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

Abstract

To provide a method for producing a large number of therapeutic extracellular vesicles (EVs).SOLUTION: There is provided a method for producing a large number of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules. The method comprises: laying 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 pulsatile electric field across the cells laid on the chip surface and a plasmid / vector buffer solution below the chip surface to strongly stimulate the cells and thereby deliver the plasmids / vectors into the cells by non-endocytic transfection; and collecting the EVs secreted by the transfected cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001]

[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-endocytic delivery of DNA plasmids and other vectors into donor cells, whereby while non-endocytic delivery of DNA plasmids / vectors results in rapid RNA transcription and protein translation in the cytoplasm, the strong stimulation caused by the delivery causes the donor cells to produce a large number of vesicles intracellularly and endogenously encapsulate those functional biomolecules into the vesicles before they are secreted from the donor cells as EVs. [Background technology]

[0002]

[0002] Extracellular vesicles (EVs), including exosomes, microvesicles, and other vesicles, are secreted by numerous cell types. In humans, >10EVs per mL of blood 12 There are 100 types of EVs, which are also present in various body fluids. Exosomes are nanovesicles (40–150 nm), while microvesicles vary in size from <100 nm to >1 μm. They contain both coding and non-coding RNAs and their fragments, DNA fragments, proteins, and other cell-associated biomolecules. EVs and their biomolecular content have been proposed as biomarkers for disease diagnosis. Furthermore, they play a key role in intercellular communication in the tumor microenvironment and circulation.

[0003]

[0003] Functional RNA- and protein-loaded EVs have also been proposed as drug and drug carriers for therapeutic applications. To deliver specific nucleic acids and / or proteins to target tissues or cell types in vivo or in vitro, methods are needed that can produce EVs with either endogenous or exogenous therapeutic cargo.

[0004]

[0004] Recently, the post-insertion of exogenous small interfering RNA (siRNA) and shRNA plasmids into existing exosomes by conventional bulk electroporation (BEP) has been developed. Although their therapeutic function has been successfully demonstrated in several mouse models of cancer and non-cancer diseases, this approach faces many limitations. First, the post-insertion of large biomolecules, such as DNA plasmids, mRNA, and proteins, into nanosized exosomes is inefficient. Second, the strong electric field generated by BEP will destroy many exosomes, resulting in a low yield of therapeutic exosomes. Furthermore, many large biomolecules, such as mRNA and proteins, are difficult and expensive to synthesize in vitro.

[0005]

[0005] It would be highly desirable to develop new methods to transfect donor cells with DNA plasmids or other vectors to produce large numbers of exosomes or other EVs that endogenously contain therapeutic RNA and protein targets.

[0006]

[0006] In our prior U.S. patent application, Ser. No. 14 / 282,630, we developed a nanochannel electroporation (NEP) biochip capable of non-endocytosing the delivery of DNA plasmids or other charged particles and molecules to individual cells with good dosage control. This patent demonstrates 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 previously described post-insertion methods. In addition to NEP, other non-endocytic delivery methods, such as gene guns and micro / nanoinjection, may be able to achieve similar performance if appropriate cell stimulation and high-speed plasmid / vector delivery can be provided. Summary of the Invention [Means for solving the problem]

[0007]

[0007] The present invention relates to the development of a novel concept and method for non-endocytic delivery of DNA plasmids and other vectors into donor cells via strong cell stimulation, such that numerous vesicles and transcribed RNAs, translated proteins, etc. are formed within the transfected cells. The cells will secrete numerous extracellular vesicles (EVs) containing specific RNA and protein targets with therapeutic functions.

[0008]

[0008] d To demonstrate the above design concept, we transfect many donor cells with pre-specified DNA plasmids to create a three-dimensional (3D) NEP biochip capable of secreting 10–100 times more EVs, including exosomes containing high copies of intact mRNA and miR targets, up to several thousand times more than the EVs secreted from non-transfected donor cells.

[0009]

[0009] Some aspects of the present invention are achieved by methods for producing large numbers of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules. Such methods include: 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 the buffer solution on the chip; applying a pulsed electric field to the cells placed on the chip surface and the entirety of the plasmid / vector buffer solution below the surface of the chip, thereby strongly stimulating the cells and delivering the plasmid / vector into the cells by non-endocytosis; and and harvesting the EVs secreted by the transfected cells.

[0010]

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

[0011]

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

[0012]

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

[0013]

[0013] In some embodiments of the methods of the present invention, the procedure includes adding measures to increase the expression of heat shock proteins and other proteins to promote vesicle formation and exocytosis in the transfected cells, including heat shock treatment of the cells or addition of heat shock proteins to the cultured cells.

[0014]

[0014] In some embodiments, the procedure includes additional steps to increase the expression of proteins that promote exosome formation in the transfected cells, including co-transfection of CD63, CD9, and other DNA plasmids.

[0015]

[0015] In some embodiments, multiple DNA plasmids and other vectors are delivered sequentially into transfected cells to facilitate colocalization of RNA / protein targets and EV secretion.

[0016]

[0016] In some embodiments, exogenous biomolecules, such as DNA plasmids, other transfection vectors, RNA, proteins / peptides, and small molecule drugs, are encapsulated in intracellular vesicles by sequential transfection of donor cells with NEP and secreted as therapeutic EVs. In some of these configurations, other cell transfection methods are used in addition to NEP to generate therapeutic EVs with similar efficacy, providing strong stimulation to donor cells for EV secretion and non-endocytic plasmid / vector delivery for rapid RNA transcription and protein translation. Furthermore, in these configurations, other cell transfection methods include gene guns and micro- or nanoinjection.

[0017]

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

[0018]

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

[0019]

[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, comprising a three-dimensional (3D) nanochannel electroporation (NEP) biochip and a chip having a receiving buffer formed thereon, the receiving buffer being suitable for receiving plasmids and other transfection vectors.

[0020]

[0020] Another aspect of the invention includes cells transfected by any of the above methods. [Brief explanation of the drawings]

[0021]

[0021] Many aspects of the present disclosure can be better understood with reference to the following drawings, in which the components are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. [Figure 1] FIG. 1 is a schematic diagram of a 3D nanochannel electroporation (NEP) biochip for donor cell transfection. [Figure 2A] This shows a comparison of BEP- and NEP-based cell transfections 1 hour after transfection using a DNA plasmid of the neural-related gene, Achaete-Scute Complex Like-1 (Ascl1), fluorescently labeled with YOYO-1. [Figure 2B] This shows a comparison of BEP- and NEP-based cell transfections 1 hour after transfection using a DNA plasmid of the neural-related gene, Achaete-Scute Complex Like-1 (Ascl1), fluorescently labeled with YOYO-1. [Figure 3]We demonstrate that NEP cell transfection with or without DNA plasmids significantly stimulates EV secretion from transfected mouse embryonic fibroblast (MEF) cells, with performance far superior to lipofectamine (Lipo) and BEP-based cell transfection. Ctrl represents untransfected MEF cells. NEP represents NEP cell transfection with DNA plasmids. NEP-PBS represents NEP cell transfection with PBS buffer alone. The DNA plasmids used are Achaete-Scute Complex Like-1 (Ascl1), Pou Domain Class 3 Transcription factor 2 (Pou3f2 or Brn2), and Myelin Transcription Factor 1 Like (Myt1l) in a weight ratio of 2 / 1 / 1. A mixture of these DNA plasmids is known to reprogram donor cells into induced neurons (iNs). [Figure 4] The effects of heat shock protein 70 (HSP70) inhibitors and heat shock protein 90 (HSP90) inhibitors on EV secretion from MEF cells transfected with NEP are shown. After NEP transfection, cell cultures were replaced with fresh medium containing an HSP70 inhibitor (VER 155008, 50 μM), an HSP90 inhibitor (NVP-HSP90, 1 μM), or a mixture of these. The medium was collected 24 h posttransfection, and the number of EVs was detected by dynamic light scattering (DLS) goniometry. [Figure 5] The 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] Figure 1 shows the size distribution measured by DLS of EVs with or without NEP harvested 24 hours after cell transfection. [Figure 7]Figure 1 shows Ascl1 mRNA expression in EVs 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a 2 / 1 / 1 ratio using various methods. [Figure 8] Figure 1 shows Brn2 mRNA expression in EVs 24 hours after transfection, measured by qRT-PCR, from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a 2 / 1 / 1 ratio using various methods. [Figure 9] Figure 1 shows Myt1l mRNA expression in EVs measured by qRT-PCR 24 hours after transfection from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a 2 / 1 / 1 ratio using various methods. [Figure 10] We show that only EVs obtained by NEP contain functional mRNA as measured by in vitro translation. [Figure 11] We show that EV-mRNA from NEP is present in exosomes, not in microvesicles. [Figure 12] We show that exosome-mRNA, but not microvesicle-RNA, from NEP cell transfection can translate proteins. [Figure 13] Figure 1 shows the secretion profile of EV-mRNA from MEF cells transfected with NEP. [Figure 14] Action potential detection by patch clamp indicates that MEF cells transfected every other day with EVs derived from NEP containing Ascl1 / Brn2 / Myt1l mRNA can be reprogrammed into functional induced neurons (iNs) after 24 days. [Figure 15] Using various methods, miR-128 expression in EVs was measured by qRT-PCR from MEF cells transfected with miR-128 DNA plasmid 24 hours after transfection. [Figure 16A]This is a comparison between secreted EVs containing miR-128 by NEP transfection of DNA plasmid into MEF cells and pre-existing EVs loaded with pre-harvested miR-128 by post-BEP insertion. [Figure 16B] This is a comparison between secreted EVs containing miR-128 by NEP transfection of DNA plasmid into MEF cells and pre-existing EVs loaded with pre-harvested miR-128 by post-BEP insertion. [Figure 16C] This is a comparison between secreted EVs containing miR-128 by NEP transfection of DNA plasmid into MEF cells and pre-existing EVs loaded with pre-harvested miR-128 by post-BEP insertion. [Figure 16D] This is a comparison between secreted EVs containing miR-128 by NEP transfection of DNA plasmid into MEF cells and pre-existing EVs loaded with pre-harvested miR-128 by post-BEP insertion. [Figure 16E] This is a comparison between secreted EVs containing miR-128 by NEP transfection of DNA plasmid into MEF cells and pre-existing EVs loaded with pre-harvested miR-128 by post-BEP insertion. [Figure 17A] This is a comparison of secreted EVs containing Brn2 mRNA by NEP transfection of DNA plasmid into MEF cells with pre-existing EVs loaded with pre-harvested Brn2 mRNA by post-BEP insertion. [Figure 17B] This is a comparison of secreted EVs containing Brn2 mRNA by NEP transfection of DNA plasmid into MEF cells with pre-existing EVs loaded with pre-harvested Brn2 mRNA by post-BEP insertion. [Figure 17C] This is a comparison of secreted EVs containing Brn2 mRNA by NEP transfection of DNA plasmid into MEF cells with pre-existing EVs loaded with pre-harvested Brn2 mRNA by post-BEP insertion. [Figure 18]Sequential NEP (Neuroprotective Epithelial Growth Promoting) demonstrates increased colocalization of mRNAs within the same EV. For NEP transfection, Ascl1, Brn2, and Myt1l plasmids were cotransfected as described above. For sequential NEP, Myt1l plasmid was transfected first, followed by Brn2 plasmid 4 h later, and Ascl1 plasmid 4 h after Brn2 transfection. 24 h after Myt1l transfection, medium was collected for TLN assay. Equal amounts of FAM-Ascl1, Cy3-Brn2, and Cy5-Myt1l MBs were encapsulated within tethered lipoplex nanoparticles for EV-mRNA detection. Yellow arrows: EVs containing three mRNAs. Blue arrows: EVs containing two mRNAs. Pink arrows: EVs containing one mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0022] Example 1: Schematic diagram of the 3D NEP biochip and comparison of EV secretion and EV mRNA content using different transfection methods.

[0022] Figure 1 shows a schematic diagram of a 3D NEP biochip with a monolayer of donor cells deposited on the chip surface. After overnight cell incubation, DNA plasmids preloaded in PBS buffer were injected into individual donor cells through the nanochannels using a 220 V electric field across the nanochannels. Various electroporation conditions, such as voltage level, pulse number, and pulse length, can be selected.

[0023]

[0023] Figures 2A and 2B show transfected cells 1 hour after transfection with BEP or NEP, using a DNA plasmid fluorescently labeled with YOYO-1 (Achaete-Scute Complex Like-1 (Ascl1)) at a wavelength of 488 nm. Fluorescence intensity was calculated using NIS software. A comparison of the fluorescence intensities of these two groups is shown in the bar graph. The results show that under the best conditions recommended by the manufacturer, BEP delivers nearly three times more plasmid than NEP at 220 V in five 10-ms 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 was already uniformly diffused within the cytoplasm. This indicates that BEP-based cell transfection primarily relies on electroporation-mediated endocytosis, whereas NEP-based cell transfection is non-endocytic.

[0024]

[0024] Figure 3 compares the number of EVs secreted from the same number of MEF cells (5E6 cells) transfected with the same Ascl1, Brn2, and Myt1l DNA plasmids at a 2 / 1 / 1 weight ratio using either Lipofectamine (Lipo), BEP, or NEP. All EVs were collected from the cell culture medium 24 h posttransfection, and the total number of EVs was measured using a NanoSight® microscope. For BEP, the transfection voltage was 1250 V with one 30-ms pulse. For NEP, the transfection voltage was 220 V with five 10-ms pulses. The concentrations of the plasmids used were 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 harvested from the cell culture medium by simple centrifugation at 1500g for 10 minutes. The results indicate that Lipofectamine (Lipo)-based cell transfection did not alter EV secretion. The EV concentration was approximately 2E9 / ml regardless of transfection. Perhaps the slow plasmid endocytosis process by nanoparticle carriers did not significantly stimulate transfected cells, resulting in little change in EV secretion. In contrast, BEP-based cell transfection increased EV secretion to nearly 6E9 / ml. A significant increase of over 1.3E11 / ml was observed following NEP cell transfection, regardless of the addition of plasmid. This indicates that transfected cells were somewhat stimulated by BEP but significantly stimulated by NEP, resulting in a highly significant increase in EVs in the latter case.

[0025]

[0025] During electroporation, Joule heating caused by the applied electric field can temporarily increase cellular temperature and induce heat shock in transfected cells. Heat shock is known to increase cellular secretion of EVs through chaperone-mediated autophagy, which is triggered by an increase in intracellular heat shock proteins (HSPs) (8-10). Indeed, NEP was found to substantially 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 with non-transfected MEF cells (Ctrl). Adding HSP inhibitors to the cell culture medium after electroporation can suppress EV secretion. Figure 4 shows a 50%, 40%, and 70% decrease in EV secretion in MEF cells transfected with NEP containing an HSP70 inhibitor (VER 155008, 50 µM), an HSP90 inhibitor (NVP-HSP990, 1 µM), and their mixture, 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 their mixture. The medium was collected 24 h posttransfection, and EV numbers were detected by dynamic light scattering (DLS) goniometry. These results imply that cellular stimuli that can increase the expression of heat shock proteins will promote EV secretion.

[0026]

[0026] Similarly, an increase in proteins required for late endosomal multivesicular body (MVB) formation in cells may also promote exosome secretion. Figure 5 shows the effect of NEP transfection of CD63 DNA plasmid on EV secretion from MEF cells. Cells were transfected with NEP with or without CD63 DNA plasmid. Cell culture medium was collected and replaced with fresh medium every 4 hours. EV counts were detected by DLS goniometry. The results show a similar EV secretion profile in both cases during the first 16 hours after NEP transfection. However, NEP transfection with CD63 DNA plasmid secreted more EVs between 16 and 44 hours. CD63 protein is essential for the reorganization of endosomal membranes into tetraspanin-enriched microdomains, which are precursors for exosome secretion.

[0027]

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

[0028]

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

[0029]

[0029] Figure 10 shows that some EV mRNAs were intact and functional, as they were capable of translating Ascl1, Brn2, and Mytl1 proteins. Here, equal amounts of total RNA (1 μg) from each transfection method were subjected to in vitro protein translation using a rabbit reticulocyte lysate system (Promega Corporation) according to the manufacturer's instructions. Samples were separated by SDS-PAGE, and proteins were detected with various antibodies, as shown in the Western blotting plots.

[0030]

[0030] From the total EVs collected, larger microvesicles were separated by ultracentrifugation at 10,000 g for 30 minutes. The supernatant was further centrifuged at 100,000 g for 2 hours to separate smaller exosomes. Total RNA was extracted from these two fractions. Total mRNA concentration was measured using Nanodrop®, and ABM expression of Ascl1, Brn2, and Myt1l mRNA was measured by qRT-PCR. Figure 11 shows that there was more than twice as much RNA in exosomes as in microvesicles, but most of the Ascl1, Brn2, and Myt1l mRNA was present in exosomes. Figure 12 shows that functional Ascl1, Brn2, and Myt1l mRNA was also present in exosomes, and these exosomes possess typical exosome protein markers, CD9, CD63, and Tsg101. In comparison, larger microvesicles have the typical protein marker Arf6.

[0031]

[0031] Figure 13 shows the EV secretion volume and content profiles as a function of time after NEP transfection with Ascl1, Brn2, and Mytl1 DNA plasmids. The Ascl1 plasmid was the smallest (7 kbp), the Mytl1 plasmid the largest (9 kbp), and the Brn2 plasmid the intermediate (8 kbp). At the indicated time points, EVs in the cell culture medium were harvested and replaced with fresh medium. EV counts were detected by DLS goniometry, and EV mRNA expression was detected by qRT-PCR as described previously. Results showed a rapid increase in EV secretion within 4 h after transfection, peaking at 8 h and resulting in continuous EV secretion for over 24 h. EVs containing Ascl1 and Brn2 mRNA also appeared within 4 h after transfection, a profile closely consistent with the EV secretion profile. EVs containing Mytl1 mRNA appeared later but within 24 h. This suggests that the time of EV secretion and the time of mRNA transcription must be synchronized within cells, a feasible outcome achieved by NEP-based cell transfection.

[0032]

[0032] To demonstrate the therapeutic function of NEP-produced EVs containing endogenous mRNA, MEF cells were treated with these EVs every other day at a concentration of 1 μg of total EV RNA per 100,000 cells. After several days, the treated MEF cells began to exhibit neuronal-like morphology, and by day 24, the treated cells exhibited electrophysiological activity, indicated by the ability to undergo induced action potentials, as shown in Figure 14. In comparison, NEP-transfected MEF cells also exhibited similar electrophysiological activity at day 21. The cells exhibited voltage-dependent currents necessary for firing action potentials. Both transient inward and sustained outward currents were observed in response to depolarizing voltage simulations. A typical response to a 20 pA current injection is shown in Figure 14, demonstrating that the cells fired action potentials in response to the depolarizing current.

[0033]

[0033] Whole-cell patch clamp recordings were used to measure excitability. 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). Glass electrodes (3-4 MΩ) were filled with a pipette solution containing 115 mM K-gluconate, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 4 mM NaCl, 0.5 mM ethylene glycol tetraacetic acid (EGTA), and 1.5 mM MgCl2 (pH 7.3). After whole-cell access, the cell's patch resistance exceeded 100 MOhm, and series resistance was compensated by 40-50%. Data were collected using an Axopatch 200B amplifier, a Digidata 1322A digitizer, and Clampex 9 software (Molecular Devices, Sunnyvale, CA). For voltage-gated current analysis, the basal holding potential was -70 mV, and cells were pulsed from -120 mV to 80 mV in 10 mV increments for 400 ms. Transient inward currents due to voltage-gated sodium channel activity were isolated from the measured peak amplitude. Sustained plateau currents, reflecting voltage-gated potassium currents, were measured as the average of the last 50 ms of the voltage step during the plateau phase of the current. Action potential induction was measured using current clamp. Currents were held at 0 pA and then pulsed at 20 pA intervals for 1 s.

[0034] Example 2: MicroRNA content of EVs using different transfection methods.

[0034] To demonstrate broader therapeutic applicability, we transfected MEF cells with DNA plasmids transcribing intracellular microRNA targets. Figure 15 shows EV miR-128 expression in EVs harvested from cell culture medium 24 hours after transfection (with miR-128 plasmid) using various methods. 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 previously described procedure. Again, NEF-based transfection was able to generate EVs containing high amounts of miR-128 (more than a 4,500-fold increase), which cannot 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 with existing EVs loaded with pre-harvested RNA by post-BEP insertion.

[0035] Here, we compared the effectiveness of NEP-based cell transfection and the post-insertion approach of BEP, which has been used by several researchers. For the former, miR-128 plasmid was cotransfected with CD63-GFP plasmid into MEF cells by NEP, following the procedure described previously, to generate EVs containing miR-128. For the latter, blank EVs were first collected from MEF cells transfected with CD63-GFP plasmid 24 h after NEP. In parallel, miR-128 was collected from MEF cells transfected with miR-128 plasmid 24 h after NEP transfection. The collected miR-128 (1 μg) was mixed with blank EVs (10E6) and electroporated with BEP (1250 V, 30 ms) according to the conditions used by other researchers. EVs from the two approaches were tested using tethered lipoplex nanoparticle (TLN) biochips under total internal reflection fluorescence (TIRF) microscopy. Figure 16A shows a schematic diagram of the TLN-TIRF assay (2, 11). Briefly, RNA-targeted molecular beacons (MBs) are engineered and encapsulated in cationic liposomal nanoparticles. These cationic lipoplex nanoparticles are tethered to a glass slide and can capture negatively charged EVs through electrostatic interactions to form larger nanoscale complexes. This lipoplex-EV fusion results in the mixing of the RNA and MBs within nanoscale limits near the interface of the biochip. TIRF microscopy can detect single biomolecules and measures signals below 300 nm near the interface where the tethered liposomal nanoparticles are located.

[0036]

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

[0037]

[0037] A similar comparison was performed using the same approach as for miR-128 with Brn2 mRNA, a much larger RNA (Brn2 mRNA is 6272 bases, compared with 21 bases for miR-128). Figures 17A-C show that the NEP approach was able to generate over 70% of EVs containing Brn2 mRNA, whereas only a small fraction of pre-existing EVs were able to be loaded with the same mRNA using the BEP post-insertion approach. The concentration of Brn2 mRNA in EVs generated with NEP was high, whereas the concentration of Brn2 mRNA in EVs generated with BEP post-insertion was very low.

[0038] Example 4: Improvement of multiple mRNAs co-localized in the same secreted EV by sequential NEP transfection of DNA plasmids into MEF cells.

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

[0039]

[0039] While the invention has been explained in relation to its preferred embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. It is, therefore, to be understood that the invention disclosed herein is intended to cover all such modifications as fall within the scope of the appended claims.

[0040] Supplementary explanation of the drawing Figure 1: Schematic of the 3D nanochannel electroporation (NEP) biochip for donor cell transfection. Figure 2: Comparison of BEP and NEP in the efficacy of YOYO-1 fluorescently labeled DNA plasmid delivery 1 hour after transfection. A. Representative cell images of BEP and NEP. B. Comparison of fluorescence intensity between the two cases. Figure 3: NEP cell transfection with or without DNA plasmids significantly stimulates EV secretion from transfected mouse embryonic fibroblast (MEF) cells, with performance far superior to lipofectamine (Lipo) and BEP-based cell transfection. Ctrl represents untransfected MEF cells. NEP represents NEP cell transfection with DNA plasmids. NEP-PBS represents NEP cell transfection with PBS buffer alone. The DNA plasmids used were Achaete-Scute Complex Like-1 (Ascl1), Pou Domain Class 3 Transcription factor 2 (Pou3f2 or Brn2), and Myelin Transcription Factor 1 Like (Myt1l) in a weight ratio of 2 / 1 / 1. A mixture of these DNA plasmids is known to reprogram donor cells into induced neurons (iNs). Equal numbers of MEF cells were transfected with DNA plasmids by various methods, and cell culture medium was collected 24 h posttransfection. EV numbers were measured by NanoSight®. For BEP, the transfection voltage was 1250 V. For NEP, the transfection voltage was 220 V with five 10-ms pulses.

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

[0042] Figure 8: Brn2 mRNA expression in EVs 24 hours post-transfection measured by qRT-PCR from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a 2 / 1 / 1 ratio using various methods. Total RNA was obtained and reverse-transcribed according to the manufacturer's instructions. The same amount of total RNA (20 ng) was used for Brn2 detection by qRT-PCR. Figure 9: Myt1l mRNA expression in EVs 24 hours post-transfection measured by qRT-PCR from MEF cells transfected with Ascl1 / Brn2 / Myt1l DNA plasmids at a 2 / 1 / 1 ratio using various methods. Total RNA was obtained and reverse-transcribed according to the manufacturer's instructions. The same amount of total RNA (20 ng) was used for Myt1l detection by qRT-PCR. Figure 10: Only EVs obtained by NEP contained functional mRNA as measured by in vitro translation. Equal amounts of total RNA (1 µg) from each transfection method were 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 is present in exosomes but not in microvesicles. EVs were harvested from cell culture media by simple centrifugation at 1500g for 10 min. Microvesicles were harvested by ultracentrifugation at 10,000g for 30 min. The supernatant was further centrifuged at 100,000g for 2 h to harvest exosomes. Total RNA was collected from these two fractions, and total mRNA concentration was measured using Nanodrop®. EV mRNA expression was measured by qRT-PCR. Figure 12: Exosome-mRNA, but not microvesicle-RNA, from NEP cell transfection can translate proteins. EVs were harvested from the cell culture medium by simple centrifugation at 1500g for 10 minutes. Microvesicles were harvested by ultracentrifugation at 10,000g for 30 minutes. The supernatant was further centrifuged at 100,000g for 2 hours to harvest exosomes. Total RNA was harvested from these two fractions, and 1 μg of total RNA was used for in vitro protein translation. Samples were separated by SDS-PAGE, and protein, exosome, and microvesicle markers were detected by Western blotting. Figure 13: Secretion profile of EV-mRNA from MEF cells transfected with NEP. MEF cells were transfected with DNA plasmids using 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: Action potential detection by patch clamp shows that MEF cells transfected every other day with EVs derived from NEP containing Ascl1 / Brn2 / Myt1l mRNA 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 in EVs 24 hours post-transfection measured by qRT-PCR from MEF cells transfected with miR-128 DNA plasmids using various methods. EVs were harvested from cell culture medium 24 hours post-transfection using various methods. 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 DNA plasmid into MEF cells with pre-existing EVs loaded with pre-harvested miR-128 by BEP post-insertion. Figure 17: Comparison of secreted EVs containing Brn2 mRNA by NEP transfection of DNA plasmid into MEF cells with pre-existing EVs loaded with pre-harvested Brn2 mRNA by BEP post-insertion. Figure 18: Sequential NEP increases colocalization of mRNAs within the same EV. For NEP transfection, Ascl1, Brn2, and Myt1l plasmids were transfected simultaneously as described above. For sequential NEP, Myt1l plasmid was transfected first, Brn2 plasmid transfected 4 hours later, and Ascl1 plasmid transfected 4 hours after Brn2 transfection. 24 hours after Myt1l transfection, medium was collected for TLN assay. Equal amounts of FAM-Ascl1, Cy3-Brn2, and Cy5-Myt1l MBs were encapsulated into tethered lipoplex nanoparticles for EV-mRNA detection. Yellow arrows: EVs containing three mRNAs. Blue arrows: EVs containing two mRNAs. Pink arrows: EVs containing one mRNA.

[0044] Claim 1: A method for producing large numbers of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules, comprising: 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 solution on the chip; Applying a pulsed electric field to the cells placed on the surface of the chip and the entirety of the plasmid / vector buffer solution below the surface of the chip, thereby strongly stimulating the cells and delivering the plasmid / vector into the cells by non-endocytosis; and The method comprises the step of harvesting EVs secreted by the transfected cells. Claim 2: The method of claim 1, wherein the nanochannel has a diameter of 50 to 900 nm. Claim 3: 3. The method of claim 1 or 2, wherein the plasmids and vectors transcribe mRNA, microRNA, shRNA and other RNAs, resulting in the translation of proteins and other biomolecules in transfected cells. Claim 4: 4. The method of claim 1, wherein the EVs secreted by the transfected cells contain transcribed mRNA, microRNA, shRNA, and other RNA, as well as translated proteins and other biomolecules. Claim 5: 5. The method of claim 1, wherein the procedure further comprises increasing the expression of heat shock proteins and other proteins that promote vesicle formation and exocytosis in the transfected cells, the increasing expression comprising heat shock treatment of the cells or addition of heat shock proteins to cultured cells. Claim 6: 6. The method of any one of claims 1 to 5, wherein the procedure further comprises increasing the expression of proteins that promote exosome formation in the transfected cells, the increasing the expression of proteins that promote exosome formation in the transfected cells comprising co-transfection of CD63, CD9 and other DNA plasmids. Claim 7: 7. The method of any one of claims 1 to 6, wherein multiple DNA plasmids and other vectors are delivered sequentially to transfected cells to promote co-localization of RNA / protein targets and EV secretion. Claim 8: The method of any one of claims 1 to 7, wherein exogenous biomolecules such as DNA plasmids, other transfection vectors, RNA, proteins / peptides, small molecule drugs, etc. are encapsulated in intracellular vesicles by sequential transfection of donor cells with NEP and secreted as therapeutic EVs. Claim 9: In addition to NEP, other cell transfection methods that strongly stimulate donor cells and promote EV secretion and non-endocytic plasmid / vector delivery for fast RNA transcription and protein translation produce therapeutic EVs with similar efficacy. Claim 10: 10. The method of claim 9, wherein other cell transfection methods include gene guns, and micro- or nano-injection. Claim 11: 11. The method of any one of claims 8 to 10, wherein the plasmids and / or other vectors are tethered to nano- or micron-sized gold or other solid particles, which are injected into donor cells under air pressure using a gene gun, resulting in strong cell stimulation and non-endocytic plasmid / vector delivery. Claim 12: 12. The method of any one of claims 8 to 11, wherein the plasmids and / or other vectors are tethered to nano- or micron-sized chip arrays and the donor cells are pulled by the chips, resulting in strong cell stimulation and non-endocytic plasmid / vector delivery to the donor cells. Claim 13: A device for producing a large number of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules, comprising a three-dimensional (3D) nanochannel electroporation (NEP) biochip and a chip having a receiving buffer formed thereon, the receiving buffer being suitable for receiving plasmids and other transfection vectors. Claim 14: A cell transfected by the method of any one of claims 1 to 12.

Claims

1. 1. A method for producing extracellular vesicles comprising copies of a functional nucleic acid, the method comprising: (a) disposing donor cells on a first surface of an electroporation biochip; (b) adding a buffer solution contacting a plurality of plasmids, vectors in a form other than a plasmid, or a combination thereof, with a second surface of the electroporation biochip, wherein a plurality of channels connects the first surface of the electroporation biochip and the second surface of the electroporation biochip, the plurality of channels being arranged such that an individual channel of the plurality of channels contacts each of the donor cells; (c) applying an electric field to the plurality of channels, thereby stimulating the cells to non-endocytose the plasmid or vector into the donor cells; and (d) collecting extracellular vesicles secreted from the donor cells; The method, wherein the extracellular vesicles comprise at least one RNA transcribed from the plasmid or vector delivered to the donor cell, wherein the at least one RNA is: (i) intact, (ii) translatable in vitro into one or more polypeptides, and (iii) encapsulated within the extracellular vesicles.

2. 10. The method of claim 1, wherein the at least one RNA is a messenger RNA.

3. The method of claim 1, further comprising the step of delivering to the donor cells a DNA plasmid or DNA vector encoding CD63, CD9, Tsg101, or a combination thereof.

4. 10. The method of claim 1, further comprising the step of delivering a biomolecule or therapeutic drug to the transfected donor cells by an additional transfection method.

5. 5. The method of claim 4, wherein the additional transfection method is selected from the group consisting of a gene gun, microinjection, and nanoinjection.

6. 2. The method of claim 1, wherein a portion of the extracellular vesicles comprises messenger RNA transcribed from a plasmid or vector delivered to the donor cell.

7. 7. The method of claim 6, wherein at least 70% of the extracellular vesicles comprise messenger RNA transcribed from a plasmid or vector delivered to the donor cells.

8. 7. The method of claim 6, wherein at least 25% of the extracellular vesicles comprise messenger RNA transcribed from a plasmid or vector delivered to the donor cells.

9. 2. The method of claim 1, wherein the extracellular vesicles comprise extracellular vesicles having a diameter of about 40 nm to about 110 nm.

10. 10. The method of claim 1, further comprising the step of culturing the donor cells on the first surface of the electroporation biochip for a period of time prior to the step of (c).

11. The method of claim 10, wherein the period of incubation is overnight.

12. 2. The method of claim 1, wherein the at least one RNA is at least one mRNA having a length of at least 21 bases and up to 7144 ribonucleotide bases.

13. 2. The method of claim 1, wherein the length of said at least one RNA is from 21 to 6272 ribonucleotide bases.

14. 2. The method of claim 1, wherein the extracellular vesicles are collected at least 4 hours and up to 24 hours after step (c).

15. 2. The method of claim 1, wherein the extracellular vesicles are collected within 24 hours after step (c).

16. 2. The method of claim 1, wherein the extracellular vesicles are collected within 44 hours after step (c).

17. The method of claim 1, wherein the extracellular vesicles have a therapeutic function.

18. 10. The method of claim 1, further comprising culturing the donor cells in fresh medium after delivery of the plasmid or vector to the donor cells.

19. 10. The method of claim 1, further comprising contacting the extracellular vesicles comprising the at least one RNA with a target cell.

20. 2. The method of claim 1, wherein one or more polypeptides translated from at least one RNA are detectable by Western blotting.

21. The method of claim 1, wherein the extracellular vesicles comprise exosomes or microvesicles.

22. The extracellular vesicles include exosomes and microvesicles, 10. The method of claim 1, further comprising separating the exosomes from the microvesicles.

23. The method of claim 22, wherein the separation of the exosomes and the microvesicles is carried out by ultracentrifugation.

24. 10. The method of claim 1, wherein the extracellular vesicles are collected at multiple time points.

25. 25. The method of any one of claims 1 to 24, wherein the extracellular vesicles comprise exosomes.

Images