Methods of cell electroporation

Abstract

1. A method of electroporation involving cell culture for the purpose of improving cell viability, comprising the steps of electroporating cells of interest with an anti-apoptotic protein, treating the cells with DNase after electroporation, and shifting the temperature of the cells from 37°C to 32°C under resting conditions after electroporation.

Description

[Technical field]

[0001] Priority This application claims priority to U.S. Provisional Application No. 63 / 311,006, filed February 16, 2022, which is incorporated by reference herein in its entirety.

[0002] The present disclosure generally relates to methods for enhancing cell viability and transgene expression from large DNA plasmids in cells, such as iPS cells, in a novel combinatorial approach of different enhancer methods for cell viability and transgene expression from exogenous large DNA plasmids. [Background technology]

[0003] Electroporation is a known method for introducing compositions into cells. The terms electroporation, electrotransfection, and electroloading have been used interchangeably in the literature to emphasize the general meaning of this technique. Electroporation can be, for example, flow electroporation or static electroporation. Methods and devices for electroporation are also described, for example, in published PCT application Nos. WO 03 / 018751 and WO 2004 / 031353; U.S. patent application Nos. 10 / 781,440, 10 / 080,272, and 10 / 675,592; and U.S. Patent Nos. 5,720,921, 6,074605, 6,773,669, 6,090,617, 6,485,961, 6,617,154, and 5,612,207, all of which are incorporated by reference.

[0004] Electroporation can damage cells, leading to a decrease in cell viability after electroporation. The prior art does not describe an electroporation method or system that improves cell viability and expression when attempting to transfect large plasmids such as DNA. The present invention provides a method of electroporation of cells that increases cell viability. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Published PCT Application No. WO 03 / 018751 [Patent Document 2] Published PCT Application No. WO 2004 / 031353 [Patent Document 3] U.S. Patent Application Serial No. 10 / 781,440 [Patent Document 4] U.S. Patent Application Serial No. 10 / 080,272 [Patent Document 5] U.S. Patent Application Serial No. 10 / 675,592 [Patent Document 6] U.S. Patent No. 5,720,921 [Patent Document 7] U.S. Patent No. 6,074,605 [Patent Document 8] U.S. Patent No. 6,773,669 [Patent Document 9] U.S. Patent No. 6,090,617 [Patent Document 10] U.S. Patent No. 6,485,961 [Patent Document 11] U.S. Patent No. 6,617,154 [Patent Document 12] U.S. Patent No. 5,612,207 Summary of the Invention [Means for solving the problem]

[0006] The present disclosure seeks to overcome the aforementioned deficiencies by a method that can be applied to other cell types and applications to improve the efficiency of cell engineering, whether related to protein production or cell therapy. Thus, a novel method is described to improve cell viability and expression of large DNA plasmids that are very difficult to transfect in order to achieve cell engineering.

[0007] The disclosed method aims to overcome one or more problems of the prior art by enhancing cell viability and transgene expression from large DNA plasmids in cells such as iPS cells.Therefore, a balanced electroporation protocol is described that uses an appropriate DNA concentration to allow cells to survive by adding enhancers.Enhancers have been shown to have no effect when DNA is highly toxic and electroporation energy is very high.

[0008] In one embodiment, a method of electroporation involving cell culture is disclosed, comprising the steps of: a) co-electroporating the cells with an anti-apoptotic protein, e.g., an mRNA form of the gene BCL-XL; b) adding DNase to the electroporated cells at a temperature T1, such as above 37°C; and c) lowering the temperature of the cells after electroporation to a temperature T2, such as below 32°C.

[0009] In one embodiment, a method for co-electroporation of BCL-XL mRNA and DNA plasmid in iPSCs is described. As used herein, iPS cells refer to "induced pluripotent stem cells". These are a type of pluripotent stem cells derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining characteristics of ES cells.

[0010] In one embodiment, modifications to electroporation protocols are described to achieve the beneficial properties described herein. Such modifications include, but are not limited to, the addition of BCL-XL mRNA to improve cell viability and enhance transgene expression. One or more of the modifications described results in increased cell stability. This requires a balance between higher efficiency and fewer cells and lower efficiency and higher cell numbers.

[0011] In one embodiment, post-electroporation modifications are described, including, but not limited to, the addition of DNase after electroporation enhances cell viability, and a cold shock such as for 48 hours appears to enhance transgene expression.

[0012] In one embodiment, the method further comprises adding a gene editing tool delivered as a DNA plasmid together with DNase.Non-limiting examples of gene editing tools that can be used herein include CRISPR-Cas9 based PRIME editing or base editors.

[0013] In one embodiment, DNase is added in an amount of 1 unit of DNase per 20 ul of volume.

[0014] The shift in temperature to lower temperatures reduces the degradation of the delivered cargo and reduces cell proliferation, ensuring higher expression of the transgene from the delivered vector into the cells.

[0015] In one embodiment, after BCL-XL mRNA is co-electroporated with a DNA plasmid in iPSCs, DNase can be added to the electroporated cells to allow for subsequent membrane recovery. Finally, the disclosed methods include a step of heat treatment, which typically involves lowering the temperature to, for example, 32°C for a desired period of time, such as 24-48 hours, before increasing the temperature to about 37°C.

[0016] Also described are electroporated cells comprising at least one added material produced by the disclosed methods. The disclosed electroporated cells comprise an added substance that enhances at least one property selected from cell viability and transgene expression.

[0017] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles disclosed herein. [Brief description of the drawings]

[0018] [Figure 1] Brightfield images / results of treating iPSC cells with DNase 2 days after electroporation are shown. [Diagram 2] Both GFP fluorescence microscopy and bright field images of iPS cells electroporated under three different conditions are shown. [Diagram 3] Both GFP fluorescence microscopy and bright field images of iPS cells electroporated under four different conditions are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As used herein, iPS cells refer to "induced pluripotent stem cells," which are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem (ES) cell-like state through the forced expression of genes and factors critical to maintaining the properties that define ES cells.

[0020] As used herein, "passage number" is the number of times a cell culture has been subcultured, i.e., harvested and re-seeded into multiple "daughter" cell culture flasks.

[0021] As used herein, "DNase" refers to deoxyribonuclease, the major nuclease present in blood and other body fluids, which is responsible for the digestion of extracellular nuclear proteins, which may be crucial for the prevention of autoimmune reactions.

[0022] Unless otherwise specifically defined herein, all technical, scientific, and other terms used herein have the same meaning as commonly understood by one of ordinary skill in the art with respect to rating-based methodologies and web-based rating systems and related science. Additional terms may be defined, as necessary, in the disclosure that follows.

[0023] The selected cells or cell lines of interest are grown and / or stimulated for a specified length of time depending on the cell type. On the day of electroporation, suspension or adherent cells are harvested and cell sampling is performed to determine cell number and viability.

[0024] In one embodiment, the cell culture includes a step of dissociating the target cells.

[0025] In one embodiment, single cell dissociation for cell culture works better for more accurate cell counting and determining cell viability.

[0026] In one embodiment, the cells are washed with a buffer or basal medium to remove residual components in the medium. A selected number of cells are concentrated into a processing assembly by centrifugation or any other cell concentration device, depending on the scope of the experiment.

[0027] In one embodiment, cell culture can be completed by using techniques known in the art specific to the cell type of interest. Cells can be passaged by clump passaging using EDTA or single cell passaging using Accutase. In one embodiment, the concentration of cells after culture is 1×10 7 ~5×10 7 The range is 1 / mL.

[0028] In one embodiment, the cells are dissociated with ethylenediaminetetraacetic acid (EDTA), and as used herein, EDTA refers to the cell dissociation solution used for clump passaging.

[0029] In one embodiment, the cells are dissociated in Accutase. As used herein, "Accutase" refers to a cell dissociation solution containing proteolytic and collagenolytic enzymes.

[0030] In one embodiment, cells are treated with DNase after being electroporated. DNase allows cell membranes to recover after electroporation. Excess DNA on the outside of cell membranes after electroporation prevents the membrane from resealing. Removing excess DNA helps cells to close the holes that were opened during electroporation. Any commercially available DNase, such as Pulmozyme (Genentech), can be used according to the embodiments described herein.

[0031] In one embodiment, anti-apoptotic genes can be added to increase cell viability. Non-limiting examples of such genes include BCL2 or BLC-XL. The methods disclosed herein can be used with a wide variety of cell populations. In some embodiments, cells can be derived from blood, interstitial fluid, and any tissue, such as bone marrow, peripheral blood, or umbilical cord blood, or any other normal or diseased tissue.

[0032] In one embodiment, the cells may be derived from whole peripheral blood or whole cord blood. In some embodiments, the cells may be derived from whole peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells may be derived from whole cord blood mononuclear cells (CBMCs). In some embodiments, the cells may be derived from a fraction of peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells may be derived from a fraction of cord blood mononuclear cells (CBMCs). In some embodiments, the cells may be derived from a specific cellular component of blood.

[0033] Non-limiting examples of PBMCs include alpha beta TCR+ T cells, gamma delta TCR+ T cells, NK cells, invariant NKT cells, B cells, dendritic cells, monocytes, macrophages, neutrophils, granulocytes, hematopoietic progenitor cells, mesenchymal progenitor cells, and stromal cells. These cells may be mature or immature. These cells may also be lineage committed and uncommitted cells.

[0034] In one embodiment, the cell type can be selected from, but is not limited to, T cells, HSC, NK cells, DC, B cells, PBMC, HEK 293, CHO, IPSC cells, and / or K652.

[0035] In one embodiment, the claimed method can be used with iPSC cells. To do so with single cell passaging, the cells can be passaged at low cell numbers and grown for 7-8 days (e.g., 15-20,000 cells in a 6-well plate, i.e., 1.5-2.0×10 cells). 3 pieces / cm 2 ). This can result in very compact colonies by days 7-8, where the cells in the center of the colony are denser than the cells in the outer parts of the colony, which can lead to less efficient uniform electroporation.

[0036] In one embodiment, cells could be passaged at a higher density, for example 1×10 cells if 80% confluency is desired by passage 3 days prior to electroporation. 4 pieces / cm 2 100,000 cells can be seeded in a 6-well plate, or 1 × 10 cells if passaged the day before electroporation. 6 pieces / cm 2 That is 1×10 5It is considered that the cells are passaged into 6-well plates. Those skilled in the art will understand that these are approximate cell seeding densities and need to be adjusted based on the growth characteristics of iPSCs. Some iPSCs will grow faster depending on the coating and medium used. For example, iMatrix-511 combined with StemFitAK03N medium generally reaches 80% confluency under the above conditions. Other combinations of coating and medium may result in different cell growth kinetics.

[0037] In one embodiment, cells can be passaged 3 days prior to electroporation using a dissociation reagent such as 0.5 mM EDTA at a ratio of 1:10. For example, from a single well in a 6-well plate, 10% of the well is seeded into a new well of the 6-well plate. Generally, this will result in approximately 80% confluency on the day of electroporation. Cell viability should be greater than 90% for iPSC lines. Generally, cells with less than 30 passages are preferred, and the doubling rate should be less than 24 hours. Cells that have just been thawed after cryopreservation should not be electroporated. Cells should be passaged 2-3 times after cryopreservation, and the cell doubling rate should be checked prior to electroporation.

[0038] In one embodiment, iPSC cells can be prepared for electroporation by first washing the cells with PBS, aspirating the PBS, repeating the washing step if the cells are floating in suspension, adding 0.5 mL of Accutase to the wells of a 6-well plate, or an appropriate amount of Accutase to cover the plate if a larger culture vessel is used, rocking the plate to ensure the surface is covered with Accutase, and placing the plate in a 37° C. incubator for 5-15 minutes, although depending on the coating agent used it may take longer for the cells to round up and be ready to be detached from the plate.

[0039] For example, in one embodiment, a product that allows cell adhesion, such as a protein, is used. In one embodiment, when using a protein-based coating (vitronectin coating), the cells tend to detach within 5 minutes. However, with i-Matrix-511, it may take longer than 10 minutes for the cells to start to dissociate. The cells are then pipetted up and down in the Accutase solution to obtain single cell dissociation. It is advisable not to use a scraper to mix the cells at this point, as this will damage the cells.

[0040] In some embodiments, the method may then include adding iPSCs to a centrifuge tube containing iPSC medium with Accutase.For example, when using a 6-well plate, add the cells pipetted with 0.5mL of Accutase to a 1.5mL centrifuge tube containing 0.9mL of iPSC medium.When combining multiple wells, a larger centrifuge tube can be used.

[0041] In one embodiment, the method then includes centrifuging the cells at 250× gravity (g) for 5 minutes with gentle interruptions. Care should be taken not to stress the cells during centrifugation, as high centrifugation speeds may sensitize the cells to electroporation damage. For example, centrifugation above 300 g may result in damage to the iPSCs. Aspirate the supernatant after centrifugation and pipette any residual medium from the tube, without disturbing the cell pellet. The cells are then resuspended in 5-10 mL of electroporation buffer using standard washing techniques. The cells are then counted and viability measured to ensure that viability does not fall below 80%.

[0042] After counting the cells, the method may then include centrifuging the cells at 250×g for 5-10 minutes with gentle pauses. While the cells are being centrifuged, the processing assembly packaging (PA) is sprayed with 70% isopropyl alcohol (IPA), the PA packaging is placed in a biosafety cabinet, and the packaging is opened.

[0043] In some embodiments, a 1.5 mL microcentrifuge tube corresponding to the DNA concentration to be evaluated can be prepared and the corresponding amount of DNA can be pipetted into this 1.5 mL microcentrifuge tube. For large DNA, it is best to use a total DNA concentration between 50-300ug / mL. Once the microcentrifuge tube is prepared, electroporation can be performed. This involves removing the cells from the centrifuge and aspirating the supernatant. Resuspend the cells in an appropriate amount of electroporation buffer to obtain the desired cell concentration. Take into account the cell pellet volume. For example, if a total volume of 1 mL is required, the cell volume may be 300ul, therefore only 700ul of buffer is added to bring the total to 1mL. The cell pellet volume can be estimated by comparing an empty 50mL tube and filling it with a known volume. Alternatively, a smaller volume can be intentionally added to the cells so that the total volume is less than 1mL. The appropriate amount of volume can then be added to bring the total to 1mL. While estimation at this stage is acceptable, it should be noted that knowing the cell density helps ensure reproducibility of experiments.

[0044] After adding the buffer, an appropriate volume of cells is aliquoted and added to a 1.5 mL centrifuge tube containing the desired amount of DNA and 50 ug / mL BCL-XL mRNA. The cells and DNA are mixed at least 5 times. Avoid introducing air bubbles. The cells and loading agent mixture is then transferred to the processing assembly, and the processing assembly is placed on the electroporation system to electroporate the cells.

[0045] After electroporation, transfer the cells from the processing assembly to a V-bottom 96-well plate containing DNase. Rinse the wells of the processing assembly with an equal volume of MaxCyte buffer and transfer it to the same wells of the 96-well plate. Use 1 unit of DNase for every 20ul volume. Mix the solution for 30-40 minutes in a humidified incubator at 37°C, 5% CO2 and recover. Transfer the cells from the processing assembly to pre-warmed culture medium at 37°C in a culture vessel.

[0046] The processing assembly can be rinsed with medium to recover any remaining cells and added to the culture vessel. The cell density after electroporation depends on the viability of the cells. For example, if an electroporation energy that prioritizes efficiency over viability is selected, fewer cells will survive, so more cells should be seeded into the culture vessel. If a lower electroporation energy with higher cell viability is selected, a lower seeding density should be selected. Finally, the cells are cultured at 32°C for 1-2 days, and then the temperature is returned to 37°C. It is believed that lowering the temperature slows down cell proliferation and enhances gene expression.

[0047] Referring to the figures, FIG. 1 shows brightfield images / results from treating iPSC cells with DNase two days after electroporation. The figure shows results from both single cell passaging using Accutase and clump passaging using EDTA. Accutase treatment was used to improve cell dissociation to make it easier to determine cell viability and cell number. Treating cells with DNase after electroporation improved cell viability in both clump and single cell passaging. The figure demonstrates this by showing a higher concentration of cells in the image for cells treated with DNase.

[0048] FIG. 2 shows both GFP fluorescence microscopy images and bright field images of iPS cells electroporated in three different conditions. Column 1 shows iPS cells electroporated without DNA plasmid or BCL-XL mRNA. iPS cells were treated with DNase after electroporation. 24 hours after electroporation, no GFP expression was observed and the cells appeared healthy. Column 2 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, but without BCL-XL mRNA. iPS cells were treated with DNase after electroporation. Weak GFP expression was observed 24 hours after electroporation, and cell viability was low. Column 3 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, and BCL-XL mRNA. iPS cells were treated with DNase after electroporation. 24 hours after electroporation, higher expression of GFP and cell viability were observed.

[0049] FIG. 3 shows both GFP fluorescence microscopy images and bright field images of iPS cells electroporated under four different conditions. Column 1 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, but without BCL-XL mRNA. iPS cells were not treated with DNase after electroporation and cultured at 32° C. for 48 hours. Column 2 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, and BCL-XL mRNA. iPS cells were treated with DNase after electroporation and cultured at 32° C. for 48 hours. Column 3 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, but without BCL-XL mRNA. iPS cells were not treated with DNase after electroporation and cultured at 37° C. for 48 hours. Column 4 shows iPS cells electroporated with a large DNA plasmid expressing Cas9 and GFP genes, and BCL-XL mRNA. iPS cells were treated with DNase after electroporation and cultured for 48 hours at 37° C. The highest expression of GFP was observed in column 2 when BCL-XL mRNA was co-transfected with DNA and iPS cells were treated with DNase after electroporation and cultured for 48 hours at 32° C. This is demonstrated by the higher concentration of GFP positive cells in column 2 compared to the other columns.

[0050] In some embodiments, the spatial and temporal control of electroporation efficiency can be altered or adjusted within a cell population. It is envisioned that a variety of unique specific parameters may be applied to transfection methods that affect one cell type but not another, for example, affecting T cells but not B cells within a sample of cells from a subject.

[0051] In some embodiments, the methods and compositions disclosed herein may be effective in many immunotherapies, including but not limited to the treatment of cancer and autoimmune diseases. The methods and compositions disclosed herein may also be used for the treatment of several other diseases, including but not limited to chronic and infectious diseases, viral, bacterial, or parasitic infections, graft-versus-host disease, lymphoproliferative disorders, and hyperproliferative diseases. It is envisioned that these methods and compositions may also be useful for additional indications not discussed herein.

[0052] In some embodiments, modulation is direct or indirect. In some embodiments, alteration is direct or indirect. In some embodiments, therapeutic efficacy or therapeutic index may range from immune response, immune activation, or immune suppression.

[0053] In one aspect of the present disclosure, a method for producing modified cells for in vitro or ex vivo cell vaccine therapy is provided. The method includes isolating the cells, introducing a composition into the cells, and administering the cells to a subject. In some embodiments, the composition includes at least one mRNA encoding at least one antigen, alone or in combination, and the modified cells may or can induce an immune response against the antigen. In some embodiments, the modified cells may or can induce an immune response against other antigens expressed by target cells in the subject through a mechanism called epitope spreading.

[0054] In some embodiments, gene editing agents include CRISPR CAS-9, RNA, plasmids, mega-TALS, gene-writing, DNase I, benzonase, exonuclease I, exonuclease III, mung bean nuclease, nuclease BAL 31, RNase I, 51 nuclease, lambda exonuclease, RecJ, T7 exonuclease, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease, or site-specific nuclease.

[0055] The term "cellular vaccine" as used herein refers to cells modified to express an antigen. In particular, cellular vaccines refer to cells modified to induce an immune response against an antigen and activate immune cells against target antigen-expressing cells. When delivered to a subject, cellular vaccines generate an inflammatory environment to induce an immune response against malignant tumors and against abnormally proliferating autoimmune cells, cells infected with viruses, bacteria, fungi, or any disease-causing biological agent, thereby providing them with the ability to specifically suppress and / or inactivate or kill diseased / infected cells or disease-causing cells. Non-limiting examples of antigens can include proteins, polypeptides, carbohydrate antigens, lipoproteins, or peptide antigens, or peptidomimetics.

[0056] In general, the molecules can include proteins, nucleotide sequences, carbohydrates, lipoproteins, or fragments thereof. Any of these molecules can be used as antigens or, in the case of nucleotide sequences, for example, can be used to generate antigens. These molecules can be natural (i.e., biological) or synthetic. In some embodiments, the antigen can be a protein, a polypeptide, a peptide multimer, a peptide avimer, a carbohydrate antigen, or a lipid protein, or a combination thereof.

[0057] The term "transduction" is used to describe the viral-mediated transfer of nucleic acids into cells. In contrast to the transfection of cells with exogenous DNA or RNA, no transfection reagent is required here. Viral vectors, also called virions by themselves, can infect cells and transport DNA directly into the nucleus, independent of further action. After the release of DNA into the nucleus, the protein of interest is produced using the cellular machinery.

[0058] Features and advantages of the methods disclosed herein are illustrated by the following examples, which should not be construed in any way as limiting the scope of the disclosure. EXAMPLES

[0059] To demonstrate that the claimed method works, iPSC cells were prepared by growing them to a density of 80% confluence after 32 passages. When the cells reached a final density of (x), they were spun down in a centrifuge at 250×g for 5 minutes with gentle pauses. While the cells were in the centrifuge, the electroporation system (here, ExPERT STx OC-25x3 processing assembly) was sprayed with 70% isopropyl alcohol in a biosafety cabinet (BSC). A 1.5 mL microcentrifuge tube containing 50 ug / mL BCL-XL mRNA was prepared, corresponding to a DNA concentration of 100 ug / mL. The electroporation system was then turned on and the Opt 4 protocol was selected. After the cells had finished spinning down in the centrifuge, they were transferred into the BSC and the supernatant was aspirated after spraying with IPA. The pellet was then resuspended in MaxCyte buffer to obtain 1×10 cells. 8 A cell density of 100 μg / mL was obtained. From this mixture, a volume of 20 ul was added to a 1.5 mL centrifuge tube with 100 μg / mL DNA and 50 μg / mL BCL-XL mRNA. The cells were then gently mixed and transferred to the processing assembly, which was then placed on the MaxCyte(x) for electroporation.

[0060] After electroporation, the cells were transferred from the processing assembly to a 96-well plate. The wells of the processing assembly were washed with an equal volume of buffer and transferred to the same wells of the 96-well plate. 40ul was then split into two wells (20ul in each well, 1x10 cells per well). 6 pcs). 2 units of DNase (Pulmozyme; Dornase alfa) were then added. The solution was then placed in a humidified incubator at 37°C with 5% CO2 for 30 min to recover. After recovery, cells were transferred from the 96-well plate to pre-warmed culture medium at 37°C in a 6-well culture vessel and cultured at 32°C for 24 h. The results were analyzed using bright field and fluorescence microscopy.

[0061] Figure 3 shows that cells that were (1) electroporated with BCL-XL mRNA, (2) treated with DNase after electroporation, and (3) subjected to a temperature shift from 37°C to 32°C after electroporation showed significantly higher GFP expression compared to cells electroporated without BCL-XL mRNA that were also subjected to a temperature shift.

[0062] It should be understood that both descriptions disclosed herein are intended to be illustrative and non-limiting.

[0063] It is intended that the specification and examples disclosed herein be considered as exemplary only, with a true scope and spirit of the invention being indicated in the following claims. Other embodiments of the compositions, devices and methods described herein will be apparent to those skilled in the art from consideration of the disclosure and practice of the various exemplary embodiments disclosed herein.

[0064] Other than in the examples, or unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, analytical measurements, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. Each numerical parameter should, at the very least, be construed in light of the number of significant digits and ordinary rounding approaches, but rather as an attempt to limit the application of the doctrine of equivalents to the scope of the claims.

[0065] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, unless otherwise indicated, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0066] As used herein, the terms "the," "a," or "an" mean "at least one" and should not be limited to "only one," unless expressly indicated otherwise. Thus, for example, "a hybrid peptide" should be interpreted to mean "at least one hybrid peptide."

[0067] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein.

Claims

1. A method of electroporation involving cell culture, comprising: (a) co-electroporating cells with an anti-apoptotic protein to form electroporated cells; (b) adding DNase to the electroporated cells at temperature T1; and (c) Reducing the temperature of the cells after electroporation to a temperature T2 The method includes:

2. The method of claim 1, wherein the cell culture comprises a step of dissociating the cells.

3. The method of claim 1, wherein the anti-apoptotic protein is the mRNA form of the gene BCL-XL.

4. The method of claim 3, wherein the BCL-XL is in a plasmid form.

5. The method of claim 1, wherein the anti-apoptotic protein is the mRNA form of the gene BCL2.

6. The method of claim 5, wherein the BCL2 is in a plasmid form.

7. 2. The method of claim 1, wherein the cell type is selected from the group consisting of iPSCs, PBMCs, B cells, HEK293 cells, and CHO cells.

8. 2. The method of claim 1, further comprising adding a gene editing tool delivered as a DNA plasmid along with DNase.

9. 9. The method of claim 8, wherein the gene editing tool is a CRISPR-Cas9-based PRIME editing or a base editor.

10. The method of claim 1, wherein 1 unit of DNase is added per 20 ul volume.

11. 2. The method of claim 1, wherein the temperature T1 is greater than 37°C.

12. 2. The method of claim 1, wherein the temperature T2 is equal to or lower than 32°C.

13. 13. The method of claim 12, wherein the temperature is maintained at T2 for a period ranging from 30 to 40 minutes.

14. Electroporated cells comprising at least one added substance, the added substance enhancing at least one characteristic selected from cell viability and introduced gene expression, the electroporated cells being produced by the method of claim 1.

Images