF cells transfected with the CD63-GFP plasmid 24 hours after NEP. In parallel, miR-128 was collected from MEF cells transfected with the miR-128 plasmid 24 hours after transfection by NEP. The collected miR-128 (1 μg) was mixed with blank EVs (10E6) and electroporated with BEP (1250 V, 30 milliseconds) according to the conditions used by other researchers. EVs from the two approaches were tested using a tethered lipoplex nanoparticle (TLN) biochip with a total internal reflection illumination fluorescence (TIRF) microscope. Figure 16A shows a schematic diagram of the TLN-TIRF assay (2, 11). Briefly, a molecular beacon (MB) of the RNA target is designed and encapsulated in cationic liposome nanoparticles. These cationic lipoplex nanoparticles are tethered on a slide glass and can capture negatively charged EVs by electrostatic interaction to form larger nanoscale complexes. By the fusion of this lipoplex and EV, RNA and MB are mixed within the nanoscale confinement near the interface of the biochip. The TIRF microscope can detect single biomolecules and measure signals less than 300 nm near the interface where the tethered liposome nanoparticles are located.

[0036] Figure 16B shows representative TLN-TIRF images of captured EVs. The green fluorescence is from EVs containing CD63-GFP, and the red fluorescence is from the hybridization of miR-128 molecules and MB of Cy5-miR-128 within the captured EVs. It is clear that the NEP method can generate more EVs containing high copies of miR-128 than the post-insertion method after BEP. Figures 16C-E show a quantitative comparison of these two methods. Both methods can generate EVs containing miR-128 (about 80% of the total captured EVs), but the concentration of EV miR-128 in EVs (about 3-fold MB fluorescence intensity) is much higher with the NEP-based direct cell transfection than with the BEP-based microRNA post-insertion. Furthermore, the post-insertion of BEP tends to destroy almost half of the blank EVs, leading to a very low yield of therapeutic EVs.

[0037] A similar comparison was also performed for the mRNA of Brn2, which is a much larger RNA, using the same method as miR-128 (Brn2 mRNA is 6272 bases while miR-128 is 21 bases). Figures 17A-C show that the NEP method was able to generate more than 70% of EVs containing Brn2 mRNA, while only a very small number of existing EVs were able to load the same mRNA by the post-insertion method of BEP. The concentration of Brn2 mRNA in EVs generated by NEP is high, while the concentration of Brn2 mRNA in EVs generated by the post-insertion of BEP is very low.

[0038] Example 4: Improvement of multiple mRNAs co-localizing in the same EVs secreted by sequential NEP transfection of DNA plasmids into MEF cells. Figure 13 shows that, due to differences in plasmid size or other reasons, even when multiple DNA plasmids are delivered to cells simultaneously, different mRNA targets can be transcribed at different times and rates in transfected cells. This can lead to individual EVs containing only one or a few mRNA targets. To obtain better therapeutic effects, it would be useful to be able to encapsulate more or all mRNA targets within the same secreted EV. By sequentially delivering each DNA plasmid to MEF cells using NEP based on the transcription time, Figure 18 shows that secreted EVs containing all three mRNAs of Ascl1, Brn2, and Myt1l required for iN reprogramming could be significantly increased (>50% vs. <25%). In the case of NEP transfection, the Ascl1, Brn2, and Myt1l plasmids were transfected simultaneously as described above. In the case of sequential NEP, the Myt1l plasmid was transfected first, the Brn2 plasmid was transfected 4 hours later, and the Ascl1 plasmid was transfected 4 hours after the Brn2 transfection. Twenty-four hours after the Myt1l transfection, the medium was collected for the TLN assay. Equal amounts of MB, FAM-Ascl1, Cy3-Brn2, and Cy5-Myt1l were encapsulated into tethered lipoplex nanoparticles for EV mRNA detection. In the figure, the yellow arrow means EVs containing all three mRNAs, the blue arrow means EVs containing two mRNAs, and the pink arrow means EVs containing only one mRNA.

[0039] Although the invention has been described in connection with its preferred embodiments, it should be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification. Accordingly, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.

[0040] Supplementary Explanation of Drawings Figure 1: Schematic diagram of a 3D nanochannel electroporation (NEP) biochip for donor cell transfection. Figure 2: Comparison of BEP and NEP in the effect of DNA plasmid delivery fluorescently labeled with YOYO-1 1 hour after transfection. A. Representative cell images of BEP and NEP. B. Comparison of fluorescence intensities in these two cases. Figure 3: Transfection of NEP cells with or without DNA plasmid significantly stimulates EV secretion from transfected mouse embryonic fibroblast (MEF) cells and has performance far superior to lipofectamine (Lipo)- and BEP-based cell transfection. Ctrl represents non-transfected MEF cells. NEP represents transfection of NEP cells with DNA plasmid. NEP-PBS represents transfection of NEP cells with only PBS buffer. The DNA plasmid used is Achaete-Scute Complex Like-1 (Ascl1), Pou Domain Class 3 Transcription factor 2 (Pou3f2 or Brn2), and Myelin Transcription Factor 1 Like (Myt1l) at a weight ratio of 2 / 1 / 1. A mixture of these DNA plasmids is known to reprogram donor cells into induced neurons (iN). The same number of MEF cells were transfected with DNA plasmid by various methods, and the cell culture medium was collected 24 hours after transfection. The number of EVs was measured by NanoSight®. In the case of BEP, the transfection voltage was 1250 V. In the case of NEP, the transfection voltage was 220 V with 5 times 10 millisecond pulses.

[0041] Figure 4: Effects of inhibitors of heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) on EV secretion from MEF cells transfected with NEP. After NEP transfection, the cell culture was switched to fresh medium containing the HSP70 inhibitor (VER 155008, 50 μM), the HSP90 inhibitor (NVP-HSP90, 1 μM), or a mixture of them. The medium was collected 24 hours after transfection, and the number of EVs was detected by dynamic light scattering (DLS) goniometry. Figure 5: Effect of NEP transfection of CD63 DNA plasmid on EV secretion from MEF cells. Cells were transfected with or without the CD63 plasmid by NEP. The cell culture medium was collected and replaced with fresh medium every 4 hours. The number of EVs was detected by DLS goniometry. Figure 6: Size distribution of EVs with or without NEP measured by DLS, collected 24 hours after cell transfection. The cell culture medium was collected 24 hours after NEP transfection, and cell debris was removed by centrifugation at 1500 g for 10 minutes. EVs in the supernatant were detected by DLS. Figure 7: mRNA expression of Ascl1 in EVs 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with DNA plasmids of Ascl1 / Brn2 / Myt1l at a ratio of 2 / 1 / 1 using various methods. After total RNA was obtained, reverse transcription was performed according to the manufacturer's instructions. The same amount of total RNA (20 ng) was used for the detection of Ascl1 by qRT-PCR.

[0042] Figure 8: mRNA expression of Brn2 in EVs 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with DNA plasmids of Ascl1 / Brn2 / Myt1l at a ratio of 2 / 1 / 1 using various methods. After total RNA was obtained, reverse transcription was performed according to the manufacturer's instructions. The same amount of total RNA (20 ng) was used for the detection of Brn2 by qRT-PCR. Figure 9: mRNA expression of Myt1l in EVs at 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a ratio of 2 / 1 / 1 using various methods. After obtaining total RNA, reverse transcription was performed according to the manufacturer's instructions. The same amount of total RNA (20 ng) was used for the detection of Myt1l by qRT-PCR. Figure 10: Only EVs obtained by NEP contained functional mRNAs measured by in vitro translation. The same amount of total RNA (1 μg) from each transfection method was used for in vitro protein translation according to the manufacturer's instructions. Samples were separated by SDS-PAGE and proteins were detected with antibodies. Figure 11: EV-mRNA from NEP exists within exosomes rather than within microvesicles. EVs were collected from cell culture medium by simple centrifugation at 1500 g for 10 minutes. Microvesicles were collected by ultracentrifugation at 10,000 g for 30 minutes. The supernatant was further centrifuged at 100,000 g for 2 hours to collect exosomes. Total RNA was collected from these two fractions and the total mRNA concentration was measured with Nanodrop (registered trademark). mRNA expression of EVs was measured by qRT-PCR. Figure 12: Exosome-mRNA rather than microvesicle-RNA from NEP cell transfection can translate proteins. EVs were collected from cell culture medium by simple centrifugation at 1500 g for 10 minutes. Microvesicles were collected by ultracentrifugation at 10,000 g for 30 minutes. The supernatant was further centrifuged at 100,000 g for 2 hours to collect exosomes. Total RNA was collected from these two aforementioned fractions and 1 μg of total RNA was used for in vitro protein translation. Samples were separated by SDS-PAGE and markers for proteins, exosomes and microvesicles were detected by Western blotting. Figure 13: Secretion profile of EV-mRNA from NEP-transfected MEF cells. MEF cells were transfected with a DNA plasmid by NEP. EVs in the cell culture medium were collected at the indicated time points, and the culture medium was replaced with fresh medium. The number of EVs was detected by DLS goniometry. mRNA expression was measured by qRT-PCR.

[0043] Figure 14: Patch-clamp detection of action potentials shows that MEF cells transfected every other day with EVs obtained from NEP containing mRNA of Ascl1 / Brn2 / Myt1l can be reprogrammed into functional induced neurons (iNs) after 24 days. NEP-transfected MEF cells were reprogrammed into iNs after 21 days. Figure 15: miR-128 expression as EVs 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with a DNA plasmid of miR-128 using various methods. After transfection with various methods (miR-128 plasmid), EVs were collected from the cell culture medium at 24 hours. Total RNA was obtained according to the manufacturer's instructions. The same amount of total RNA (30 ng) was used for miR-128 detection by qRT-PCR. Figure 16: Comparison of secreted EVs containing miR-128 by NEP transfection of a DNA plasmid into MEF cells with pre-collected EVs loaded with miR-128 by BEP post-insertion. Figure 17: Comparison of secreted EVs containing Brn2 mRNA by NEP transfection of a DNA plasmid into MEF cells with pre-collected EVs loaded with Brn2 mRNA by BEP post-insertion. Figure 18: Increase in the co-localization of mRNAs within the same EV by sequential NEP. In the case of NEP transfection, the Ascl1, Brn2, and Myt1l plasmids were co-transfected simultaneously as described above. In sequential NEP, the Myt1l plasmid was transfected first, the Brn2 plasmid was transfected 4 hours later, and the Ascl1 plasmid was transfected 4 hours after the transfection of Brn2. 24 hours after the Myt1l transfection, the medium was harvested for the TLN assay. Equal amounts of MB of FAM-Ascl1, Cy3-Brn2, and Cy5-Myt1l respectively were encapsulated into tethered lipoplex nanoparticles for EV-mRNA detection. Yellow arrow: EV containing three mRNAs. Blue arrow: EV containing two mRNAs. Pink arrow: EV containing one mRNA.

Claims

1. A method for producing extracellular vesicles (EVs), the method comprising: a) providing an electroporation biochip, wherein the electroporation biochip comprises i) a cell compartment that contacts a first surface of the electroporation biochip, ii) a buffer compartment that contacts a second surface of the electroporation biochip, and iii) a plurality of channels that connect the first surface of the electroporation biochip and the second surface of the electroporation biochip, wherein individual channels of the plurality of channels are arranged to contact individual cells within the cell compartment that contacts the first surface of the electroporation biochip, the plurality of channels comprising; said step; b) sequentially delivering a plurality of DNAs encoding different RNA targets to donor cells using the electroporation biochip, such that different RNAs transcribed from the plurality of DNAs encoding different RNA targets are co-localized in the extracellular vesicles secreted by the donor cells by sequentially delivering the plurality of DNAs encoding different RNA targets to the donor cells based on the transcription rate of each of the different RNA targets; and c) collecting the extracellular vesicles secreted by the donor cells, the extracellular vesicles comprising the different RNAs transcribed from the plurality of DNAs encoding different RNA targets; the method comprising.

2. The method according to claim 1, wherein sequentially delivering the plurality of DNAs encoding different RNA targets to the donor cells comprises delivering the plurality of DNAs encoding different RNA targets to the donor cells in descending order according to the size of each DNA of the plurality of DNAs encoding different RNA targets.

3. The method according to claim 1, wherein the plurality of DNAs encoding different RNA targets are DNA plasmids, DNA vectors or mixtures thereof.

4. The method according to claim 1, wherein the extracellular vesicles (EVs) are collected within 4 hours after delivery of the plurality of DNAs encoding different RNA targets to the donor cells. **Claim 5**: The method according to claim 1, wherein the extracellular vesicles (EVs) are collected within 24 hours after delivery of the plurality of DNAs encoding the different RNA targets to the donor cells. **Claim 6**: The method according to claim 1, wherein the extracellular vesicles (EVs) are collected within 44 hours after delivery of the plurality of DNAs encoding the different RNA targets to the donor cells. **Claim 7** The method according to claim 1, wherein a DNA plasmid or DNA vector encoding CD63, CD9 or a mixture thereof is delivered to the donor cells. **Claim 8** The method according to claim 1, further comprising the step of delivering a biomolecule or therapeutic agent to the donor cells by another cell transfection method. **Claim 9** The method according to claim 8, wherein the other cell transfection method is selected from the group consisting of gene gun, microinjection and nanoinjection. **Claim 10** The method according to claim 1, wherein the extracellular vesicles have a median diameter greater than 47.5 nm. **Claim 11**: The method according to claim 1, wherein the different RNA targets transcribed from the plurality of DNAs encoding the different RNA targets include different messenger RNA (mRNA) targets. **Claim 12**: The method according to claim 1, wherein the different RNA targets transcribed from the plurality of DNAs encoding the different RNA targets include different non-coding RNA targets, different microRNA (miRNA) targets, different short hairpin RNA (shRNA) targets or a mixture thereof. **Claim 13**: The method according to claim 1, wherein at least one of the different RNA targets transcribed from the plurality of DNAs encoding the different RNA targets includes at least one intact mRNA target, and the at least one intact mRNA target can produce a protein measurable by Western blot. **Claim 14** The method according to claim 13, wherein the at least one intact mRNA target can produce the protein in vivo. **Claim 15** The method according to claim 14, wherein the protein has a therapeutic function. **Claim 16** The method according to claim 1, wherein the extracellular vesicles (EVs) include exosomes. **Claim 17** The method according to claim 16, wherein the exosomes express Tsg101, CD9 or CD63. **Claim 18** The method according to claim 1, wherein the extracellular vesicles (EVs) comprise exosomes or microvesicles.

19. The method according to claim 1, wherein the extracellular vesicles (EVs) comprise exosomes and microvesicles.

20. The method according to claim 19, further comprising the step of isolating the exosomes from the microvesicles.

21. The method according to claim 20, wherein the isolation comprises sorting the extracellular vesicles (EVs) by centrifugation.

22. The method according to claim 16, wherein the exosomes have a diameter in the range of 40 nm to 110 nm.

23. The method according to claim 1, further comprising the step of culturing the donor cells in fresh medium after delivery of the plurality of DNAs encoding the different RNA targets to the donor cells.

24. The method according to claim 1, wherein the extracellular vesicles (EVs) are collected at multiple time points.

25. The method according to claim 1, wherein the extracellular vesicles (EVs) are quantified.

26. The method according to claim 1, wherein the extracellular vesicles (EVs) have a therapeutic function.

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