Methods for improving genetic engineering in adoptive cell therapy

Abstract

The present disclosure provides methods for electroporating cells, particularly cells useful in cell-based therapies, that increase cell viability, cell proliferation, and gene transfer efficiency, and that utilize appropriate post-electroporation adjuvants to improve the genetic manipulation of various cells.

Description

[Technical Field]

[0001] The present disclosure provides methods for electroporating cells, particularly cells useful in cell-based therapies, that increase cell viability, cell proliferation, and gene transfer efficiency, and that utilize appropriate post-electroporation adjuvants to improve the genetic manipulation of various cells. [Background technology]

[0002] Genetic engineering is an essential yet challenging role in creating safe and effective cell therapies. A common approach to genetically modifying cells involves using electroporation (EP) to create transient pores in the cell membrane and introduce mRNA, DNA, endonucleases, and / or transposon systems into cells. While electroporation can achieve high gene editing and transduction efficiencies, it often results in high cell death and a significant decrease in cell viability.

[0003] What is needed are methods to improve cell resilience and viability, as well as gene transfer efficiency, to improve the adoption and success of cell-based therapies. The present invention meets these needs. Summary of the Invention

[0004] In embodiments, provided herein are methods of electroporating cells, comprising suspending cells in an electroporation buffer to form a cell suspension, subjecting the cells to one or more electroporation pulses, and then adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0005] In a further embodiment, provided herein is a method for introducing a genetic construct into cells, comprising suspending the cells in an electroporation buffer to form a cell suspension, introducing the genetic construct into the cell suspension, subjecting the cells to one or more electroporation pulses, and adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0006] Also provided herein is a composition comprising lipid-enriched human AB serum, which composition improves cell viability of an electroporated cell population. [Brief explanation of the drawings]

[0007] [Figure 1] Cell viability 24 hours after electroporation is shown. [Figure 2] Cell numbers 24 hours after electroporation are shown. [Figure 3] Transgene expression 4 days after electroporation is shown. DETAILED DESCRIPTION OF THE INVENTION

[0008] The use of the terms "a" or "an" in the claims and / or specification when used in conjunction with the term "comprising" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0009] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being used to determine the value. Typically, the term is meant to encompass a variability of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0010] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0011] As used in this specification and claim(s), the terms "comprising" (and any of the comprising forms such as "comprise" and "comprises"), "having" (and any of the having forms such as "have" and "has"), "including" (and any of the including forms such as "includes" and "include"), or "containing" (and any of the containing forms such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0012] Electroporation is a widely used method for permeabilizing cell membranes by generating transient pores in the membrane using electrical stimulation. Applications of electroporation include the introduction of various gene constructs, including DNA, RNA, and siRNA, as well as peptides, proteins, antibodies, drugs, and other substances into a wide variety of cells.

[0013] During electroporation, cells are suspended in a buffer or medium. The cell suspension is then placed in a cuvette, chamber, cartridge, or other device, and an electrical discharge is applied to the cell suspension, and the cells within the suspension, to induce a permeabilization effect. Various parameters for performing electroporation, including the strength of the electrical discharge, the length and number of pulses of the discharge, and the waiting period between discharges, are well known to those skilled in the art.

[0014] The ability to effectively deliver target substances into cells while maintaining cell viability and proliferation is a challenge in the field of electroporation. The electroporation process is typically toxic to cells. First, excessively high electric field strength can irreversibly damage cell membranes. Second, while electrically induced membrane pores allow the uptake of target substances into cells, these pores can also allow the escape of cellular contents or the inflow of other unintended substances, which can adversely affect cell viability. Third, heat generated by the electric current can damage cells. Finally, electrochemically generated toxic substances, such as free radicals, gases, and metal ions near the electrodes, can be harmful to cells.

[0015] As described herein, it has been found that the use of post-electroporation adjuvants can significantly improve cell recovery and survival rates while maintaining or even improving the delivery efficacy of targeted substances.

[0016] In embodiments, provided herein are methods of electroporating cells, comprising suspending cells in an electroporation buffer to form a cell suspension, subjecting the cells to one or more electroporation pulses, and then adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0017] As used herein, "electroporating" or "electroporation" refers to the act or process of introducing a target substance into a cell using one or more pulses of electricity to briefly open pores in the cell membrane. As described herein, a population of cells, preferably of the same type and / or derived from the same source, are suspended in an electroporation buffer. Various electroporation buffers are known to those skilled in the art and suitably contain a base buffer system (e.g., HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), sodium phosphate, phosphate-buffered saline (PBS), etc.), as well as sugars (e.g., sucrose, trehalose), and various salts such as MgCl, KCl, MgSO, or a MgCl / KCl mixture. Other additives may also be included.

[0018] The cell suspension, and thus the cells themselves, are subjected to one or more electroporation pulses. The intensity, duration, and timing of the pulses, as well as the interval between pulses, can be determined by one of ordinary skill in the art and are generally adjusted depending on the type of cell being electroporated, the buffer used, the target substance being introduced, and other factors. Generally, small cells (e.g., 10-25 microns in diameter) require a higher applied voltage (e.g., 500-1200 volts, depending on the temperature of the environment and the solution) for successful membrane permeation, while larger cells (e.g., 25-50 microns in diameter) require a lower applied voltage (e.g., 100-500 volts, depending on the temperature of the environment and the solution) for successful membrane permeation. The pulse duration is generally between 5 and 800 microseconds.

[0019] Following electroporation, a post-electroporation supplement is added to the cell suspension as described herein, hi embodiments, the post-electroporation supplement comprises lipid-enriched human serum.

[0020] Suitably, the post-electroporation supplement is added within about 30 minutes after completion of the electroporation protocol (i.e., before 30 minutes have elapsed since completion of the electroporation protocol). More suitably, the post-electroporation supplement is added within about 25 minutes, or within about 20 minutes, or within about 15 minutes, or within about 10 minutes, or within about 5 minutes, or between about 5 and about 30 minutes, or between about 5 and about 20 minutes, or between about 5 and about 10 minutes, after completion of the electroporation protocol. In other embodiments, the post-electroporation supplement is added more than about 30 minutes after completion of the electroporation protocol, but within about 1 hour of completion of the protocol.

[0021] The methods described herein can be utilized with any cells, including mammalian cells (including human cells) and insect cells. As used herein, cell and cell population comprising a plurality of cells are used interchangeably. In preferred embodiments, the cells are cells useful for therapeutic applications in mammals, such as human cell-based therapy. Exemplary cells include T cells, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), natural killer cells (NK cells), and the like. Suitably, the source of the human cells is a patient undergoing the same therapy (autologous therapy). In other embodiments, the cells can be from a donor and used in one or more other patients (allogeneic therapy).

[0022] Various methods can be used to produce the post-electroporation adjuvant. For example, lipid-enriched human serum can be prepared by heat-inactivating human serum to concentrate lipids in the serum. For example, human serum can be heated to a temperature of about 50°C to about 80°C for about 10 minutes to about 50 minutes. Following heating, the human serum can be passed through a filter and centrifuged to collect the lipid-enriched human serum. Suitably, human serum is heated to a temperature of about 60°C to about 80°C for about 20 minutes to about 40 minutes, and more suitably, human serum is heated to a temperature of about 70°C for about 30 minutes. In an exemplary embodiment, heat-inactivated human serum is passed through a 0.2 mm filter to obtain heat-inactivated lipid-enriched human serum.

[0023] In another embodiment, human serum is passed through a column to concentrate lipids in the serum, and lipid-enriched human serum is collected. For example, human serum is passed through a silica column. In an embodiment, the column is prepared from fumed silica powder, and human serum is added to the column. The human serum is then centrifuged through the column, and the delipidated serum (the upper aqueous layer) is removed, thereby obtaining a lipid-rich human serum layer.

[0024] In an exemplary embodiment, the human serum utilized in the preparation of lipid-enriched human serum is human AB serum, i.e., human serum from an AB-positive human donor that lacks antibodies specific for both A and B blood group antigens, and is suitable for transplantation and cell therapy. Other human sera may also be used.

[0025] In a suitable embodiment, the post-electroporation supplement is added after electroporation of the cells at a ratio of about 1% to about 20% by volume of the cell suspension, more suitably about 2% to about 15%, or about 2% to about 10%, about 3% to about 7%, about 4% to about 6%, or about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by volume of the cell suspension.

[0026] As described herein, it has surprisingly been found that the addition of a post-electroporation supplement increases cell recovery and improves cell viability following electroporation.

[0027] For example, in embodiments, the viability of a cell (or population of cells) receiving a post-electroporation supplement is at least about 10% higher after electroporation than cells that do not receive the post-electroporation supplement. In embodiments, the viability of cells receiving a post-electroporation supplement is at least about 20% higher, at least about 30% higher, at least about 40% higher, or about 5% to about 40% higher, about 10% to about 30% higher, about 20% to about 40% higher, about 20% to about 30% higher, about 30% to about 40% higher, or about 20% higher, about 30% higher, or about 40% higher than cells that do not receive the post-electroporation supplement.

[0028] The methods described herein have been found to surprisingly increase the number of viable cells recovered after electroporation compared to cells that did not receive post-electroporation supplements, with results nearly comparable to the number of viable cells recovered in the absence of electroporation. In embodiments, the number of surviving cells may be at least about 1.1 times or more, or at least about 1.2 times or more, at least about 1.3 times or more, at least about 1.4 times or more, at least about 1.5 times or more, at least about 1.6 times or more, at least about 1.7 times or more, at least about 1.8 times or more, at least about 1.9 times or more, or at least about 2 times or more, or about 1.1 times to about 3 times, about 1.2 times to about 2 times, about 1.3 times to about 1.8 times, about 1.4 times to about 1.6 times, or about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, or about 2 times, when compared to a cell population to which a post-electroporation adjuvant is not administered after electroporation.

[0029] In a further embodiment, provided herein is a method for introducing a genetic construct into cells, comprising suspending the cells in an electroporation buffer to form a cell suspension, introducing the genetic construct into the cell suspension, subjecting the cells to one or more electroporation pulses, and adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0030] In addition to increased cell recovery and viability, it was surprisingly found that cells receiving post-electroporation supplements had higher gene transfer efficiency after electroporation than cells not receiving post-electroporation supplements.

[0031] As used herein, the term "transfecting" refers to the introduction of a genetic construct into a cell, but does not require the use of a viral vector or plasmid to enter the cell. A "genetic construct" refers to an artificially designed or engineered nucleic acid. Examples of genetic constructs that can be transfected into cells include, but are not limited to, RNA, messenger RNA (mRNA), antisense RNA, small inhibitory RNA (siRNA), microRNA, DNA, and DNA contained within a viral vector or viral plasmid. In embodiments, the genetic construct can include a transposon (transposable element), including DNA transposons, retrotransposons, autonomous or non-autonomous transposons, and the like. In additional embodiments, the genetic construct can include an endonuclease, i.e., an enzyme that cleaves a polynucleotide chain, including type I, type II, and type III endonucleases.

[0032] The methods described herein can also be used to deliver other targeted substances into cells, such as proteins, peptides, amino acids, endosomes, etc.

[0033] As described herein, the viability of cells receiving post-electroporation supplements is increased, suitably at least 30% higher than cells not receiving post-electroporation supplements, hi other embodiments, the number of viable cells is at least 1.5 times higher than cells not receiving post-electroporation supplements.

[0034] In embodiments in which cells are transduced with a genetic construct, it has surprisingly been found that the transduction efficiency of cells after electroporation is at least 5% higher than cells that do not receive a post-electroporation supplement. As used herein, transduction efficiency refers to the percentage of cells in a population that express or contain the protein expressed by the delivered target substance, suitably the genetic construct, after the electroporation procedure. For example, gene transfer efficiency is at least 7% higher, at least 8% higher, at least 9% higher, at least 10% higher, at least 11% higher, at least 12% higher, at least 13% higher, at least 14% higher, at least 15% higher, at least 16% higher, at least 17% higher, at least 18% higher, at least 19% higher, at least 20% higher, or about 5% to about 25% higher, about 5% to about 20% higher, about 5% to about 15% higher, about 10% to about 15% higher, or about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% higher after electroporation compared to cells that did not receive the supplement after electroporation.

[0035] Examples of cells that can be transduced using the methods of the present disclosure are described herein and include, for example, T cells and hematopoietic stem cells, as well as MSCs, NK cells, and the like.

[0036] Exemplary post-electroporation supplements for use in gene transfer methods include the lipid-enriched human AB serum described herein.Methods for preparing such lipid-enriched human AB serum are also described herein.Appropriately, the post-electroporation supplement is added to the cell suspension at a volume ratio of about 2% to about 10%, and suitably, the volume ratio is added to the cell suspension at a volume ratio of about 3% to about 7%.

[0037] Also provided herein is a composition comprising heat-inactivated, lipid-enriched human AB serum, which improves the viability of electroporated cell populations. A method for preparing heat-inactivated, lipid-enriched human AB serum includes heating human AB serum to a temperature of about 60°C to about 80°C for about 20 to 45 minutes. Another method includes passing human AB serum through a silica column and recovering lipid-enriched human AB serum. These methods are described herein and in the Examples.

[0038] Embodiment In embodiments, in embodiment 1 herein, a method of electroporating cells is provided, comprising suspending cells in an electroporation buffer to form a cell suspension, subjecting the cells to one or more electroporation pulses, and then adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0039] Embodiment 2 includes the method of embodiment 1, wherein the viability of the cells is at least 30% higher than cells that do not receive the supplement after electroporation.

[0040] Embodiment 3 includes the method of embodiment 1 or embodiment 2, wherein the number of viable cells is at least 1.5 times greater than cells that do not receive the supplement after electroporation.

[0041] Embodiment 4 includes the method of any one of embodiments 1 to 3, wherein the cell is a T cell.

[0042] Embodiment 5 includes the method of any one of Embodiments 1 to 3, wherein the cells are hematopoietic stem cells.

[0043] Embodiment 6 includes the method of any one of embodiments 1-5, wherein the post-electroporation adjuvant comprises lipid-enriched human AB serum.

[0044] Embodiment 7 includes the method of any one of Embodiments 1 to 6, wherein the post-electroporation adjuvant is added in a proportion of about 2% to about 10% by volume of the cell suspension.

[0045] Embodiment 8 is a method of introducing a genetic construct into cells, comprising suspending the cells in an electroporation buffer to form a cell suspension, introducing the genetic construct into the cell suspension, subjecting the cells to one or more electroporation pulses, and adding a post-electroporation supplement comprising lipid-enriched human serum to the cell suspension.

[0046] Embodiment 9 includes the method of embodiment 8, wherein the viability of the cells is at least 30% higher than cells that do not receive the supplement after electroporation.

[0047] Embodiment 10 includes the method of embodiment 8 or embodiment 9, wherein the number of viable cells is at least 1.5 times greater than cells that do not receive the supplement after electroporation.

[0048] Embodiment 11 includes the method of any of embodiments 8 to 10, wherein the gene transfer efficiency of the cells is at least 15% higher than cells that do not receive the adjuvant after electroporation.

[0049] Embodiment 12 includes the method of any of embodiments 8 to 11, wherein the cell is a T cell.

[0050] Embodiment 13 includes the method of any of embodiments 8 to 11, wherein the cells are hematopoietic stem cells.

[0051] Embodiment 14 includes the method of any of embodiments 8-13, wherein the post-electroporation adjuvant comprises lipid-enriched human AB serum.

[0052] Embodiment 15 includes the method of any one of embodiments 8 to 14, wherein the genetic construct is RNA or mRNA. Embodiment 16 includes the method of embodiment 15, wherein the RNA is antisense RNA, siRNA, or microRNA.

[0053] Embodiment 17 includes the method of any one of embodiments 8 to 14, wherein the genetic construct is DNA.

[0054] Embodiment 18 includes the method of any one of embodiments 8 to 14, wherein the genetic construct is a viral vector or a viral plasmid.

[0055] Embodiment 19 includes the method of any one of embodiments 8 to 14, wherein the genetic construct comprises a transposon.

[0056] Embodiment 20 includes the method of any one of embodiments 8 to 14, wherein the genetic construct comprises an endonuclease.

[0057] Embodiment 21 includes the method of any one of Embodiments 8 to 20, wherein the post-electroporation adjuvant is added in a proportion of about 2% to about 10% by volume of the cell suspension.

[0058] Embodiment 22 is a composition comprising lipid-enriched human AB serum, wherein the composition improves cell viability of an electroporated cell population.

[0059] Embodiment 23 comprises the composition of embodiment 22, prepared by heating human AB serum to a temperature of about 60°C to about 80°C for about 20 to 45 minutes.

[0060] Embodiment 24 comprises the composition of embodiment 22, prepared by passing human AB serum through a silica column and recovering lipid-enriched human AB serum. [Example]

[0061] Example 1: Preparation of lipid-enriched human serum As described herein, two methods have been developed for the preparation of lipid-enriched human serum for use as a post-electroporation adjuvant.

[0062] material The following human AB sera were utilized in the following manner: Human AB serum (SIGMA ALDRICH®), Catalog No. H4522-100mL cGMP Human AB Serum (AKRON®), Catalog Number AR1010-0100

[0063] Additional materials include: Silica, Fumed Power (SIGMA ALDRICH®), Catalog Number S5130-25G

[0064] protocol Heat inactivation of human AB serum for the production of lipid-enriched human AB serum Lipid-enriched human AB serum was prepared by heat inactivation. The human AB serum was incubated in a water bath at 70°C for 30 minutes, then passed through a 0.2 μM sterile filter and centrifuged at 500 × g for 10 minutes. The resulting lipid-enriched serum was ready for immediate use or stored at 4°C.

[0065] Silica extraction of human AB serum for the production of lipid-enriched human AB serum 0.5 g of fumed silica powder was added to a 50 mL conical tube, followed by 10 mL of human AB serum. The tube was gently inverted and rotated intermittently (every 15–30 min) at room temperature for 2 h.

[0066] The tubes were then centrifuged at 4000 rpm for 10 minutes.

[0067] The delipidated plasma (upper aqueous layer) was then removed by gentle aspiration.

[0068] Lipid-enriched human AB serum will form a layer at the bottom of the tube and can be used immediately or stored at 4°C for up to one week before use.

[0069] Example 2: Electroporation and addition of post-electroporation adjuvants Two cryopreserved human Pan T cell populations were supplied by Charles River Laboratories Cell Supply (catalog number PB03C-3). Cryopreserved hematopoietic stem cells (HSCs) were obtained from an internal Charles River Laboratories source (isolated via CliniMACS® Prodigy; >90% CD34+).

[0070] Human T cell culture medium contained: LONZA® XVivo 15 Media (BE02-060Q) 100 IU recombinant human IL-2 (MILTENYI®, 130-097-743) Human HSC medium contained: CELLGENIX® GMP SCGM Media (20802-0500) The cells were thawed, diluted with the respective medium (10-25 mL), and centrifuged at 350 × g for 5 minutes. Next, the cells were washed twice with the respective medium and seeded into 6-well plates (FALCON®) at the following densities: T cells at 1 e 6 cells / mL, 0.5e for HSCs 6 cells / mL.

[0071] One day after thawing, cells were centrifuged and resuspended in 100 mL of supplemented P3 buffer according to the manufacturer's Nucleofection protocol (LONZA® P3 Nucleofection Kit, V4XP-3024). 10 ng of pMax green fluorescent protein (GFP) expression plasmid was added to each reaction tube.

[0072] 5-10e in 100+ / -10ml of P3 buffer (+GFP) 6 T cells or HCS were loaded into a Lonza Nucleofector cuvette, and the cells were electroporated using the protocol recommended by the Lonza Nucleofector (high-frequency voltage pulse stimulation in both cases).

[0073] T cells: Unstimulated T cell program (EO-115) HSC: Human HSC Program (X) For each cell type, as a non-transduced control, add 5 e to a non-electroporated cuvette. 6 The cells were loaded.

[0074] Immediately after nucleofection, the cells were collected into 15 mL conical tubes, and 10 mL of each medium was added to the tubes.

[0075] Then, the cells, T cells, 6 cells / mL, 0.5e for HSCs 6 The respective densities of cells / mL were replated into 6-well plates (FALCON®) in a volume of 2 mL per well.

[0076] The following additives were then added to each well of T cells and HSCs at the concentrations indicated: Lipid-enriched human AB serum, 100ul (5%) No supplement (control) 24 hours after electroporation, cells were harvested for cell density and viability (NC200). Four days after electroporation, cells were harvested and run on a BD FACS Lyric flow cytometer to confirm GFP expression.

[0077] Cell viability, cell number, and gene transfer efficiency were then calculated.

[0078] As shown in Figure 1, 24 hours after electroporation, the viability of the T cell population in the post-electroporation adjuvant group was 30–40% higher than that of cells without adjuvant. HSCs also showed a 30–40% increase in viability when comparing cells with and without adjuvant.

[0079] Figure 2 shows the viable cell counts for the mock (no electroporation), supplement-added group (group that received supplement after electroporation), and non-supplement-added group. As shown, 24 hours after electroporation, the viable cell counts of T cells and HSCs in the supplement-added group were more than 1.5-fold higher than those in the non-supplement-added group, nearly reaching the level of non-electroporated cells. Therefore, cell recovery increased from 50% to 80%-100% when supplement was added. The survival-promoting effect of supplements was not cell type specific; similar improvements in cell viability and recovery were observed for both T cells and HSCs.

[0080] Finally, Figure 3 shows the transfection efficiency for T cells and HSCs 4 days after electroporation. As previously mentioned, GFP expression (transgene expression) was greater than 15% in both cell types when comparing supplemented and unsupplemented cells, reaching greater than 20% in the supplemented HSC population. The improved cell recovery in supplemented media was also associated with up to two-fold higher transgene expression in both T cells and HSCs, due to the improved survival and proliferation of the transformed cells.

[0081] In summary, the addition of the post-electroporation adjuvants described herein to cells has been shown to result in significant and unexpected improvements in cell recovery, viability, and gene transfer efficiency following electroporation, providing an important mechanism for improving genetic engineering of adoptive cell therapies for the treatment of cancer and other diseases.

[0082] Although specific embodiments have been illustrated and described herein, it should be understood that the claims are not limited to the specific forms and configurations so described and illustrated. Although exemplary embodiments have been disclosed herein, and specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It should therefore be understood that the embodiments may be practiced otherwise than as specifically described.

[0083] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method of electroporating a cell, comprising: a. suspending the cells in an electroporation buffer to form a cell suspension; b. subjecting the cells to one or more electroporation pulses; and c) adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises lipid-enriched human serum.

2. 10. The method of claim 1, wherein the survival rate of the cells is at least 30% higher than cells that do not receive the supplement after electroporation.

3. 3. The method of claim 1 or claim 2, wherein the number of surviving cells is at least 1.5 times greater than cells that have not received the supplement after electroporation.

4. The method of any one of claims 1 to 3, wherein the cell is a T cell.

5. The method according to any one of claims 1 to 3, wherein the cells are hematopoietic stem cells.

6. The method of any one of claims 1 to 5, wherein the post-electroporation adjuvant comprises lipid-enriched human AB serum.

7. The method according to any one of claims 1 to 6, wherein the post-electroporation adjuvant is added in a proportion of about 2% to about 10% by volume of the cell suspension.

8. A method for transfecting a gene construct into a cell, comprising: a. suspending the cells in an electroporation buffer to form a cell suspension; b. introducing the genetic construct into the cell suspension; c. subjecting the cells to one or more electroporation pulses; and d. adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises lipid-enriched human serum.

9. 9. The method of claim 8, wherein the survival rate of the cells is at least 30% higher than cells that do not receive the supplement after electroporation.

10. 10. The method of claim 8 or claim 9, wherein the number of surviving cells is at least 1.5 times greater than cells that have not received the supplement after electroporation.

11. The method according to any one of claims 8 to 10, wherein the gene transfer efficiency of the cells is at least 15% higher than that of cells that have not received an adjuvant after electroporation.

12. The method of any one of claims 8 to 11, wherein the cell is a T cell.

13. The method according to any one of claims 8 to 11, wherein the cells are hematopoietic stem cells.

14. The method of any one of claims 8 to 13, wherein the post-electroporation adjuvant comprises lipid-enriched human AB serum.

15. The method according to any one of claims 8 to 14, wherein the gene construct is RNA or mRNA.

16. 16. The method of claim 15, wherein the RNA is an antisense RNA, an siRNA, or a microRNA.

17. The method according to any one of claims 8 to 14, wherein the genetic construct is DNA.

18. The method according to any one of claims 8 to 14, wherein the genetic construct is a viral vector or a viral plasmid.

19. The method of any one of claims 8 to 14, wherein the genetic construct comprises a transposon.

20. The method of any one of claims 8 to 14, wherein the genetic construct comprises an endonuclease.

21. The method according to any one of claims 8 to 20, wherein the post-electroporation adjuvant is added in a proportion of about 2% to about 10% by volume of the cell suspension.

22. A composition comprising lipid-enriched human AB serum that improves cell viability of an electroporated cell population.

23. 23. The composition of claim 22, prepared by heating human AB serum to a temperature of about 60°C to about 80°C for about 20 to 45 minutes.

24. 23. The composition of claim 22, prepared by passing human AB serum through a silica column and recovering lipid-enriched human AB serum.

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