Genetically engineered methods for improving adoptive cell therapy

Abstract

The present disclosure provides methods for electroporation of cells, particularly cells useful for cell-based therapy, that increase cell viability, cell proliferation and transfection efficiency. The methods suitably utilize post-electroporation supplements to improve genetic engineering of various cells.

Description

TECHNICAL FIELD

[0001] The present disclosure provides methods for electroporating cells, particularly cells useful for cell-based therapies, that increase cell viability, cell proliferation, and transfection efficiency. The methods suitably utilize a post-electroporation supplement to improve genetic engineering of various cells. BACKGROUND

[0002] Genetic engineering is an essential but challenging part of creating safe and effective cell therapies. A popular method for genetically modifying cells is to use electroporation (EP) to create transient pores in the cell membrane and introduce mRNA, DNA, endonucleases, and / or transposon systems into the cells. While electroporation can result in high gene editing and transfection efficiency, it often results in high cell death and a significant decrease in cell viability.

[0003] What is needed are methods to improve cell recovery and viability and transfection efficiency to increase the adoption and success of cell-based therapies. The present invention meets these needs. SUMMARY

[0004] In embodiments, provided herein is a method of electroporating a cell, the method comprising suspending the cell in an electroporation buffer to produce a cell suspension, subjecting the cell to one or more electroporation pulses, and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises lipid-rich human serum.

[0005] In further embodiments, provided herein is a method of transfecting a gene construct into a cell, the method comprising suspending the cell in an electroporation buffer to produce a cell suspension, introducing the gene construct into the cell suspension, subjecting the cell to one or more electroporation pulses, and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises lipid-rich human serum.

[0006] Also provided herein is a composition comprising lipid-rich human AB serum, wherein the composition increases cell viability of a population of electroporated cells. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 Cell viability is shown 24 hours post-electroporation.

[0008] Figure 2 Cell number is shown 24 hours post-electroporation.

[0009] Figure 3 Transgene expression is shown 4 days post-electroporation. DETAILED DESCRIPTION

[0010] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0011] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being employed to determine the value. Unless otherwise stated, the term intends to cover a range of values that are approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the value as the case can be.

[0012] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Additionally, the use of "one" or "another" to refer to an element or implementation can be understood as referring to "at least one" or "one or more" unless otherwise indicated by context.

[0013] As used in this specification and claims, the terms "comprising," "having," "including," or "containing" and any form of these terms are open-ended and do not exclude the addition of further unrecited elements or method steps.

[0014] Electroporation is a widely used method of permeabilizing cell membranes by temporarily creating membrane pores via electrical stimulation. Applications of electroporation include delivery of various genetic constructs, including DNA, RNA, and siRNA, as well as peptides, proteins, antibodies, drugs, or other substances into a variety of cells.

[0015] During electroporation, cells are suspended in a buffer or culture 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 thus to the cells within the suspension to induce the permeabilization effect. Various parameters for performing electroporation, including the strength of the electrical discharge, the length and number of discharge pulses, waiting periods between discharges, and the like, are known in the art.

[0016] The ability to effectively deliver target material into cells while still maintaining cell viability and proliferative capacity is a challenge in the field of electroporation. The electroporation process is generally toxic to cells. First, the cell membrane can be irreversibly damaged when the electric field strength is too high. Second, while the electrically induced membrane pores allow the target material to enter the cell, the pores can also allow cell contents to flow out and other unintended materials to flow in, which can negatively impact cell viability. Third, the heat generated by the electric current can damage the cell. Finally, toxic agents generated by the electrochemistry, such as free radicals, gases, and metal ions near the electrodes, can be harmful to the cell.

[0017] As described herein, it has been determined that the use of a post- electroporation supplement can significantly increase cell recovery and viability while still maintaining or even increasing the effectiveness of target material delivery.

[0018] In embodiments, provided herein are methods of electroporating cells, the methods comprising suspending the cells in an electroporation buffer to produce a cell suspension; subjecting the cells to one or more electroporation pulses; and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises human serum enriched for lipids.

[0019] As used herein, “electroporating” or “electroporation” refers to the act or process of using one or more electric pulses to transiently hole the cell membrane, thereby introducing target material into the cell. As described herein, cells, suitably a population of all identical cell types and / or from the same source, are suspended in an electroporation buffer. Various electroporation buffers are known in the art and suitably include a buffer system (e.g., HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), sodium phosphate, phosphate buffered saline (PBS), etc.) as a base, as well as sugars (e.g., sucrose, trehalose) and various salts, such as MgCl2, KCl, MgSO4, or a MgCl2 / KCl mixture. Other additives can also be included.

[0020] The cell suspension, and thus the cells themselves, are subjected to one or more electroporation pulses. The pulse strength, duration, timing, and pauses between pulses can be determined by one of skill in the art and are generally tailored to the type of cell being electroporated, the buffer used, the target material being introduced, and other factors. Generally, smaller sized cells (e.g., between 10-25 microns in diameter) require higher applied voltages (e.g., between 500-1200 volts, depending on the temperature of the environment / aqueous solution) to achieve successful membrane penetration, while larger sized cells (e.g., between 25-50 microns in diameter) require lower applied voltages (e.g., between 100 and 500 volts, depending on the temperature of the environment / aqueous solution) to achieve successful membrane penetration. The pulse duration is generally between 5 and 800 microseconds.

[0021] As described herein, a post-electroporation supplement is added to the cell suspension following electroporation. In embodiments, the post-electroporation supplement comprises human serum that is enriched for lipids.

[0022] Suitably, the post-electroporation supplement is added within about 30 minutes of the end of the electroporation protocol (i.e., before 30 minutes has elapsed since the end of the electroporation protocol). More suitably, the post-electroporation supplement is added within about 25 minutes, or within about 20 minutes, within about 15 minutes, within about 10 minutes, within about 5 minutes, or between about 5 minutes and about 30 minutes, between about 5 minutes and about 20 minutes, or between about 5 minutes and about 10 minutes of the end of the electroporation protocol. In other embodiments, the post-electroporation supplement is added after about 30 minutes of the end of the electroporation protocol, but before about 1 hour of the end of the protocol.

[0023] The methods described herein can be used for any cell, including mammalian cells (including human cells) as well as insect cells. As used herein, a cell and a population of cells comprising multiple cells are used interchangeably. In suitable embodiments, the cells are cells that can be used in therapeutic applications for mammals, such as human cell-based therapies. 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 the patient that is also being treated with the therapy (autologous therapy). In other embodiments, the cells can be from a donor and can be used for one or more other patients (allogeneic therapy).

[0024] Various methods can be used to produce post-electroporation supplements. For example, lipid-rich human serum can be prepared by heat-inactivating human serum, thereby enriching the serum with lipids. For example, the human serum can be heated to a temperature of about 50°C to about 80°C and maintained for about 10 minutes to about 50 minutes. After heating, the human serum can be centrifuged through a filter to collect the lipid-rich human serum. Suitably, the human serum is heated to a temperature of about 60°C to about 80°C and maintained for about 20 minutes to about 40 minutes, more suitably, the human serum is heated to a temperature of about 70°C and maintained for about 30 minutes. In an exemplary embodiment, the heat-inactivated human serum is passed through a 0.2 mm filter to produce heat-inactivated lipid-rich human serum.

[0025] In other embodiments, human serum is passed through a column to concentrate the lipids in the serum, and the lipid-rich human serum is collected. For example, human serum is passed through a silica column. In embodiments, a 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 defatted serum (aqueous top layer) is removed to produce a lipid-rich human serum layer.

[0026] In an exemplary embodiment, the human serum used to prepare 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, making it suitable for transplantation and cell therapy. Other human sera may also be used.

[0027] In suitable embodiments, the post-electroporation supplement is added after cell electroporation at about 1% to about 20% by volume of the cell suspension. More suitably, the post-electroporation supplement is added at 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.

[0028] As described herein, it has been surprisingly discovered that, following electroporation, the addition of a post-electroporation supplement increases cell recovery and results in greater cell viability.

[0029] For example, in embodiments, after electroporation, the viability of the cells (or cell mass) receiving the supplement after electroporation is at least about 10% higher than the cells that do not receive the supplement after electroporation. In embodiments, after electroporation, the viability of the cells receiving the supplement after electroporation is at least about 20%, at least about 30%, at least about 40%, or about 5% to about 40%, about 10% to about 30%, about 20% to about 40%, about 20% to about 30%, about 30% to about 40%, or about 20%, about 30%, or about 40%.

[0030] It was also surprisingly discovered that the methods described herein increase the number of viable cells recovered after electroporation and are comparable to the number of viable cells recovered without any electroporation, as compared to cells that did not receive a post-electroporation supplement after electroporation. In embodiments, the number of viable cells is at least about 1.1 fold, or at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, or about 1.1 fold to about 3 fold, about 1.2 fold to about 2 fold, about 1.3 fold to about 1.8 fold, about 1.4 fold to about 1.6 fold, or about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, or about 2 fold, of a population of cells that did not receive a post-electroporation supplement after electroporation, as compared to cells that did not receive a post-electroporation supplement after electroporation.

[0031] In further embodiments, provided herein is a method of transfecting a genetic construct into a cell, the method comprising suspending the cell in an electroporation buffer to produce a cell suspension, introducing the genetic construct into the cell suspension, subjecting the cell to one or more electroporation pulses, and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises human serum that is enriched for lipids.

[0032] In addition to the increase in cell recovery and viability, it was also surprisingly discovered that the transfection efficiency of cells that received a post-electroporation supplement after electroporation was greater than cells that did not receive a post-electroporation supplement after electroporation.

[0033] As used herein, the term “transfection” 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 a nucleic acid that is artificially designed or engineered. Examples of genetic constructs that can be transfected into a cell 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 also include a transposon (transposable element), including a DNA transposon, a retrotransposon, an autonomous or non-autonomous transposon, and the like. In further embodiments, the genetic construct can include an endonuclease, i.e., an enzyme that cleaves a polynucleotide strand, including Type I, Type II, and Type III endonucleases.

[0034] The methods described herein can also be used to deliver other target substances, such as proteins, peptides, amino acids, endosomes, and the like, into a cell.

[0035] As described herein, the viability of cells receiving the post-electroporation supplement is increased, suitably, the viability of the cells is at least 30% greater than cells that do not receive the post-electroporation supplement. In other embodiments, the number of viable cells is at least 1.5 times that of cells that do not receive the post-electroporation supplement.

[0036] In embodiments where the cells are transfected with a genetic construct, it is surprisingly found that the transfection efficiency of the cells is at least 5% greater than cells that do not receive the post-electroporation supplement after electroporation. Transfection efficiency, as used herein, refers to the percentage of cells within a population that express or contain the delivered target material (suitably, a protein expressed from the genetic construct) after the electroporation procedure. For example, the transfection efficiency is at least 7% greater, at least 8% greater, at least 9% greater, at least 10% greater, at least 11% greater, at least 12% greater, at least 13% greater, at least 14% greater, at least 15% greater, at least 16% greater, at least 17% greater, at least 18% greater, at least 19% greater, at least 20% greater, or from about 5% to about 25% greater, from about 5% to about 20% greater, from about 5% to about 15% greater, from about 10% to about 15% greater, or about 5% greater, about 6% greater, about 7% greater, about 8% greater, about 9% greater, about 10% greater, about 11% greater, about 12% greater, about 13% greater, about 14% greater, about 15% greater, about 16% greater, about 17% greater, about 18% greater, about 19% greater, or about 20% greater than cells that do not receive the post-electroporation supplement after electroporation.

[0037] Examples of cells that can be transfected using the disclosed methods are described herein and include, for example, T cells and hematopoietic stem cells, as well as MSCs, NK cells, and the like.

[0038] Exemplary post-electroporation supplements for use in the transfection methods include lipid- enriched human AB serum as described herein. Methods of preparing such lipid-enriched human AB serum are also described herein. Suitably, the post-electroporation supplement is added at about 2% to about 10% by volume of the cell suspension, suitably about 3% to 7% by volume.

[0039] Also provided herein is a composition comprising heat-inactivated lipid-enriched human AB serum, wherein the composition increases cell viability of a population of electroporated cells. Methods of preparing heat-inactivated lipid-enriched human AB serum include heating human AB serum to a temperature of about 60 °C to about 80 °C for about 20-45 minutes. Other methods include passing human AB serum through a silica column and recovering the lipid-enriched human AB serum. These methods are described herein and in the examples.

[0040] Embodiments

[0041] In embodiments, provided herein in embodiment 1 is a method of electroporating a cell, the method comprising suspending the cell in an electroporation buffer to produce a cell suspension, subjecting the cell to one or more electroporation pulses; and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises human serum that is enriched in lipids.

[0042] Embodiment 2 includes the method of embodiment 1, wherein the viability of the cell is at least 30% higher than a cell that did not receive the post-electroporation supplement.

[0043] Embodiment 3 includes the method of embodiment 1 or embodiment 2, wherein the number of viable cells is at least 1.5 times that of a cell that did not receive the post-electroporation supplement.

[0044] Embodiment 4 includes the method of any one of embodiments 1-3, wherein the cell is a T cell.

[0045] Embodiment 5 includes the method of any one of embodiments 1-3, wherein the cell is a hematopoietic stem cell.

[0046] Embodiment 6 includes the method of any one of embodiments 1-5, wherein the post- electroporation supplement comprises human AB serum that is enriched in lipids.

[0047] Embodiment 7 includes the method of any one of embodiments 1-6, wherein the post- electroporation supplement is added at about 2% to about 10% by volume of the cell suspension.

[0048] Embodiment 8 is a method of transfecting a gene construct into a cell, the method comprising suspending the cell in an electroporation buffer to produce a cell suspension; introducing the gene construct into the cell suspension; subjecting the cell to one or more electroporation pulses; and adding a post-electroporation supplement to the cell suspension, wherein the post-electroporation supplement comprises human serum that is enriched in lipids.

[0049] Embodiment 9 includes the method of embodiment 8, wherein the viability of the cell is at least 30% higher than a cell that did not receive the post-electroporation supplement.

[0050] Embodiment 10 includes the method of embodiment 8 or embodiment 9, wherein the number of viable cells is at least 1.5 times that of a cell that did not receive the post- electroporation supplement.

[0051] Embodiment 11 includes the method of any one of embodiments 8-10, wherein the transfection efficiency of the cell is at least 15% higher than a cell that did not receive the post-electroporation supplement.

[0052] Embodiment 12 includes the method of any one of embodiments 8 to 11, wherein the cell is a T cell.

[0053] Embodiment 13 includes the method of any one of embodiments 8 to 11, wherein the cell is a hematopoietic stem cell.

[0054] Embodiment 14 includes the method of any one of embodiments 8 to 13, wherein the post-electroporation supplement comprises lipid-rich human AB serum.

[0055] Embodiment 15 includes the method of any one of embodiments 8 to 14, wherein the genetic construct is RNA or mRNA.

[0056] Embodiment 16 includes the method of embodiment 15, wherein the RNA is antisense RNA, siRNA, or microRNA.

[0057] Embodiment 17 includes the method of any one of embodiments 8 to 14, wherein the genetic construct is DNA.

[0058] 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.

[0059] Embodiment 19 includes the method of any one of embodiments 8 to 14, wherein the genetic construct comprises a transposon.

[0060] Embodiment 20 includes the method of any one of embodiments 8 to 14, wherein the genetic construct comprises an endonuclease.

[0061] Embodiment 21 includes the method of any one of embodiments 8 to 20, wherein the post-electroporation supplement is added at about 2% to about 10% by volume of the cell suspension.

[0062] Embodiment 22 is a composition comprising lipid-rich human AB serum, wherein the composition increases cell viability of an electroporated cell population.

[0063] Embodiment 23 includes the composition of embodiment 22, prepared by heating human AB serum to a temperature of about 60°C to about 80°C for about 20-45 minutes.

[0064] Embodiment 24 includes the composition of embodiment 22, prepared by passing human AB serum through a silica column and recovering the lipid-enriched human AB serum.

[0065] Embodiments

[0066] Example 1: Preparation of lipid-enriched human serum

[0067] Two methods for preparing lipid-enriched human serum for use as a post- electroporation supplement as described herein have been developed.

[0068] Materials

[0069] The following human AB serum was used in the following methods:

[0070] Human AB Serum (SIGMA ) Catalog # H4522-100 mL

[0071] cGMP Human AB Serum Catalog # AR1010-0100

[0072] Additional materials included:

[0073] Silica, fumed powder (SIGMA ) Catalog # S5130-25G

[0074] Procedure

[0075] Heat inactivation of human AB serum to produce lipid-enriched human AB serum

[0076] Lipid-enriched human AB serum was prepared by heat inactivation. Human AB serum was incubated at 70°C for 30 minutes in a water bath and then centrifuged at 500 x g through a 0.2 uM sterile filter for 10 minutes. The resulting serum was lipid-enriched and could be used immediately or stored at 4°C.

[0077] Silica extraction of human AB serum to produce lipid-enriched human AB serum

[0078] 0.5 g of fumed silica powder was added to a 50 mL conical tube. 10 mL of human AB serum was then added to the same tube. At room temperature, the tube was gently inverted and swirled intermittently (every 15-30 minutes) for 2 hours.

[0079] The tube was then centrifuged at 4000 rpm for 10 minutes.

[0080] The delipidated plasma (top aqueous layer) was then removed by gentle suction.

[0081] Lipid-enriched human AB serum formed a layer at the bottom of the tube. The resulting serum could be used immediately or stored at 4°C for up to one week prior to use.

[0082] Example 2: Electroporation and post-electroporation supplement addition

[0083] Two cryopreserved human pan T cell populations were obtained from Charles River Laboratories Cell Supply (Cat. No. PB03C-3). Cryopreserved hematopoietic stem cells (HSCs) were obtained from Charles River Laboratories internal supply (via Prodigy isolation; >90% CD34+).

[0084] Human T cell culture medium contains:

[0085] XVivo 15 Medium (BE02-060Q)

[0086] 100IU recombinant human IL-2 ( 130-097-743)

[0087] Human HSC Medium contains:

[0088] GMP SCGM Medium (20802-0500)

[0089] Thaw the cells, dilute in the corresponding culture medium (10-25 mL), and centrifuge at 350 x g for 5 minutes. Then wash the cells twice in the corresponding culture medium and then seed into 6-well plates at the following density: Medium: 1e 6 cells / mL T cells; 0.5e 6 cells / mL HSC.

[0090] One day after thawing, the cells were transfected according to the manufacturer's nucleofection protocol ( P3 Nucleofection Kit, V4XP-3024) The cells were centrifuged and resuspended in 100 ml of supplemented P3 buffer. 10 ng of pMax green fluorescent protein (GFP) expression plasmid was added to each reaction tube.

[0091] 5-10e in 100+ / -10ml of P3 buffer (+GFP) 6 T cells or HCS were loaded into Lonza Nucleofector cuvettes. The cells were electroporated on the Lonza Nucleofector using the recommended protocol (high-frequency voltage pulse stimulation for both):

[0092] T cells: Non-simulated T cell program (EO-115)

[0093] HSC: Human HSC Program (X)

[0094] For each cell type, 5e6 Cells were loaded into a cuvette and not electroporated to serve as a non-transfected control.

[0095] Immediately after nucleofection, collect the cells into a 15 mL conical tube and add 10 mL of each corresponding culture medium to the tube.

[0096] Then, the cells were reseeded into 6-well plates at the following density: Middle: 1e for T cells 6 cells / mL and 0.5e for HSC 6 cells / mL, and the volume of both was 2 mL per well.

[0097] The following supplements were then added to the corresponding wells of T cells and HSCs at the indicated concentrations:

[0098] Lipid-rich human AB serum, 100ul (5%)

[0099] No supplement (control)

[0100] 24 hours after electroporation, cells were sampled to determine cell density and viability (NC200). 4 days after electroporation, cells were sampled and run on a BD FACS Lyric flow cytometer to examine GFP expression.

[0101] Cell viability, cell number, and transfection efficiency were then calculated.

[0102] like Figure 1 As shown, 24 hours after electroporation, the viability of the T cell population in the supplemented group was greater than 30-40% higher than that of the unsupplemented cells. HCS also showed that the viability of the supplemented cells increased by more than 30-40% relative to the unsupplemented cells.

[0103] Figure 2 Shown are the numbers of viable cells for the simulated (non-electroporated) and supplemented (received post-electroporation supplements) and unsupplemented cell groups. As shown, 24 hours after electroporation, the number of viable cells in the supplemented groups of T cells and HSCs was more than 1.5 times that of the unsupplemented cells and almost reached the level of non-electroporated cells. Therefore, when the cells were supplemented, the cell recovery rate increased from 50% to 80%-100%. The pro-survival effect of the supplements was not cell type specific, as similar improvements in cell viability and recovery were observed for both T cells and HSCs.

[0104] at last, Figure 3Transfection efficiency of T cells and HSCs is shown 4 days after transfection by electroporation. GFP expression (transgene expression) was over 15% higher for both cell types when comparing supplemented cells to non-supplemented cells as described above, and for HSCs supplemented populations, close to over 20%+. The higher cell recovery in supplemented media also correlated with up to 2-fold higher transgene expression in T cells and HSCs as transfected cells were able to survive and expand better.

[0105] In summary, these results demonstrate that supplementing cells with the post- electroporation supplement described herein can have a significant and unexpected improvement on cell recovery, viability, and transfection efficiency after electroporation. This provides an important mechanism for improving genetic engineering of adoptive cell therapies for treating cancer and other diseases.

[0106] It is understood that while certain embodiments have been illustrated and described, the claims are not limited to the precise form disclosed and that workments modifications and variations are possible therefrom. It is intended that the embodiments encompass all technical, structural, and functional equivalents that are within the scope of the appended claims. Other embodiments can be apparent to those of ordinary skill in the art from the further consideration of the specification and drawings. It is intended that the specification and examples be considered as exemplary only, with the true scope being indicated by the following claims.

[0107] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the extent allowable under the patent laws. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference is prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

Claims

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

2. The method of claim 1, wherein the cell has at least 30% greater viability than a cell that did not receive the electroporation post-supplement.

3. The method of claim 1 or claim 2, wherein the number of viable cells is at least 1.5 times greater than a cell that did not receive the electroporation post-supplement.

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

5. The method of any one of claims 1 to 3, wherein the cell is a hematopoietic stem cell.

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

7. The method of any one of claims 1 to 6, wherein the electroporation post-supplement is added at about 2% to about 10% by volume of the cell suspension.

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

9. The method of claim 8, wherein the cell has at least 30% greater viability than a cell that did not receive the electroporation post-supplement.

10. The method of claim 8 or claim 9, wherein the number of viable cells is at least 1.5 times greater than a cell that did not receive the electroporation post-supplement.

11. The method of any one of claims 8 to 10, wherein the cell has at least 15% greater transfection efficiency than a cell that did not receive the electroporation post-supplement.

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

13. The method of any one of claims 8 to 11, wherein the cell is a hematopoietic stem cell.

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

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

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

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

18. The method of any one of claims 8 to 14, wherein the genetic construct is a viral vector or 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 of any one of claims 8 to 20, wherein the electroporation post- supplement is added at about 2% to about 10% by volume of the cell suspension.

22. A composition comprising lipid-enriched human AB serum, wherein the composition increases cell viability of an electroporated cell population.

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 20-45 minutes.

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

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