Methods for improving virus-like particle production per cell in suspension

Abstract

A method of producing virus-like particle production using electroporation. The method comprises cultivating a population of cells in a log growth phase in a cell culture vessel using media, cultivating the population of cells to a desired level of confluency, removing the media from the cell culture vessel and adding a transfection buffer. The method further comprises adding to the cell culture vessel at least one vector, transfecting the cell population, culturing the cell population and seeding the cells located in the cell culture vessel. The disclosed method may further include adding an endonuclease to the cell culture vessel, adding a compound that increases transcription efficiency and subjecting the cell culture to a temperature shift.

Description

METHODS FOR IMPROVING VIRUS-LIKE PARTICLE PRODUCTION PER CELL IN SUSPENSIONCross-Reference to Related Applications

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 63 / 615,176, filed December 27, 2023; 63 / 549,643, filed February 5, 2024; 63 / 640,421, filed April 30, 2024; and 63 / 706,166, filed October 11, 2024. The contents of each of these applications are incorporated herein by reference in their entirety.Technical Field

[0002] The present disclosure relates to a method and device for improving the production of virus-like particles or protein nanoparticles per cell in suspension by, for example, using electroporation, DNase, sodium butyrate, and temperature shifting the cells during cell culture.Background

[0003] Delivery systems for purposes of gene editing, gene expression changes, exon skipping, imaging, and other varied cell, tissue, organ, and system modulation for research and treatments have traditionally employed the use of viral vectors. While useful, the use of viral vectors is not always ideal as these are infectious and can lead to long term expression of the vectors, leading to undesired mutations, other off-target effects, and immune response complications. Virus-like particles (“VLPs”) or protein nanoparticles can help overcome these challenges as they are not infections and can be employed transiently, limiting mutations and other off-target effects.

[0004] VLPs are similar to viral vectors as they are covered by a protein shell that can selfassemble and contain cargo for delivery into target cells or systems. Contrary to viral vectors, however, VLPs do not contain viral genetic material, which results in VLPs being non- infectious as they cannot replicate after they are inserted into a cell. VLPs can have bio-active molecules, such as lipids, proteins, and RNA that are ready to perform a specific function oncedelivered inside of a cell. This allows VLPs to shortcut the reverse transcription, transcription, and translation that are usually necessary for a viral vector to effectuate its function.

[0005] Traditional methods and devices to produce VLPs include chemical transfections. These protocols for producing VLPs involve the use of expensive chemical mediated expression in VLP producer cells or employ the use of toxic chemicals that may lead to nonideal levels of VLP produced or reduced levels of viable VLP.

[0006] In addition, traditional methods of VLP production usually restrict the VLP producer cell to adherent cells, not including cells in suspension, thus limiting the potential benefits of using suspension cells in VLP production systems. Some potential benefits of using cells in suspension to produce VLPs include their scalability. Cells in suspension are not limited by the surface area of the vessel as is the case with adherent cells. As a result, under the right conditions, cells in suspension can be grown to a much higher cell number in a single bioreactor, not needing potentially more complicated growth vessels like multilayer culture vessels such as Coming’s Cell STACK or HYPER stack culture chambers.

[0007] Together, these hindrances lead to increased cost, limited applications, excessive use of reagents, poor reproducibility, limited scalability, and scientists’ time that results in slower scientific progress.

[0008] Therefore, there is a need for methods and devices to increase the production of VLPs per suspension cell.Summary

[0009] Consistent with some disclosed embodiments, methods of producing vims-like particle production using electroporation are disclosed. In one embodiment, the method comprises cultivating a population of cells in a log growth phase in a cell culture vessel using media, cultivating the population of cells to a desired level of confluency, removing the media from the cell culture vessel and adding a transfection buffer.

[0010] The method further comprises adding to the cell culture vessel at least one vector, transfecting the cell population, culturing the cell population and seeding the cells located in the cell culture vessel. In some embodiments, the disclosed method may further include adding an endonuclease to the cell culture vessel, such as prior to the seeding step.

[0011] In some embodiments, the method may further include adding a compound that increases transcription efficiency. In some embodiments, the method may additionally or alternatively comprise subjecting the cell culture to a temperature shift for a desired period of time.Brief Description of the Figures

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some disclosed embodiments and, together with the description, serve to explain the disclosed embodiments. The particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the present disclosure. The description taken with the drawings makes apparent to those skilled in the art how embodiments of the present disclosure may be practiced.

[0013] Figures 1A-1B illustrate that electroporation parameters enable high transfection efficiency and high production yields of VLPs from VLP producer HEK293 cells in suspension.

[0014] Figure 2 illustrates that VLP activity from suspension HEK cells increases in an electroporation energy dependent manner.

[0015] Figures 3A-3B illustrate that prolonged harvest of VLPs results in a reduction in VLP activity in the absence of sodium butyrate.

[0016] Figure 4 illustrates that the addition of sodium butyrate increases VLP production.

[0017] Figures 5A-5D illustrate that VLP activity decreases when harvested at day 2 without sodium butyrate but sodium butyrate allows for increased VLP activity at day 1 or day 2 harvest.

[0018] Figures 6A-6B illustrate that suspension HEK293 cells can be cultured up to 12xl06cells / mL after electroporation to increase VLP activity.

[0019] Figures 7A-7C illustrate that VLP Production is higher than chemical transfection on day 1 harvest with and without a chemical enhancer.

[0020] Figures 8A-8F illustrate that Electroporation is superior to chemical transfection kinetically and in total VLP activity.

[0021] Figure 9 illustrates that electroporation enables the packaging of adenine base editor (“ABE”) RNPs into VLPs to target HEK3 loci and induce high rates of base substitutions.

[0022] Figures 10A-10B illustrate that a temperature shift after electroporation improves ABE8e-VLP editing activity and capsid titer.

[0023] Figures 11A-11B illustrate that primary immune cells are efficiently edited with VLPs produced in suspension HEK293 cells using the disclosed method.

[0024] Figures 12A-12C show a comparison of producer cell viability (12A), density (12B), and expansion (12C) following electroporation or chemical transfection.

[0025] Figures 13A-13B show B2M gene knockout (13A) and indel formation in cells transduced with VLPs generated from producer cells that were electroporated or chemically transfected.

[0026] Figures 14A-14B show VLP-mediated knockout of B2M in activated T-cells using conservative (14A) and non-conservative gating (14B).

[0027] Figures 15A-15D characterize VLP titers (15 A), percentage of full titers (15B), VLP size (15C), and aggregation (15D) in VLPs generated from producer cells that were electroporated or chemically transfected.Detailed DescriptionDefinitions:

[0028] As used herein, “virus-like particles” (“VLPs”) or “virus nanoparticles” refer to natural or artificial structures that closely resemble viruses by having, for example, a protein shell that envelops cargo material and protects said cargo from degradation from outside influences. As described herein, “VLPs” and “virus nanoparticles” include nanoparticles, such as a pseudovirus or pseudovirions (“PsV”), nanoblades, and quasi-virions (“QV”). VLPs are distinct from viruses in part because they have no genetic material as part of their cargo, making VLPs non-infectious as these cannot replicate. Their non-infectious feature may be preferable as it presents a lower biosafety risk and may be better suited for applications where a transient use of the cargo is desirable. Instead of having genetic material as their cargo, VLPs can have active biomolecules as cargo. For example, VLPs can contain lipids, proteins, and other organic compounds. Once the VLP is inside the cell, the cargo gets released and the active biomolecules can rapidly begin to exert their functions without the added steps of transcription, translation, and post translation modifications.

[0029] Those skilled in the art understand that the disclosed method would also work to create extracellular vesicles (“EVs”) as both VLPs and EVs are lipid bilayered particles secreted by cells.

[0030] As used herein, “suspension cells” refers to cells free floating in culture media.

[0031] As used herein, “log growth phase” or “logarithmic growth phase” refers to the phase in which cells are actively proliferating and their cell density and or quantity increases exponentially. This is in contrast to the other growth phases, lag, stationary, decline, and senescence, in which the cells grow and proliferate at a slower rate, stop growing, or begin to decline in total number and or density.

[0032] As used herein, “cell culture vessel” or “vessel” refers to the vessel where cells are grown. The vessel may be made of glass, plastic, thermoplastic, polyolefins, polycarbonate, polystyrene, or other materials. The a “vessel” can be, for example, non-baffled shake flasks of volumes that include 125 mL, 250 mL, 500 mL, 1000 mL, 2000 mL, and of volumes in between the ones described as well as above and below the disclosed maximum and minimum volumes disclosed. As used herein, a “vessel” can be wave bags of volumes that include 1000 mL, 2000 mL, 5000 mL, 10,0000 mL, and of volumes in between the ones described as well as above and below the disclosed maximum and minimum volumes disclosed. As used herein, vessel includes bioreactors of volumes that include 10 mL, 15 mL, 100 mL, 150 mL, 200 mL, 250 mL, 1 L, 2 L, 5 L, 10 L, 25 L, 50 L, 100 L, 200 L, 250 L, 300 L, 350 L, 500 L, 800 L, 1000 L, 2000 L, 2500 L, 5000 L, 7500 L, and 10000 L.

[0033] As used herein, “confluency” includes a value representing how much of the vessel surface area adherent cells are occupying. For suspension cells, confluency refers to the density of cells per volume.

[0034] As used herein, “transfection buffer” includes buffers made for electroporation, for example, electroporation buffer (“EP buffer,” “EP,” or “EB”), lipofection, and chemical transfection, including but not limited to Opti -MEM™ Reduced Serum Medium.

[0035] As used herein, “vector” or “vectors” include distinct pieces of non-genomic genetic material that may be in a closed circular conformation, like a plasmid.

[0036] As used herein, “electroporation” is meant to encompass the process of facilitating the insertion of foreign materials, like genetic material, into cells by using a combination of electricity in combination with other reagents and solutions. Any electroporation system may be used to carry out the claimed invention including but not limited to the ExPERT ATx™, the EXPERT GTX™, the EXPERT STX™, and the ExPERT VLx™.

[0037] As used herein, “cell culture conditions” include standard cell culture conditions for cell lines. Although the following parameters can vary per cell line and other factors, these usually include standard cell culturing conditions of CO2 at a range of 37% and humidity at about 80% culture density. Standard cell culture conditions can include the use of a shaker or other mechanisms to help oxygenate and disseminate nutrients to the cultured cells. Additionally, cells can be cultured at a temperature of 32 °C to 37 °C, with a CO2 concentration of 4% to 7%, and a humidity of about 80% to 95%. The cell culture conditions can vary beyond the limitations described depending on the cell type, the downstream applications, and according to what a person of ordinary skill in the art would determine applicable under the circumstances.

[0038] As used herein, “transcription efficiency” refers to the efficacy of transcribing DNA into messenger RNA (“mRNA”).

[0039] As used herein, “endonucleases” are enzymes or other molecules that cleave DNA in a nonspecific manner, usually to cause degradation of undesired DNA or genetic material. Examples of reagents that can achieve these results are DNase, Pulmozyme®, and Benzonase®. As disclosed herein, where appropriate the amount of endonuclease used ranges from 0.1 units to 5 units per 20 pL of transfection volume. A “unit” is defined as a microliter of the prepared solution of endonuclease.Description of Embodiments:

[0040] The following is a detailed description with several non-limiting embodiments and accompanying figures, and substitutions, additions, or modifications may be made to the components and steps provided.

[0041] Embodiments of the present disclosure are directed to methods of improving VLP production per cell by using, for example, a suspension culture of HEK293, a suspension culture of HEK293T, or other suspension culture of HEK293 -derived cell line. Embodimentsof the present disclosure include using cell culture media that can be, but is not limited to, Dulbecco's Modified Eagle Medium (“DMEM”), Roswell Park Memorial Institute (“RPMI”) 1640 Medium, Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (“DMEM F12”), TexMACS™, Minimum Essential Medium (“MEM”), Iscove’s Modified Dulbecco’s Medium (“IMDM”), CTS™ OpTmizer™, Opti-MEM™ Reduced Serum Medium, or others. The present disclosure also includes supplementing with industry standard serum such as but not limited to, fetal bovine serum, horse serum, human serum, or other synthetic serum, at an amount of, for example, 2%, 5%, 10%, 15%, or 20% or any value between those percentages, as well as above and below the maximum and minimum values disclosed, taking into account variations in cell line, upstream and downstream applications, as well as other variables. The present embodiment and disclosure also include culturing the cells with media that is chemically defined or otherwise suited as compliant for good manufacturing procedures (“GMP”). Furthermore, embodiments of the present disclosure include culturing the cells until they have achieved a logarithmic growth state. Determining a logarithmic growth state can be done by estimating the number of cells at a particular point (for example 24 hours after seeding) and comparing the number of cells at later time points (for example, 48 hours and 72 hours after seeding). If the change in cell growth has increased logarithmically and in a non-linear fashion, then the cells can be said to be in a logarithmic phase. Further, embodiments of the present disclosure involve removing the media by using, for example, a vacuum aspirator, decanting the media, a serological pipette, or a micropipette. Embodiments of the present disclosure also include adding a transfection buffer to the cell culture that can be an electroporation buffer, such as MaxCyte’s® commercially available electroporation buffers, in an amount at least sufficient to cover the surface area of the vessel.

[0042] The present disclosure and embodiments include mixing the desired vectors in a tube, for example, an Eppendorf 1.5 mL tube, containing electroporation buffer described above.The amount of each vector may vary depending on the application and it requires taking into account the number of cells to be transfected, the cell line, the application, and the vessel used. Similarly, the ratios of each of the different vectors used to generate VLPs are not limited to the ratios of each vector disclosed herein and will vary depending on the application, cell type, and other relevant factors. Per mL, the total amount of vector can add to a total of 0.1 pg to 300 pg of vector. For example, per mL, the total amount of vector can be 0.1 pg, 10 pg, 50 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, and any value in between the disclosed values as well as higher and lower than the maximum and minimum values disclosed.

[0043] The number of distinct vectors can also vary from 1 vector to 10 different vectors. By different vectors, it is understood that it can mean vectors that have the same or different backbones, genes, open reading frames, variants of a gene or genes, reporters, selection markers, and features of a particular vector. A non-limiting example of the identity of these vectors adding up to about 300 ug / mL of vector for transfecting 15 / | 06cells, and the amount that can be used per vector is: (1) MLV-Gag (109 pg / mL or 10.9 pg in 100 pl of EP volume); (2) MLV-Gag-Cas9 (36 pg / mL or 3.6 pg in 100 pl of EP volume); (3) VSV-G (13 pg / mL or 1.3 pg in 100 pL of EP volume); and (4) sgRNA (142 pg / mL or 14.2 pg in 100 pL of EP volume).

[0044] In some embodiments, the present disclosure includes adding the desired combination and amount of vector to concentrated cells in electroporation buffer and subjecting the cells to a transfection device, like an electroporation device, and applying a transfection protocol to transfect the cells. The device and method can include using electricity at a particular voltage for a particular time, and other particular parameters and reagents to effectively transfect the cells. The present disclosure includes transferring the cells to, for example, an Eppendorf tube, a Falcon tube, or a culture vessel, after completing a transfection protocol adding an optional washing step with EP buffer and adding fresh EP buffer to an appropriate volume. Anappropriate volume can be, for example, an amount equivalent to the electroporation volume, such as, 100 pL of buffer when 100 pL of electroporation volume was used.

[0045] In additional embodiments, there is described gently resuspending the residual cells in the EP buffer and transferring them to an Eppendorf tube, a Falcon tube, or culture vessel. Other examples of transferring the cells post-transfection and resuspending them should take into account the total number of cells transfected. For example, for transfecting 3 x io6cells, resuspending the cells in 1000 pL of media may be appropriate, while transfecting a much larger number of cells, for example, 5 x 1011cells may require resuspending the cells up to 16.7 L of culture media.

[0046] In some embodiments, there is described a lower cell number range for transfection of about 10 x io5cells. The present embodiment also encompasses the following cell numbers for transfection: 10 x io6cells, 10 x io7cells, 10 x 108cells, 10 x io9cells, 10 x io10cells, and 1011cells. The present disclosure also envisions using for electroporation any number of cells in between the numbers described above as well as amounts that go above and below the number disclosed.

[0047] In some embodiments, there is disclosed a method of adding an appropriate amount of the electroporated cells in EP buffer into a non-baffled shake flask ranging in size from 125, 250, 500, 1000, 2000 mL, a bioreactor, or another vessel, and providing the cells with a recovery period that can be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, or 120 minutes or any amount of time in between the disclosed values as well as more and less amount of time than the maximum and minimum amounts of time disclosed.

[0048] In some embodiments, the present disclosure also encompasses adding an appropriate amount of cell culture media like, for example, DMEM media to the cell culture vessels containing electroporated cells and EP buffer. The disclosed media can be supplemented withserum, for example, fetal bovine serum, horse serum, human serum, or other synthetic serum. The level of serum supplementation in the media can be, for example, 2%, 5%, 10%, 15%, or 20% or any value between those percentages, as well as above and below the maximum and minimum values disclosed.

[0049] In some embodiments, the present embodiment and disclosure also include culturing the cells with media that is chemically defined or otherwise suited as compliant for good manufacturing procedures (“GMP”). Additionally, the cells could be grown in culture medium without serum.

[0050]

[0045] In some embodiments, the present disclosure contemplates adding compounds to the cell culturing process to increase transcription efficiency in the cells. These compounds may include, but are not limited to, histone deacetylase (HDACs) inhibitors such as sodium butyrate, valproic acid, and / or caffeine. Sodium butyrate may be added to increase transcription efficiency at a final concentration of 0.4 mM, 1.0 mM, 1.5 mM, 2 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, or 5 mM, or any value between those disclosed as well as a concentration that goes below or above the disclosed minimum and maximum. The sodium butyrate can be substituted by caffeine at a concentration that includes 0.4 mM, 1.0 mM, 1.5 mM, 2 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, or 5 mM, or any value between those disclosed as well as a concentration that goes below or above the disclosed minimum and maximum.

[0051] The present disclosure also encompasses sodium butyrate combined with caffeine to increase transcription efficiency. The present disclosure encompasses the combination of sodium butyrate and caffeine wherein the concentration of each reagent is applied using any of the concentrations described above for each reagent. The present disclosure encompasses applying the combination of caffeine and sodium butyrate at a ratio of, for example, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, or others that go above and below the disclosed ratios. Similarly, the presentdisclosure encompasses applying the combination of sodium butyrate and caffeine at a ratio of for example 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, or others that go above and below the disclosed ratios. In some embodiments, the present disclosure includes adding sodium butyrate or caffeine, or the combination or none, after about 1 hour after adding media to the electroporated cells. Instead of sodium butyrate or caffeine, other compounds known to increase transcription efficiency can be added to the cell culture in an amount and concentration sufficient to increase transcription efficiency in the cells.

[0052] In some embodiments, the present disclosure also includes adding sodium butyrate or caffeine, or the combination for 1.5 hours, 2.0 hours, 3.0 hours, 4.0 hours, 8.0 hours, or 10 hours after media was added to the electroporated cells. The addition of sodium butyrate or caffeine, or the combination of the two can also be added at any value of time between those disclosed as well as at a time below or above the disclosed minimum and maximum.

[0053] In some embodiments, the present disclosure also encompasses incubating the electroporated cells cultured with media and sodium butyrate or caffeine (or both) for a period of time including but not limited to 18 hours, 20 hours, 24 hours, 30 hours, 38 hours, 48 hours as well as a number of hours that go above and below the numbers disclosed.

[0054] In some embodiments, the method contemplates subjecting the cells cultured with media to a temperature shift that can include a delta of 15° C of a difference. In some embodiments, the method contemplates subjecting the cells cultured with media and a compound that increases transcription efficiency, including but not limited to sodium butyrate and caffeine, to a temperature shift that can include a delta of 15° C of a difference. For example, the temperature shift can change a starting temperature from 37° C to 28° C, from 37° C to 30° C, 37° C to 32° C, 37° C to 34° C, or 37° C to 36° C. The temperature shift can also change the starting temperature from 35° C to 28° C, from 35° C to 30° C, 35° C to 32° C, or 35° C to 34° C. The temperature shift can also change the starting temperature from 33° C to28° C, from 33° C to 30° C, or 33° C to 32°. The temperature shift can also include a starting temperature above or below those explicitly disclosed above and a final temperature above or below those explicitly disclosed above. The present disclosure encompasses culturing the cells in the final temperature of the temperature shift 12 hours, 18 hours, 24, hours, 30 hours, 36 hours, 42 hours, 48 hours, 72 hours, or 96 hours. The amount of time culturing the cells at the final temperature of the temperature shift can also include any amount of time between the times disclosed, as well as more and less time than the maximum and minimum amounts of time disclosed.

[0055] The present disclosure encompasses collecting the VLPs released by the cells to the media using a combination of syringes and filters, wherein the filters include those with pores of 0.45 microns or larger. The present disclosure encompasses concentrating the collected VLPs using Lenti-X™, using standard protocols available, using for example 9 mL VLP + 3 mL Lenti-X™. In addition, the present disclosure encompasses using ultracentrifugation to concentrate the VLPs, with, for example, 100,000 g for 2 hours at 4° C, with 20% sucrose weight by volume as a cushion. The present disclosure encompasses using the concentrated VLP solution for downstream applications or storing the VLP solution at 4° C.

[0056] The present disclosure includes embodiments that generate VLPs for the purpose of generating non-targeted gene editing with, for example, Cas9 WT protein or any other suitable gene editing technology. The efficiency of the non-targeted gene edits can be quantified with tools like Inference of CRISPR Edits (“ICE”), Tracking of Indels by Decomposition (“TIDE”), Tracking of Insertion, Deletions and Recombination events (“TIDER”), or others.

[0057] The present disclosure includes embodiments wherein the VLPs are generated for the purpose of producing targeted gene editing. For such embodiments, the vectors used to generate the VLPs may differ from those disclosed above. For example, instead of a non-specific sgRNA vector as the guide sequence for gene editing, the vector guide sequence could include a guidesequence ranging from 15 nucleotides to 30 nucleotides that target a specific region of the DNA meant to be targeted and or edited.

[0058] The present disclosure includes embodiments wherein the VLPs are generated in such a way that they target specific cell types by, for example, replacing the VSV-G vector described above with another vector that synthetizes a different VLP envelope that targets a specific cell type, such as rabies virus envelop (“RV-G”) that targets neural cells, or a viral glycoprotein bearing a cell-specific ligand or single chain antibodies.

[0059] The present disclosure includes embodiments wherein the VLPs are generated to function as a reporter system or visualization or imaging system for target cells. Such embodiments would include the VLPs encapsulating green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, or other similar naturally occurring or altered fluorescent proteins. In another example, the protein encapsulated by the VLP is luciferase or a similar protein that allows measurements of light emitted by the protein acting on a particular target molecule.

[0060] The present disclosure includes embodiments wherein the VLPs are used to suppress transcription of a target sequence by, for example replacing the Cas9 vector disclosed with a catalytically inactive Cas9 protein (e.g., dCas9) and providing a corresponding guide sequence targeting the desired DNA sequence, such that the Cas9 protein blocks the transcription of corresponding RNA molecules, thus suppressing the resulting protein mRNA alone and, where applicable, the corresponding protein.

[0061] The present disclosure includes embodiments wherein the VLPs are used to activate transcription of a target sequence by, for example, replacing the disclosed vectors coding for the Cas9 protein and the vector containing the guide sequence and replacing it with a vector containing dCas9 fused with an activation domain that recruits transcription activation proteins (e.g., VP64 or others alone or in combination) and a corresponding guide sequence that targets,for example, the promoter or enhancer region of the desired gene, to activate transcription of the desired gene.

[0062] The present disclosure includes embodiments wherein the VLPs are used to effectuate prime editing. Prime editing includes gene editing that introduces base to base conversions as well as small insertions or deletions without creating double-strand breaks in the process. The embodiments supporting the use of VLPs for prime editing include, for example, replacing the vector coding for the Cas9 protein disclosed above with a vector that codes for Cas9 nickase fused with a reverse transcriptase. Similarly, the embodiments supporting the use of VLPs for prime editing include replacing the guide sequence vector disclosed above with another guide sequence that is longer, for example, a guide sequence that is more than 90 or more than 100 nucleotides in length.

[0063] The present disclosure includes embodiments wherein the VLPs are used to effectuate base editing. Base editing includes gene editing that introduces precise changes to nucleotides without creating double-strand breaks. The embodiments supporting the use of VLPs for base editing include, for example, replacing the vector coding for the Cas9 protein discussed above with a vector that codes for an inactive Cas9 enzyme (e.g., dCas9) fused with a base modifying enzyme. Suitable base modifying enzymes include cytosine base editors (e.g., cytidine deaminase) and adenine base editors (e.g., adenine deaminase).

[0064] The present disclosure includes embodiments wherein the VLPs are used to reprogram cells by, for example, replacing the vector coding for the Cas9 protein described above with vectors coding for the Yamanaka factors (e.g., Oct3 / 4, Sox2, Klf4, c-Myc) or other combination of proteins or molecules that help induce pluripotency, multipotency, unipotency on target cells, or that can help differentiate a target cell into another cell type such as neurons and skeletal muscle cells.

[0065] In one embodiment, the cells can be passaged up to 19 times prior to electroporation.

[0066] In one embodiment, the cells are grown to a level between 60%-100% confluent prior to electroporation.

[0067] The present disclosure includes embodiments wherein the VLPs are produced for the purpose of inserting a target molecule into a target cell, by replacing for example the Gag vector or vectors described above with another Gag vector fused to the genetic sequence of the protein of interest.

[0068] In one embodiment, cells are harvested on day 1 or 2 following electroporation.

[0069] In one embodiment, cells are harvested between 20 hours to 50 hours following electroporation.

[0070] It is understood that the disclosed method can be used to electroplate up to 1 x 1011cells.

[0071] Figure 1 shows electroporation enables high transfection efficiency and high production yields of VLPs harvested from cells in suspension. VLP production was tested by using LV MAX™ from ThermoFisher and the protocol described in example 1. Figure 1A shows three different MaxCyte electroporation protocols using HEK293 cells with the VLP plasmids as described above to produce VLPs containing CRISPR-ribonucleoprotein (“RNP”). The target locus was the HEK3 gene in target HEK293T cells. In all electroporation protocols tested, VLP production reached a peak of activity with only 0.5 pL of concentrated VLPs. With the 2x LV1 protocol, up to 60% editing activity in target cells could be obtained, suggesting that this electroporation produced higher amounts of VLPs (compared to protocols Opt 9 - DNase and Opt 2-4, which resulted in less than 10% of editing activity for each group). Figure IB shows the changes in viable cell number of the VLP producer cells 48 hours after electroporation. The results show an increase in viable cell number for all three protocols tested, said increase being up to 83% of the initial cell number when using the 2x LV 1 protocol, suggesting that electroporation of suspension cells is not impairing cell growth.

[0072] Figure 2 shows that VLP production in suspension HEK293 cells increased in an electroporation energy dependent manner. VLP production was tested using the protocol detailed in example 1 and three separate MaxCyte electroporation protocols were applied to the samples, Opt 9, Opt 2-4, and LV-lx2 each protocol applied a different amount of overall energy with Opt9 exerting the least amount of energy and LV-lx2 exerting the most.

[0073] Figure 3A illustrates that VLP activity decreased as VLPs were harvested after day 1 and that VLP activity was not detected at 0.5 pL of concentrated VLPs when harvested 3 days after electroporation in the absence of an enhancer molecule. Figure 3B illustrates that VLP producer cell numbers expanded 2-fold by day 2 and 3 -fold by day 3 and indicates that the VLP producer cell number over time is inversely correlated with VLP activity and production.

[0074] Figure 4 shows that sodium butyrate boosts VLP Production up to 10 mM concentration VLP editing activity from VLPs produced at a cell density of 1.5 x 108cells / mL for a total of 6 x io7cells per sample in 400 pL of electroporation volume. The DNA concentration used during electroporation was 300 pg / mL for a total of 120 pg in 400 pL of electroporation volume. 6 x io7cells were cultured in a 125 mL shake flask at a cell culture density of 3 x 106cells / mL. A range of sodium butyrate (0 mM, 4 mM, 6 mM, 8 mM, and 10 mM) was added to the electroporated cells 4 hours after electroporation. VLPs were harvested at day 1 and processed as described in Example 2. 40,000 adherent HEK293 cells were inoculated with a range of volumes of concentrated VLPs and the CRISPR-Cas9 editing activity was evaluated 3 days after inoculation. All sodium butyrate concentrations increased VLP activity, but 6 mM sodium butyrate concentration was the optimal concentration.

[0075] Figures 5A-D illustrate that VLP activity decreases when harvested at day 2 without sodium butyrate but sodium butyrate allows for increased VLP activity on day 1 or day 2 harvest. VLP editing activity was measured from VLPs produced using the method stated in Example 2. The DNA concentration used during electroporation was 300 pg / mL for a total of120 pg in 400 pL of electroporation volume. 6 x 107cells were cultured in a 125 mL shake flask at a cell culture density of 3 x io6cells / mL. 4mM sodium butyrate was added or not added to the electroporated cells 4 hours after electroporation. VLPs were harvested on day 1 or day 2 after electroporation and processed as described in the protocol. 40,000 adherent HEK293 cells were inoculated with a range of volumes of concentrated VLPs and the CRISPR- Cas9 editing activity was evaluated 3-5 days after inoculation. Figure 5A illustrates that when no sodium butyrate is added VLP producer cells, VLP activity from produced VLPs drops on day 2 compared with Day 1. Figure 5B illustrates that when sodium butyrate is added to VLP producer cells, the overall activity of VLPS increases compared to without sodium butyrate as indicated in Figure 3. However, at Day 2 harvest, there was an increase in VLP activity. This is the opposite of what is seen when VLPs are produced in the absence of sodium butyrate. Figure 5C illustrates that the total VLP producer cell number increases from Day 0 to Day 2 without sodium butyrate. However, although the VLP producer cell number increases from Day 0 to Day 1, it drops from Day 2 to Day 3. The difference in cell number affects VLP production and there is a more significant impact when the cell number increases on Day 2 in the absence of sodium butyrate. Controlling the VLP producer cell number increase during the VLP production process is a critical factor in getting higher VLP activity. Figure 5D is similar to Figure 5C, but further illustrates that the VLP producer cell viability drops more on day 2 when sodium butyrate is added compared to without sodium butyrate treated VLP producer cells.

[0076] Figures 6A-C illustrate that suspension HEK293 cells can be cultured up to 12 x 106cells / mL after electroporation to increase VLP activity. VLP editing activity from VLPs was measured using VLPs produced from the protocol detailed in Example 2. The figures illustrate that increasing the cell culture density after electroporation increases VLP activity for all cell culture densities tested up to 12 x io6cells / mL. Figure 6A shows the increase of VLP activitywith the addition of sodium butyrate. Figure 6B shows that increasing the cell culture density up to 12 x 106cells / mL increases VLP activity.

[0077] Figures 7A-C illustrate that VLP Production is higher than chemical transfection on day 1 harvest with and without chemical enhancer VLP editing activity from VLPs produced pursuant to the protocol disclosed by Example 2. The effect of sodium butyrate was evaluated. For samples with sodium butyrate, 4 mM sodium butyrate was added to the electroporated cells 4 hours after electroporation. LV MAX transfected cells were transfected according to the manufacturer’s instructions and described in the protocol section. The effect of the chemical enhancer from the LV MAX transfection kit was evaluated and added 5 hours to 6 hours after transfection. VLPs were harvested on day 1 after electroporation or chemical transfection and processed as described in the protocol. 40,000 adherent HEK293 cells were inoculated with a range of volumes of concentrated VLPs and the CRISPR-Cas9 editing activity was evaluated 3 days after inoculation. Figure 7A shows that VLP activity increased when a chemical enhancer from the LV Max transfection kit was added to chemical transfected VLP producer cells. Figure 7B shows that VLP activity increased when sodium butyrate was added to electroporated VLP producer cells. The activity of VLPs produced from electroporated cells was higher than chemical transfection in both the absence and presence of the sodium butyrate enhancer. Figure 7C illustrates that the gag p30 levels, which indicate VLP particle number, increased with either chemical enhancer or sodium butyrate was added in chemical transfection and electroporation generated VLPs, respectively. The p30 levels were higher in electroporation generated VLPs compared with chemical transfection.

[0078] Figures 8A-F illustrate that electroporation is superior to chemical transfection kinetically and in total VLP activity. Figures 8A and 8B measured VLP editing activity from VLPs produced in the protocol described in Example 2. Chemical transfection with the LV MAX transfection kit was performed using 1.2 x 108cells according to the manufacturer’sinstructions. 6 mM of sodium butyrate was added to the electroporated cells 4 hours after electroporation and LV MAX enhancer was added 5 hours to 6 hours after transfection. VLP activity was measured by using Sanger sequencing via Synthego ICE analysis and showed that electroporated VLPs showed higher activity than chemical transfection on day 1 and day 2. Importantly, the kinetics of VLP activity were faster with electroporation compared with that of chemical transfection indicating that electroporation generates more efficient VLPs faster. Figures 8C and 8D measured VLP editing activity from VLPs produced using the protocol described in Example 2 with and without sodium butyrate added to show that both were higher than that of chemical transfection. Although 1 * 109cells were electroporated, 1.2 x 108cells were cultured in a 125 mL shake flask to produce the VLPs at the same culture scale as chemical transfection to remove any influence of VLP production by the culture vessel. Chemical transfection with the LV MAX transfection kit was performed using 1.2 x 108cells according to the manufacturer’s instructions. 6 mM of sodium butyrate was not added or added to the electroporated cells 4 hours after electroporation and LV MAX enhancer was added 5 hours to 6 hours after transfection. VLP activity from electroporated VLPs showed higher activity than chemical transfection on day 1 and day 2 in the absence or presence of enhancer. Figures 8E and 8F measure VLP activity from the same VLP samples in Figures 8C and 8D, the p30 gag levels were measured by ELISA. Gag levels indicate the amount of secreted particle numbers. Similar to VLP activity results, the levels of p30 measured were higher for electroporation generated VLPs on Day 1 and Day 2 compared with chemical transfection.

[0079] Figure 9 illustrates that electroporation enables packaging of ABE-RNPs into VLPs to target HEK3 loci and induce high rates of base substitutions. VLPs were produced in suspension HEK293 cells with sodium butyrate pursuant to the disclosed method. 5 pL of VLPs produced without sodium butyrate and titrated onto adherent HEK293T cells could induce 42% base substitution at Ae (A:T to G:C). The addition of sodium butyrate to productionmedia improved editing, up to 71% at Ae. Base editing at As occurred at 48% and 75%, without and with sodium butyrate, respectively.

[0080] Figures 10A and 10B illustrate that a temperature shift after electroporation improved ABE8e-VLP editing activity and capsid titer. Suspension HEK293 cells were transfected via electroporation. 4 hours after electroporation, 6 mM of sodium butyrate was added to the cells and the cells were shifted from 37° C to 32° C. Figure 10A illustrates shifting producer cells to 32° C 4 hours after electroporation improved ABE8e-BLP editing activity at the HEK3 locus. Figure 10B illustrates p30 levels measured by ELISA and shows the temperature shift 4 hours after electroporation improved capsid titer.

[0081] Figures 11A and 11B illustrate that primary immune cells are efficiently edited with VLPs produced in suspension HEK293 cells using electroporation. T cell viability and B2M expression were measured using the protocol detailed in Example 3 to measure efficiency. Figure 11A illustrates that VLPs produced following electroporation do not negatively affect T cell viability when harvested 5 days post-transduction. Figure 11B illustrates that VLPs produced following electroporation disrupt B2M expression in T cells more efficiently compared to VLPs produced following chemical transfection.Examples

[0082] What follows are a series of examples that illustrate the present invention in a nonlimiting matter. Accordingly, the following detailed description is not limited to the disclosed embodiments and examples. Instead, the proper scope is defined by the appended claims.

[0083] Example 1: A method of improving virus-like particle production (“VLP”) per cell: 2.5 x 106HEK293 cells / mL were seeded in suspension using standard cell culture conditions a day before electroporation in a non-baffled shake flask. On the day of electroporation, without discarding the old media, the cells were diluted with fresh media to 2.5 x io6cells / mL. Viability of the cells on the day of electroporation was above 90% but those skilled in the art understandthat viability can range from 70%-100% confluency. 55 mL of the cell culture was centrifuged at 250 x g for 10 minutes at room temperature. The media was aspirated, and the cell pellet was resuspended with MaxCyte buffer (MaxCyte EP Buffer and Opti-MEM) at a cell density of 1.5 x io8cells / mL. 400 pL of the resuspended cells were pipetted out and placed in a separate sterile tube and DNA was mixed in at a concentration of 300 pg / mL (a total of 120 pg of DNA used for the 400 pL of resuspended cells in electroporation buffer). The amount of each plasmid used is listed in Table 1. 400 pL of the cells (1.5 x io7cells) were mixed with DNA onto an optimization chamber (“OC”)-100 processing assembly. The cells were then electroporated using a MaxCyte ExPERT STx™. Following electroporation, the cells were transferred from the processing assembly to a culture vessel. The culture vessel was placed in a 37° C humidified incubator with 5% CO2 for 30 minutes to 40 minutes for recovery. After the recovery incubation, 20 mL of 37° C pre-warmed complete media (“LV MAX” media) was added to the cells (resulting in an approximate cell density of 3 x 106cells / mL. The cells were cultured in a 37° C humidified incubator with 5% CO2 for 48 hours. After the 48-hour incubation, the cells were counted and then centrifuged at 250 x g. The supernatant was collected and spun at 400 g for 5 minutes to remove large cell debris. The media was then filtered through a 0.45 pm syringe filter to further remove debris. Lenti-X™ concentrator (Takara) was used to precipitate the VLPs. To test the VLP activity, 0.1 pL, 0.5 pL, 1 pL, 5 pL, 10 pL, and 20 pL of concentrated VLPs were inoculated onto target cells. 40,000 HEK293 cells were used as the target cells to measure editing activity of the VLPs and seeded onto a 48-well plate.Table 1. Plasmids and Amounts of DNA Used in Experiment.

[0084] Example 2: To test VLP production in suspension HEK293 cells with the addition of sodium butyrate, suspension HEK293 cells were grown in suspension to a density of 2.5 x 106cells / mL. On the day of electroporation, the culture cell density was increased to 4.6 x io6cells / mL. 55 mL of the cells (1.29 x 109cells) were then centrifuged at 350 x g for 10 minutes at room temperature, the supernatant was decanted, and the cell pellet was resuspended with a mix of 300 pL of MaxCyte electroporation buffer (MaxCyte EP Buffer and Opti-MEM) with 4 plasmids (VSV-G, MMLVgag-pro, MMLVgag-Cas9, and HEK3 sgRNA) with a DNA concentration of 300 pg / mL of total DNA (12.9 pg / mL VSV-G, 108.9 pg / mL MMLVgag-pro, 36.3 pg / mL, 36.3 pg / mL MMLVgag-Cas9, and 141.9 pg / mL HEK3 sgRNA). The cell density of the resuspended cells was 1.5 x 108cells / mL. 400 pL of the cells were transferred to 4 x OC-400 processing assemblies and electroporated using a MaxCyte Gen2 STX™. After electroporation, cells were transferred from the processing assembly into a 125 mL shake flask (6 x 107cells) which was then recovered for 35 minutes in a humidified 37° C incubator at 5% CO2. After recovery, 20mL of LV MAX ™ Media and 6mM sodium butyrate were added and allowed to culture for 24 hours or 48 hours. The cell mix in the flask was then centrifuged for 10 minutes at 250 x g. The supernatant was removed and centrifuged to remove cell debris at for 5 minutes at 400 x g. The supernatant of the second spin was then filtered using a syringe filter (0.45 pm) and concentrated by adding 6mL of Lenti X concentrator to 20 mL of the filtered supernatant. The mixture was then inverted several times and incubated at 4° C overnight. The following day, the remaining mixture was centrifuged at 1500 x g for 45 minutes to concentrate the VLPs. The pelleted VLPs were resuspended in 100 pL of PBS at 10° C. To measure VLP activity, 0.1 pl or 0.5 pl of concentrated VLPs were inoculated onto40,000 adherent HEK293 cells. PCR amplification and Sanger sequencing was then performed to analyze the quantification of insertion and deletion of the Synthego ICE.

[0085] Example 3: To test the ability to target primary immune cells for gene editing, suspension HEK293 cells were transfected by electroporation or with chemical transfection. 4 hours after electroporation, 6 mM of sodium butyrate was added to the electroporated cells. The VLPs were harvested on day 1 or day 2 after electroporation. T cells were isolated from PBMCs with EasySep™ Human CD8+ T cell isolation kit and activated with CD3 / CD28 Dynabeads™ (cells:beads, 1 :2) for 48 hours. 5 * 104cells were seeded in RetroNectin®-coated 96-well plate in 200 pL media supplemented with IL-2, IL-7, and IL- 15 and transduced with concentrated VLPs. 48 hours later, cells were expanded into 1 mL complete media, transferred to 48-well plate, and re-transduced with the same volume concentrated VLP. On day 5, cells were harvested and analyzed for cell viability (AO / PI) and B2M surface expression (FACS). The results of Example 3 are illustrated in FIG 11 A and FIG 1 IB.

[0086] Example 4: The methods for VLP production described herein using electroporation were evaluated against chemical transfection to assess the efficiency of VLP production. HEK293 suspension cells (1.2 x 108cells) were prepared and electroporated as described herein (or transfected using LV-MAX™ according to the manufacturer’s instructions). Experimental groups and culturing conditions are described below and in Table 2. Following electroporation / transfection, cells were transferred in 20 mL (for electroporation) or 30 mL (for chemical transfection) production volumes to a 125 mL shake flask. For cells that were electroporated, following electroporation, the cells recovered at 37°C for 20 minutes (static), and then production media was added to the flask, and the cells were cultured for four hours with shaking. After four hours, 6 mM NaB (1 : 165), an enhancer, was added and the cells were cultured for another 20 hours. The VLPs were then harvested from the supernatant and concentrated into a 100 pL volume (200X concentration). For chemically transfected producercells, the cells, plasmids, and transfection reagents were all combined in the 125 mL shake flask and the flask was cultured at 37°C with shaking. After five hours, the enhancer (LV MAX™ kit enhancer) was added at a 1 :25 ratio, and the cells were cultured for another 43 hours. The VLPs were then harvested from the supernatant and concentrated into a 100 pL volume (300X concentration).Table 2. Experimental groups and conditions for Example 2.

[0087] Following electroporation, producer cell viability remained largely unchanged from Day 0 to Day 1 when VLPs were harvested, whereas viability of producer cells that were chemically transfected decreased by more than 20% (FIG 12A). Producer cells that were electroporated also maintained an increased cell density at Day 1 relative to Day 0, indicating that cells were actively expanding (FIGs. 12B-12C).

[0088] Once VLPs were collected and concentrated, HEK293T cells were cultured with VLPs for 3 days to assess the ECso of the VLPs. Briefly, 40,000 HEK293T cells were seeded in a tissue-culture treated 48-well plates and the concentrated VLPs were added in 10-fold dilutions (ranging from 5 pL to 0.00005 pL). After three days, cells were harvested, stained with a mouse anti-B2M APC / Fire 750 antibody, and fixed with BD Fix / Perm Medium A. Cells were then analyzed by flow cytometry on a BD CytoFLEX-S instrument and raw data was analyzed using FlowJo. Flow sorting for HEK293T cells that were B2M negative (i.e., cells where the B2M gene had been knocked out due to delivery of the cargo) at three days post-transduction revealed that the ECso of VLPs produced by the electroporated cells was similar (approximately2.17x higher) to that of VLPs produced by chemically transfected producer cells (FIG 13 A and Table 3 A). Further, VLPs produced by electroporated cells exhibited an ECso that was similar (2.42x higher) to that of VLPs produced by chemically transfected producer cells when assessing the rate of indel formation in cells (FIG. 13B and Table 3B).Table 3A, Knockout of B2M by VLPsTable 3B. Indel formation by VLPs

[0089] To assess the knockdown of B2M in activated T cells, T cells were isolated fromPBMCs using commercially available kits (Akadeum and Stem Cell Technologies) and isolated T cells were activated with CD3 / CD28 Dynabeads™ for 48 hours. Cells were seeded into 96-well RetroNectin®-coated (10 pg / cm2) plates (5* 104cells in 200 pL) and transduced with 2.5 pL of the VLP for three days. The cells were then expanded in 1 mL into 48-well plates, and re-transduced for an additional two days. Five days after transduction of activated T cells, the knockout of B2M in activated T cells was assessed by flow cytometry (FIGs. 14A and 14B). VLP produced by producer cells that were electroporated and cultured with an enhancer (EP2) exhibited editing of activated T cells that was approximately two times as efficient as VLPs produced by producer cells that had been chemically transfected.

[0090] The electroporation-based methods for VLP production described herein proved to be an effective and efficient alternative to VLP production via chemical transfection, shortening the production timeline by one day. Electroporated producer cells also produced VLPs with ahigher “full” titer relative to VLPs produced by chemically transfected producer cells (1.45 x 1011compared to 3.67 x 1011; FIG. 15A-15B), exhibited a comparable size (FIG. 15C), and had undetectable aggregation (FIG. 15D).

[0091] The foregoing description is presented for purposes of illustration. It is not exhaustive and is not limited to precise forms or embodiments disclosed. Modifications and adaptations of the embodiments will be apparent from consideration of the specification and practice of the disclosed embodiments. While certain components have been described as being coupled to one another, such components may be integrated with one another or distributed in any suitable fashion.

[0092] Moreover, while illustrative embodiments have been described herein, the scope includes any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., of aspects across various embodiments), adaptations and / or alterations based on the present disclosure. The elements in the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application, which examples are to be construed as nonexclusive. Further, the steps of the disclosed methods can be modified in any manner, including reordering steps and / or inserting or deleting steps.

[0093] The features and advantages of this disclosure are apparent from this detailed specification, and thus, it is intended that the appended claims cover all systems and methods falling within the true spirit and scope of the disclosure. As used herein, the indefinite articles “a” and “an” mean “one or more.” Similarly, the use of a plural term does not necessarily denote a plurality unless it is unambiguous in the given context. Words such as “and” or “or” mean “and / or” unless specifically directed otherwise. Further, since numerous modifications and variations will readily occur from studying the present disclosure, it is not desired to limit the disclosure to the exact construction and operation illustrated and described, andaccordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the disclosure.

[0094] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numeric values within that range. For example, description of a range such as from 1 to 6 should be considered to include subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and so forth, as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0095] Other embodiments will be apparent from consideration of the specification and practice of the embodiments disclosed herein. It is intended that the specification and examples be considered as example only, with a true scope and spirit of the disclosed embodiments being indicated by the following claims.

Claims

ClaimsWhat is claimed is:

1. A method of producing virus-like particle production using electroporation, comprising: cultivating a population of cells in a log growth phase in a cell culture vessel using media; cultivating the population of cells to a desired level of confluency; removing the media from the cell culture vessel and adding a transfection buffer; adding to the cell culture vessel at least one vector in a desired concentration and a desired amount; transfecting the cell population via electroporation; culturing the cell population at a desired temperature for a desired period of time; and seeding cells located in the cell culture vessel to a desired density.

2. The method of claim 1, further comprising adding an endonuclease to the cell culture vessel prior to seeding.

3. The method of claim 1, further comprising adding a compound that increases transcription efficiency at a desired concentration and amount to the cell culture vessel after the seeding.

4. The method of claim 1, further comprising subjecting the cell culture to a temperature shift for a desired period of time.

5. The method of claim 1, wherein the population of cells is comprised of the cell line HEK293 in suspension.

6. The method of claim 1, wherein the population of cells is derived from the cell line HEK293 in suspension.

7. The method of claim 1, wherein the media comprises cell culture media supplemented with serum.

8. The method of claim 1, wherein the cell culture vessel comprises a non-baffled shake flask, a bioreactor, or another vessel.

9. The method of claim 8, wherein the cell culture vessel comprises a non-baffled shake flask having a volume ranging from 100 mL to 2000 mL.

10. The method of claim 1, wherein the desired level of confluence comprises at least 3 x 106cells / mL.

11. The method of claim 1, wherein the transfection buffer is electroporation buffer.

12. The method of claim 1, wherein the desired final concentration of the at least one vector ranges from 50 mg / mL to 400 pg / mL.

13. The method of claim 1, wherein the desired amount of the at least one vector ranges from 0.1 pg to 300 pg.

14. The method of claim 1, wherein the desired amount of the at least one vector ranges from 300 pg to 1000 pg.

15. The method of claim 1, comprising adding multiple desired vectors to the cell culture vessel, which are not identical.

16. The method of claim 15, wherein the amount of each vector added is not identical.

17. The method of claim 15, wherein the desired vectors added to the cell culture comprise combinations of identical and non-identical vectors.

18. The method of claim 1, wherein the at least one vector added to the cell culture vessel ranges from about 1 to 10.

19. The method of claim 1, wherein transfecting the cell population is performed using an appropriate device that includes one or more electroporation machines.

20. The method of claim 1, wherein the desired temperature ranges from about 32° C to 37° C.

21. The method of claim 1, wherein the seeding density after transfection comprises an amount equal to or less than 12 x 106cells / mL.

22. The method of claim 3, wherein the compound that increases transcription efficiency comprises histone deacetylase (HDACs) inhibitors selected from sodium butyrate and valproic acid sodium butyrate.

23. The method of claim 22, wherein sodium butyrate is found in a concentration ranging from 0.1 mM to 10 mM.

24. The method of claim 3, wherein the compound that increases transcription efficiency comprises caffeine.

25. The method of claim 24, wherein the caffeine is found in a concentration ranging from 0.1 mM to 10 mM.

26. The method of claim 3, wherein the compound that increases transcription efficiency comprises a combination of sodium butyrate and caffeine.

27. The method of claim 4, wherein the temperature shift comprises a reduction in temperature of about 10° C.

28. The method of claim 4, wherein the temperature shift comprises a reduction in temperature from about 37° C to about 28° C.

29. The method of claim 4, wherein the temperature shift comprises a reduction from a higher temperature to a lower temperature.

30. The method of claim 4, wherein the temperature shift comprises a reduction in temperature from about 37° C to about 33° C.

31. The method of claim 4, wherein the desired period of time for a temperature shift comprises about 18 hours to 72 hours.

32. The method of claim 2, wherein the endonuclease is DNase.

33. The method of claim 1, wherein the cells are passaged up to 19 times prior to electroporation.

Images