Vector-free intracellular delivery by reversible permeabilization

Abstract

Compositions and methods are provided for facilitating transfection of suspension cells, such as non-adherent cells. [Solution] A method for delivering a payload across the plasma membrane of non-adherent cells is provided, comprising the steps of providing a population of non-adherent cells and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising the payload and an alcohol at a concentration greater than 5% (v / v).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 62 / 438,298, filed December 22, 2016, U.S. Provisional Patent Application No. 62 / 528,963, filed July 5, 2017, and U.S. Provisional Patent Application No. 62 / 536,831, filed July 25, 2017, each of which is expressly incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to the delivery of agents into mammalian cells. [Background technology]

[0003] 2. Background of the Invention There is a variability in cell transfection efficiency between different cell types. Transfection of suspension cells, such as non-adherent cells, has proven to be very difficult with conventional methods. Therefore, there is a need for compositions and methods to facilitate the transfection of such cells. Summary of the Invention

[0004] The present invention provides a solution to the problem of delivering payload / cargo compounds and compositions into non-adherent cells. Thus, a method of delivering a payload across the plasma membrane of non-adherent cells includes providing a population of non-adherent cells and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising a payload and an alcohol at a concentration greater than 5% (v / v). For example, the alcohol comprises ethanol, for example greater than 10% ethanol. In some examples, the aqueous solution comprises 20-30% ethanol, for example 27% ethanol.

[0005] Aqueous solutions for delivering cargo to cells include a salt, such as 12.5-500 mM potassium chloride (KCl). For example, the solution is isotonic to the cytoplasm of a mammalian cell, such as a human T cell. One example of such an isotonic delivery solution is 106 mM KCl.

[0006] The method can be used to deliver any one or more cargo molecules to adherent or non-adherent mammalian cells, and is particularly useful for delivering cargo to non-adherent cells, since it was difficult to do so before the present invention. In some examples, the non-adherent cells include peripheral blood mononuclear cells, for example, the non-adherent cells include immune cells such as T cells (T lymphocytes). The immune cells such as T cells are optionally activated by a ligand for CD3, a ligand for CD28, or a combination thereof. For example, the ligand is an antibody or antibody fragment that binds to CD3 or CD28, or both.

[0007] In this method, cargo in a delivery solution is delivered to a population of non-adherent cells that constitute a monolayer. For example, the monolayer is contacted with a spray of an aqueous delivery solution. This method delivers payload / cargo (compound or composition) into the cytoplasm of cells, where the population of cells forms a greater survival percentage compared to the delivery of payload by electroporation or nucleofection. This is a major advantage of the Soluporation system.

[0008] Any compound or composition can be delivered. For example, the payload comprises a messenger ribonucleic acid (mRNA), such as an mRNA encoding a gene editing composition. For example, the gene editing composition reduces the expression of an immune checkpoint inhibitor, such as PD-1 or PD-L1. In some examples, the mRNA encodes a chimeric antigen receptor (CAR).

[0009] In certain embodiments, a monolayer of non-adherent / suspension cells is present on a membrane filter. In some embodiments, the membrane filter is vibrated after the cell monolayer is contacted with a spray of delivery solution. The membrane filter can be vibrated or agitated before, during, and / or after spraying the delivery solution onto the cells.

[0010] The present invention also encompasses a system comprising a housing configured to receive a plate containing wells, a pressure difference application device configured to apply a pressure difference to the wells, a delivery solution application device configured to deliver atomized delivery solution to the wells, a stop solution application device configured to deliver a stop solution to the wells, and a culture medium application device configured to deliver a culture medium to the wells. The stop solution is devoid of a cell membrane permeabilizing agent, such as ethanol. An example is phosphate buffered saline or any physiologically compatible buffer solution. The system optionally further comprises an addressable well assembly configured to align the pressure difference application device adjacent to the wells for applying a pressure difference to the wells, align the delivery solution application device adjacent to the wells for delivering atomized delivery solution to the wells, align the stop solution application device adjacent to the wells for delivering the stop solution to the wells, and / or align the culture medium application device adjacent to the wells for delivering the culture medium to the wells.

[0011] The addressable well assembly can include a movable base plate configured to receive a plate including wells and move the plate in at least one direction. The addressable well assembly can include a mounting assembly configured to couple to a delivery solution application device, to a stop solution application device, and to a culture medium application device.

[0012] The delivery solution application device can include a nebulizer. The delivery solution application device can be configured to deliver between 10 and 300 microliters of delivery solution per actuation.

[0013] The system can include a temperature control system configured to control the temperature of the delivery solution and / or the temperature of the plate containing the wells.

[0014] The system can include an enclosure configured to control the environment of the plate containing the wells.

[0015] The pressure differential application device can include a nozzle assembly configured to form a seal with an opening of the well and configured to deliver steam to the well to increase or decrease pressure within the well, thereby forcing a liquid portion of the culture medium out of the well such that the layer of cells remains within the well.

[0016] The stop solution application device can include a needle emitter configured to couple to a stop solution reservoir.

[0017] The culture medium application device can include a needle emitter configured to couple to a culture medium reservoir.

[0018] The system may further comprise a controller configured to receive user input, to operate a delivery solution applicator to deliver atomized delivery solution to the cell monolayer in the well, to incubate the cell monolayer for a first incubation period after application of the delivery solution, to operate the stop solution applicator in response to expiration of the first incubation period to deliver a stop solution to the cell monolayer, and to incubate the cell monolayer for a second incubation period and in response to application of the stop solution. The controller may be further configured to repeat the operation of the delivery solution applicator, incubation for the first incubation period, operation of the stop solution applicator, and incubation for the second incubation period a predetermined number of iterations.

[0019] The system may further comprise a controller configured to operate the positive pressure system to remove supernatant from the well and generate a cell monolayer in the well.

[0020] The delivery solution application device can include a spray head and a collar surrounding a distal end of the spray head, where the collar is configured to prevent contamination between wells in a single multi-well plate and where the collar is configured to provide a gap between the plate and the collar.

[0021] The delivery solution application device can include a spray head and a film surrounding a distal end of the spray head.

[0022] The system may further include a vibration system coupled to the membrane holder and configured to vibrate the membrane.

[0023] The system can further comprise a plate in which the wells are configured to contain a population of non-adherent cells.

[0024] The delivery solution includes an isotonic aqueous solution, the aqueous solution including a payload and an alcohol at a concentration greater than 5 percent (v / v). The alcohol can include ethanol. The aqueous solution can include greater than 10% ethanol. The aqueous solution can include 20-30% ethanol. The aqueous solution can include 27% ethanol. The aqueous solution can include 12.5-500 mM KCl. The aqueous solution can include 106 mM KCl.

[0025] The non-adherent cells can comprise peripheral blood mononuclear cells. The non-adherent cells can comprise immune cells. The non-adherent cells can comprise T lymphocytes. The population of non-adherent cells can comprise a monolayer.

[0026] The payload can include messenger ribonucleic acid (mRNA). The mRNA can encode a gene editing composition. For example, the gene editing composition reduces expression of PD-1. The mRNA can encode a chimeric antigen receptor.

[0027] The system can be used to deliver a cargo compound or cargo composition to a mammalian cell.

[0028] In another aspect, a composition comprises an isotonic aqueous solution comprising KCl at a concentration of 10-500 mM and ethanol at a concentration of greater than 5 percent (v / v) for use in delivering a cargo compound or cargo composition to a mammalian cell. The KCl concentration can be 106 mM and the alcohol concentration can be 27%.

[0029] The compounds to be loaded into the MPS composition are processed or purified. For example, polynucleotides, polypeptides or other agents are purified and / or isolated. Specifically, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide or protein as used herein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound. Purity is measured by any appropriate standard method, such as by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its natural state. Purified also refers to a degree of sterility that makes it safe for administration to a human subject, e.g., lacking infectious or toxic agents. In the case of tumor antigens, the antigen can be purified or a processed preparation, such as a tumor cell lysate.

[0030] Similarly, "substantially pure" refers to a nucleotide or polypeptide that has been separated from components that naturally accompany it. Typically, nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules that naturally accompany them.

[0031] A small molecule is a compound with a mass of less than 2000 Daltons. The molecular mass of a small molecule is preferably less than 1000 Daltons, more preferably less than 600 Daltons, for example, the compound is less than 500 Daltons, less than 400 Daltons, less than 300 Daltons, less than 200 Daltons, or less than 100 Daltons.

[0032] The transitional phrase "comprising," which is synonymous with "comprising," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional elements or method steps not recited. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, as well as those that "do not materially affect the basic and novel characteristics" of the claimed invention.

[0033] [The present invention 1001] Providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising a payload and an alcohol at a concentration greater than 5% (v / v); A method for delivering said payload across the plasma membrane of a non-adherent cell comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the alcohol comprises ethanol. [The present invention 1003] The method of claim 1002, wherein the aqueous solution contains more than 10% ethanol. [The present invention 1004] The method of claim 1002, wherein the aqueous solution contains 20 to 30% ethanol. [The present invention 1005] 1001. The method of claim 1001, wherein the aqueous solution contains 27% ethanol. [The present invention 1006] 1001. The method of claim 1001, wherein the aqueous solution contains 12.5 to 500 mM KCl. [The present invention 1007] 1001. The method of claim 1001, wherein the aqueous solution contains 106 mM KCl. [The present invention 1008] 1001. The method of claim 1001, wherein said non-adherent cells comprise peripheral blood mononuclear cells. [The present invention 1009] 1001. The method of claim 1001, wherein said non-adherent cells comprise immune cells. [The present invention 1010] 1001. The method of claim 1001, wherein said non-adherent cells comprise T lymphocytes. [The present invention 1011] The method of claim 1009, wherein said immune cells are activated by a ligand for CD3, a ligand for CD28, or a combination thereof. [The present invention 1012] 1001. The method of claim 1001, wherein said population of non-adherent cells constitutes a monolayer. [The present invention 1013] The method of claim 10, wherein said monolayer is contacted with a spray of said aqueous solution. [The present invention 1014] 1001. The method of claim 1001, wherein said method delivers said payload into the cytoplasm of said cells and said population of cells comprises a greater survival percentage compared to delivery of said payload by electroporation. [The present invention 1015] 1001. The method of claim 1001, wherein the payload comprises messenger ribonucleic acid (mRNA). [The present invention 1016] The method of claim 1005, wherein the mRNA encodes a gene editing composition. [The present invention 1017] The method of claim 1016, wherein the gene editing composition reduces expression of PD-1. [The present invention 1018] The method of claim 1013, wherein said monolayer is present on a membrane filter. [The present invention 1019] The method of claim 1013, wherein the membrane filter is vibrated after contact with the spray. [The present invention 1020] The method of any one of claims 1 to 5, wherein the mRNA encodes a chimeric antigen receptor. [The present invention 1021] a housing configured to receive a plate containing wells; a pressure differential application device configured to apply a pressure differential to the well; a delivery solution application device configured to deliver atomized delivery solution to the well; a stop solution application device configured to deliver a stop solution to the well; and a culture medium application device configured to deliver culture medium to said well. A system comprising: [The present invention 1022] Addressable Well Assembly Further equipped with the addressable well assembly comprising: aligning the pressure differential application device adjacent to the well for applying the pressure differential to the well; aligning the delivery solution application device adjacent to the well for delivering the atomized delivery solution to the well; aligning the stop solution application device adjacent to the well for delivering the stop solution to the well; and / or Aligning the culture medium application device adjacent to the well for delivering the culture medium to the well. The system of the present invention 1021 is configured as follows. [The present invention 1023] The system of claim 1022, wherein the addressable well assembly comprises a movable base plate configured to receive the plate containing the wells and move the plate in at least one direction. [The present invention 1024] The system of claim 1022, wherein the addressable well assembly comprises a mounting assembly configured to couple to the delivery solution application device, to the stop solution application device, and to the culture medium application device. [The present invention 1025] The system of claim 1021, wherein the delivery solution application device comprises a nebulizer. [The present invention 1026] The system of claim 1021, wherein the delivery solution application device is configured to deliver 10 to 300 microliters of the delivery solution per actuation. [The present invention 1027] The system of the present invention 1021 further comprising a temperature control system configured to control the temperature of the delivery solution and / or the temperature of the plate containing the wells. [The present invention 1028] The system of claim 1021 further comprising an enclosure configured to control the environment of the plate containing the wells. [The present invention 1029] The system of claim 1021, wherein the pressure differential application device includes a nozzle assembly configured to form a seal with an opening of the well and configured to deliver steam to the well to increase or decrease the pressure within the well, thereby pushing a liquid portion of the culture medium out of the well such that a layer of cells remains within the well. [The present invention 1030] The system of claim 1021, wherein the stop solution application device includes a needle emitter configured to couple to a stop solution reservoir. [The present invention 1031] The system of claim 1021, wherein the culture medium application device comprises a needle emitter configured to couple to a culture medium reservoir. [The present invention 1032] Receive user input, activating the delivery solution application device to deliver the atomized delivery solution to the cell monolayer in the well; incubating the cell monolayer for a first incubation period after application of the delivery solution; activating the stop solution application device in response to expiration of the first incubation period to deliver the stop solution to the cell monolayer; and The cell monolayer is incubated for a second incubation period and in response to application of the stop solution. The system of the present invention 1021 further comprises a control device configured to: [The present invention 1033] The control device, repeating the actuation of the delivery solution applying device, the incubation for the first incubation period, the actuation of the stop solution applying device, and the incubation for the second incubation period a predetermined number of iterations. The system of the present invention 1022 further configured as follows. [The present invention 1034] Activating a positive pressure system to remove the supernatant from the well and generate a cell monolayer within the well. The system of the present invention 1021 further comprises a control device configured to: [The present invention 1035] The system of the present invention 1021, wherein the delivery solution application device includes a spray head and a collar surrounding a distal end of the spray head, wherein the collar is configured to prevent contamination between wells in a multi-well plate, and the collar is configured to provide a gap between the plate and the collar. [The present invention 1036] The system of claim 1035, wherein the delivery solution application device comprises a spray head and a film surrounding a distal end of the spray head. [The present invention 1037] The system of claim 1021, further comprising a vibration system coupled to the membrane holder and configured to vibrate the membrane. [The present invention 1038] a plate, the wells of which are configured to contain a population of non-adherent cells. The system of the present invention 1021 further comprises: [The present invention 1039] The system of the present invention 1021, wherein the delivery solution comprises an isotonic aqueous solution, the aqueous solution comprising a payload and alcohol at a concentration greater than 5 percent (v / v). [The present invention 1040] The system of claim 1038, wherein the alcohol comprises ethanol. [The present invention 1041] The system of claim 1039, wherein the aqueous solution contains more than 10% ethanol. [The present invention 1042] The system of claim 1039, wherein the aqueous solution contains 20 to 30% ethanol. [The present invention 1043] The system of claim 1038, wherein the aqueous solution contains 27% ethanol. [The present invention 1044] The system of claim 1038, wherein the aqueous solution contains 12.5 to 500 mM KCl. [The present invention 1045] The system of claim 1038, wherein the aqueous solution contains 106 mM KCl. [The present invention 1046] The system of claim 1037, wherein the non-adherent cells comprise peripheral blood mononuclear cells. [The present invention 1047] The system of claim 1037, wherein the non-adherent cells comprise immune cells. [The present invention 1048] The system of claim 1037, wherein the non-adherent cells comprise T lymphocytes. [The present invention 1049] The system of claim 1038, wherein the payload comprises messenger ribonucleic acid (mRNA). [The present invention 1050] The system of claim 1049, wherein the mRNA encodes a gene editing composition. [The present invention 1051] The system of the present invention 1050, wherein the gene editing composition reduces the expression of PD-1. [The present invention 1052] The system of claim 1049, wherein the mRNA encodes a chimeric antigen receptor. [The present invention 1053] The system of the present invention 1021 for use in delivering a cargo compound or cargo composition to a mammalian cell. [The present invention 1054] The system of the present invention, wherein the population of non-adherent cells forms a monolayer. [The present invention 1055] A composition comprising an isotonic aqueous solution comprising KCl at a concentration of 10 to 500 mM and ethanol at a concentration greater than 5 percent (v / v) for use in delivering a cargo compound or cargo composition to a mammalian cell. [The present invention 1056] 1055. The composition of claim 10, wherein said KCl concentration is 106 mM and said alcohol concentration is 27%. [The present invention 1057] Apparatuses, systems, techniques, compositions, and articles described or illustrated herein. Other features and advantages of the present invention will be apparent from the following description of its preferred embodiments and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In carrying out or testing the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. All published foreign patents and patent applications mentioned herein are incorporated herein by reference. Genbank and NCBI registrations referenced herein by accession numbers are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature mentioned herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will take precedence. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. [Brief description of the drawings]

[0034] [Figure 1] 1 is a bar graph illustrating the evaluation of feeder cells as a method of T cell activation. GFP mRNA was delivered to T cells cultured in cRPMI medium. [Diagram 2]Figure 2A is a bar graph illustrating the results of RPMI medium comparing StemCellTech reagent with two concentrations of Dynabeads. Figure 2B is a bar graph illustrating that the experiment (from Figure 2A) was repeated to include 3x the recommended concentration of StemCell tech reagent, but there was no effect. [Diagram 3] 1 is a bar graph illustrating improved uptake using Prime XV culture medium and activation with Dynabeads (3:1 beads to cells). [Figure 4] Figure 4A is a bar graph illustrating the low cell recovery when cultured in Prime XV medium. Figure 4B is an image illustrating the low cell recovery and variability of cell growth when cultured in Prime XV. Cells were activated using Dynabeads (3:1). [Diagram 5] 1 is a bar graph illustrating the results of Dynabead-activated T cells cultured in Immunocult culture medium. [Figure 6] FIG. 1 is a bar graph illustrating the optimal post-activation window when Dynabeads are used as activation reagents to deliver mRNA to T cells. [Figure 7] 1 is a bar graph illustrating Immunocult TransAct vs. Dynabead over time. [Figure 8] 1 is a bar graph illustrating the results of TexMACS versus Immunocult using TransAct as a T cell activator. [Figure 9] 1 is a bar graph illustrating the results that cells allowed to recover before activation showed higher uptake efficiency than those activated immediately after thawing. [Figure 10] 1 is a bar graph illustrating the results of culturing cells at higher cell densities prior to delivery using vector-free delivery techniques. Higher cell densities (5×106 cells / ml) correlated with higher uptake efficiency. [Figure 11] 1 is a bar graph illustrating that addition of zinc chloride to the culture medium resulted in higher uptake efficiency. [Figure 12] Bar graph showing that multiple hits in CD4+ T cells were negatively selected using CD8 microbeads. Cells were transfected with GFP mRNA by sorboration. Multiple hits improved uptake in the three donors tested. [Figure 13] Bar graph showing expression of transfected mRNA by PBMC-initiated T cells. T cells were enriched from PBMCs for 2-3 days. Cells were transfected with GFP mRNA by sorbolation. [Figure 14] The diagram in Figure 14A, the photograph in Figure 14B, and the photograph in Figure 14C represent the membrane insert. An exemplary image of a ThinCert 12-well insert (Figure 14A). An image of the insert device where the insert sits in the O-ring indicated by the arrow (Figure 14B). The device is attached to a syringe to allow for the application of a vacuum to the insert to remove media (Figure 14C). [Figure 15] Bar graph showing Solupore delivery of cargo to suspension (non-adherent) cells. Fluorescently labeled β-lactoglobulin (BLG), bovine serum albumin (BSA) or ovalbumin (OVA) were delivered to monolayers of Jurkat cells formed using the insert device. Expression levels were analyzed by flow cytometry. [Figure 16] Figure 16A is a photograph of a microtiter plate (PES filter plate), and Figures 16B and 16C are dot plots showing expression of products from transfected mRNA. Exemplary images of Acroprep Advance, Supor membrane, 96-well filter plate (Figure 16A). GFP expression levels from five random donors in five experiments 24 hours after mRNA delivery to T cells (Figure 16B). Relative viability of T cells 24 hours after mRNA delivery to T cells (Figure 16C). [Figure 17]1 is a bar graph showing the results of cargo delivery using PCTE filter plates. Viability and GFP expression 24 hours after mRNA delivery to T cells using PCTE 96-well plates. [Figure 18] 1 is a bar graph showing mRNA expression after cargo delivery to cells (comparison of 96-well filter plates). GFP expression in T cells after mRNA delivery. Cell monolayers were formed on either PCTE or PES filter plates. [Figure 19] Figures 19A-B are bar graphs showing expression of transfected mRNA. Comparison of media removal methods. Media was removed from cells seeded in 96-well filter plates by either centrifugation or vacuum pressure (Figure 19A). Media was removed from cells seeded in 96-well filter plates by either centrifugation or positive pressure (Figure 19B). GFP mRNA was delivered by solporation and expression was assessed by flow cytometry. [Figure 20] Figures 20A and 20B are photographs of cells. Monolayer formation using cells bound to Dynabeads (Figure 20A). The medium was removed by pipetting and GFP mRNA was delivered by solporation. GFP expression was detected by fluorescence microscopy after 24 hours (Figure 20B). [Figure 21] Figure 21A is a series of photographs illustrating the delivery of GFP mRNA to MSCs. GFP RNA was delivered to BM-MSCs and iPSC-MSCs by vector-free reversible permeabilization method and analyzed by fluorescence microscopy (10x magnification). Figure 21B is a bar graph illustrating the delivery of GFP mRNA to MSCs analyzed by flow cytometry; n=3, data are expressed as mean ± standard deviation. [Figure 22]Figure 22A is a table of cells displaying green fluorescent markers, and Figure 22B is a photograph of cells displaying green fluorescent markers. Uptake of Alexa488 dextran 10KD using ultrasonic nebulizer. Representative data showing uptake of dextran into U2OS cells using ultrasonic nebulizer. (a) Table summarizing the data where a maximum of 64% dextran uptake was achieved and (b) fluorescent microscopy image showing green dextran-Alex488. [Diagram 23] Photograph of Ari Mist nebulizer payload fluidic control using a pinch valve. The image shows the fluidic control of payload solution to an Ari Mist nebulizer in a test rig used to optimize atomization of the delivery solution. The Ari Mist nebulizer (1) is connected to a pinch valve (2) which is connected to an Elveflow delivery solution reservoir (3). [Figure 24] Dot plots representing Ari Mist test rig longitudinal data. The graph shows GFP mRNA delivery efficiency to T cells using the Ari Mist nebulizer collected during optimization experiments. Data shows an average uptake efficiency of up to 25% and high reproducibility between multiple replicates. [Diagram 25] Dot plots representing ultrasound 180 kHz test rig longitudinal data. The graph shows GFP mRNA delivery efficiency to T cells using an ultrasonic nebulizer collected during optimization experiments. Data shows average uptake efficiency of up to 30% and high reproducibility between replicates. [Figure 26] Dot plots representing nasal head test rig longitudinal data. The graph shows GFP mRNA efficiency into T cells using the MAD nasal head nebulizer collected during optimization experiments. Data shows an average uptake efficiency of up to 22%, with reduced reproducibility between multiple replicates compared to the Ari Mist and ultrasonic spray head. [Figure 27]Figures 27A and 27B are bar graphs comparing atomizer heights above cells. GFP expression was assessed in T cells after mRNA delivery using atomizer heights set at 31, 26, 12, or 11 mm. [Figure 28] 1 is a bar graph depicting a comparison of delivery solution volumes.GFP expression in T cells following delivery of GFP mRNA in different volumes of delivery solution (4, 1, or 0.5 μl). [Figure 29] 1 is a bar graph depicting a comparison of salt concentrations. GFP mRNA was added to a delivery solution containing 12 mM KCl (hypotonic) or 106 mM KCl (isotonic). "Hypotonic" and "isotonic" refer to tonicity relative to the cytoplasm of the cells. Expression levels at 24 hours were assessed by flow cytometry. [Diagram 30] Bar graph showing comparison of the number of "hits." T cells were sprayed (hits) once, twice or three times with a 2-hour incubation between each spray. GFP expression was assessed by flow cytometry after 24 hours. [Diagram 31] FIG. 1 is a bar graph depicting a comparison of T cell seeding densities. 1.25, 2.5, 3.5, 5, and 7.5×105 T cells were seeded per well and GFP mRNA was delivered by sorbolation. GFP expression was assessed after 24 hours by flow cytometry. [Diagram 32] Line graph showing distribution of cell diameters: Mean T cell diameter (9.5 μm) 24 hours after addition of DynaBeads (beads to cells ratio 3:1). [Diagram 33]Figures 33A-C are bar graphs depicting delivery and viability of Avectas (solporation) compared to electroporation. In (Figure 33A), delivery efficiency of 10 kDa dextran-Alexa488 into A549 cells was 52.8% (±2.7%) as quantified by flow cytometry, and cell viability compared to untreated controls was 78.3% (±4.1%) as determined by propidium iodide exclusion and flow cytometry. (Figure 33B) For electroporation, delivery efficiency was 92.9% (±0.6%) and cell viability was 73.0% (±9.8%). (FIG. 33C) The transfection score ((number of transfected cells / total cells)×(number of viable cells / total cells)) was 0.33 (±0.05) for the subject technique and 0.51 (±0.13) for electroporation, with no significant difference between the scores, p=0.25. [Diagram 34] Bar graph showing a comparison of Solupore and Nucleofection. Human T cells were transfected with GFP mRNA using either Solupore or Nucleofection. GFP expression and cell viability (7-AAD) were analyzed by flow cytometry after 24 hours. [Diagram 35] The bar graph in Figure 35A, the line graph in Figure 35B, and the photographs in Figures 35C and 35D represent a comparison of the fluorescence intensity between Solupore- and Nucleofection-mediated delivery of cargo. The median fluorescence intensity generated by human T cells 24 hours after GFP mRNA delivery was compared between Solupore and Nucleofection (Figure 35A). The histogram represents the increase in intensity (Figure 35B). Exemplary images of T cell aggregates expressing GFP after Solupore (light) and Nucleofection. [Diagram 36] Dot plots showing a comparison of fluorescence intensity and GFP expression between Solupore and nucleofection delivered with various amounts of mRNA. The dose response in median fluorescence intensity produced by human T cells 24 hours after delivery of various amounts of GFP mRNA was observed after Solupore and after nucleofection. [Figure 37] The photographs in Figure 37A and the bar graphs in Figures 37B, 37C, and 37D represent the evaluation of cargo delivery to cells. Diffusion of cargo into cells and resealing of plasma membrane. (Figure 37A) 10kDa dextran-Alexa488 was delivered into A549 cells and analyzed by fluorescence microscopy 30 seconds, 1 minute, 2 minutes, and 3 minutes after delivery (10x magnification). (Figure 37B) A549 cells were pretreated with Dynasore (4mM) or chloropromazine (20μM) to inhibit clathrin-mediated endocytosis, or with nystatin (20μg / ml) or EIPA (100μM) to inhibit caveolae-mediated endocytosis and micropinocytosis, respectively (n=3). None of these inhibitors prevented the uptake of EGFP mRNA. (Figure 37C) Lipofectamine 2000 was used as a positive control for endocytosis-mediated delivery. GFP expression was significantly reduced in lipofected cells treated with Dynasore (n=3). (Figure 37D) To examine cell membrane recovery after permeabilization, delivery solution was sprayed onto A549 cells in the absence of cargo, and at later time points (0-182.5 min), media was removed and 50 μl of propidium iodide (100 μg / ml) in PBS was added. After 2 min of incubation, PI solution was removed and cells were harvested. For basal levels of PI uptake, untreated cells were given 50 μl of PI in PBS. PI uptake was analyzed by flow cytometry. Data show that cells remain permeable to PI up to 6 min after treatment, but then form a reseal, preventing further uptake (n=3). Error bars represent standard deviation (SE) from three experiments (n=3).***p<0.001, Student's t-test for independent means. [Figure 38]Figure 38A is a bar graph illustrating PD-1 surface expression on T cells. Data shown are the average of 5 independent experiments with control group normalized to 100%. Figure 38B is a photographic image illustrating PD-1 surface expression on T cells as determined by flow cytometry 72 hours after transfection. Data shown are from one independent experiment. [Figure 39] Photographic images of T cell clones 4 days after treatment (see arrows) by light microscopy, showing good proliferation and activation of T cells in untreated controls and cells treated with the vector-free intracellular delivery method described herein. Significantly fewer clones were observed in electroporated cells. Data shown are from one independent experiment. [Diagram 40] Schematic diagram of the CAR construct of the lenti-T7-CD19-3rd-CAR vector. [Diagram 41] Figure 41A is a schematic diagram illustrating the sequence of the single chain variable fragment (scFv). Figure 41B is a schematic diagram illustrating the nucleotide sequence (codon optimized) of the CAR cassette. Figure 41C is a schematic diagram illustrating the protein sequence of the CAR cassette. [Diagram 42] 1 is a photographic image showing a restriction map of the CAR vector. [Diagram 43] 1 is a table depicting the QC results and certificate of analysis of commercially available CAR vectors. [Diagram 44] Figures 44A-B are schematic diagrams illustrating CAR sequence alignment validation of commercially available CAR vectors. [Diagram 45] 1 is a dot plot of flow cytometry data showing CAR expression in human primary T cells following mRNA delivery by the vector-free delivery technique described herein. [Figure 46] FIG. 46A is a photograph of an electrophoretic gel, and FIG. 46B is a bar graph depicting supercoiled versus open circular nucleic acid. [Figure 47]Figures 47A-B are dot plots depicting the effect of transfection method on CD4 and CD8 expression in T cells. Cell surface CD4 and CD8 expression was examined 6 and 24 hours after either nucleofection, electroporation (Neon), or solporation. At 6 hours, expression levels were similar to untreated control cells. However, at 24 hours, expression in soluporated and nucleofected cells was similar to control untreated cells, whereas expression was significantly reduced in electroporated cells. [Figure 48] Figures 48A-C are dot plots representing mRNA microarray analysis. Cells were transfected with GFP mRNA. The highest level of gene expression changes occurred with Neon electroporation treatment. Of the 20,893 genes analyzed, Solupore altered 32 genes, Nucleofection altered 24 genes, and electroporation altered 317 genes. [Figure 49] Figure 1 is a line graph showing cell proliferation. T cells were transfected with GFP mRNA and then counted daily for 7 days. The proliferation rates of sorbolated and nucleofected cells were similar to untreated control cells, whereas the potency of Neon-electroporated cells was reduced compared to control cells. [Figure 50] Figure 1 is a line graph showing cell proliferation after cryopreservation. T cells were transfected with GFP mRNA and 24 hours after transfection, cells were cryopreserved in 10% DMSO and fetal bovine serum. Cells were thawed and seeded at 0.5x106 cells / ml in Immunocult medium + IL-2 on day 0. Cells were counted and reseeded by adding medium daily for 5 days. Proliferation rates in sorborated and nucleofected cells were similar to untreated control cells, while electroporated (e.g. Neon) cells had reduced potency compared to control cells. [Figure 51] 1 is a series of bar graphs depicting interferon-γ (IFNg) production. IFNg production measured 14 days after transfection was not reduced in T cells following solporation, nucleofection, or electroporation compared to control cells. [Figure 52] Figure 1 is a bar graph showing IFNg production. Sorprated, nucleofected, electroporated, and untreated cells were cryopreserved 24 hours after transfection. Cells were then restimulated with either Dynabeads or PMA / ionomycin cocktail for 4 hours, and their supernatants were analyzed for IFN-γ production. [Figure 53] Figures 53A-C are bar graphs depicting the assessment of CD45+ cells after treatment. Analysis of blood 14 days after injection of human PBMCs into NSG mice. Figure 53A. Human CD45+ cells were detected in mice fed untreated (UT) and sorbolated (Sol) PBMCs. Fewer human CD45+ cells were detected in mice fed nucleofected (Nuc) PBMCs. Figure 53B. The presence of CD4+ cells was confirmed in groups 2, 5, and 6. Figure 53C. The presence of CD8+ cells was confirmed in groups 2, 5, and 6. [Figure 54] 1 is a line graph showing calibration data for delivery solution using the elveflow-pinch valve system. The graph shows calibration data for repeated atomization of a 4 μl volume of delivery solution. The orange bars represent the volumes measured from 11 replicate sprays of delivery solution, where the expected volume was 4 μl. The data shows a relative standard deviation (%RSD) of 9.38% over this volume range. The pink bars represent the volumes measured from 9 replicate sprays after refilling the sample reservoir. These data clearly show the limitations of the elveflow-pinch valve system, including a lack of precision and accuracy when spraying a 4 μl volume, as well as a lack of calibration retention after refilling the system. [Figure 55]1 is a line graph illustrating force sensor parameterization 1. The force sensor was placed 31, 26, or 11 mm below the Ari Mist atomizer and the pressure of the air pushed out by the atomizer was adjusted from 0.5 bar to 2 bar. Adjusting the amount of pressure applied to the Elveflow system also changed the amount of liquid dispensed. The peak pressure felt by the sensor is measured in volts. [Figure 56] 1 is a series of line graphs illustrating force sensor parameterization 2. The force sensor was placed 31, 26, or 11 mm below the Arimist atomizer and the pressure of the air being pushed by the atomizer was adjusted from 1.15 bar to 2.15 bar. The peak pressure felt by the sensor is measured in volts. [Figure 57] 1 is a bar graph depicting force sensor GFP delivery, showing GFP expression, viability (survival), and peak pressure (volts) following GFP mRNA delivery with various air pressures and volumes. [Figure 58] Photograph of the Certus Flex mechanism. The image shows the Certus flex mechanism with delivery solution loaded into channel 1, stop solution loaded into channel 2, and culture media loaded into channel 3. [Figure 59] FIG. 1 is a diagram of droplet array patterns. The diagram shows the droplet array patterns that were tested and are further detailed in Table 1. The total volume delivered into the well was either 2 μl or 7 μl, and the number of droplets was increased from 4 to 25. This corresponded to droplet volumes ranging from 0.08 to 0.5 μl. [Figure 60] The line diagram in Figure 60A and the bar graph in Figure 60B show the effect of seeding mask and seeding mask, respectively. Comparison of GFP mRNA expression in cells seeded in wells of PCTE plate in the presence or absence of seeding mask (Figure 60A). GFP expression was measured 24 hours after delivery (Figure 60B). [Figure 61]The bar graph in Figure 61A and table in Figure 61B present data showing the lack of GFP mRNA delivery using Certus Flex digital dispensing technology. (Figure 61A) The graph illustrates representative data showing the lack of mRNA delivery using Certus Flex. There was no detrimental effect on cell viability using this system. (Figure 61B) The corresponding data set is detailed in the table. [Figure 62] FIG. 62A is a bar graph depicting the effect of the collar, and FIG. 62B is a photograph depicting the collar. Testing of an enclosing collar on an Ari Mist spray head (FIG. 62A). The graph shows data comparing delivery efficiency without an enclosing collar (- collar), with an enclosing collar (+ collar), and with an enclosing collar and a 1 mm gap between the collar and the 96-well plate (+ collar and 1 mm gap). The results show that the addition of an enclosing collar had a negative effect on the spray, which was countered by leaving a 1 mm gap between the collar and the well plate. (FIG. 62B) The image shows the enclosing collar mechanism. The collar is inserted onto the Ari Mist spray head. The spray head is positioned 27 mm above the well of the double height plate, leaving a 1 mm gap between the collar and the top of the well plate. Delivery efficiency was not affected by this mechanism. [Figure 63]FIG. 63A is a bar graph showing the results of testing with the Gyger valve, and FIG. 63B is a photographic image of the X-pierce film. The graphs represent data from three independent experiments comparing the delivery efficiency of Test Rig 1 (with Clippard pinch valve), Test Rig 3 (with Gyger microvalve), and Test Rig 3 with X-pierce film on PCTE filter plates. Note: Only two experiments were performed comparing TR3 and Gyger valves alone (TR3 Gyger). The results show that using the Gyger microvalve (TR3 Gyger) instead of the Clippard valve (TR1) increased the delivery efficiency. The addition of the X-pierce film on the PCTE plate had no effect on this delivery efficiency (TR3 Gyger + Enclosure). Each bar represents one experiment with a minimum of four replicates. [Figure 64] 1 is a process flow diagram illustrating an example process according to some aspects of the present subject matter. [Figure 65] FIG. 1 is a diagram illustrating one embodiment of a delivery system. [Figure 66] FIG. 1 is a diagram illustrating the nine elements of the delivery system. [Figure 67] FIG. 1 is a side view of one embodiment of a delivery system. [Figure 68] FIG. 1 is a perspective view of a portion of one embodiment of a delivery system. [Figure 69] FIG. 69 is an enlarged perspective view of the delivery system shown in FIG. [Figure 70] FIG. 69 is an exploded top perspective view of a vacuum manifold assembly of the delivery system shown in FIG. [Figure 71] FIG. 69 is an exploded bottom perspective view of the vacuum manifold assembly of the delivery system shown in FIG. [Figure 72] FIG. 71 illustrates a top view of the base plate of the vacuum manifold assembly shown in FIG. [Figure 73] FIG. 73 is a side cross-sectional view of the base plate shown in FIG. 72. [Figure 74] FIG. 73 is a bottom view of the top plate of the vacuum manifold assembly shown in FIG. 72. [Figure 75] FIG. 75 is a side cross-sectional view of the top plate shown in FIG. 74. [Figure 76] FIG. 75 is a plan view of the top plate shown in FIG. 74. [Figure 77] FIG. 73 is a top view of the well filter plate of the vacuum manifold assembly shown in FIG. [Figure 78] FIG. 73 is another plan view of the well filter plate shown in FIG. 72, where the well filter plate has been rotated 180 degrees. [Figure 79] FIG. 68 is a front perspective view of a portion of the high precision rig system shown in FIG. [Figure 80] FIG. 68 is a rear perspective view of a portion of the high precision rig system shown in FIG. [Figure 81] FIG. 68 is a plan view of a portion of the high precision rig system shown in FIG. [Figure 82] 1. Distribution of GlowGerm particles observed after centrifugation and vacuum extraction. [Figure 83] FIG. 1 is a diagram illustrating the six elements of a positive pressure delivery system. [Figure 84] FIG. 1 is a perspective view of one embodiment of a positive pressure delivery system including a manifold assembly, a mounted array with modular fluid head modules, an XY actuator, and a control system. [Figure 85] FIG. 85 is an enlarged view of a portion of the positive pressure delivery system shown in FIG. [Figure 86] FIG. 13 is a close-up view of a modular fluid head module that allows vertical movement. [Figure 87] 87. The modular fluid head module shown in Fig. 86 with the needle assembly attached. The needle assembly is used to dispense culture media and stop solution. [Figure 88] 87. The modular fluid head module shown in FIG. 86 with the nebulizer assembly attached. The nebulizer assembly is used to atomize the payload and delivery solution and deliver the solution to the cells. [Figure 89]87. The modular fluid head module shown in Fig. 86 with the positive pressure nozzle assembly attached. The positive pressure nozzle assembly is used for removal of culture media. [Figure 90] FIG 90 is a side view of the positive pressure nozzle of the positive pressure system shown in FIG 84-85 used to remove culture medium. FIG 90 illustrates the distal end of the nozzle in contact with the wells of a 96-well filter plate. [Figure 91] FIG. 91 is an enlarged view of a portion of the positive pressure nozzle shown in Figure 90. The positive pressure nozzle is shown forming a seal with the well. [Figure 92] 1 is an exemplary embodiment of a portion of a nozzle assembly including a valve for controlling the delivery of air to the wells of a filter plate. [Figure 93] One embodiment of a nebulizer assembly used to optimize atomization of a delivery solution. The nebulizer assembly includes a syringe, a microvalve, and a nebulizer. The nebulizer is coupled to a coupling element (e.g., a pre-column coupling). The microvalve is retained and / or coupled within a valve holder that is coupled to a syringe via an adapter. The nebulizer assembly allows for the delivery of payload solutions to the nebulizer with high accuracy and precision. [Figure 94] 1 is a plot depicting the efficiency of payload delivery for an ultrasonic emitter operating at 180 kHz. [Figure 95] 1 is a plot representing a series of data characterizing GFP uptake for an ultrasound emitter, an Ari Mist nebulizer, and a MAD nasal spray emitter. The data show GFP delivery efficiencies of 32.7%, 24.8%, and 16.9% for the ultrasound emitter (180 Hz), the Ari Mist nebulizer, and the MAD nasal spray emitter, respectively. [Figure 96]1 is a plot representing a series of data characterizing cell viability for an ultrasonic emitter, an Ari Mist nebulizer, and a MAD nasal spray emitter. The data show relative cell viability of 72.3%, 85.3%, and 99.7% for the ultrasonic (180 Hz) nebulizer, the Ari Mist nebulizer, and the MAD nasal nebulizer, respectively. Data represents a minimum of three technical replicates for each spray head tested. [Figure 97] 1 is an exemplary embodiment of a nebulizer assembly including an enclosing collar that is positioned around the spray head of an Ari Mist nebulizer. The collar is inserted onto the Ari Mist spray head such that the spray head is positioned 27 mm from the bottom of the well of the filter plate, leaving a 1 mm gap between the collar and the top of the filter plate. [Figure 98] 1 is a plot representing data characterizing the efficiency (GFP incorporation) corresponding to a spray head without a collar, a spray head with a collar that forms a seal with the filter plate, and a spray head with a collar where there is a 1 mm gap between the collar and the filter plate. The data shows that the use of a collar that forms a seal with the filter plate has a negative effect on spraying. A collar located 1 mm above the filter plate did not affect spraying. [Figure 99] FIG. 1 is an exemplary embodiment of a 96-well PCTE filter plate with an X-pierce film adhered to the top surface of the filter plate. [Figure 100]FIG. 13 is a plot representing data characterizing the efficiency (GFP uptake) corresponding to a study performed in Rig 1 (R1) using a filter plate without an enclosure, a study performed in Rig 3 (R3) using a filter plate without an enclosure, and a study performed in R3 using a filter plate containing an X-pierce film enclosure on the wells of the filter plate. R1 uses a clippard pinch valve to control the flow, while R3 uses a Gyger microvalve. The data shows an increase in delivery efficiency when a Gyger microvalve (R3 Gyger) is used instead of a Clippard valve (R1). The addition of X-Pierce film to the PCTE filter plate had no effect on delivery efficiency. Each bar represents one experiment with a minimum of four replicates. [Figure 101] FIG. 1 is an embodiment of a heating system that can be used to heat the delivery solution, the stop solution, and the culture medium. [Figure 102] FIG. 1 is a side view of one embodiment of a cooling system that can be used to cool the delivery solution, the stop solution, and the culture medium. [Figure 103] FIG. 1 is a perspective view of one embodiment of a cooling system that can be used to cool the delivery solution, the stop solution, and the culture medium. [Figure 104] FIG. 1 is a perspective view of one embodiment of a mounting assembly capable of releasably retaining a needle emitter and an ultrasonic atomizer. [Figure 105] FIG. 91 is an exploded perspective view of a portion of the mounting assembly shown in FIG. 90. [Fig. 106] FIG. 13 is a perspective view of another embodiment of a mounting assembly capable of releasably retaining a needle emitter and an ultrasonic atomizer. [Figure 107] FIG. 1 is a perspective view of one embodiment of a mounting assembly capable of releasably retaining a needle emitter and a nebulizer. [Figure 108]FIG. 1 is an exploded view of an exemplary embodiment of a stirred cell system configured to facilitate the formation of a cell monolayer. The stirred cell system is assembled by inserting a membrane into a membrane holder, inserting the membrane holder into a base, and screwing the body of the system onto the base. Cells (e.g., culture medium containing cells) are delivered to a chamber formed by the base and body, and a cap is screwed onto the body opposite the base to close the chamber. A positive pressure in the range of 50-100 mbar is delivered to the chamber via a pressure line connected to the cap. Pressure is applied for 10-60 seconds. [Fig. 109] FIG. 109 is a plan view of the membrane holder of the stirred cell system shown in FIG. 108. [Figure 110] FIG. 13 is a plan view of another embodiment of a membrane holder that includes holes but no ridges. [Figure 111] FIG. 111 is a plan view of a membrane used with the membrane holder shown in FIG. 110 in a study to evaluate the effect of membrane holder design on cell monolayer formation. Dynabeads were used instead of cells. The results show that the Dynabeads accumulated in locations on the membrane that corresponded to the locations of the holes in the membrane holder. [Figure 112] FIG. 13 is a plan view of another embodiment of a membrane holder containing holes, concentric channels, and straight channels. [Figure 113] FIG. 113 is a plan view of a membrane used with the membrane holder shown in FIG. 112 in a study to evaluate the effect of membrane holder design on cell monolayer formation. Dynabeads were used instead of cells. Results show that this membrane holder produces an even distribution of Dynabeads. [Fig. 114] FIG. 13 is a perspective view of another embodiment of a membrane holder configured to facilitate efficient removal of culture medium and formation of a cell monolayer. [Figure 115]1 is an exemplary embodiment of a nebulizer assembly for a solution delivery system, also referred to as Rig 4 (R4), that includes a nebulizer (e.g., an LB-100 spray head), a coupling element (e.g., an IDEX fitting) configured to facilitate delivery of air and liquid (e.g., permeabilization solution) to the nebulizer, a solution reservoir (e.g., an Elveflow sample reservoir) configured to provide permeabilization solution to the nebulizer, and a pinch valve configured to control delivery of the permeabilization solution to the nebulizer. [Fig. 116] FIG. 116 is a perspective view of a mounting system for a solution delivery system to which the nebulizer assembly shown in Fig. 115 can be attached. The mounting system can include a valve and reservoir mounting, a spray head mounting, and a nebulizer retention collar for receiving the nebulizer. [Figure 117] 117 is a perspective view of a solution delivery system illustrating the nebulizer assembly shown in FIG. 115 mounted in the mounting system shown in FIG. 116. [Figure 118] A side view of a solution delivery system illustrating the nebulizer assembly shown in FIG. 115 mounted in the mounting system shown in FIG. 116. [Figure 119]FIG. 1 is a side view of an exemplary closed stirred cell system. Three components are shown: (a) chamber lid, (b) chamber wall, and (c) chamber base. Part (a) lid is made of 316 stainless steel and houses an LB-100 spray head and inlets for adding cell suspension, stop solution, and culture media. Part b) wall is made of glass and contains an inlet that can allow for the introduction of cell suspension via cell introduction and includes ports that can allow for the introduction of instruments to allow humidity or gas monitoring. The wall is designed to allow for air expansion by incorporation of an expansion port that allows for the addition of an expansion chamber or by designing with a larger diameter in one area of ​​the wall. Part c) base is made of 316 stainless steel and will house a custom membrane holder. The connections of part a to part b and part b to part c are mating NW-KF flanges fastened with stainless steel clasps that comply with GMP manufacturing. [Figure 120] 1 is a plot representing data characterizing the force profile of the LB-100 nebulizer in response to various test parameters. [Figure 121] Figure 1 is a plot showing data characterizing cell recovery and viability after monolayer formation. Representative data shows cell recovery from PES and PCTE filter membranes. Cell suspensions were applied to a 63 mm stirred cell unit at two different concentrations. Pressures ranging from 100 to 250 mbar were applied for 10 seconds. After cell monolayer formation, filters were rinsed and cells were counted and viability analyzed. Data shows that PCTE (track-edged) filters provide improved cell recovery and viability. [Figure 122] 13 is another plot depicting data characterizing cell recovery and viability following cell monolayer formation. [Figure 123]FIG. 11 is another plot showing data characterizing the delivery efficiency using the LB-100 nebulizer. Representative data shows the delivery efficiency of GFP mRNA into T cells using the LB-100 nebulizer at various parameters tested (distance 100 mm, spray duration 420 ms, pressure 600 mBar corresponding to a delivered volume of 100 μl, and air pressure 2.5 bar). In addition, the presence of walls and enclosing films was tested. 100 μl of delivery solution was atomized using the LB-100 to a monolayer of 20×106 T cells in a target area of ​​40 mm diameter. A maximum delivery efficiency of 50% was achieved with the single spray protocol and 58% with the double spray protocol. The average survival rate among treatment groups was 60%. [Figure 124] FIG. 1 is a diagram illustrating the mechanism of action. [Fig. 125] An embodiment of the Ari Mist nebulizer. Here the liquid and air connections to the nebulizer are shown. As described in U.S. Patent Nos. 5,411,208, 6,634,572, and Canadian Patent Nos. 2,112,093 and 2,384,201. [Fig. 126] 1 is an exemplary embodiment of a payload delivery system (Rig 1). [Figure 127] 1 is an exemplary embodiment of a payload delivery system (Rig 3). [Figure 128] 1 is an exemplary embodiment of a payload delivery system (Rig 4). [Figure 129] FIG. 1 is a schematic diagram of a software platform including a graphical user interface used to output programs to a PLC that provides local control of the mechanical and electrical systems. [Fig. 130]1 is an example of a graphical user interface experiment canvas according to some aspects of the present subject matter. The user interface includes an experiment canvas that allows a user to modify parameters, including the location and number of wells to be addressed, vacuum or positive pressure, payload, sequence of steps including dispensing of stop solution and culture medium, and volume delivered. The user can also modify actuator speed and incubation time. [Fig. 131] 1 is an exemplary embodiment of a closed stirred cell system configured to facilitate the formation of a cell monolayer. As shown in the illustrated example, the closed stirred cell system 12200 includes a cap 12202, a body 12204 forming a chamber wall, a cell introduction portion 12203, and a chamber base 12208 including a membrane holder. [Fig. 132] An exemplary Midi system. Shown is a 63 mm stirred cell unit containing a 44 mm membrane holder (modified to include additional holes to promote better filtration). The enclosing film can be seen adhered to the top of the stirred cell unit. The spray head holder containing the LB-100 is inserted into the chamber such that the distance from the emitter tip to the surface of the filter membrane is 82 mm. [Fig. 133] FIG. 1 is a plot representing data characterizing delivery efficiency and viability using the LB-100 nebulizer. The graph shows average delivery efficiency of 59.63%±1.2 and average viability data of 74.6%±5.3 across three technical replicates. A cell monolayer was formed using a 0.4 μm PCTE filter membrane and 20×107 cells at 120 mbar pressure for 40 seconds. The LB-100 spray parameters were 2.5 bar pressure, 600 mbar 400 ms spray duration (volume 100 μl) and 82 mm distance. The data represents results from three replicates characterizing delivery efficiency and viability. [Fig. 134] 1 is one embodiment of an exemplary clinical delivery system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description The difficulty of transfecting molecules into non-adherent cells has plagued research and therapy, such as cell therapy, gene therapy, and genetic modification, for decades. The reason for the difficulty in transfecting such cells is that non-adherent cells lack cell surface heparan sulfate proteoglycans, the molecules responsible for the adhesion of cells to the extracellular matrix. Transfection methods such as electroporation and / or nucleofection have the drawback that they impair cell viability, the ability of cells to resume proliferation after treatment, and cell function, such as the immune activity of lymphocytes. The transfection composition and transfection method (solporation) described herein are considered to have a great advantage over previous methods of introducing cargo molecules into mammalian cells, such as non-adherent / suspension cells, which are difficult to transfect, since they do not have such drawbacks.

[0036] The present invention is based on the surprising discovery that a compound or a mixture of compounds (compositions) is delivered into the cytoplasm of eukaryotic cells by contacting the cells with a solution containing the compound (e.g., payload) to be delivered and an agent that reversibly permeabilizes or dissolves the cell membrane.Preferably, the solution is delivered to the cells in the form of a spray, for example, in the form of aqueous particles (see, for example, PCT / US2015 / 057247 and PCT / IB2016 / 001895, which are incorporated herein by reference).For example, the cells are coated with the spray, but are not immersed or submerged in the solution containing the delivery compound.Exemplary agents that permeate or dissolve eukaryotic cell membranes include alcohols and detergents, for example, ethanol and Triton X-100, respectively. Other exemplary cleaning agents, e.g., surfactants, include polysorbate 20 (e.g., Tween 20), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), sodium dodecyl sulfate (SDS), and octylglucoside.

[0037] An example of a condition for achieving coverage of a population of coated cells includes delivery of a fine particle spray, excluding, for example, dripping or pipetting a bolus volume of solution onto the cells, such that a significant number of cells are submerged or submerged by the bolus volume of fluid. Thus, the mist or spray includes a ratio of fluid volume to cell volume. Alternatively, the condition includes a ratio of mist or spray volume to exposed cell area, for example, to exposed cell membrane area when the cells are present as a confluent or substantially confluent layer on a substantially flat surface, for example, on the bottom of a tissue culture vessel, such as a well of a tissue culture plate, such as a microtiter tissue culture plate.

[0038] "Cargo" or "payload" is a term used to describe a compound or composition that is delivered in an aqueous solution across the cell plasma membrane to the interior of the cell.

[0039] In one aspect, delivery of a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution comprising a payload and an alcohol content greater than 5 percent concentration. The volume of the aqueous solution can be a function of the exposed surface area of ​​the population of cells or can be a function of the number of cells in the population of cells.

[0040] In another aspect, a composition for delivering a payload across the plasma membrane of a cell includes an aqueous solution containing a payload, an alcohol at a concentration greater than 5 percent, more than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffer, wherein the concentration of the alcohol, e.g., ethanol, does not exceed 50%.

[0041] One or more of the following features may be included in any possible combination: The volume of solution delivered to the cells is in multiple units, e.g., multiple droplets on a spray, e.g., aqueous particles. The volume is stated relative to an individual cell, or relative to the exposed surface area of ​​a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100% confluent) cell population. For example, the volume is 6.0×10 per cell. -7 Microliters ~ 7.4 x 10 per cell -4 The volume can be 4.9 x 10 per cell. -6 Microliters to 2.2 x 10 per cell -3 The volume is 9.3 x 10 per cell. -6 Microliters to 2.8 x 10 per cell -5 The volume can be in microliters, approximately 1.9 x 10 per cell. -5 The volume can be in microliters, within 10% of about 6.0 x 10 per cell. -7 Microliters to 2.2 x 10 per cell -3The volume is 2.6 × 10 per square micrometer of exposed surface area. -9 Microliters ~ 1.1 x 10 per square micrometer of exposed surface area -6 The volume can be 5.3 x 10 microliters per square micrometer of exposed surface area to 1.6 x 10 microliters per square micrometer of exposed surface area. -7 The volume can be in microliters, approximately 1.1×10 per square micrometer of exposed surface area. -7 It can be in microliters. It can be within about 10%.

[0042] Cell confluency refers to cells that are in contact with each other on a surface. For example, confluency can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of a surface, e.g., the surface of a tissue culture vessel, is covered with cells, and 100% means that the surface is completely covered. For example, adherent cells grow two-dimensionally on the surface of a tissue culture well, plate, or flask. Non-adherent cells can be removed by centrifugation, settling by vacuum, or by aspirating the tissue culture medium from the top of the cell mass or by aspirating or vacuuming the tissue culture medium from the bottom of the vessel.

[0043] The contact of the population of cells with the volume of aqueous solution can be achieved by propelling the aqueous solution with a gas to form a spray. The gas can include nitrogen, ambient air, or an inert gas. The spray can include discrete volumetric units having diameters ranging from 1 nm to 100 μm, for example 30 to 100 μm. The spray can include discrete volumetric units having diameters of about 30 to 50 μm. A total volume of 20 μl of aqueous solution can be sprayed to a volume of about 1.9 cm. 2 A total volume of 10 μl of aqueous solution can be delivered to a cell footprint of approximately 0.95 cm, for example, to one well of a 24-well culture plate. 2The cell population is delivered to a cell footprint of 1000 μg / ml, for example, to one well of a 48-well culture plate. Typically, the aqueous solution contains the payload to be delivered across the cell membrane into the cells, and the second volume is a buffer or culture medium that does not contain the payload. Alternatively, the second volume (buffer or medium) can also contain the payload. In some embodiments, the aqueous solution contains the payload and the alcohol, and the second volume does not contain the alcohol (and optionally does not contain the payload). The population of cells can be contacted with the aqueous solution for 0.1 to 10 minutes before adding a second volume of buffer or culture medium to submerge or suspend the population of cells. The buffer or culture medium can be phosphate buffered saline (PBS). The population of cells can be contacted with the aqueous solution for 2 seconds to 5 minutes before adding a second volume of buffer or culture medium to submerge or suspend the population of cells. The population of cells can be contacted with the aqueous solution, e.g., an aqueous solution containing a payload, for 30 seconds to 2 minutes before a second volume of buffer or culture medium, e.g., one without a payload, is added to submerge or suspend the population of cells. The population of cells can be contacted with the spray for about 1 to 2 minutes before a second volume of buffer or culture medium is added to submerge or suspend the population of cells. During the time between spraying the cells and adding the buffer or culture medium, the cells remain hydrated by a layer of water from the spray volume.

[0044] The aqueous solution can contain 5-30% ethanol. The aqueous solution can contain 75-98% H 2 The delivery solution may contain one or more of the following: 2H2O, 2-45% ethanol, 6-91 mM sucrose, 2-500 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). For example, the delivery solution contains 106 mM KCl and 27% ethanol.

[0045] The cell population can include adherent cells or non-adherent cells. The adherent cells can include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, nerve cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or immortalized cells, such as cell lines. In a preferred embodiment, the cell population includes non-adherent cells, such as the non-adherent cell rate in the population is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99% or 100% non-adherent cells. The non-adherent cells can be primary cells as well as immortalized cells, such as cell lines. Exemplary non-adherent / suspension cells include cell lines such as primary hematopoietic stem cells (HSCs), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or Jurkat T cells.

[0046] The payload can include small chemical molecules, peptides or proteins, or nucleic acids. Small chemical molecules can be less than 1,000 Da. Chemical molecules can include MitoTracker® Red CMXRos, propidium iodide, methotrexate, and / or DAPI (4',6-diamidino-2-phenylindole). Peptides can be about 5,000 Da. Peptides can include ecallantide (known under the trade name Kalbitor, which is a 60 amino acid polypeptide for the treatment of hereditary angioedema and for preventing blood loss in cardiothoracic surgery), liraglutide (sold under the trade name Victoza and used to treat type II diabetes, and sold under the trade name Saxenda for the treatment of obesity), and icatibant (sold under the trade name Firazyer, a peptidomimetic for treating acute attacks of hereditary angioedema). Small interfering ribonucleic acid (siRNA) molecules can be about 20-25 base pairs in length or about 10,000-15,000 Da. siRNA molecules can reduce expression of any gene product, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC, cyclophilin B siRNA, and / or lamin siRNA, and can knock down gene expression of clinically important target genes or model genes. Protein therapeutics can include peptides, enzymes, structural proteins, receptors, cellular or circulating proteins, or fragments thereof. Proteins or polypeptides can be about 100-500,000 Da, for example, 1,000-150,000 Da. Proteins can include any therapeutic, diagnostic or research protein or peptide, for example, β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and / or horseradish peroxidase. In other examples, the protein can include a cancer-specific apoptotic protein, such as tumor necrosis factor-related apoptosis-inducing protein (TRAIL).

[0047] The antibody generally has a molecular mass of about 150,000 Da. The antibody can include anti-actin antibody, anti-GAPDH antibody, anti-Src antibody, anti-Myc ab, and / or anti-Raf antibody. The antibody can include green fluorescent protein (GFP) plasmid, GLuc plasmid, and BATEM plasmid. The DNA molecule can be greater than 5,000,000 Da. In some examples, the antibody can be a mouse-derived monoclonal antibody, such as ibritumomab tiuxetin, muromomab-CD3, tositumomab, a human antibody, or a humanized mouse (or other species-derived) antibody. In other examples, the antibody can be a chimeric monoclonal antibody, such as abciximab, basiliximab, cetuximab, infliximab, or rituximab. In yet another example, the antibody can be a humanized monoclonal antibody, such as alemtuzamab, bevacizumab, certolizumab pegol, daclizumab, gentuzumab ozogamicin, trastuzumab, tocilizumab, ipilimumamb, or panitumumab. The antibody can include an antibody fragment, such as abatecept, aflibercept, alefacept, or etanercept. The present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments, modified single chain Fv molecules, or chimeric molecules, such as antibodies that contain the binding specificity of an antibody derived from a mouse or the like and the remaining portion of another antibody derived from a human or the like.

[0048] The payload can include a therapeutic agent. A therapeutic agent, such as a drug or active agent, can refer to any compound that serves a therapeutic or diagnostic purpose, and the term can be understood to mean any compound administered to a patient for the treatment of a condition. Thus, a therapeutic agent can include proteins, peptides, antibodies, antibody fragments, and small molecules. The methods described herein can use therapeutic agents described in U.S. Pat. No. 7,667,004, which is incorporated herein by reference. The therapeutic agent can include at least one of cisplatin, aspirin, statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chlorpromazine HCl, thioridazine HCl, polymyxin B sulfate, chloroxine, benfluorex HCl, and phenazopyridine HCl), and fluoxetine. The payload can include a diagnostic agent. The diagnostic agent can include a detectable label or marker, such as at least one of methylene blue, patent blue V, and indocyanine green. The payload can include a fluorescent molecule. The payload can include a detectable nanoparticle. The nanoparticle can include a quantum dot.

[0049] The population of non-adherent cells can be substantially confluent, e.g., greater than 75% confluent. Cell confluency refers to cells in contact with each other on a surface. For example, confluency can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of a surface, e.g., the surface of a tissue culture vessel, is covered with cells, and 100% means that the surface is completely covered. For example, adherent cells grow two-dimensionally on the surface of a tissue culture well, plate, or flask. The non-adherent cells can be spun down, sedimented by vacuum, or tissue culture medium can be aspirated from the top of the cell population or removed by aspiration or vacuum from the bottom of the vessel. The population of cells can form a monolayer of cells.

[0050] The alcohol may be selected from methanol, ethanol, isopropyl alcohol, butanol, and benzyl alcohol. The salt may be selected from NaCl, KCl, Na 2 HPO 4 , K.H. 2 PO 4 , and C 2 H 3 O 2 NH. In a preferred embodiment, the salt is KCl. The sugar can include sucrose. The buffer can include 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid.

[0051] The present subject matter relates to methods for delivering molecules across plasma membranes. The present subject matter is useful in the field of intracellular delivery, and has application in the delivery of molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues or organs. The subject method includes the steps of introducing the molecule into an aqueous composition to form a matrix, atomizing the matrix into a spray, and contacting the matrix with the plasma membrane.

[0052] The present subject matter relates to compositions used to deliver molecules across plasma membranes. The present subject matter is useful in the field of intracellular delivery, and has applications in the delivery of molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues, or organs. The present subject matter compositions include alcohol, salts, sugars, and / or buffers.

[0053] In some embodiments, a permeabilization technique is presented that facilitates intracellular delivery of molecules independent of the molecule and cell type. Nanoparticles, small molecules, nucleic acids, proteins, and other molecules can be efficiently delivered to suspension or adherent cells in situ, including primary cells and stem cells, with low cytotoxicity, and the technique is compatible with high-throughput and automated cell-based assays.

[0054] Exemplary methods described herein include a payload, where the payload includes an alcohol. The term "alcohol" refers to a polyatomic organic compound that includes a hydroxyl (-OH) functional group attached to at least one carbon atom. The alcohol may be a monohydric alcohol and may include at least one carbon atom, such as methanol. The alcohol may include at least two carbon atoms (e.g., ethanol). In another aspect, the alcohol may include at least three carbons (e.g., isopropyl alcohol). The alcohol may include at least four carbon atoms (e.g., butanol) or at least seven carbon atoms (e.g., benzyl alcohol). Exemplary payloads may include up to 50% (v / v) alcohol, more preferably, the payload includes 2-45% (v / v) alcohol, 5-40% alcohol, and 10-40% alcohol. The payload may include 20-30% (v / v) alcohol.

[0055] Most preferably, the payload delivery solution comprises 25% (v / v) alcohol. Alternatively, the payload may comprise 2-8% (v / v) alcohol or 2% alcohol. The alcohol may comprise ethanol, and the payload may comprise 5%, 10%, 20%, 25%, 30%, and up to 40% or 50% (v / v) ethanol, for example 27% ethanol. An exemplary method may comprise methanol as the alcohol, and the payload may comprise 5, 10, 20, 25, 30, or 40% (v / v) methanol. The payload may comprise 2-45% (v / v) methanol, 20-30% (v / v) or 25% (v / v) methanol. Preferably, the payload comprises 20-30% (v / v) methanol. Further alternatively, the alcohol is butanol, and the payload comprises 2, 4, or 8% (v / v) butanol.

[0056] In some aspects of the present subject matter, the payload is an isotonic solution or an isotonic buffer solution.

[0057] According to the present subject matter, the payload can include at least one salt. The salt can be NaCl, KCl, Na2 HPO 4 , C 2 H 3 O 2 NH 4 , and K.H. 2 PO 4 For example, the KCl concentration is in the range of 2 mM to 500 mM. In some preferred embodiments, the concentration is greater than 100 mM, for example 106 mM.

[0058] According to exemplary methods of the present subject matter, the payload can include a sugar (e.g., sucrose or a disaccharide). According to exemplary methods, the payload includes less than 121 mM sugar, 6-91 mM, or 26-39 mM sugar. Further, the payload includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload includes 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

[0059] According to an exemplary method of the present subject matter, the payload can include a buffering agent (e.g., a weak acid or a weak base). The buffering agent can include a zwitterion. According to an exemplary method, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The payload can include less than 19 mM of the buffering agent (e.g., 1-15 mM or 4-6 mM or 5 mM of the buffering agent). According to an exemplary method, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payload includes 1, 2, 3, 4, 5, 10, 12, 14 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. More preferably, the payload includes 5 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0060] According to an exemplary method of the present subject matter, the payload comprises ammonium acetate. The payload can comprise less than 46 mM ammonium acetate (e.g., 2-35 mM, 10-15 mM, or 12 mM ammonium acetate). The payload can comprise 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.

[0061] The volume of aqueous solution that is injected with the gas can include compressed air (e.g., ambient air). Other embodiments can include inert gases, such as helium, neon, and argon.

[0062] In certain aspects of the present subject matter, the population of cells can include adherent cells (eg, lung, kidney, immune cells such as macrophages, etc.) or non-adherent cells (eg, suspension cells).

[0063] In certain aspects of the present subject matter, the population of cells can be substantially confluent, and can be substantially greater than 75 percent confluent. In preferred embodiments, the population of cells can form a single monolayer.

[0064] According to an exemplary method, the payload to be delivered has an average molecular weight of up to 20,000,000 Da. In some instances, the payload to be delivered can have an average molecular weight of up to 2,000,000 Da. In some embodiments, the payload to be delivered can have an average molecular weight of up to 150,000 Da. In further embodiments, the payload to be delivered can have an average molecular weight of up to 15,000 Da, 5,000 Da, or 1,000 Da.

[0065] The payload to be delivered across the plasma membrane of cells can comprise small chemical molecules, peptides or proteins, polysaccharides or nucleic acids or nanoparticles.Small chemical molecules can be less than 1,000 Da, peptides can have molecular weights of about 5,000 Da, siRNAs can have molecular weights around 15,000 Da, antibodies can have molecular weights of about 150,000 Da, and DNA can have molecular weights of more than or equal to 5,000,000 Da.In a preferred embodiment, the payload comprises mRNA.

[0066] According to an exemplary method, the payload comprises 3.0 to 150.0 μM of the molecule to be delivered, more preferably 6.6 to 150.0 μM of the molecule to be delivered (e.g., 3.0, 3.3, 6.6, or 150.0 μM of the molecule to be delivered). In some embodiments, the payload to be delivered has an average molecular weight of up to 15,000 Da, and the payload comprises 3.3 μM of the molecule to be delivered.

[0067] According to an exemplary method, the payload to be delivered has an average molecular weight of up to 15,000 Da and the payload comprises 6.6 μM to be delivered. In some embodiments, the payload to be delivered has an average molecular weight of up to 1,000 Da and the payload comprises 150.0 μM to be delivered.

[0068] According to a further aspect of the present subject matter, there is provided a method for delivering molecules of multiple molecular weights across a plasma membrane, the method including the steps of introducing molecules of multiple molecular weights into an aqueous solution and contacting the aqueous solution with the plasma membrane.

[0069] In some embodiments, the method includes introducing a first molecule having a first molecular weight and a second molecule having a second molecular weight into the payload, where the first molecule and the second molecule can have different molecular weights or the first molecule and the second molecule can have the same molecular weight. According to an exemplary method, the first molecule and the second molecule can be different molecules.

[0070] In some embodiments, the payload to be delivered may include therapeutic or diagnostic agents, such as cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chlorpromazine HCl, thioridazine HCl, polymyxin B sulfate, chloroxine, benfluorex HCl, and phenazopyridine HCl), and fluoxetine. Other therapeutic agents include antimicrobials (aminoglycosides (e.g., gentamicin, neomycin, streptomycin), penicillins (e.g., amoxicillin, ampicillin), glycopeptides (e.g., avoparcin, vancomycin), macrolides (e.g., erythromycin, tilmicosin, tylosin), quinolones (e.g., sarafloxacin, enrofloxin), streptogramins (e.g., viginiamycin, quinupristin-dalfoprisitin), carbapenems, lipopeptides, oxazolidinones, cycloserine, ethambutol, ethionamide, isoniazrid, para-aminosalicylic acid, and pyrazinamide). In some instances, antiviral drugs (e.g., abacavir, acyclovir, enfuvirtide, entecavir, nelfinavir, nevirapine, Nexavir, oseltamivir raltegravir, ritonavir, stavudine, and valacyclovir). Therapeutic agents can include protein-based therapies for treating various diseases, such as cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B or C.

[0071] Further exemplary payloads can also include detectable markers or labels, such as methylene blue, patent blue V, and indocyanine green.

[0072] The methods described herein can also include a payload that includes a detectable moiety or a detectable nanoparticle (e.g., a quantum dot). The detectable moiety can be a fluorescent molecule or a radioactive agent (e.g., 125 When a fluorescent molecule is exposed to the proper wavelength of light, its presence can be detected via fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde, and fluorescamine. Molecules can be 152 Molecules can also be detectably labeled with fluorescent metals such as Eu or other elements of the lanthanide series. These metals can be attached to the molecule using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). A molecule can also be detectably labeled by linking it to a chemiluminescent compound. The presence of the chemiluminescent-tagged molecule is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0073] In further embodiments, the payload to be delivered may include a composition (i.e., a gene editing tool) that edits genomic DNA. For example, the gene editing composition may include a compound or complex that cuts genomic DNA, nicks genomic DNA, splices, rearranges, moves, recombines, or otherwise modifies genomic DNA. Alternatively or additionally, the gene editing composition may include (i) a compound that can be included in a gene editing complex that cuts genomic DNA, nicks genomic DNA, splices, rearranges, moves, recombines, or otherwise modifies genomic DNA, or (ii) a compound that can be processed or modified into a compound that can be included in a gene editing complex that cuts genomic DNA, nicks genomic DNA, splices, rearranges, moves, recombines, or otherwise modifies genomic DNA. In various embodiments, the gene editing composition includes one or more of (a) a gene editing protein, (b) an RNA molecule, and / or (c) a ribonucleoprotein (RNP).

[0074] In some embodiments, the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or an Flp recombinase.In further embodiments, the gene editing protein can be a fusion protein that combines a homing endonuclease with the modular DNA binding domain of TALEN (megaTAL).For example, the megaTAL can be delivered as a protein, or the mRNA that codes for the megaTAL protein can be delivered to the cell.

[0075] In various embodiments, the gene editing composition comprises an RNA molecule, wherein the RNA molecule comprises an sgRNA, a crRNA, and / or a tracrRNA.

[0076] In certain embodiments, the gene editing composition comprises an RNP, and the RNP comprises a Cas protein and a sgRNA or a crRNA and a tracrRNA. Aspects of the present subject matter are particularly useful for controlling when or how long a particular gene editing compound is present in a cell.

[0077] In various embodiments of the subject matter, the gene editing composition is detectable in the population of cells or their progeny (a) for about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours after contacting the population of cells with the aqueous solution, or (b) for less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours after contacting the population of cells with the aqueous solution.

[0078] In some embodiments, the genome of a cell in the population of cells or its progeny comprises at least one site-specific recombination site for Cre recombinase, Hin recombinase, or Flp recombinase.

[0079] Aspects of the present invention relate to a cell that contains one gene editing compound and inserts another gene editing compound into the cell. For example, one component of RNP can be introduced into a cell that expresses or otherwise already contains another component of RNP. For example, a cell in a population of cells or its progeny can contain sgRNA, crRNA, and / or tracrRNA. In some embodiments, a population of cells or its progeny expresses sgRNA, crRNA, and / or tracrRNA. Alternatively or additionally, a cell in a population of cells or its progeny expresses Cas protein.

[0080] Various embodiments of the subject matter herein include a Cas protein. In some embodiments, the Cas protein is a Cas9 protein or a variant thereof. Exemplary Cas proteins, including non-limiting examples of Cas9 and Cas9 variants, are described herein.

[0081] In various aspects, the concentration of Cas9 protein can range from about 0.1 to about 25 μg. For example, the concentration of Cas9 can be about 1 μg, about 5 μg, about 10 μg, about 15 μg, or about 20 μg. Alternatively, the concentration of Cas9 can range from about 10 ng / μL to about 300 ng / μL, for example, from about 10 ng / μL to about 200 ng / μL, or from about 10 ng / μL to about 100 ng / μL, or from about 10 ng / μL to about 50 ng / μL.

[0082] In certain embodiments, the gene editing composition comprises: (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; or (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule. the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule; (d) comprises a first crRNA molecule and a second crRNA molecule, where the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule, and further comprises a tracrRNA molecule; or (e) comprises a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, where the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

[0083] In aspects, the ratio of Cas9 protein to guide RNA can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0084] In embodiments, the rate of editing can be increased by increasing the number of times the cells undergo the delivery process (or increasing the number of administrations), hi some embodiments, the number of administrations can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations.

[0085] In various embodiments, the first and second sgRNA or first and second crRNA molecules collectively comprise a nucleic acid sequence complementary to a target sequence adjacent to a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, where the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length or is at least about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length. In some embodiments, the use of a pair of RNPs comprising the first and second sgRNA or first and second crRNA molecules may be used to generate a polynucleotide molecule comprising a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.

[0086] In certain embodiments, the target sequence of the sgRNA or crRNA is about 12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17 to 23, or 18 to 22 nucleotides in length. In some embodiments, the target sequence is 20 nucleotides in length or about 20 nucleotides in length.

[0087] In various embodiments, the first and second sgRNA or the first and second crRNA molecules are complementary to sequences flanking an extrachromosomal sequence located within the expression vector.

[0088] Aspects of the subject matter relate to delivery of multiple components of a gene editing complex, where the multiple components are not complexed with each other. In some embodiments, the gene editing composition comprises at least one gene editing protein and at least one nucleic acid, where the gene editing protein and the nucleic acid are not bound to or complexed with each other.

[0089] The subject matter allows for high gene editing efficiency while maintaining high cell viability. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the population of cells or their progeny become genetically modified after contact with the aqueous solution. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the population of cells or their progeny are viable after contact with the aqueous solution.

[0090] In certain embodiments, the gene editing composition induces a single-stranded or double-stranded break in DNA in the cell. In some embodiments, the gene editing composition further comprises a repair template polynucleotide. In various embodiments, the repair template comprises (a) a first flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on one side of the single-stranded or double-stranded break and a second flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on the other side of the single-stranded or double-stranded break, or (b) a first flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-stranded or double-stranded break and a second flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-stranded or double-stranded break. A non-limiting description of gene editing using the CRISPR-Cas system (including repair templates) is discussed in Ran et al. (2013) Nat Protoc. 2013 Nov; 8(11):2281-2308, the entire contents of which are incorporated herein by reference. Embodiments involving repair templates are not limited to those involving the CRISPR-Cas system.

[0091] In various embodiments of the present subject matter, a volume of the aqueous solution is delivered to a population of cells in the form of a spray. In some embodiments, the volume is 6.0×10 per cell. -7 Microliters ~ 7.4 x 10 per cell -4 In certain embodiments, the spray comprises colloidal particles or subparticles with diameters between 10 nm and 100 μm. In various embodiments, the volume is 2.6×10 per square micrometer of exposed surface. -9 Microliters ~ 1.1 x 10 per square micrometer of exposed surface area -6 It is a microliter.

[0092] In some embodiments, the RNPs have a size of approximately 100 Å×100 Å×50 Å, i.e., 10 nm×10 nm×5 nm. In various embodiments, the size of the spray particles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNPs per spray particle.

[0093] For example, the step of contacting the population of cells with the volume of aqueous solution may be performed by a gas that propels the aqueous solution to form a spray. In certain embodiments, the population of cells is contacted with the aqueous solution for 0.01 to 10 minutes (e.g., 0.1 to 10 minutes) before adding a second volume of buffer or culture medium to submerge or suspend the population of cells.

[0094] In various embodiments, the population of cells comprises at least one of primary cells or immortalized cells. For example, the population of cells can comprise mesenchymal stem cells, lung cells, neural cells, fibroblasts, human umbilical vein (HUVEC) cells and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells. Non-limiting examples of T cells can include CD8+ or CD4+ T cells. In some aspects, CD3 + The CD8+ subpopulation of T cells is used. + T cells can be purified from the PBMC population by positive isolation using anti-CD8 beads. In some aspects, primary NK cells can be isolated from PBMCs and delivered with GFP mRNA by platform delivery technology (i.e., 3% expression and 96% viability at 24 hours). In further aspects, NK cell lines such as NK92 can be used.

[0095] Cell types also include cells that have already been modified to enhance therapeutic efficacy, such as T cells, NK cells and MSCs. For example, T cells or NK cells expressing chimeric antigen receptors (CAR T cells, CAR NK cells, respectively); T cells expressing modified T cell receptors (TCR); MSCs that have been virally or non-virally modified to overexpress therapeutic proteins that complement their innate properties (e.g. delivery of Epo using lentiviral vectors or BMP-2 using AAV-6) (reviewed in Park et al, Methods, 2015 Aug;84-16); MSCs primed with non-peptide drugs or magnetic nanoparticles, respectively, to enhance efficacy and exogenously modulate targeting (Park et al., 2015); MSCs that have been modified to express therapeutic proteins that complement their innate properties by enzymatic modification (e.g. fucosyltransferase), chemical conjugation (e.g. modification of SLeX on MSCs using N-hydroxy-succinimide (NHS) chemistry) or non-covalent interactions (e.g. palmitated proteins acting as hydrophobic anchors for subsequent antibody conjugation). MSCs functionalized with targeting moieties to enhance homing to the treatment site using cell surface engineering with a gene editing protein (Park et al., 2015). For example, T cells, e.g., primary T cells or T cell lines, modified to express a chimeric antigen receptor (CAR T cells) can be further treated with a complex containing a gene editing protein and / or a guide nucleic acid specific for the CAR coding sequence, according to the invention, to edit the gene encoding the CAR and thereby reduce or stop expression of the CAR in the modified T cells.

[0096] Aspects of the present invention relate to the expression vector-free delivery of gene editing compounds and gene editing complexes to cells and tissues, such as the delivery of Cas-gRNA ribonucleoproteins for genome editing in primary human T cells, hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs). In some examples, the mRNA encoding the above-mentioned proteins is delivered to cells.

[0097] Various aspects of the CRISPR-Cas system are known in the art. Non-limiting aspects of this system are described, for example, in U.S. Patent No. 9,023,649, issued May 5, 2015; U.S. Patent No. 9,074,199, issued July 7, 2015; U.S. Patent No. 8,697,359, issued April 15, 2014; U.S. Patent No. 8,932,814, issued January 13, 2015; PCT International Patent Application Publication No. WO 2015 / 071474, published August 27, 2015; Cho et al., (2013) Nature Biotechnology Vol 31 No 3 pp 230-232 (including supplementary information); and Jinek et al., (2012) Science Vol 337 No 6096 pp 816-821, the entire contents of each of which are incorporated herein by reference.

[0098] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csx1 ... The Cas9 enzymes include sm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified forms thereof. These enzymes are known. For example, the amino acid sequence of S. pyogenes Cas9 protein can be found in SwissProt database under accession number Q99ZW2 and in NCBI database under accession number Q99ZW2.1. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 are other examples of Cas9 protein amino acid sequences. Another non-limiting example is the Streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme, such as Cas9, has DNA cleavage activity. In certain embodiments, the CRISPR enzyme is Cas9, which can be Cas9 from Streptococcus pyogenes or S. pneumoniae. In various embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, for example, within the target sequence and / or within the complementary strand of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.In some embodiments, the vector encodes a mutated CRISPR enzyme, so that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence, compared with the corresponding wild-type enzyme.For example, the aspartic acid to alanine substitution in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cuts one strand).Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A.In aspects of the present invention, nickases can be used for genome editing by homologous recombination.

[0099] In certain embodiments, Cas9 nickase may be used in combination with a guide sequence, for example, two guide sequences that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ.

[0100] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to generate mutant Cas9 that substantially lacks all DNA cleavage activity. D10A mutation can be combined with one or more of H840A, N854A, or N863A mutations to generate mutant Cas9 that substantially lacks all DNA cleavage activity. In certain embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less compared to the non-mutated form. Other mutations can also be useful. If Cas9 or other CRISPR enzyme is derived from a species other than Streptococcus pyogenes, mutations in the corresponding amino acids can be introduced to achieve a similar effect.

[0101] In certain embodiments, the delivered protein (e.g., a Cas protein or variant thereof) may include an intracellular localization signal. For example, a Cas protein in an RNP may include an intracellular localization signal. Depending on the context, for example, a fusion protein including Cas9 and a nuclear localization signal may be referred to herein as "Cas9" without specifying that a nuclear localization signal is included. In some embodiments, the payload (such as an RNP) includes a fusion protein that includes a localization signal. For example, the fusion protein may contain a nuclear localization signal, a nucleolar localization signal, or a mitochondrial targeting signal. Such signals are known in the art and non-limiting examples are described in Kalderon et al., (1984) Cell 39(3 Pt 2):499-509, Makkerh et al., (1996) Curr Biol. 6(8):1025-7, Dingwall et al., (1991) Trends in Biochemical Sciences 16(12):478-81, Scott et al., (2011) BMC Bioinfomatics 12:317(page 7), Omura T (1998) J Biochem. 123(6):1010-6, Rapaport D (2003) EMBO Rep. 4(10):948-52 and Brocard & Hartig (2006) Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1763(12):1565-1573, the contents of each of which are incorporated herein by reference. In various embodiments, the Cas protein can contain multiple localization signals, for example, two, three, four, five, or more nuclear localization signals. In some embodiments, the localization signals are at the N-terminus of the Cas protein, and in other embodiments, the localization signals are at the C-terminus of the Cas protein.

[0102] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as, for example, a mammal, including but not limited to, human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a native sequence with the codon that is more frequently or most frequently used in the gene of the host cell, while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit a certain bias for a certain codon of a certain amino acid. Codon bias (differences in codon usage among organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, and is thought to depend, among other things, on the properties of the codons being translated and on the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis.

[0103] Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, such as in the "Codon Usage Database," and these tables can be adapted in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms can also be used to codon-optimize a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen, Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons or all codons) in the sequence encoding the CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0104] In general, guide sequence is any polynucleotide sequence that has sufficient complementarity with target polynucleotide sequence to hybridize with target sequence and direct sequence-specific binding of CRISPR complex to target sequence.In some embodiments, the degree of complementarity between guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, when optimally aligned using a suitable alignment algorithm.In some embodiments, the degree of complementarity is 100%. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is at least about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 42, 44, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, , 45, 50, 75 or more nucleotides in length, or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In certain embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length, or even less.The ability of guide sequence to direct sequence-specific binding of CRISPR complex to target sequence can be evaluated by any suitable assay.For example, sufficient components of CRISPR system to form CRISPR complex containing guide sequence to be tested can be provided to host cell with corresponding target sequence, for example by transfection with vector encoding components of CRISPR sequence, and then evaluation of preferential cleavage in target sequence can be performed, for example by Surveyor assay as described herein.Similarly, cleavage of target polynucleotide sequence can be evaluated in vitro by providing target sequence, components of CRISPR complex containing guide sequence to be tested, and control guide sequence different from test guide sequence, and comparing binding or cleavage rate in target sequence between test guide sequence reaction and control guide sequence reaction.

[0105] CRISPR-Cas technology is rapidly developing to facilitate genome engineering in a wide range of cell types. It has recently been revealed that delivery of Cas9-gRNA editing tools in the form of ribonucleoproteins (RNPs) offers several benefits compared to delivery of plasmids encoding Cas9 and gRNAs. The benefits include faster and more efficient editing, fewer off-target effects, and less toxicity. RNPs have been delivered by lipofection and electroporation, but these delivery methods still have limitations, including toxicity and low efficiency, especially for certain clinically important cell types. Therefore, there is a need to provide a vector-free, e.g., viral vector-free, approach to deliver biologically important payloads, such as RNPs, across the plasma membrane into cells. "Cargo" or "payload" is a term used to describe a compound or composition that is delivered as an aqueous solution across the cell plasma membrane to the interior of the cell.

[0106] The subject matter relates to a delivery technique that facilitates the delivery of a wide range of payloads to cells with low toxicity. Genome editing can be achieved by using some aspects of the subject matter to deliver RNP to cells. The level then decreases until Cas9 is no longer detectable. The delivery technique itself does not adversely affect the viability or functionality of Jurkat T cells and primary T cells. The subject matter allows gene editing with Cas9 RNP in clinically important cell types with minimal toxicity.

[0107] Transient direct delivery of CRISPR / Cas components such as Cas and / or gRNA has advantages compared to delivery by expression vectors. For example, compared to using expression vectors, a certain amount of Cas, gRNA, or RNP can be added at a more precise time and for a limited length of time. Components expressed from vectors can be produced in different amounts and for different lengths of time, which makes it difficult to achieve consistent gene editing without off-target editing. In addition, preformed complexes of Cas and gRNA (RNP) cannot be delivered by expression vectors.

[0108] In one aspect, the subject matter describes cells attached to a solid support (e.g., a strip, a polymer, a bead, or a nanoparticle). The support or scaffold can be a porous or non-porous solid support. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. For the purposes of the subject matter, the nature of the carrier can be soluble to some degree or insoluble. The support material can have virtually any conceivable structural configuration. Thus, the configuration of the support can be spherical, as in the case of beads, or cylindrical, as in the case of the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, for example, a sheet or a test strip. Preferred supports include polystyrene beads.

[0109] In another aspect, the solid support comprises a polymer, and the cells are chemically bonded, immobilized, dispersed or associated therewith. The polymer support may be a polymer network, and may be prepared in a beaded form (e.g., by suspension polymerization). The cells on such scaffolds may be sprayed with an aqueous solution containing the payload of the present invention to deliver the desired compound to the cytoplasm of the scaffold. Exemplary scaffolds include stents and other implantable medical devices or structures.

[0110] The subject matter further relates to devices, systems, techniques, and articles for delivering a payload across a plasma membrane. The subject matter also relates to devices for delivering a payload, such as a protein or protein complex, across a plasma membrane. The subject matter can be used in the field of intracellular delivery, and has applications in the delivery of molecular biological and pharmacological therapeutic agents to a target site, such as a cell, tissue, or organ.

[0111] In some embodiments, the device for delivering a payload across a plasma membrane can include an atomizer having at least one atomizer emitter and a support adapted for the atomizer. The method further includes atomizing the payload prior to contacting the plasma membrane with the payload.

[0112] The atomizer can be selected from a mechanical atomizer, an ultrasonic atomizer, an electrospray, a nebulizer, and a venturi. The atomizer can be a commercially available atomizer. The atomizer can be an intranasal mucosal atomization device. The atomizer can be an intranasal mucosal atomization device available from LMA Teleflex, North Carolina, USA. The atomizer can be an intranasal mucosal atomization device available from LMA Teleflex, North Carolina, USA under catalog number MAD300.

[0113] The atomizer may be adapted to provide a colloidal suspension of particles having a diameter of 30-100 μm prior to contacting the plasma membrane with the payload. The atomizer may be adapted to provide a colloidal suspension of particles having a diameter of 30-80 μm. The atomizer may be adapted to provide a colloidal suspension of particles having a diameter of 50-80 μm.

[0114] The atomizer may include a gas reservoir. The atomizer may include a gas reservoir in which a gas is maintained under pressure. The gas may be selected from air, carbon dioxide, and helium. The gas reservoir may include a fixed pressure head generator. The gas reservoir may be in fluid communication with the atomizer emitter. The gas reservoir may include a gas guide, which may be in fluid communication with the atomizer emitter. The gas guide may be adapted to allow gas to pass therethrough. The gas guide may include a hollow body. The gas guide may be a hollow body having an open end. The gas guide may include a hollow body having first and second open ends. The gas guide may be a hollow body having first and second opposed open ends. The diameter of the first open end may be different from the diameter of the second open end. The diameter of the first open end may be different from the diameter of the second open end. The diameter of the first open end may be greater than the diameter of the second open end. The first open end can be in fluid communication with the gas reservoir, and the second open end can be in fluid communication with the atomizer emitter.

[0115] The device may include a sample reservoir. The sample reservoir may be in fluid communication with an atomizer. The sample reservoir may be in fluid communication with an atomizer emitter. Both the gas reservoir and the sample reservoir may be in fluid communication with the atomizer emitter.

[0116] The apparatus may include a sample valve located between the sample reservoir and the gas reservoir. The apparatus may include a sample valve located between the sample reservoir and the gas guide. The sample valve may be adapted to regulate sample flow from the sample reservoir. The sample valve may be adapted to allow for continuous or semi-continuous sample flow. The sample valve may be adapted to allow for semi-continuous sample flow. The sample valve may be adapted to allow for a defined volume of semi-continuous sample flow. The sample valve is adapted to allow for a semi-continuous sample flow of 0.5-100 μL. The sample valve may be adapted to allow for a semi-continuous sample flow of 10 μL. The sample valve may be adapted to allow for a semi-continuous sample flow of 0.065-0.085 cm. 2 The device can be adapted to allow semi-continuous sample flow of 1 μL onto an area of ​​10 μL.

[0117] The atomizer and the support can be spaced apart. The support can include a solid support. The support can include a plate including sample wells. The support can include a plate including sample wells selected from 1, 6, 9, 12, 24, 48, 384, 1536, or more wells. Alternatively, the support includes a scaled-up configuration that can accommodate a monolayer of more cells than a plate, e.g., a microtiter plate. The solid support can be formed from an inert material. The solid support can be formed from a plastic material or a metal or alloy or combinations thereof. The support can include a heating element. The support can include a resistive element. The support can be reciprocally mountable to the device. The support can be reciprocally movable to the device. The support can be reciprocally movable to the atomizer. The support can be reciprocally movable to the atomizer emitter. The support can include a support actuator for reciprocating the support to the atomizer. The support can include a support actuator for reciprocating the support to the atomizer emitter. The support may include a support actuator for reciprocating the support relative to a longitudinal axis of the atomizer emitter.The support may include a support actuator for reciprocating the support transversely to the longitudinal axis of the atomizer emitter.

[0118] The longitudinal axis of the spray zone can be coaxial with the longitudinal axis or center point of the support and / or the circular well of the support to which the payload is delivered. The longitudinal axis of the atomizer emitter can be coaxial with the longitudinal axis or center point of the support and / or the circular well of the support. The longitudinal axis of the atomizer emitter, the longitudinal axis of the support and the longitudinal axis of the spray zone can each be coaxial. The longitudinal length of the spray zone can be greater (more than twice) than the diameter of the circular base of the spray zone (e.g., the area of ​​the cells to which the payload is to be delivered).

[0119] The device may comprise a valve located between the gas reservoir and the atomizer. The valve may be an electromagnetically actuated valve. The valve may be a solenoid valve. The valve may be a pneumatic valve. The valve may be located in the gas guide. The valve may be adapted to regulate gas flow in the gas guide. The valve may be adapted to allow for continuous or semi-continuous gas flow. The valve may be adapted to allow for semi-continuous gas flow. The valve may be adapted to allow for semi-continuous gas flow for defined time intervals. The valve may be adapted to allow for semi-continuous gas flow for 1 second intervals. The device may comprise at least one filter. The filter may comprise a pore size of less than 10 μm. The filter may have a pore size of 10 μm. The filter may be located in the gas guide. The filter may be in fluid communication with the gas guide.

[0120] The device may comprise at least one regulator. The regulator may be an electrical regulator. The regulator may be a mechanical regulator. The regulator may be located in the gas guide. The regulator may be in fluid communication with the gas guide. The regulator may be an adjustment valve. The pressure in the gas guide may be 1.0-2.0 bar. The pressure in the gas guide may be 1.5 bar. The pressure in the gas guide may be 1.0-2.0 bar and the distance between the atomizer and the support may be less than or equal to 31 mm. The pressure in the gas guide may be 1.5 bar and the distance between the atomizer and the support may be 31 mm. The pressure in the gas guide may be 0.05 bar per millimeter of distance between the atomizer and the support. The adjustment valve may be adapted to adjust the pressure in the gas guide to 1.0-2.0 bar. The adjustment valve may be adapted to adjust the pressure in the gas guide to 1.5 bar. The or each regulating valve may be adapted to maintain a pressure in the gas guide of between 1.0 and 2.0 bar.The or each regulating valve may be adapted to maintain a pressure in the gas guide of 1.5 bar.

[0121] The apparatus may include two regulators. The apparatus may include a first and a second regulator. The first and second regulators may be located in the gas guide. The first and second regulators may be in fluid communication with the gas guide. The first regulator may be located between the gas reservoir and the filter. The first regulator may be adapted to regulate pressure from the gas reservoir in the gas guide to 2.0 bar. The first regulator may be adapted to maintain pressure in the gas guide at 2.0 bar. The second regulator may be located between the filter and the valve.

[0122] The atomizer emitter can be adapted to provide a cone-shaped spray zone (e.g., a generally cone-shaped spray zone). The atomizer emitter can be adapted to provide a 30° cone-shaped spray zone. The device can further include a microprocessor for controlling any or all parts of the device. The microprocessor can be arranged to control any or all of the sample valve, the support actuator, the valve, and the regulator. The device can include an atomizer having at least one atomizer emitter and a support adapted for the atomizer, the atomizer can be selected from a mechanical atomizer, an ultrasonic atomizer, an electrospray, a nebulizer, and a Venturi tube. The atomizer can be adapted to provide a colloidal suspension of particles having a diameter of 30 to 100 μm. The device can include a sample reservoir and a gas guide and a sample valve located between the sample reservoir and the gas guide. The sample valve can be adapted to allow a semi-continuous sample flow of 10 to 100 μL. The atomizer and the support can be spaced apart to define a generally cone-shaped spray zone therebetween. Also, the distance between the atomizer and the support can be about twice the diameter of the circular base of the area of ​​cells to which the molecules are to be delivered, and the distance between the atomizer and the support can be 31 mm and the diameter of the circular base of the area of ​​cells to which the molecules are to be delivered can be 15.5 mm. The apparatus can include a gas guide, and the pressure in the gas guide is 1.0-2.0 bar. The apparatus can include at least one filter having a pore size of less than 10 μm.

[0123] The aqueous solution and / or composition may be saponin-free.

[0124] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will become apparent from the following description and drawings, and from the claims.

[0125] Optimization of T cell preparations for sorption Optimization of T cell culture conditions to maximize the efficiency of mRNA delivery using a vector-free reversible permeabilization method is described below. The vector-free method for intracellular delivery of macromolecules and nucleic acids described herein successfully facilitates the delivery of gene editing tools such as CRISPR / Cas9 and mRNA into mammalian cells, such as primary human immune cells. Culture conditions were determined to efficiently introduce mRNA into human T cells using a vector-free delivery platform.

[0126] Considerable variation exists in the ex vivo activation of human lymphocytes among clinical groups and T cell engineering companies. There is no single standardized protocol for the expansion of primary human T cells. Therefore, we undertook a comprehensive evaluation of culture media, additives, activation methods, and timing schedules to identify optimal ex vivo culture conditions that promote maximal efficiency of mRNA delivery using vector-free reversible permeabilization technology. Here, we describe the cell isolation protocols and results / conclusions of each experiment undertaken.

[0127] To address suspension cells (i.e., non-adherent cells), a centrifugation step was developed as part of the delivery process. This step was developed to allow for the formation of an exposed monolayer of suspension cells. This centrifugation step allows for both the formation of the monolayer and the removal of the supernatant in one step. In contrast, for adherent cells, the cells had already formed a monolayer and the medium was removed by pipetting.

[0128] material and method Cell culture and transfection. Human peripheral blood mononuclear cells (PBMCs) were harvested from Leuko Pak (AllCells, Alameda, CA) by centrifugation on a Percoll gradient. CD3 +Enriched lymphocytes were isolated. Cells were cryopreserved in 10% dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). After the first thaw from a storage aliquot, CD3 + T cells were cultured at 37°C and 5% CO 2 The cells were cultured in human recombinant interleukin-2 (IL-2) in a humidified tissue culture incubator at 37 °C, along with primary and costimulatory antibody activation using various protocols (see below).

[0129] Delivery technique. Activated T cells were plated onto 96-well filter plates (Acroprep, 1.2 μm Supor membrane, Pall, USA) at 1.5 × 10 per well. 6 Cells were seeded into wells. The medium was removed from the wells by centrifugation at 300×g for 5 min. Then, 7 μl of delivery solution (32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 27% ethanol in molecular grade water (all from Sigma-Aldrich)) containing 4 μg of GFP mRNA was sprayed into each well using a vector-free delivery spraying device. The atomizer used in this device is the MAD Nasal™ Intranasal Mucosa Atomization Device (Wolfe Tory Medical Inc, Salt Lake City, USA). The atomizer was held on a retort stand 26 mm above the bottom of the well and connected to a 6 bar compressor (Circuit Imprime Francais, Bagneux-Cedex, France) via polyurethane tubing (6 mm outer diameter, 4 mm inner diameter, SMC, Tokyo, Japan). The delivery solution containing the cargo was pipetted into the delivery port located at the top of the atomizer and a spray was generated at 1.5 bar using a spray actuator button (SMC, Tokyo, Japan). After delivery, cells were incubated in this solution for 2 min before adding 50 μl of stop solution (0.5× PBS). After 30 s, T cell medium was added (100 μl) and the cells were incubated at 37° C. and 5% CO. 2 The cells were allowed to recover overnight at RT. Uptake and viability were assessed 24 hours after delivery.

[0130] Cell viability, FACS sample preparation, and analysis. To assess cell viability after the vector-free delivery method, cells were stained using 7-aminoactinomycin D (7-AAD) (Sigma). Briefly, cells were washed in PBS + 1% fetal bovine serum (FACS buffer) and then incubated with 7-AAD (1:40, 5-10 min at room temperature, protected from light), then resuspended in PBS + 1% FBS (FACS buffer). Samples were processed on a BD Accuri C6 flow cytometer (Becton Dickinson, USA) and data were analyzed using C6 software. Cell debris was excluded from total cells using forward and side scatter parameters. Single cells were selected by excluding doublets in a plot of FSC height vs. FSC area. GFP expression was analyzed in gated live cells.

[0131] Media, activation reagents, and timing Four media for the culture and expansion of T cells were evaluated using mRNA as cargo, e.g., the model cargo GFP mRNA, and GFP expression as an indicator of successful culture methods. cRPMI, a serum-containing medium typically used for the culture of primary immune cells, was tested. However, because serum is a highly variable supplement in cell culture media, three serum-free and xeno-free expansion media optimized for the in vitro culture of human T cells were also evaluated.

[0132] cRPMI was prepared using RPMI, heat-inactivated fetal bovine serum (FBS) (10% v / v), penicillin-streptomycin, L-glutamine, and supplemented with IL-2 (100 U / ml). cRPMI was used as the culture medium in experiments evaluating the performance of various T cell expansion protocols. The first expansion protocol tested was ImmunoCult™ Human CD3 / CD28 T Cell Activator, which consists of a soluble tetrameric antibody complex that binds to the CD3 and CD28 cell surface ligands on T lymphocytes. This was evaluated alongside an alternative activation method that uses "feeder" cells as a means of presenting antigen to the T cell receptor (TCR) to induce T cell proliferation (Figure 1).

[0133] T cell activation using PBMC or A549 as feeder cells Autologous PBMCs were transferred to tissue culture plates. After 2 hours of incubation, the supernatant was removed, leaving the adherent monocytes. T cells cultured in cRPMI were added to the plates and allowed to co-incubate with the monocytes for several days. In a similar protocol, A549 cell lines were allowed to adhere for up to 2 hours. Media was removed and T cells were added to the plates and allowed to co-incubate before mRNA was delivered using vector-free delivery technology. As uptake efficiency varied within and between experiments when feeder cells were used (Figure 1), further evaluations used ImmunoCult™ Human CD3 / CD28 T Cell Activator Reagent.

[0134] T Cell Activation Using Dynabeads® T cells are activated using methods known in the art, for example, using antibodies that bind cell surface proteins such as CD3 and / or CD28, feeder cells, and / or magnetic beads containing immunostimulatory molecules. For example, Dynabeads® were investigated as an alternative to ImmunoCult™ human CD3 / CD28 T cell activator. Dynabeads® are superparamagnetic beads coated with antibodies against human CD3 and CD28 that provide the primary and costimulatory signals required for T cell activation and expansion. Although the recommended bead-to-cell ratio is 1:1, other conditions using 3 beads per cell were also used to evaluate whether more efficient and rapid expansion would positively affect the uptake of mRNA into T cells using solvolysis delivery. Increasing the bead-to-cell ratio significantly improved the uptake rate (Figure 2A). Next, a repeat experiment was performed in which ImmunoCult™ Human CD3 / CD28 T Cell Activator was included at three times the concentration recommended by the manufacturer, but this concentration did not result in the same improvement in efficiency as observed with Dynabeads (Figure 2B).

[0135] Culture medium: Prime XV The delivery method used a culture medium compatible with the cells. For example, Prime-XV (Irvine Scientific) was the first serum-free and animal component-free medium tested in a vector-free delivery technology. Based on the positive data observed using Dynabeads at a 3:1 bead-to-cell ratio (Figure 2 and Figures 2A and 2B), we decided to continue testing alternative culture media using this activation method. We tested this medium using Dynabeads to induce proliferation along with an alternative activation method in which T cell proliferation was stimulated by binding anti-human CD3 antibodies to cell culture plates followed by the addition of soluble anti-CD28 to the medium. Dynabead-activated T cells cultured in Prime XV showed significantly improved uptake efficiency (up to 50%) compared to cells stimulated with soluble α-CD3 / CD28 (<20%) (Figure 3).

[0136] Although delivery to T cells cultured in Prime XV improved mRNA uptake using vector-free technology, this was accompanied by cell handling issues, such as removing cells from culture 24 hours after initial plating and cell recovery from the Dynabeads upon washing (Figure 4A and Figure 4B).

[0137] Culture medium: Supplementary cytokines In some cases, cell culture medium was supplemented with higher concentrations of IL-2 (200U / ml instead of 100U / ml) to enhance proliferation rates (Tumeh P, et al., J Immunother 2010. 33(6):759-768 and Besser MJ, et al., Cytotherapy 2009. 11:206-217).

[0138] Culture medium: Immunocult Immunocult™-XF Expansion Medium (StemCell Technologies) was evaluated. Like Prime XV, it is also a serum-free, xeno-free medium for T cell culture. In this example, T cells were cultured in Immunocult Expansion Medium and activated using Dynabeads® at a bead-to-cell ratio of 3:1. GFP mRNA was delivered to cells on days 1 and 2 post-activation and evaluated 24 hours later (Figure 5).

[0139] Timing of delivery after T cell activation The cells are activated for 15-21 hours, with 19 hours being preferred.

[0140] A preferred "window" for delivery after activation following the addition of Dynabeads was identified. mRNA was delivered at several time points using vector-free technology. Optimal GFP expression was observed when mRNA payload, e.g., model payload GFP mRNA, was delivered 19 hours after activation compared to 17 and 21 hours (Figure 6). Studies were undertaken to determine whether there was a correlation between increased cell size and time after activation. Cell size was estimated using forward scatter (FSC) data obtained from flow cytometry analysis. Maximum transfection efficiency was observed to correlate with the time during which cells were actively increasing in size. Exemplary results are illustrated in the table below.

[0141] (Table) Timing of delivery after T cell activation TIFF0007644048000001.tif35128

[0142] T Cell Activation using TransAct T cell TransAct™ (Miltenyi) is a colloidal reagent consisting of a nanomatrix conjugated to CD3 and CD28 agonists that provide signals for T cell activation and expansion. This is advantageous compared to Dynabeads® because excess reagent can be removed by centrifugation, eliminating all the need for magnetic separation of the beads from the cells and subsequent washing. Thus, this reagent was evaluated over a 3-day period using Immunocult as the medium to determine the preferred day for delivery of mRNA by the vector-free delivery technique described herein. TransAct initiated T cell proliferation less vigorously than Dynabeads. Therefore, optimal delivery of mRNA to T cells was observed 24 hours later than that shown with bead activation, but this was accompanied by enhanced transfection efficiency (Figure 7).

[0143] Culture medium: TexMACS We also tested TexMACS (Miltenyi Biotech) as an alternative serum-free medium for T cell culture. This T cell stimulation and expansion reagent was useful but was not subsequently used routinely (Figure 8).

[0144] Effect of a post-thaw recovery period before activation The benefit to delivery of recovering cells overnight before addition of activation reagent was evaluated compared to adding recovery agent immediately after thawing with no recovery period. Upon thawing T cells from liquid nitrogen storage, cells were allowed to recover overnight in culture medium alone before addition of activation reagent. This step resulted in a 30% improvement in transfection efficiency. In some examples, fresh primary non-adherent cells are used in the method, while in other examples, primary non-adherent cells are frozen, e.g., for storage, and then thawed prior to the payload delivery method. Thus, the method may optionally include a freeze / thaw step of primary non-adherent cells. These data indicate that a recovery period (after thawing) prior to activation is useful.

[0145] cell culture density Prior to vector-free delivery, cells were cultured at various seeding densities (1 × 10 6 / ml and 5×10 6 The cell density before sorption was 1–5 × 10 6 Increasing the seeding number led to improved uptake efficiency (Figure 10).

[0146] T cell activation using zinc Zinc influx can support T cell activation (Yu M, et al., J. Exp. Med. 208(4):775-785) but can also improve nucleic acid transfection (Niedzinski EJ, et al. Mol Ther 2003 7(3):396-400). Zinc improved T cell proliferation in two independent experiments (Figure 11). There was a trend towards improved transfection efficiency. Thus, zinc is an optional component of cell culture media for the activation step. The zinc concentration range is 0.03 mM to 3 mM.

[0147] Optimization of T cell culture conditions to maximize the efficiency of mRNA delivery Evaluation of multiple culture media, activation methods, supplements, and time courses resulted in a preconditioning protocol that maximizes transfection efficiency of mRNA, e.g., model payload GFP mRNA, into human primary T cells using vector-free delivery technology. This evaluation involved the incubation of 5×10 6 Cells were plated at a density of 100 / ml. Cells were allowed to recover overnight after thawing and then 1× T cell TransAct™ (Miltenyi) was added for 48 hours prior to vector-free delivery of nucleic acid.

[0148] Human peripheral blood mononuclear cells (PBMCs) were harvested from Leuko Pak (AllCells, Alameda, CA) by centrifugation on a Percoll gradient. CD4-enriched T cells were isolated by negative selection and the flow-through was collected from an LD column (Miltenyi) to harvest purified populations using anti-CD8 microbeads. Cells were cultured in standard cell culture medium, e.g., complete RPMI with basal medium, heat-inactivated fetal bovine serum (FBS) (10% v / v), penicillin-streptomycin, L-glutamine, and supplemented with IL-2 (200 U / ml). Cells were allowed to recover for 4 hours before adding Dynabeads® at a 3:1 bead-to-cell ratio. mRNA was delivered to cells 1 day after activation and uptake was assessed 24 hours later. Improved uptake efficiency was achieved with multiple hits in this cell type, >30% (Figure 12).

[0149] T cells were enriched from PBMCs cultured in X-VIVO 15 supplemented with 2 mM GlutaMAX, 10 mM HEPES, and 5% human AB serum and 250 IU / ml IL-2. Cells were cultured at 1×10 6Cells were seeded at a density of 100 / mL and supplemented with anti-CD3 and anti-CD28 antibodies (Miltenyi) prior to culture. Cells were delivered with mRNA, e.g., test payload GFP mRNA, by solporation on either day 2 or day 3 after initiation and assessed for uptake 24 hours later (Figure 13).

[0150] Optimization of T cell monolayer formation A cell monolayer is a culture in which cells are oriented in a single layer on a substrate. The substrate is typically a plate, such as a microtiter plate, flask, petri dish, membrane or filter, on which the cells are placed. In cell culture, a monolayer refers to a layer of cells in which the cells are substantially side-by-side on the same surface, often in contact with each other. The cells are adherent cells (cells that attach to a substrate) or non-adherent cells (cells that float or are suspended in culture medium). Adherent cells grow and attach on a substrate, thereby forming a monolayer. A monolayer can also be generated from non-adherent cells or "suspension cells". The terms "non-adherent cells" and "suspension cells" are used interchangeably herein.

[0151] A number of techniques may be used to generate cell monolayers from non-adherent or suspension cells prior to delivery of a payload to the cells, including settling cultured suspension cells onto a substrate, centrifugation, exposure to vacuum, exposure to positive pressure, use of magnetic T cell activation beads, and / or deposition onto a membrane (e.g., using the transwell insert system described below).

[0152] Transwell Insert A transwell insert system was used to form a monolayer of suspension cells that allowed the cells to be optimally presented to the spray. 1 × 10 cells were cultured in 400 μl per insert. 6Cells were seeded at 100 μl and the medium was removed by placing a transwell insert (Greiner bio-one; Cat. No. 655640; 12-well ThinCert; PET 0.4 μm) into a device that allows applying a vacuum (-0.5 bar to -0.65 bar) to the bottom of the insert (see Figure 14A, B, C). Once the medium was removed, the remaining cells formed a monolayer onto which a spray could be applied. Non-adherent cells such as PBMCs, primary T cells or cell lines, e.g. Jurkat T cells, were added to the insert system, a vacuum was applied, the insert was placed in a 12-well plate and sprayed with a delivery solution (10 μl) containing a test payload such as either fluorescently labeled β-lactoglobulin (BLG), bovine serum albumin (BSA) or ovalbumin (OVA). Stop solution (50 μl) was applied after 2 min of incubation and normal medium (100 μl) was applied after 30 s. Expression levels of 55.6, 28.5, and 15.3% were achieved (Figure 15). The insert system proved useful as an exemplary technique for generating monolayers using non-adherent cells.

[0153] 96-well polyethersulfone (PES) plate Permeable membranes that allow for filtration of culture medium are also useful for generating cell monolayers, including cellulose nitrate membranes, cellulose acetate or PES membranes.

[0154] Such membrane-based systems were evaluated to generate suspension cell monolayers. 96-well filter bottom plates provide a 96-well format with filter bottoms in the wells. Pall Supor filter plates (AcroPrep Advance; PES 1.2 μm, Cat. No. 8039) were evaluated. 1 × 10 6Human primary T cells were seeded at 100 μl per well and the plate was centrifuged at 300×g for 5 min. Once the medium was removed by centrifugation and the remaining cells had formed a monolayer, the plate was placed in a Solupore device and the cells were sprayed with delivery solution containing the mRNA. After 2 min of incubation, stop solution (50 μl) was applied, followed 30 s later by regular medium (100 μl). The cells were incubated for 2 h before the process was repeated. At the end of this spraying, the cells were incubated at 37° C. and 5% CO in a humidified incubator. 2 Cells were incubated overnight in 500 mL of PBS and assessed for GFP fluorescence by flow cytometry. A GFP expression level of 52% ± 3.6 was achieved across five donors and five experiments, with a viability of 97% ± 3.3 (Figure 16A,B,C). PES plates contain a mesh-like filter that may render cells unrecoverable. Other filter types were evaluated, such as track-etched filters. Track-etched membranes are thin (approximately 5-25 micrometers) polymer membranes whose pores are formed by irradiating an initial non-porous material with high-energy particles and then etching the latent tracks (usually with a caustic etchant, e.g. NaOH) to form pores of a given diameter through the membrane.

[0155] 96-well polycarbonate track etched (PCTE) plates Alternative membrane filter systems may be used to generate cell monolayers of non-adherent cells. For example, an alternative filter plate with a 0.4 μm hydrophilic PCTE filter was obtained from Agilent technologies. 2.5×10 per 100 μl per well 5Human primary T cells were seeded with 100 cells and centrifuged at 350 x g for 2 min. Once the medium was removed and a cell monolayer formed, the plate was placed in a Solupore device and the cells were sprayed with a delivery solution containing a test payload, such as GFP mRNA. After 2 min of incubation, stop solution (50 μl) was applied, followed 30 s later by regular medium (100 μl). The cells were incubated for 2 h before repeating the process. At the end of this spraying, the cells were incubated at 37 °C and 5% CO in a humidified incubator. 2 The cells were incubated overnight at 25°C and assessed for GFP fluorescence by flow cytometry. A GFP expression level of 72%±5 was achieved, with a viability of 75.0%±3.5 (FIG. 17). The results comparing PES and PCTE plates are shown in FIG. 18. The results show enhanced uptake using PCTE plates. Thus, hydrophilic membrane filters, optionally track-etched filters, are useful exemplary membranes and are preferred in some embodiments.

[0156] Solvent Removal Methods for removing media from cells were also addressed. Centrifugation, vacuum pressure, and positive pressure were evaluated using filter plates. 1 × 10 cells per well were plated onto a 96-well filter plate (Pall; Supor, 1.2 μm; Cat. No. 8039). 6 Human primary T cells were seeded. The medium was removed by centrifugation at 300×g for 5 min or by vacuum pressure (−20 mBar, 30 s; see Figures 105-107). 4 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. Stop solution (50 μl) was applied after 2 min incubation, and normal medium (100 μl) was applied after 30 s. Incubation was continued at 37° C. and 5% CO in a humidified incubator. 2 Cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry (Figure 19A,B). In a separate experiment, Agilent PCTE filter plates (Agilent; PCTE 0.4 μm) were seeded with 2.5 × 10 5Human primary T cells were seeded at 1000 x g. The medium was removed by either centrifugation (350 x g for 2 min) or positive pressure (200 mBar for 1 min). Once the medium was removed and a cell monolayer had formed, the plate was placed in a Solupore device and the cells were sprayed with delivery solution containing GFP mRNA. After 2 min of incubation, stop solution (50 μl) was applied, followed 30 s later by regular medium (100 μl). The cells were incubated for 2 h before the process was repeated. At the end of this spraying, the cells were incubated at 37 °C and 5% CO in a humidified incubator. 2 The cells were incubated overnight at 25° C. and assessed for GFP fluorescence by flow cytometry (FIG. 19A,B). A cell monolayer is obtained by removing the medium from the wells using vacuum pressure. Optionally, both positive pressure and centrifugation are used to generate the monolayer. In some embodiments, the latter technique is preferred.

[0157] Magnetic beads An alternative method combines the use of T cell activation beads (e.g. DynaBeads 3:1 ratio) with a magnet. After overnight activation, T cells (bound to DynaBeads) were seeded into a 96-well plate. A magnet was placed under the well and the medium was removed with a pipette. The magnet holds the beads and cells in place while the medium is removed. The mRNA was then delivered by solporation. GFP expression was detected by light microscopy after 24 hours.

[0158] mRNA delivery to MSCs To confirm delivery to cells in a monolayer, mesenchymal stromal cells (primary human cells or iPSC-derived cells) were seeded into 96-well plates such that confluency was 80-90% by 24 h.

[0159] Delivery of mRNA, e.g., test payload / cargo GFP mRNA, into BM-MSCs and iPSC-MSCs was evaluated. Delivery of various card compounds, such as 10kDa dextran, into primary BM-MSCs using a vector-free intracellular delivery method with reversible permeabilization has been previously reported (O'Dea S, et al., PLoS One. 2017. 30;12(3):e0174779). Delivery methods for functional molecules, such as mRNA, were also evaluated. Reporter GFP mRNA was used to evaluate the efficiency of mRNA delivery into BM-derived MSCs and iPSC-derived MSCs.

[0160] Multiple treatments, for example, three GFP mRNAs, were delivered to BM-MSCs and iPSC-MSCs over a period of two days. Fluorescence microscopy confirmed the expression of GFP protein in cells 24 hours after the last mRNA was delivered (Figure 72A). Flow cytometry analysis showed a delivery efficiency of 29.5±10.4% and 31.0±2.2% (n=3) in BM-MSCs and iPSC-MSCs, respectively (Figure 21A,B).

[0161] Optimizing atomization of delivery solutions The Mucosal Atomization Device (MAD Nasal™) spray head used to atomize the payload solution dispenses milliliter volumes, whereas solporation works in microliter volumes. Use of a microliter volume atomizer / droplet delivery system is preferred.

[0162] Alternative spray heads were investigated with two main objectives: to increase uptake and improve reproducibility across multiple replicates (within an experiment) and across multiple experiments (between experiments). Evaluation of alternative atomizer devices was undertaken to find which was optimal for mRNA delivery to T cells. The atomizer allows the delivery solution to be applied to the cell monolayer in a dropletized form. In addition to identifying the atomizer, a controller device was designed and constructed that allows fine control of the atomization process. Different parameter sets were tested to find the optimal parameters for delivering mRNA to T cells.

[0163] The results showed that microliter volume delivery devices such as the Ari Mist nebulizer and the 180kHz ultrasonic nebulizer gave comparable uptake and reproducibility, with cell viability being slightly higher with the Ari Mist. Such microliter volume delivery devices, such as the Ari Mist head, are preferred due to their small size, easy handling, and do not require a power box to operate, and can be integrated into automated solutions. Other units such as the Burgener nebulization technology (U.S. Patent No. 6,634,572, incorporated by reference) are also suitable for scaling up solporation. Taken together, the Ari Mist and other Burgener nebulizers were used for automation and scaling.

[0164] Atomization of a delivery solution to generate monodisperse droplets A variety of methods were used to deliver the cell membrane permeabilization solution onto the cell monolayer, for example, the permeabilization solution can be atomized using ultrasound or nebulized using a pneumatic nebulizer.

[0165] Both air-assisted sonication and pneumatic nebulization were tested as delivery methods. A total of eight different spray heads were tested: three ultrasonic heads, 60 KHz (Sonaer), 130 kHz (Sonaer), and 180 kHz (Sonotek Echo), and five pneumatic nebulizers, Ari Mist, X-175, PFA250, T2100, and Peek Mira Mist (Burgener Research).

[0166] Sonication tests were performed at 60 kHz, 130 kHz, and 180 kHz. Liquid can be forced through an ultrasonic nozzle by a pumping system and atomized into a fine mist spray using high frequency sonic vibrations.

[0167] A curtain of gas (air) can aid this process. An auxiliary piece called a shaper is fitted around the ultrasonic head, and the function of the air is to shape the mist of the sonicated liquid. For example, the air had a dual function of not only shaping the spray but also facilitating the entry of the payload into the cells. This was possible because the air delivered through the shaper was at a pressure that exceeded the pressure used for the shaping function.

[0168] Tests with ultrasonic nebulizers have provided useful results for the delivery of cargo / payload to mammalian cells, e.g., non-adherent cells. Up to 60% of dextran-Alexa488 (model cargo) was delivered to U2OS cells using a 180 kHz ultrasonic head (Figure 22A, B).

[0169] A piezoelectric transducer can be used to transfer electrical input into mechanical energy in the form of vibrations. When introduced into a nozzle, these vibrations create capillary waves in the liquid, resulting in atomization of the liquid. Each ultrasonic probe was operated at a given resonant frequency. The operating frequency can determine the size of the droplets produced. The size of the droplets can also be affected by the power at which the ultrasonic probe is operated, although to a lesser extent by the operating frequency. An auxiliary air flow can be used to help control and shape the spray.

[0170] Exemplary ultrasonic spray emitters produce fine sprays with narrow droplet size distributions, which results in uniform deposition of the delivery solution and payload on the cells. For example, with respect to the preferred parameters evaluated for the MAD nasal spray head, reducing the delivered volume and reducing the ethanol concentration improved delivery efficiency and improved viability with the ultrasonic spray head.

[0171] Additional nebulizers for generating small droplets (microliter volumes) were tested, including the Ari Mist, Peek Mira Mist, T2100, X175, and PFA250 nebulizers (Burgener Research). These exemplary nebulizers run on compressed gas and require a pump to deliver the sample solution. These exemplary atomizers have two parallel channels, one for the gas (air) and the other for the liquid to be nebulized. Both paths terminate at the tip of the nebulizer with an orifice for the gas and an outlet for the liquid. The gas flow can draw the liquid into the gas flow. Collisions with gas molecules can break the liquid into small droplets, resulting in nebulization.

[0172] The Burgener nebulizers (US Pat. No. 6,634,572) tested differed in internal diameter, material, and optimal flow rate. In preliminary studies, the nebulizers gave comparable expression of cargo mRNA. Droplet size characterization revealed that the droplets ranged from 1 to 20 μm, with peak droplet numbers in the 5 to 7 μm range. The Sauter mean (D32 The mean particle size, defined as the mean particle diameter, is 13 μm (see http: / / www.burgener.com / EnhancedData.html). Of the set of Burgener nebulizers tested, the Ari Mist was selected as the preferred spray head based on several factors: it had good uptake and viability, its specifications (optimum flow rate, internal diameter) were matched to the pumping system features, and its internal diameter (225 μm) was small enough to handle low volumes of liquid without the inconvenience of clogging.

[0173] Certus Digital Dispensing Technology The Certus Flex liquid dispensing instrument was equipped with an 8-channel dispensing head (cat. no. D196057) and two valve sizes: nozzle diameter 0.10, travel 0.03 (cat. no. 21765) and nozzle diameter 0.15, travel 0.03 (cat. no. 21766). Each channel is individually controlled using Certus' proprietary software and electronics. Certus Flex enables contact-free dispensing of liquids and large molecules using Gyger microvalve technology and pneumatic control. Volumes in the nanoliter (nl) range can be delivered with high precision (CV 5% at 100nl; CV stands for coefficient of variation or relative standard deviation).

[0174] To assess the feasibility of mRNA delivery to T cells using the Certus Flex microfluidic platform, we investigated the delivery of mRNA to T cells by generating small droplets in the nanoliter (nl) to μl size range.

[0175] CD3 + T cells were activated using, for example, either Dynabeads or TransAct. 1.5 × 10 cells per well were placed in a 96-well filter plate (PES). 6Cells were seeded with 100 μl of cells. The plate was centrifuged, for example, at 300×g for 5 minutes to remove cell culture medium. Delivery solution was dispensed into each well via channel 1 with the parameters listed in Table 1. A total volume of microliters, for example 2 μl or 7 μl, was delivered as droplets ranging in volume from 7 to 0.08 μl. The volume of the droplets was determined by the number of droplets dispensed in the well. Figure 58 represents the droplet array patterns tested (Figure 58). Valve type, pressure and height were varied as outlined in Table 2. Cells were incubated for 2 minutes after application of delivery solution. 50 μl of stop solution was added via channel 2 and incubated for 30 seconds. 100 μl of culture medium was added via channel 3. Plates were incubated at 37 degrees for 24 hours prior to analysis (Figure 58).

[0176] The results show that cell viability was comparable to untreated cells using this system. No delivery of GFP mRNA was observed using this system. This was seen for all parameters tested (Figure 61). Thus, the use of Certus digital dispensing technology to deliver droplets in the nl-μl range did not result in uptake of GFP mRNA into T cells.

[0177] (Table 1) Plate delivery template parameters TIFF0007644048000002.tif55146

[0178] (Table 2) Dispensing Head Channel Configuration TIFF0007644048000003.tif62147

[0179] Use of instruments to allow fine control of spray A test rig was assembled to control key spray parameters and allow for mechanization of spraying. Plates containing cell suspensions were centrifuged before being placed in the test rig. Delivery solutions containing the payload were loaded into elveflow fluid reservoirs or syringe systems. Fluidic control of the delivery solutions containing the payload was achieved using either pinch valves or microvalves. Stopping and addition of culture medium were performed manually.

[0180] Fluidic control of the delivery solution containing the payload was achieved using two systems: an elveflow-pinch valve system and a syringe-microvalve system. The syringe-microvalve system was found to have advantages over the elveflow-pinch valve system.

[0181] (a) Fluid control of the delivery solution containing the payload. (i) Elveflow-Pinch Valve Elveflow refers to a microfluidic reservoir used with 1.5 ml Eppendorf tubes or 50 ml Falcon tubes depending on the sample reservoir size required (Elvesys, Innovation Center, Avenue Philippe Auguste 83, 75011 Paris, France). Pinch valve can refer to any pinch valve, an example is the electronic Clippard pinch valve (Clippard, 7390 Colerain Avenue, Cincinnati, OH 45239, USA). Fluid control can be achieved by a fluid control system that can apply a constant pressure to the elveflow fluid reservoir to push the fluid through the pinch valve (Figure 23). The volume of fluid that can be dispensed can be controlled by the amount of pressure applied, the length of time the valve is left open, and / or the diameter of the tubing used.

[0182] The valves can be actuated by metal oxide semiconductor field effect transistors (MOS FETs) which can be controlled by a microprocessor.

[0183] (ii) Syringe-microvalve The above-mentioned Elveflow-pinch valve system had the following limitations: - The system did not retain calibration when refilling the elveflow sample reservoir. - The accuracy and precision when dispensing volumes below 5 μl was insufficient (for low volumes (<5 μl), the relative standard deviation for repeated dispenses was approximately 9%. These data were generated at Avectas and are summarized in Figure 54). Calibration data for delivery solutions used the Elveflow-pinch valve system.

[0184] A new fluidic system was used to address these limitations.

[0185] This may involve the use of a microvalve fluidics system such as the Gyger microvalve (SMLD300, Fritz Gyger AG, Bodmarstrasse 12, 3645 Gwat (Thun), Switzerland). This system consists of a syringe sample reservoir connected to a microvalve which is connected to an Air Mist nebulizer. This system was more accurate and precise when delivering volumes in the range of 1 μl to 100 μl. When comparing the delivery efficiency using the microvalve and the pinch valve, there is no difference in the delivery efficiency (Figure 63A,B).

[0186] (b) Air flow control The air pressure is optionally controlled by a solenoid valve.

[0187] (c) Electronic devices for controlling spray operation To allow for electronically controlled spray actuation, a system was designed using a microprocessor-based development board to allow easy development of time-controlled sequences. The development board used a microprocessor, e.g., PIC16F1619. The spray actuation time and fluid delivery time can be manipulated by the development board's interface software. This microprocessor development board allows for pulsing of the nebulizer spray.

[0188] The system was then upgraded to utilize repeatable high speed PLC (Programmable Logic Controller) technology to better align with industry standards and serve as a demonstrator for the automated Solupore™ technology, which is based on very high speed Programmable Logic Controller (PLC) technology. The test rig controller consisted of a PLC and a Gyger controller and a program that communicated between these two pieces of hardware. The hardware had operator interaction via momentary push buttons.

[0189] (d) Spray head alignment Spray heads, such as the Ari Mist nebulizer parallel channel design, produce an off-center spray from the tip of the nebulizer. A custom spray head holder fitted with a goniometer can be used to adjust the alignment of the spray head.

[0190] Identifying optimal parameters for delivery of mRNA to T cells Work was carried out to characterize and optimize the spray from three different ultrasonic heads and the Ari Mist head. The different spray characteristics were assessed using high-speed camera recordings. The spray forces experienced by the cell monolayer were measured by force sensor analysis. In some cases, the volume delivered to the wells of a 96-well plate was assessed using a colorimetric assay.

[0191] For the optimization study, the following parameters were tested with the ranges indicated: (a) Air pressure: 0.5 to 2 bar; (b) Volume delivered: 1-7 μl; (c) Atomizer height to target area: 26mm and 31mm, (d) Length of spray actuation: 50-900 ms; (e) Flow rate: 1~20μl / s, (f) Ultrasonic probe power: 40-80%, (g) Spray head: Ari Mist, Ultrasonic 60kHz, Ultrasonic 130kHz, Ultrasonic 180kHz.

[0192] Using EGFP mRNA as the payload, a large set of parameter combinations was tested using three types of ultrasound probes and an Ari Mist nebulizer.

[0193] All parameter sets resulted in GFP expression, with uptake varying from 5% to 30%.

[0194] Among the ultrasound emitters, the 180kHz was found to deliver payloads to T cells more effectively compared to the 130kHz and 60kHz ultrasound heads. Test results showed that the payload was successfully delivered to T cells with an efficiency of approximately 15-28% and a high level of consistency (±1%) between multiple repetitions. Cell health was maintained after delivery (relative viability 85%).

[0195] The uptake and reproducibility obtained with Ari Mist, 180 kHz ultrasound, and the Mad Nasal spray head were compared. Results are presented in longitudinal data plots for Ari Mist, 180 kHz ultrasound, and the Mad Nasal spray head (Figure 24, Figure 25, and Figure 26, respectively). The longitudinal data also show the progression of optimization of delivery parameters over several weeks. Uptake improved with 180 kHz ultrasound (Figure 25) and Ari Mist (Figure 24), reaching 20-30% positive cells, but fluctuated by less than 20% when cells were sorbolated using the MAD nasal spray head (Figure 26).

[0196] Improved reproducibility was observed when using an ultrasonic or Burgener nebulizer (Figure 24, Figure 25) compared to the MAD nasal spray head (Figure 26). Reproducibility of uptake was expressed in terms of standard deviation (StDev). The primary objective of evaluating the other spray heads was to identify a nebulizer that would give the narrowest possible standard deviation of uptake within multiple repetitions of an experiment. When longitudinal data were examined by averaging the standard deviation of uptake across all experiments, the average standard deviation of uptake was 2.4% for the 180 kHz ultrasonic spray head and 2.1% for the Ari Mist, while it was 4.5% for the MAD nasal. In both cases it was an improvement over the MAD nasal nebulizer.

[0197] The results showed that Ari Mist and 180kHz ultrasonic nebulizers gave comparable levels of delivery efficiency, with 20-30% GFP-positive cells detected. Cell viability was higher with the Ari Mist head. Furthermore, this nebulizer is smaller, easier to handle, and does not need to be powered to operate. All these reasons contributed to the selection of a nebulizer, e.g., the Ari Mist nebulizer, as the spray head for solporation, and the optimal delivery parameters evaluated for mRNA delivery, e.g., as shown in Table 4.1. This set of parameters was the result of an extensive screening and became the starting point for further studies of sophisticated optimization, fine-tuning other parameters such as delivery solution components, cell number, and filter plate, in addition to volume, spray distance and length to further improve mRNA delivery. Table 4.1 lists the parameters and ranges tested, including the preferred parameters for delivery of mRNA (e.g., model cargo GFP mRNA) to T cells. Table 4.1 includes the ranges tested and the preferred parameters for both the benchtop Flexi (benchtop) and Midi (scale-up) systems.

[0198] Optimizing the process for applying the delivery solution to cells Atomizer Height Several parameters were evaluated to increase the uptake efficiency and expression of mRNA in human primary T cells. The height of the AriMist atomizer was evaluated to see if there was an effect on the delivery of GFP mRNA to T cells. 1 × 10 per well was added to a 96-well filter plate (Pall; Supor, 1.2 μm; Cat. No. 8039). 6 Human primary T cells were seeded. Plates were centrifuged at 300×g for 5 min and 4 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. Atomizer heights were evaluated at 31, 26, and 11 mm above the bottom of the well (a separate experiment evaluated 26 mm vs. 12 mm). Stop solution (50 μl) was applied after 2 min of incubation and regular medium (100 μl) was applied 30 s later. Cells were incubated in a humidified incubator at 37° C. and 5% CO. 2 The wells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry. Uptake of mRNA by T cells was achieved when the atomizer was placed 31 mm and 26 mm above the bottom of the well. In some cases, not all of the 4 μl delivered entered the well. A preferred height of 12 mm above the bottom of the well was chosen (Figure 27A,B). This allowed for precise dosing of the payload and prevented overspray contaminating other wells. The reduced height also allowed for a reduced delivered volume. The height range was from 11 mm above the bottom of the well (filter) to 31 mm above.

[0199] Volume delivered A volume comparison was undertaken to determine the optimal delivered volume that would maximize mRNA uptake by T cells. 1 × 10 cells were delivered per well to a 96-well filter plate (Pall; Supor, 1.2 μm; Cat. No. 8039). 6Human primary T cells were seeded. Plates were centrifuged at 300×g for 5 min and 4, 1 or 0.5 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. Stop solution (50 μl) was applied after 2 min incubation and regular medium (100 μl) was applied 30 s later. Cells were incubated in a humidified incubator at 37° C. and 5% CO 2 The wells were incubated overnight at 4°C and evaluated for GFP fluorescence by flow cytometry. An optimal volume of 1 μl per well was determined (FIG. 28). In the Solupore test rig system, the volume sprayed can be adjusted by varying the pressure applied to the ElveFlow or by the duration the valve is left open. In the first condition, the duration of the valve open was set to 280 ms and the pressure to 70 mBar. In the second condition, the valve open time was reduced to 140 ms and the pressure was set to 140 mBar. The optimal method was to reduce the valve open time to 140 ms.

[0200] Tonicity of the Delivery Solution Previous experiments used a delivery solution that was hypotonic relative to the cells. Delivery solutions with altered tonicity by the further addition of KCl were evaluated. 1 × 10 cells were added per well to a 96-well filter plate (Pall; Supor, 1.2 μm; Cat. No. 8039). 6 Human primary T cells were seeded on the plates. The plates were centrifuged at 300×g for 5 minutes and 4 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. In the first condition, the delivery solution contained 12.5 mM KCl, which results in a hypotonic solution for the cells. In the second condition, the delivery solution contained 106 mM KCl, which results in an isotonic solution for the cytoplasm of the cells. Other concentrations between 10 mM and 500 mM were also tested, such as 12.5 mM KCl, 328 mM and 500 mM KCl. High tonicity, 328 mM and 500 mM KCl, showed reduced levels of GFP expression. Thus, useful ranges are 12.5-500 mM, such as 50-150 mM, such as 100-125 mM, such as 100-110 mM, with 106 mM being the preferred KCl concentration.

[0201] Once the cells were sprayed, stop solution (50 μl) was applied after 2 min incubation, followed 30 s later by normal medium (100 μl). The cells were incubated at 37°C and 5% CO in a humidified incubator. 2 The cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry. An optimal concentration of 106 mM KCl, which is isotonic with the cells, was determined (Figure 29).

[0202] Multiple "hits" The gentle nature of the Solupore technology allows cells to be treated multiple times without loss of cell viability or functionality. We set out to evaluate the preferred number of "hits" (treatments), for example, with one-hit, two-hit, and three-hit strategies. 1 × 10 cells were plated per well onto a 96-well filter plate (Pall; PES, 1.2 μm). 6 Human primary T cells were seeded at 1000 x g. The plate was centrifuged at 300 x g for 5 min and 1 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. Once the cells were sprayed, stop solution (50 μl) was applied after 2 min incubation and normal medium (100 μl) was applied after 30 s. For the two-hit strategy, the cells were incubated for 2 h before the spraying process was repeated, and for the three-hit strategy, it was repeated once more after 2 h incubation. Before each additional hit and at the end of the two-h incubation, the wells containing the cell suspension were sealed using a film (e.g. Parafilm M) and the plate was placed on a stirring device such as a vortex mixer and held for 15 s. The cell suspension was then mixed three times with a pipette. The vortexing and mixing allowed the orientation of the cells to be "shuffled" before the next hit. The cells were incubated in a humidified incubator at 37 °C and 5% CO 2 The cells were incubated at RT overnight and assessed for GFP fluorescence by flow cytometry. The three-hit strategy appeared to be optimal in terms of uptake, viability and cell yield (Figure 30).

[0203] Cell seeding density We began by evaluating the optimal T cell seeding density using Agilent PCTE plates. 96-well filter plates (Agilent; PCTE, 0.4 μm) were seeded with 1.25, 2.5, 3.5, 5, and 7.5 × 10 cells per well. 5 Human primary T cells were seeded at 1000 x 100 cells. The plate was centrifuged at 350 x g for 2 min and 1 μl of delivery solution containing 0.57 μg / μl of mRNA was sprayed onto the cell monolayer. Once the cells were sprayed, stop solution (50 μl) was applied after 2 min of incubation, followed 30 s later by regular medium (100 μl). The cells were incubated for 2 h before repeating the process. At the end of this spraying, the cells were incubated at 37°C and 5% CO in a humidified incubator. 2 Cells were incubated overnight at 3.5×10 5 A seeding density of 100 cells was shown to be optimal (Figure 31). The average T cell size after Dynabead activation was approximately 9.5 μm (70.9 μm 2 32). To confirm the seeding density, the mean diameter of activated T cells was compared with the area of ​​the addressable zone of the filter well (19.6 mm 2 From this calculation, the number of cells forming a monolayer on the filter was approximately 2.77 × 10 5 There are 10.

[0204] masking It was previously noticed that there was an area of ​​negative cells near the edge of the well. The reason this edge effect exists could be due to either poor spray targeting, increased volume near the edge due to the meniscus, or ineffective droplet impingement at the edge, pressure disturbance at the edge (or a combination). A strategy to overcome the edge effect would be to create a seeding mask that is present during seeding and removed after centrifugation to prevent cells from seeding near the edge of the well. To test this theory, a mask was placed in a PCTE plate well that reduced the well diameter from 5.2 mm to 4 mm. 2.5 × 10 5Human T cells were seeded in the mask at 3.5 x 10 cells as a control. 5 Cells were seeded into unmasked wells. Plates were centrifuged at 350×g for 2 min to remove the mask before spraying the cells. This means that cells were only seeded up to about 0.5 mm from the well wall. Delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cells using a single hit strategy. At the end of this process, the cells were incubated at 37° C. and 5% CO in a humidified incubator. 2 Cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry. Results showed that samples from masked wells gave 67.7% uptake, whereas unmasked samples gave 53.1% uptake. This suggests that there is an edge effect, and that the presence of a mask for seeding counteracts it (Figure 60A,B). Thus, optionally, preventing cells from seeding to the edge of the well results in increased transfection efficiency.

[0205] force To examine the correlation between the force exerted by the spray and uptake, a force sensor analysis was performed at different heights and pressures to establish a baseline of results. The pressure pushing air through the atomizer was adjusted from 0.5 to 2 bar and the force sensor was used to measure the force experienced by the bottom of the well. The height was also adjusted to 31, 26, and 11 mm. The results showed that air pressure was the single largest factor affecting the force exerted by the spray, with a small drop in force occurring at the low height of 11 mm. At 11 mm and a force of 2.0 bar, the same force is experienced at the larger height and at normal pressure of 1.65 bar (Figure 55). This experiment was repeated to provide a more robust analysis of the factors affecting the force exerted by the spray. Again, air pressure was the largest contributor to the force exerted by the spray (Figure 56). At low pressure, the force experienced at different heights becomes negligible, with the forces at 1.15 bar and heights of 11, 26, and 31 mm indistinguishable. At 2.15 bar, the forces at heights 26mm and 31mm were greater than the force at 11mm. This can be explained by the fact that at high pressure, the higher heights drag more air from the atmosphere into the spray path, hence increasing the mass of the spray, since force = mass x acceleration. Next, the same parameters as in Figure 4.11 were tested to correlate the force profile with GFP mRNA uptake in CD3+ T cells. 1x10 per well were plated onto a 96-well filter plate (Pall; Supor, 1.2μm; Cat. No. 8039). 6 Human primary T cells were seeded. Plates were centrifuged at 300×g for 5 min and 4, 2, 1 or 0.67 μl of delivery solution containing 0.57 μg / μl GFP mRNA was sprayed onto the cell monolayer. Stop solution (50 μl) was applied after 2 min incubation and regular medium (100 μl) was applied 30 s later. Cells were incubated in a humidified incubator at 37° C. and 5% CO. 2The cells were incubated overnight at 4°C and evaluated for GFP fluorescence by flow cytometry. GFP expression results showed that GFP mRNA uptake could be achieved under a wide range of conditions with different delivery volumes, distances, and air pressures (Figure 57). The force from the spray does not directly correlate with uptake. There may be a minimum force required to achieve uptake and a maximum force at which no loss of viability is observed, but this range is wide (1-2 bar).

[0206] The range of conditions suitable for delivery of mRNA to non-adherent cells, e.g., T cells, such as primary human T cells, are summarized below. TIFF0007644048000004.tif125128

[0207] Preferred, e.g., optimal, conditions for delivering mRNA to T cells are outlined in Table 4.1.

[0208] (Table 4.1) Conditions for sequential delivery of mRNA to T cells TIFF0007644048000005.tif157146

[0209] Transfection Comparison Delivery and viability compared to electroporation - Electroporation is a widely used method of vector-free intracellular delivery, therefore, the delivery efficiency and cell viability levels using the subject delivery method were compared to electroporation.

[0210] When the subject technology was used to deliver 3 μM of a model payload, 10 kDa dextran-Alexa488, to A549 cells, the delivery efficiency was 52.8% (± 2.7%) compared to 92.9% (± 0.6%) for electroporation (FIG. 33A,B,C). The percentage of cells that survived the delivery process was analyzed by propidium iodide exclusion and flow cytometry analysis. For the subject technology, cell viability compared to untreated control cells was 78.3% (± 4.1%) compared to 73.0% (± 9.8%) for electroporation (FIG. 33A,B,C).

[0211] Most delivery methods require a balance between effective delivery and maintaining cell viability. To examine this balance, a transfection score ((transfected cells / total cells) x (viable cells / total cells)) was used to obtain a composite characterization of cell loss, cell viability, and transfection efficiency for the subject delivery technique compared to electroporation. A score of 1.0 indicates 100% transfection efficiency and 100% cell viability, indicating that no cells are lost during the procedure. The transfection score was 0.33 (±0.05) for the subject technique and 0.51 (±0.13) for electroporation, with no significant difference between the scores (Figure 33C).

[0212] To benchmark this technology, we undertook a comparison of Solupore and nucleofection (4D; Lonza) delivery of mRNA into human T cells. The amount of mRNA delivered (μg) per cell was matched. For Solupore, 1 × 10 cells were delivered per well onto a 96-well filter plate (Pall; PES, 1.2 μm). 6 Human primary T cells were seeded at 100x100 cells. Plates were centrifuged at 300xg for 5 min and 1 μl of delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. Once the cells were sprayed, stop solution (50 μl) was applied after 2 min of incubation and regular medium (100 μl) was applied 30 s later. Cells were incubated at 37°C and 5% CO in a humidified incubator. 2 The cells were incubated at 4 °C overnight and evaluated for GFP fluorescence by flow cytometry. For nucleofection, 5 × 10 6 Human primary T cells were washed in PBS and resuspended in 40 μl of P3 buffer containing 2 μg of GFP mRNA (Lonza). Cells were then added to nucleocuvette strips and nucleofected according to the manufacturer's instructions. 100 μl of medium was added to each well and transferred to a recovery flask containing 10 ml of medium. Cells were incubated at 37°C and 5% CO in a humidified incubator. 2The cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry. GFP mRNA expression levels were 40.3% and 89.3%, respectively (Figure 34). The average median fluorescence intensity from five experiments and two donors was 203,059 and 113,895, respectively (Figure 35A,B,C,D). The dose response of mRNA delivered by each technique is shown in Figure 36.

[0213] Endocytosis-independent Diffusion of cargo into cells and resealing of plasma membrane. Having demonstrated the ability of this method to deliver a wide range of cargo to a range of cell types, the mechanism of cargo uptake into cells and reversal of cell permeability were investigated. A limitation of other delivery techniques is that they rely on active uptake pathways such as endocytosis, which can lead to sequestration of the cargo making it unavailable for function in the cell. For example, liposome-mediated delivery requires both clathrin- and caveolae-mediated endocytosis (Cui S, Wang B, Zhao Y, Chen H, Ding H, Zhi D, et al. Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells. Biotechnol Lett.2014;36(1):1-7.doi:10.1007 / s10529-013-1325-0.PubMed PMID:24068499;PubMed Central PMCID:PMCPMC3889874), whereas iTOP delivery requires macropinocytosis (D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V, et al. Efficient intracellular delivery of native proteins.Cell.2015;161(3):674-90. doi:10.1016 / j.cell.2015.03.028.PubMed PMID:25910214.).

[0214] During experiments using sorbulation, immediate uptake of the card into the cells was observed. Using 10 kDa dextran-FITC as a model cargo, the cargo could be seen inside the cells within 30 seconds of application of the delivery solution, before the addition of the stop solution (Figure 37A,B,C,C). The rapid influx of the cargo into the cells indicates that its delivery is unlikely to involve endocytosis. The results showing the loading of a wide range of molecular species into a range of cell types indicate that a diffusion mechanism through the cell membrane is the mechanism of entry of macromolecules into the cells. To investigate the contribution of alternative uptake mechanisms, such as active pathways and internalization into endocytic vesicles, A549 cells were pretreated with Dynasore (4 mM) or chloropromazine (20 μM) to inhibit clathrin-mediated endocytosis, or with nystatin (20 μg / ml) or EIPA (100 μM) to inhibit caveolae-mediated endocytosis and micropinocytosis, respectively. EGFP mRNA expression was unchanged in the presence of these inhibitors, indicating that the method results in direct delivery into the cell cytoplasm and does not rely on endocytosis (Figure 37C).Furthermore, in addition to following the methodology reported by D'Astolfo et al. (D'Astolfo et al. 2015), we included Lipofectamine 2000 as a positive control to confirm Dynasore-mediated inhibition of clathrin-mediated endocytosis (Figure 37C).

[0215] The delivery method was found to be extremely gentle on cells, causing little, if any, visible cell death or damage. The method allows the permeabilized plasma membrane to rapidly reseal, thus preserving high levels of cell viability. To examine the rate of cell membrane recovery after permeabilization, the delivery solution was applied to A549 cells without cargo. At subsequent time points (0-182.5 min), the delivery solution was removed and 50 μl of PBS containing propidium iodide (PI) (100 μg / ml) was added. After 2 min of incubation, the PI solution was removed and the cells were harvested. PI uptake was analyzed by flow cytometry. For basal levels of PI uptake, untreated cells were given 50 μl of PI in PBS. The results show that the cells remain permeable to PI for several minutes, but reseal over 6 min after treatment (Figure 37C). There is no further uptake after 6 min. Thus, not only were cells loaded within 2 min of exposure to the delivery solution, but the membrane efficiently regained its integrity within 6 min of initiating the procedure. These data indicate that delivery of agents using sorbolation does not involve endocytosis.

[0216] Gene editing in T cells To demonstrate further functional output of T cells following cargo delivery, gene editing in T cells was assessed using CRISPR / Cas9 RNP delivery.

[0217] CRISPR / Cas9 RNP delivery. PDCD1 gene encoding PD-1 protein A two-guide RNA strategy was used to knock down TIFF0007644048000006.tif105150.

[0218] TIFF0007644048000007.tif30150 (PD1-1 protein amino acid sequence, SEQ ID NO:2). Equimolar amounts of crisprRNA (also equimolar amounts TIFF0007644048000008.tif4128crisprRNA and TIFF0007644048000009.tif4128The mixture of tracrRNA and crisprRNA (Su, S.et al. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci.Rep.6, 20070;doi:10.1038 / srep20070(2016)) was incubated with tracrRNA for 10 min at room temperature. 5 μg of Cas9 (IDT) was then added to a final molar ratio of Cas9 to guide RNA of 1:3 and incubated for another 10 min at room temperature. Cas9 RNP (containing α-crystallin (220 μM) and ethanol (25% v / v) in buffer) was delivered to T cells using the vector-free intracellular delivery method described herein. 1.5×10 per treatment was delivered using the vector-free delivery method described herein. 6 RNP was delivered to cells and PD-1 expression was analyzed 72 hours after transfection.

[0219] Cell viability. FACS sample preparation and analysis: Cell viability after the vector-free intracellular delivery method was assessed using 7-AAD viability staining solution (Sigma). Cells were washed in PBS + 1% fetal bovine serum (FACS buffer) before incubation with 7-AAD (1:40, 5-10 min at room temperature, protected from light) and then resuspended in PBS + 1% FBS (FACS buffer). PD-1 labeling was performed using APC-conjugated anti-human CD279 (PD-1) (Biolegend) and processed on a BD Accuri C6 flow cytometer (Becton Dickinson, USA). Data were analyzed using C6 software. Cell debris was excluded from total cells using forward and side scatter parameters. Single cells were selected by excluding doublets in a plot of FSC height vs. FSC area. GFP expression was analyzed in gated live cells.

[0220] Knockdown of immune checkpoint gene expression, such as PD-1, in T cells following vector-free delivery of CRISPR / Cas9 RNPs CRISPR / Cas9 RNPs targeting the PDCD1 gene were delivered to T cells by vector-free intracellular delivery methods described herein or by electroporation. PD-1 expression was analyzed by flow cytometry 72 hours after transfection.

[0221] Delivery of CRISPR / Cas9 gene editing tools into activated T cells resulted in a 28% reduction in PD-1 expression with vector-free intracellular delivery as described herein, compared to a 53% inhibition in electroporated cells (Figure 38A,B).

[0222] The proliferation potential of gene-edited cells was assessed over a 4-day period by observing the formation of T cell aggregates in culture. After delivery of RNPs, cells were returned to culture. After 4 days, cells were observed under a microscope. Cell aggregation in cells transfected with the vector-free intracellular method was similar to that of untreated control activated cells, indicating that they were not affected by the described vector-free intracellular delivery method. In contrast, the proliferation rate of electroporated cells was significantly affected, as indicated by significantly less cell aggregation (Figure 39).

[0223] CAR-T: Expression of chimeric antigen receptors (CARs) in primary T cells following mRNA delivery mRNA generated from a commercially available CD19 CAR plasmid was successfully delivered to human-derived activated T cells. Surface expression of the CAR was detected by flow cytometry, with up to 50% of the population positive for CAR expression.

[0224] material and method Cell culture. Human peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation on a Percoll gradient from Leuko Pak (AllCells, Alameda, CA). CD3 +Enriched lymphocytes were isolated. Cells were cryopreserved in 10% dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). After the first thaw from a storage aliquot, CD3 + T cells were cultured at 37°C and 5% CO 2 The cells were cultured in human recombinant interleukin-2 (IL-2) in a humidified tissue culture incubator at 37 °C, along with primary and costimulatory antibody activation using various protocols (see below).

[0225] Construction of CD19 CAR plasmid CD19 CAR plasmid was obtained commercially (Creative Biolabs, NY, USA) and mRNA was generated from the plasmid. Full length chimeric antigen receptor (CAR) was synthesized and subcloned into lentiviral vector. Insert was confirmed by Sanger sequencing. The structure of CAR vector is illustrated in Figure 86. Amino acid sequence of scFv (anti-CD19 scFv VL-linker-VH) is illustrated in Figure 87A. Nucleotide sequence of CAR cassette (codon optimized) is illustrated in Figure 87B. Amino acid sequence of CAR cassette is illustrated in Figure 87C. Restriction digestion map is illustrated in Figure 88. Quality control result of vector design is illustrated in Figure 89. CAR sequence alignment validation is illustrated in Figure 90. Sequence alignment result showed that the sequence of constructed plasmid was consistent with the design.

[0226] CAR mRNA delivery 4 μg of mRNA (from the CD19 CAR plasmid described above) was added to the buffer, ethanol (27% v / v) was also added, and 1.5 × 10 6 T cells were harvested 24 hours later and analyzed for mRNA expression using flow cytometry.

[0227] Flow cytometry Biotinylated protein L (AcroBiosystems) was reconstituted at 1 mg / ml in phosphate-buffered saline (PBS). For FACS staining, 1 × 106 Cells were harvested and washed three times with a wash buffer of ice-cold PBS containing 4% bovine serum albumin (BSA). After washing, cells were resuspended in 0.2 ml of wash buffer and incubated with 1 μg protein L for 45 min at 4 °C. Cells were washed three more times and then incubated with 10 μl of PE-conjugated streptavidin in 0.2 ml of wash buffer in the dark. To assess cell viability following the vector-free delivery method described herein, cells were stained using 7-AAD (Sigma). Briefly, cells were washed in PBS + 1% fetal bovine serum (FACS buffer) and then incubated with 7-AAD (1:40, 5-10 min at room temperature, protected from light) and then resuspended in PBS + 1% FBS (FACS buffer). Samples were processed on a BD Accuri C6 flow cytometer (Becton Dickinson, USA) and data were analyzed using C6 software. Cell debris was excluded from total cells using forward and side scatter parameters. Single cells were selected by excluding doublets on FSC height vs. FSC area plots. CAR-T expression was analyzed in gated live cells.

[0228] Non-transfected cells were used as untreated controls (UT). In treated cells, the shift in fluorescence intensity observed using this delivery method indicated CAR expression after mRNA delivery (Figure 45).

[0229] Assessment of T cell functionality after sorbolation To demonstrate that functionality of T cells transfected using Solupore technology is equal to or better than cells transfected using electroporation, e.g., with a Neon electroporator, and nucleofection, e.g., with a Lonza 4D nucleofector, assays of cell membrane protein expression, gene expression, cell proliferation rate, in vitro functionality, and in vivo functionality were performed.

[0230] (i) T cell membrane protein expression analysis The objective of this study was to determine whether the expression of cell surface proteins in T cells is affected by the solubility process and to compare it with electroporation and nucleofection.

[0231] Method: 1.5 × 10 cells were cultured per well in a 96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). 6 Activated T cells were seeded at 100 cells. Media was removed from the wells by centrifugation at 300×g for 5 min. Then, 7 μl of delivery solution (32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 27% ethanol in molecular grade water (all from Sigma-Aldrich)) containing 4 μg of GFP mRNA was sprayed into each well using a vector-free delivery spray instrument. After delivery, cells were incubated in this solution for 2 min before adding 50 μl of stop solution (0.5×PBS). After 30 s, T cell media was added (100 μl) and cells were incubated at 37° C. and 5% CO. 2 For electroporation and nucleofection, 5 × 10 cells were used per transfection. 6 pcs and 2.5×10 6 Each cell was used. GFP expression and viability were assessed 6 and 24 hours after delivery.

[0232] Cell surface CD4 and CD8 expression was examined 6 and 24 hours after either nucleofection, electroporation, or solporation. Six hours after transfection, the percentage of T cells expressing CD4 and CD8 was unchanged compared to untreated control cells for each transfection method. However, at 24 hours, expression was significantly reduced in electroporated cells, whereas expression in solporated and nucleofected cells was similar to control untreated cells (Figure 47A,B).

[0233] (ii) Global mRNA expression analysis The aim of this study was to obtain the molecular signature of cellular perturbations induced by the solubilization process and to compare it with electroporation and nucleofection. Sixteen samples were analyzed using mRNA microarrays. Two donor T cells were harvested and samples included untreated controls and cells transfected with neon, nucleofector, and Solupore with GFP-mRNA at 6 and 24 hours.

[0234] Method: 1.5 × 10 cells were cultured per well on a 96-well filter plate (1.2 μm PES membrane; Pall, USA). 6 Activated T cells were seeded at 100 cells. The medium was removed from the wells by centrifugation at 300×g for 5 min. Then, 1 μl of delivery solution (32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 27% ethanol in molecular grade water (all from Sigma-Aldrich)) containing 0.2 μg of GFP mRNA was sprayed into each well using a vector-free delivery sprayer. After spraying, the cells were incubated in this solution for 2 min before adding 50 μl of stop solution (0.5×PBS). After 30 s, T cell medium was added (100 μl). A second spray was performed 2 h after the first spray. The cells were incubated at 37° C. and 5% CO 2 For electroporation and nucleofection, 5 × 10 cells were transferred to a 30-°C incubator. 6 pcs and 2.5×10 6 Each cell was used. GFP expression and viability were assessed 6 and 24 hours after delivery.

[0235] The highest level of gene expression changes occurred with the Neon electroporation treatment. However, a drawback of electroporation is the reduced viability and functionality of treated cells after electroporation. Of the 20,893 mRNAs analyzed, Neon electroporation altered a total of 317 across all time points (6 and 24 hours) and both donors, Solupore altered 32 (Tables 5.1 and 5.2), and Nucleofector altered 24 (Tables 5.3 and 5.4; Figure 48A, B, C). Notably, the background level of false positives for this microarray analysis was approximately 10%. In the case of Solupore and Nucleofection, the number of altered genes was only slightly higher than this threshold, demonstrating that Solupore and Nucleofector cause only a minimal level of perturbation to cellular gene expression. The high level of gene expression changes indicates that the Neon electroporation process induces more perturbations to cells than either Solupore or Nucleofection. Because cellular perturbation is undesirable, this data indicates that Neon electroporation is a less desirable transfection method compared to solporation and nucleofection. Solporation carries with it several major advantages over electroporation and nucleofection, including high levels and reliable delivery of cargo, e.g., mRNA, to primary human cells while preserving the integrity, function, and proliferation potential of the treated cells.

[0236] (Table 5.1) List of gene expression changes in sorbed cells compared to untreated control cells 6 hours after transfection (Numbers indicate fold change compared to untreated control cells) TIFF0007644048000010.tif112150

[0237] The data indicate that only minor (eg, negligible) changes in gene expression occur in sorbolated cells 6 hours after transfection.

[0238] (Table 5.2) List of gene expression changes in sorbated cells compared to untreated control cells 24 hours after transfection Numbers indicate fold change compared to untreated control cells. TIFF0007644048000011.tif155150

[0239] The data indicate that only minor (eg, negligible) changes in gene expression occur in sorbolated cells 24 hours after transfection.

[0240] (Table 5.3) List of gene expression changes in nucleofected cells compared to untreated control cells 6 hours after transfection Numbers indicate fold change compared to untreated control cells. TIFF0007644048000012.tif61148

[0241] The data indicate that only minor (eg, negligible) changes in gene expression occur in nucleofected cells 6 hours post-transfection.

[0242] Table 5.4. List of gene expression changes in nucleofected cells compared to untreated control cells 24 hours after transfection. Numbers indicate fold change compared to untreated control cells. TIFF0007644048000013.tif96151

[0243] The data indicate that only minor (eg, negligible) changes in gene expression occur in nucleofected cells 24 hours after transfection.

[0244] (iii) Cell proliferation analysis For therapeutic applications, T cells need to be able to proliferate after modification. Therefore, we investigated the proliferation potential of T cells after solporation, electroporation and nucleofection. We also tested the proliferation potential of cells after cryopreservation and thawing.

[0245] Method: 1.5 × 10 cells were cultured per well in a 96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). 6 Activated T cells were seeded at 1000x10 cells. Each well was then sprayed with 1 μl of delivery solution containing 0.2 μg of GFP mRNA. A second spray was performed 2 hours later. For electroporation and nucleofection, 5x10 cells were used per transfection. 6 pcs and 2.5×10 6 Cells were incubated at 37°C and 5% CO 2 The next day, cells were harvested and counted. Then, 0.5×10 6 In another experiment, cells were cryopreserved in 10% DMSO and fetal bovine serum 24 hours after transfection. Cells were thawed and on day 0, 0.5 × 10 6 Cells were seeded at 100 / ml in Immunocult medium + IL-2. Cells were counted and reseeded by adding medium daily for 5 days.

[0246] T cells were transfected and then counted daily for 7 days. The proliferation rates in soluporated and nucleofected cells were similar to untreated control cells, but the potency of Neon-electroporated cells was reduced compared to control cells (Figure 49). Soluporation did not affect proliferation after cryopreservation and subsequent thawing (Figure 50). These data indicate that a major drawback of electroporation and / or nucleofection is the stall in cell proliferation. A major advantage of the Solupore system is the absence of stall in cell proliferation.

[0247] (iv) Interferon-γ (IFNg) secretion assay For therapeutic applications, T cells need to be able to produce IFNg after modification. Therefore, we investigated the IFNg production capacity of T cells after sorboration, electroporation and nucleofection. Two different activation methods, phorbol myristate acetate / ionomycin (PMA / I) and Dynabeads, were examined. The cells were also tested for their IFN-γ secretion capacity after sorboration followed by cryopreservation and thawing.

[0248] Method: 1.5 × 10 cells were cultured per well in a 96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). 6 Activated T cells were seeded at 1000x10 cells. Each well was then sprayed with 1 μl of delivery solution containing 0.2 μg of GFP mRNA. A second spray was performed 2 hours later. For electroporation and nucleofection, 5x10 cells were used per transfection. 6 pcs and 2.5×10 6 Each cell was incubated at 37°C and 5% CO 2 The next day, cells were harvested and counted. Cells were then diluted to 0.5×10 6 Cells were allowed to return to a quiescent state by reseeding at 0.5 × 10 cells / ml and then monitoring cell size. This was approximately 2 weeks after initial activation. Cells were then restimulated with either Dynabeads or a PMA / ionomycin cocktail for 4 hours, after which the supernatants were collected and stored at -20°C until cytokine analysis. IFN-γ ELISA (Biotechne) was performed on all samples. In a separate experiment, sorborated, nucleofected, and electroporated cells were harvested and counted 24 hours after transfection. Cells were then re-stimulated with 0.5 × 10 cells / ml by daily addition of medium + IL-2 for 7 days. 6Cells were allowed to return to a quiescent state by reseeding at 100 / ml and then monitoring cell size. This was approximately 2 weeks after initial activation. Cells were then restimulated with either Dynabeads or a PMA / ionomycin cocktail for 4 hours, after which supernatants were harvested and stored at -20°C until cytokine analysis. IFN-γ ELISA (Biotechne) was performed on all samples.

[0249] IFNg production was not reduced in T cells following sorboration, nucleofection, or electroporation compared to control cells (Figure 51). Freshly thawed sorborated cells did not lose their IFN-γ secretion capacity when compared to untreated controls (Figure 52).

[0250] (v) In vivo engraftment of T cells after delivery of 3 kDa dextran To determine the effect of the Solupore technology on T cell functionality, the ability of transfected PBMCs to induce GvHD in the NSG mouse model was studied. NOD scidγ mice (NSG mice) are an art-accepted immunodeficient experimental mouse strain from The Jackson Laboratory.

[0251] If transfected cells were adversely affected by the Solupore delivery technique, their ability to engraft and induce graft-versus-host disease (GvHD) would be compromised. A comparison with nucleofection was also performed. It was impractical to include Neon electroporation as a comparator because the cell loss in this process was so high that the number of cells that would need to be electroporated would be unfeasibly large.

[0252] Method: 3 kDa dextran-Alexa488 at 20 × 10 6 pcs and 5×10 6PBMCs were delivered by sorbulation and nucleofection, respectively. Sorption was performed using a 44.45 mm stirred cell system. A cell monolayer was formed on a 1 μm PCTE hydrophilic membrane (Sterlitech) by applying 100 mbar pressure for 15-25 seconds until all medium was removed. 5 ml of delivery solution containing 3 μM dextran 3000 was prepared and loaded into an LP100 atomizer. The cells were sorbed and after 1 min 30 s the membrane was gently transferred to a 60 mm cell culture Petri dish. After 2 min, 1 ml of stop solution was added to the membrane and left to incubate for 30 seconds before adding 4 ml of medium to the cells. The Petri dish was incubated at 37 °C and 5% CO 2 For nucleofection, 5 × 10 cells were transferred to a 30-μl incubator for 30 min. 6 Cells were harvested, washed twice with 1x PBS, and diluted according to mouse weight, i.e. 1 x 10 cells per gram. 6 Resuspend and centrifuge at 100x the concentration based on body weight per mouse (1 x 10 per gram). 6 Mice were weighed 2–3 times a week and monitored for the appearance of GvHD-like symptoms. Peripheral blood was collected 9–12 days after injection and processed for flow cytometry analysis. Blood and spleens were also collected at the end of the study and prepared for analysis.

[0253] PBMCs were isolated from three different donors (D119, D120, D121) and cells from each donor were subjected to solporation or nucleofection. For this study, there were four groups of NOD scid gamma (NSG) mice with five per group. Groups were: 1. No cells, 2. UT, 3. Sorption, 4. Nucleofection (4 groups x 5 mice p / group x 3 donors = 60 mice). Cells were injected into mice on day 0. Animals were monitored daily and all animals that received cells had GvHD symptoms present by day 14 post-injection, indicating that the cells were viable and functional. Blood samples were taken from mice on day 14 and analyzed by flow cytometry for the presence of human PBMCs and T cell subsets as indicated by CD45 expression. Results showed the presence of CD45+ cells in the untreated and sorbolated groups at this time point, confirming that the cells were viable and functional (Figure 53A, B, C). Fewer CD45+ cells were detected in the nucleofected cells at this time point. These data indicate that sorbolated human PBMCs remain viable and functional in vivo and have the ability to induce GvHD in NSG mice. These results demonstrate the successful engraftment of sorbolated cells into NSG mice, showing that they remain viable and functional after sorbolation.

[0254] Filter Membrane Conditions for Delivery of Payload Compositions to Cells To facilitate and enhance the exposure of cells (non-adherent or adherent) to the permeabilization solution, the filter membrane is optionally vibrated before, after, during, or in any of these arrangements. To support the formation of a monolayer of cells on the filter membrane, the membrane can be vibrated before, after, during, or in any of these arrangements. Vibration of the filter membrane can be performed using a readily available device, for example, a piezoelectric accelerometer, such as the Miniature Triaxial DeltaTron® accelerometer (available from Bruel and Kjaer, www.bksv.com). Some suitable vibration motors are also available from Precision Microdrives (www.precisionmicrodrives.com).

[0255] For example, the vibration can be provided by an eccentric rotating mass (ERM) system or a linear resonant actuator (LRA) system. Preferably, one, two, or three actuators (LRAs) corresponding to the X, Y, and Z axes can be attached to the membrane or membrane holder such that the membrane vibrates by mechanical coupling to the actuators.

[0256] The advantage of the LRA system is that each axis can be driven independently. Thus, complex but controllable vibration patterns can be generated in the membrane. In addition, identification of mechanical resonance points due to the physical characteristics of the membrane will improve the degree of control that can be exhibited over the membrane. A three-axis accelerometer device will be mechanically coupled to the filter membrane or holder to provide feedback on the membrane-induced deflection. The accelerometer system can be used to monitor or as a control feedback signal to the vibration system to generate an error signal between the desired and achieved vibration patterns. The selection of the driving vibration frequency is based on the stiffness of the membrane and the size of the cells on the membrane. An exemplary vibration pattern is provided with 3000 Hz sinusoidal signals on the x and y axes and no signal on the z axis. The deflection is 1 mm peak-to-peak, there is no phase difference between the x and y driving waveforms, and both are coherent. Many other patterns are possible, including those that provide rotations and shaking in the x, y, and / or z axes.

[0257] Device for delivering cargo molecules to mammalian cells - Patents.com The present subject matter generally relates to the delivery of biological payloads to cells. The delivery of biological payloads to cells can involve atomization and delivery of a permeabilization solution to a monolayer of cells. Current techniques can be deficient in several areas, which are detailed below.

[0258] Before being loaded with the payload, the cells may be submerged or suspended in the culture medium. To achieve effective payload uptake, it may be beneficial to remove the culture medium so that the cells can be exposed to the permeabilization solution, and to organize the cells into a monolayer. In some current techniques, the medium is removed by centrifugation. Although this technique is effective in removing the medium, it does not usually result in the formation of a uniform monolayer of cells.

[0259] Cells can be aligned under a solution atomizer or nebulizer to ensure proper distribution of the permeabilization solution onto the cell monolayer. Current techniques generally use manual systems to align cells under the atomizer, which is prone to operator variability. Atomizers / nebulizers can be used to dispense permeabilization solutions onto cell monolayers. However, while certain atomizers are designed to dispense milliliter amounts of solution, it may be preferable or even necessary for the dispensed volume to be on the order of microliters. In addition, transfection protocols for payload delivery can involve several time-critical steps. Currently, fluid handling is generally manually controlled and is therefore inherently unstable.

[0260] Due to these shortcomings, data may be inherently inconsistent. Lack of reproducibility of data may hinder further development of payload delivery processes. To address the above-mentioned issues, some aspects of the present subject matter provide a delivery system that can increase the consistency and delivery efficiency of the delivery process while maintaining cell health.

[0261] Exemplary Delivery Systems 65 illustrates an example of a delivery system 8800 configured to deliver a payload to cells. The delivery system 8800 may include a housing 8802 configured to receive a plate 8804 including wells. The delivery system 8800 may include a pressure differential applicator 8806 configured to apply a pressure differential to the wells, a delivery solution applicator 8808 configured to deliver atomized delivery solution to the wells, a stop solution applicator 8810 configured to deliver a stop solution to the wells, and a culture medium applicator 8812 configured to deliver culture medium to the wells.

[0262] As an example, in some embodiments, the pressure differential applicator 8806 is or can include a nozzle valve assembly, such as nozzle valve assembly 9310, described below with respect to Figures 84 and 89-91. As another example, the pressure differential applicator 8806 is or can include a vacuum manifold assembly, such as vacuum manifold assembly 9008, described below with respect to Figures 93-107. In some embodiments, the delivery solution applicator 8808 is or can include a nebulizer, such as nebulizers 9304, 9804, described below with respect to Figures 85, 88, 93, and 115-118. In some embodiments, the stop solution applicator 8810 is or can include a needle emitter, such as needle emitter 9303, described below with respect to Figures 85, 87. As another example, in some embodiments, the culture medium applicator 8812 is or can include a needle emitter 9303.

[0263] The delivery system 8800 may also include a control system 8814, an actuation system 8816, a support frame 8818, and a sensing and management system 8820. In some embodiments, the pressure differential applicator 8806, the delivery solution applicator 8808, the stop solution applicator 8810, and / or the culture medium applicator 8812 can be coupled to the support frame 8818. The actuation system 8816 can be coupled to the support frame 8818, the housing 8802, and / or the plate 8804 and configured to move the support frame 8818, the housing 8802, and / or the plate 8804. As another example, the actuation system 8816 can be coupled to and configured to move the pressure differential applicator 8806, the delivery solution applicator 8808, the stop solution applicator 8810, and / or the culture medium applicator 8812. For example, the support frame 8818 may be or may include a fluid head module 9308, described below with respect to Figures 84-89. The actuation system may be or may include an actuator 9319, described below with respect to Figures 84, 85. In some embodiments, a vibration system may be included to vibrate a membrane (e.g., one located within a well of a plate).

[0264] The sensing and management system 8820 can include sensors and / or thermal management systems. By way of example, the sensors and / or thermal management systems can be coupled to the pressure differential applicator 8806, the delivery solution applicator 8808, the stop solution applicator 8810, and / or the culture medium applicator 8812 and can be configured to measure and / or control pressure, temperature, position, and flow rate.

[0265] The control system 8814 can include at least one data processor and can be electrically coupled to the actuation system 8816 and the sensing and management system 8820. The control system can be configured to control the actuation system 8816 and the sensing and management system 8820. As an example, the control system 8814 can be or include the control system 9306 described below with respect to FIG.

[0266] Exemplary Vacuum System Figure 66 shows a diagram 8900 illustrating the nine elements of the delivery system. The system includes an addressable well vacuum manifold assembly, atomization, fluid control, temperature control of the delivery solution, mounting, automation, and software, enclosure, filter, and temperature control of the base plate.

[0267] The delivery system can address the following areas of variability associated with payload delivery to cells: monolayer formation, atomization, automation of payload delivery, and temperature control of solutions and culture vessels.

[0268] To improve data consistency and delivery efficiency of payload delivery, the system removes certain known sources of variability from the process. The system can address removal of media using vacuum and generation of a monolayer of cells, atomization of permeabilization solution to generate monodisperse droplets, fluidic control of solutions to enable automation, temperature control of solutions, mounting of spray heads and temperature reservoirs, automation and software design, instrument enclosure, and temperature control of the base plate.

[0269] An exemplary embodiment of a high precision rig system 9000 is shown in Figures 67-69. The high precision rig system 9000 can include a needle emitter 9002, an atomizer 9004, and a vacuum manifold system 9006. The vacuum manifold system 9006 can include a vacuum manifold assembly 9008, a translation stage 9010, a valve 9011 (as shown in Figures 68-69), and a manifold 9024. In some embodiments, the vacuum manifold assembly 9008 can be coupled to the translation stage 9010 via a coupling member 9012. In some embodiments, the valve 9011 can be a pinch valve. The vacuum manifold assembly 9008 can include a filter plate 9014, a base plate 9016, and a top plate 9018 (as shown in Figures 68-69). As shown in Figures 68-69, the filter plate can be a 96-well filter plate. The filter plate 9014 can be seated within a recess in the base plate 9016, and a top plate can be positioned over the filter plate 9014 and coupled to the base plate 9016 to secure the filter plate 9014 in place.

[0270] The needle emitter 9002 can function to deliver culture medium, which can contain cells, to the wells of the filter plate 9014. The base plate 9016 can have a vacuum fitting 9020 extending from its bottom surface. The vacuum fitting 9020 can be in the form of a generally cylindrical tube and can allow for the application of vacuum pressure to the corresponding wells of the filter plate 9014. The vacuum pressure can be delivered to each valve 9011 via a port 9024a of a manifold 9024 and delivered to the vacuum fitting 9020. The atomizer 9004 can atomize the permeabilization solution and deliver it to the cells in the wells of the filter plate 9014. Systems, devices, and methods relating to the delivery of the permeabilization solution to a monolayer of cells are discussed in more detail below.

[0271] 68-69, the high precision rig system 9000 can include a guide rail 9022 that can extend along an X-axis. In some embodiments, the translation stage 9010 can be coupled to the guide rail 9022, which allows the vacuum manifold assembly 9008 to be translated along the X-axis. This allows the vacuum manifold assembly 9008 to be moved relative to other components, such as the needle emitter 9002 and / or the atomizer 9004.

[0272] Figures 70-71 show exploded views of the vacuum manifold assembly 9008. Figures 72-73 show plan and side cross-sectional views, respectively, of the base plate 9016. As shown in Figures 70-71, the vacuum manifold assembly can include a filter plate 9014, a base plate 9016, a gasket 9026, and a top plate 9018, which can have a connecting hole 9018b.

[0273] 70-73, the base plate 9016 can include a first and a second set of coupling holes 9016b, 9016c. The first set of coupling holes 9016b can be aligned with coupling holes 9018b in the top plate 9018 such that the base plate 9016 can be coupled to the top plate 9018 by coupling members, such as bolts or screws, that can extend through the coupling holes 9016b, 9018b. The coupling members 9012 can be coupled to the base plate 9016 via coupling elements that can extend into the second set of couplings 9016c.

[0274] The base plate 9016 can include a first recess 9028 that can receive or seat the filter plate 9014, as well as secondary recesses 9030 that can receive the gasket 9026. Each of the secondary recesses can have an opening 9030b or a passageway that can connect with a corresponding vacuum fitting 9020.

[0275] In addition to having wells with inactive openings 9014c, filter plate 9014 can have wells with active openings 9014b. Active openings 9014b can be located above and fluidly connected to openings 9030b in base plate 9016. In other words, certain wells of filter plate 9014 can be active and other wells of filter plate 9014 can be inactive.

[0276] The gasket 9026 can have holes 9026b that can align with the effective openings 9014b of the wells in the filter plate 9014 and the openings 9030b in the base plate 9016. The gasket 9026 can function to form a seal between the base plate 9016 and the filter plate 9014, thereby allowing each effective opening 9014b to be isolated from the other effective openings 9014b and the non-effective openings 9014c, while allowing fluid communication between the effective openings 9014b of the wells and the corresponding vacuum fittings 9020.

[0277] Figures 74-76 show various views of the top plate 9018. As shown in Figures 70-71 and 9-11, the top plate 9018 can include a recess 9032 that can accommodate a portion of the filter plate 9014. When assembling the vacuum manifold assembly 9008, the gasket 9026 can be seated in the secondary recess 9030 of the base plate 9016, the filter plate 9014 can be received within the first recess 9028, and the top plate can be positioned above the filter plate 9014 and coupled to the base plate 9016 as described above.

[0278] In some embodiments, all of the wells of the filter plate 9014 can be connected to different valves 9011 and manifolds so that all wells can be utilized.

[0279] Removal of medium using vacuum and generation of cell monolayer The vacuum manifold system 9006 can remove culture medium from 1-12 wells of the filter plate 9014, addressing a total of six wells at one time. A top view of the filter plate 9014 is shown in FIGS. 77-78. In FIG. 77, the filter plate 9014 is in a first position such that wells F2, F4, F6, F8, F10, and F12 are available when the filter plate 9014 is received in a first recess 9028 of the base plate 9016. The filter plate 9014 can be rotated 180° to enable wells C1, C3, C5, C7, C9, and C11, as shown in FIG. 78.

[0280] As discussed above, vacuum pressure may be routed through the manifold 9024 to each valve 9011 and to the vacuum fittings 9020. Various views of a portion of the high precision rig system 9000 are shown in FIGS. 79-81. As shown in FIGS. 79-81, the manifold may include eight ports 9024a and tubing 9034 may extend from six ports 9024a on the manifold to the six valves 9011. Tubing may also connect a port on each valve 9011 to a vacuum fitting 9020 connected to the base plate 9016. A vacuum line (not shown) may be connected to one of the two remaining open ports 9024a on the manifold 9024 to seal the remaining port 9024a.

[0281] The vacuum manifold system 9006 can be used to allow the formation of a monolayer of suspension cells. As described above, the needle emitter 9002 can deliver culture medium, which can contain cells, to the wells of the filter plate 9014. Vacuum pressure can be applied to the 8-channel manifold via a vacuum line. The valves 9011 can be opened and closed individually. Hence, access to each active well can be controlled individually. When the valve 9011 is opened, the applied vacuum pressure can be sufficient for effective removal of the culture medium in which the cells are suspended, without causing shear stresses that can lead to damage. The extracted medium passes through the tube 9034 and the valve 9011, through the manifold 9024, and out of the port 9024a to which the vacuum line is attached. In this way, a cell monolayer can be formed and cell viability can be maintained. The formation of the cell monolayer can be achieved without damaging the cells.

[0282] A vacuum manifold system 9006 was assembled and tested. During testing, culture medium containing cells was added to the wells of a 96-well filter plate 9014. Vacuum pressure was applied via manifold 9024. Vacuum pressures ranging from 10 bar to 100 mbar were applied to individual wells by opening valves 9011 associated with the wells.

[0283] At higher vacuum pressures of -100 mBar to -75 mBar, the culture medium was removed far enough from the wells that it would not "wick" back into the wells when the filter plate 9014 was removed.

[0284] At lower pressures of -50 to -10 mBar, the medium remained close enough to the wells that some of the medium "wicked" back into the wells when the filter plate 9014 was removed from the vacuum manifold assembly 9008. The amount of liquid that wicked back into the wells was approximately 5 to 10 μl. This is not an issue when the vacuum manifold assembly 9008 is part of the precision rig system 9000, as the wells are sprayed and the culture medium replaced before the filter plate 9014 is removed from the manifold.

[0285] In some embodiments, filter paper or other materials can be used to prevent wicking, for example, filter paper can be placed between the gasket 9026 and the base plate 9016 to prevent media from wicking back into the wells.

[0286] At pressures as low as -10 mBar, the culture medium was removed at a gentle, controlled rate that was more gentle on the cells, both in the presence and absence of cells, which may lead to improved viability and recovery of cells from the wells.

[0287] Pulsing valve 9011 on and off during testing did not appear to facilitate removal of culture medium.

[0288] Centrifugation was also investigated as a method to form a cell monolayer; however, this proved to be ineffective as it formed an uneven cell layer. Figure 82 shows the distribution of Glow Germ particles observed after centrifugation and after vacuum extraction. Glow Germ particles include. The top three images show the distribution of Glow Germ particles obtained when the culture medium was removed using vacuum pressure, and the bottom three images show the distribution of Glow Germ particles obtained when the culture medium was removed using centrifugation. The centrifugation samples were centrifuged at 350 x g for 5 minutes, and the vacuum pressure samples were subjected to a vacuum of -800 mbar for 15 seconds.

[0289] As shown in FIG. 82, the vacuum pressure sample showed a more even distribution of glow germ particles, whereas after centrifugation, an uneven crescent-shaped distribution of glow germ particles was observed.

[0290] At -10 mBar, comparable results were achieved for mRNA uptake using the vacuum and centrifugation methods. The GFP MFI was found to be higher in samples prepared using the vacuum method compared to the centrifugation method (approximately 300,000 units vs. 50,000 units), indicating that more mRNA entered the positive cells.

[0291] The time required to extract the culture medium from the wells is estimated to be between approximately 3 and 20 seconds. However, the amount of time required to remove the culture medium from the wells may depend on the amount of vacuum pressure applied. With more work, the length of time required to remove the medium at lower vacuum pressures can be explored.

[0292] In some embodiments, it could be possible to drain all wells simultaneously with low pressure (no plates on manifold) to remove residual medium from the outlet.

[0293] The vacuum manifold system 9006 allows for the application of vacuum pressure to individual wells of the filter plate 9014. By applying vacuum pressure to individual wells on the filter plate 9014, more precise control of the vacuum pressure, and greater consistency of the vacuum pressure applied to each well can be achieved. The existing vacuum manifold applies vacuum to the entire 96-well filter plate. During testing, a low vacuum pressure was effective in removing the culture medium and producing an even monolayer of cells within the wells.

[0294] Exemplary Positive Pressure System 83 shows a diagram 9200 illustrating six elements of an exemplary positive pressure delivery system. These elements include atomization, fluid control, positive pressure, modular head, automation and software, and enclosure.

[0295] The positive pressure delivery system can address the following areas of variability associated with payload delivery to cells: monolayer formation, atomization, automation of payload delivery, and temperature control of solutions and culture vessels.

[0296] To improve data consistency and delivery efficiency of payload delivery, the system removes certain known sources of variability from the process. The system can address the removal of media using positive pressure and generation of a monolayer of cells, atomization of permeabilization solutions to generate monodisperse droplets, fluidic control of solutions to enable automation, temperature control of solutions, attachment of emitters, atomizers, nebulizers and temperature reservoirs, automation and software design, instrument enclosure, and temperature control of a base plate configured to hold the well plate.

[0297] An exemplary embodiment of a positive pressure system 9300 is shown in Figures 84-85. This positive pressure system 9300 uses positive pressure as an alternative to vacuum to remove culture medium. The positive pressure increases the accuracy and precision of the delivery of small volumes (1 μl-100 μl) of fluid to push culture medium out of the wells of a well plate. The positive pressure system 9300 can include a mounting array 9302, a manifold assembly 9305, and an actuation and control system 9306.

[0298] The manifold assembly 9305 can include a base 9312, also referred to as a plate holder, that can extend from a housing 9314 of the manifold assembly 9305. The base 9312 can be configured to receive a filter plate 9316. As shown in the illustrated example, the filter plate 9316 can be a 96-well filter plate.

[0299] The mounting array 9302 can include a fluid head module 9308 to which a needle emitter 9303, a nebulizer 9304 and / or a positive pressure nozzle assembly 9310 are attached. The needle emitter 9303 can be configured to deliver culture medium and a stop solution, which can contain cells, to the wells of the filter plate 9316. The nebulizer 9304 can be configured to atomize the permeabilization solution and deliver it to the cells in the wells of the filter plate 9316. The positive pressure nozzle assembly 9310 can be configured to apply positive pressure to the wells of the well filter plate 9316 to remove a liquid portion of the culture medium from the wells, thereby generating a monolayer of cells. Systems, devices and methods related to the delivery of the permeabilization solution to the monolayer of cells are discussed in more detail below.

[0300] In some embodiments, the positive pressure system 9300 can include guide rails 9318. In the illustrated example, the filter plate 9316 is held stationary and the position of the mounting array 9302 can be adjusted relative to the position of the filter plate 9316. For example, the mounting array 9302 can be coupled to the guide rails 9318, which can allow the mounting array 9302 to be translated along an axis X2 defined by the guide rails 9318. For example, an actuator 9319 can move the mounting array 9302 along the guide rails 9318. The guide rails 9318 can also move along an axis Y2, which can be perpendicular to the axis X2. The actuator 9319 can also move the mounting array 9302 along the axis Y2. Thus, the mounting array 9302, including the needle emitters 9303, nebulizers 9304 and / or positive pressure nozzle assemblies 9310, can be selectively positioned over the wells of the filter plate 9316 when the filter plate 9316 is positioned within the housing 9314.

[0301] As an example, a user can insert a filter plate 9316 into the base 9312, which can then be positioned within the housing 9314. The filter plate 9316 can remain stationary after being received within the housing 9314. The filter plate 9316 can be selectively addressed by needle emitters 9303, nebulizers 9304 and / or positive pressure nozzle assemblies 9310 coupled to the fluid head modules 9308 of the mounting array 9302. In the illustrated example, the mounting array 9302 includes six fluid head modules 9308. Two fluid head modules 9308 include needle emitters 9303, three fluid head modules 9308 include nebulizers 9304, and one fluid head module 9308 includes a positive pressure nozzle assembly 9310. The mounting array 9302 can be moved along axes X2, Y2 so that each of the fluid head modules 9308, including the needle emitters 9303, nebulizers 9304, and / or positive pressure nozzle assemblies 9310, can be addressed to any location on the filter plate 9316.

[0302] In some embodiments, each of the individual fluid head modules 9308 can have the ability to move up and down parallel to axis Z2, which allows for independent actuation of the fluid head modules 9308. In some embodiments, the movement of the fluid head modules 9308 and the actuation of the needle emitter 9303, nebulizer 9304, and / or positive pressure nozzle assembly 9310 are independently controlled. The movement of one or more of the fluid head modules 9308 and the actuation of the needle emitter 9303, nebulizer 9304, and / or positive pressure nozzle assembly 9310 attached thereto can occur simultaneously. In some embodiments, the fluid head modules 9308 actuate the needle emitter 9303, nebulizer 9304, and / or positive pressure nozzle assembly 9310 as they move towards the filter plate 9316. The fluid head module 9308 can house a variety of fluid dispensing assemblies, including but not limited to needle assemblies, Ari Mist nebulizers, and positive pressure nozzle assemblies, as shown in Figures 84-85.

[0303] The actuation and control system (ACS) 9306 can include at least one data processor and can control the movement of the mounted array 9302 and / or the fluid head module 9308. The ACS 9306 can also control the actuation of the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310 attached to the fluid head module 9308. For example, the ACS 9306 can include software having instructions that can be interpreted by a data processor of the ACS 9306. The data processor can receive, process, and execute the instructions. For example, the data processor can deliver control signals to actuators and / or motors of the ACS 9306 to adjust the position of the mounted array 9302 and / or to actuate the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310.

[0304] Exemplary Fluid Head Module As described above, the fluid head module 9308 provides a mounting point for various fluid dispensing assemblies (e.g., needle emitter 9303, nebulizer 9304 and / or positive pressure nozzle assembly 9310) so that they can be coupled to the mounting array 9302. The fluid head module 9308 also provides actuation along axis Z2 so that the dispensing assemblies can be selectively actuated.

[0305] 86 shows a close-up view of the fluid head module 9308. The fluid head module 9308 can have a frame 9350. That is, it includes a series of machined aluminum pieces that form the frame 9350. For example, the frame 9350 can include a base plate 9352, a back plate 9354, and upper and lower mounting elements 9356, 9358. The machined aluminum pieces can allow for high tolerance locating of the fluid head module 9308.

[0306] The fluid head module 9308 can include a shaft 9360 extending between the base plate 9352 and the upper mounting element 9356. The shaft 9360 can extend through an opening in the lower mounting element 9358. A guide 9362, such as, for example, a ball spline, can be mounted on the shaft 9360 and coupled to the lower mounting element 9358. The guide 9362 facilitates up and down movement of the fluid dispensing assembly (e.g., needle emitter 9303, nebulizer 9304 and / or positive pressure nozzle assembly 9310) along axis Z2. For example, a pneumatic actuator 9364 located between the upper and lower mounting elements 9356, 9358 can drive the lower mounting element 9358 up and down along the shaft 9360. The pneumatic actuator 9364 can drive associated pneumatic equipment as well as two proximity sensors. For example, the sensors can be embedded in the wall of the cylinder. 87, 88 and 89 respectively show a fluid head module 9308 fitted with a needle emitter 9303, a nebulizer assembly 9301 including a nebulizer 9304, and a positive pressure nozzle assembly 9310.

[0307] Removal of medium using positive pressure and generation of cell monolayers As discussed above, the positive pressure nozzle can be configured to apply positive pressure to the wells of a well filter plate to remove culture media and generate a monolayer of cells. A close-up view of a positive pressure nozzle assembly 9310 used in the positive pressure system 9300 shown in FIGS. 84-85 is shown in FIGS. 90-91. In the illustrated example, the nozzle assembly 9310 includes an internal lumen that can be formed from a tube 9320 located within a housing 9322. The tube 9320 can have openings 9321, 9323 adjacent to first and second ends 9326, 9327 of the positive pressure nozzle assembly 9310. In some embodiments, the housing 9322 can provide structural stability to the tube 9320.

[0308] As shown in FIG. 90, the nozzle assembly 9310 includes a coupling member 9324 coupled to a first end 9326 of the tube 9320. The coupling member 9324 can be configured to form a seal with an opening 9321 adjacent the first end 9326 of the tube 9320 such that pressurized steam (e.g., air) can be delivered to the well 9316a of the filter plate 9316. For example, compressed air can be delivered from a compressor to an inner lumen 9325 of the coupling member 9324. The inner lumen 9325 of the coupling member 9324 can be in fluid communication with the lumen of the tube 9320 via the opening 9321, which can be in fluid communication with the well 9316a via the opening 9323. The compressed air can increase the pressure in the tube 9320, thereby increasing the pressure in the well 9316a. The positive pressure nozzle assembly 9310 thus controls the flow and pressure of the compressed air.

[0309] As shown in FIG. 91 , the second end 9327 of the tube 9320 includes a seal-forming element 9328 located on the exterior of the tube 9320 adjacent the opening 9323. The seal-forming element 9328 is configured to contain the well 9316a and form a seal with the top surface 9330 of the filter plate 9316, thereby isolating the well 9316a. The seal-forming element 9328 is configured to prevent air from leaking between the second end 9327 of the nozzle assembly 9310 and the top surface 9330 of the filter plate 9316, thereby improving the accuracy and precision of the control of pressure in the well 9316a. For example, when the nozzle assembly 9310 is engaged with the filter plate 9316, application of compressed air will cause liquid to flow through the filter base 9332 of the individual wells.

[0310] In some embodiments, the positive pressure nozzle assembly can include a valve located along its length. The valve can function as a manual control to control the pressure of air delivered to the wells of the filter plate. FIG. 92 shows an example of a portion of a nozzle assembly 9410 including a valve 9432. In the illustrated example, the nozzle assembly 9410 includes a first tube portion 9420a and a second tube portion 9420b coupled to the valve 9432. The second tube portion 9420b can include a seal-forming element located adjacent a distal opening 9423 of the second tube portion 9420b. This nozzle can generally function similarly to the nozzle assembly 9310 described above with respect to FIGS. 90-91. For example, the distal end of the second tube portion can be located adjacent to the top surface of the filter plate such that the seal-forming element 9428 encompasses the opening of the well to form a seal with the top surface of the filter plate. Compressed air can be delivered to the interior lumen of the first tube portion 9420a. When the valve is in the open position, air can pass through the valve 9432 via the second tube portion 9420b and into the wells of the filter plate. Alternatively, the valve 9432 can be adjusted to the off position to prevent airflow into the wells. The valve 9432 can be adjusted to control the air pressure in the wells.

[0311] Atomization of a delivery solution to generate monodisperse droplets 93 shows an expanded view of a nebulizer assembly 9301 that can be coupled to a fluid head module 9308. In the illustrated example, the nebulizer assembly 9301 includes a syringe 9366, a microvalve 9368, and a nebulizer 9304. The nebulizer 9304 can be coupled to the microvalve 9368 via a coupling member 9370 (e.g., a pre-column coupling). The microvalve 9368 can be retained and / or coupled within a valve holder 9372 that can be coupled to the syringe 9366 via an adapter 9374. The nebulizer assembly 9301 allows for the delivery of payload solution to the nebulizer 9304 with high accuracy and precision.

[0312] There are a number of different ways in which the permeabilization solution can be delivered to the cell monolayer: for example, the permeabilization solution can be atomized using ultrasound or can be nebulized using a nebulizer.

[0313] Both sonication and nebulization were tested as delivery methods. A total of four different spray heads were tested.

[0314] The following parameters were evaluated for each spray head: air pressure, flow rate, distance, delivered volume, cell number, spray time, ultrasonic probe frequency, and ultrasonic probe power.

[0315] High speed camera recording was used to assess the effect on the spray characteristics. The force experienced by the cells was measured by force sensor analysis. The volume delivered into the well was assessed using a colorimetric assay.

[0316] Sonication tests were performed at 60 kHz, 130 kHz, and 180 kHz. Liquid can be forced through an ultrasonic nozzle by a pumping system and atomized into a fine mist spray using high frequency sonic vibrations.

[0317] A piezoelectric transducer was used to convert electrical input into mechanical energy in the form of vibrations that, when introduced into the nozzle, create capillary waves in the liquid, resulting in atomization of the liquid. Each ultrasonic probe was operated at a given resonant frequency. The operating frequency can determine the size of the droplets produced. The droplet size can also be affected by the power at which the ultrasonic probe is operated, although to a lesser extent by the operating frequency. An auxiliary air flow can be used to help control and shape the spray.

[0318] Nebulization tests were performed using Ari Mist nebulizer 9304 (Figure 125). As shown in Figure 125, nebulizer 9304 includes a liquid fitting 9307 and an air fitting 9309. The Ari Mist nebulizer runs on compressed gas and requires a pump to supply the sample solution. This atomizer has two channels, one for the gas (air) and the other for the liquid to be nebulized, both flowing along parallel paths. Both paths terminate at the tip of the nebulizer with an orifice for the gas and an outlet for the liquid. The gas flow can draw the liquid into the gas flow. Collisions with the gas molecules can break up the liquid into small droplets resulting in nebulization.

[0319] The ultrasound emitter operating at 180 kHz was found to be more effective in delivering payload to T cells compared to the 130 kHz, 60 kHz ultrasound head, and the Ari Mist nebulizer. Figure 94 shows a set of results characterizing the efficiency of payload delivery for an ultrasound emitter operating at 180 kHz. The test results showed that the payload was successfully delivered to T cells with an efficiency of approximately 15-25% and a high level of consistency (±1%) between multiple repetitions. Cell health was maintained after delivery (85% viability).

[0320] These results indicate that the ultrasonic spray emitter produces a monodisperse spray that results in uniform deposition of the delivery solution and payload on the cells. Furthermore, reducing the delivered volume and lowering the ethanol concentration improved the delivery efficiency with the ultrasonic spray head.

[0321] Figure 95 shows another set of results characterizing GFP uptake for the ultrasound emitter, Ari Mist nebulizer, and MAD nasal spray emitter. A MAD Nasal™ intranasal mucosal atomization device (Teleflex, 3015 Carrington Mill Blvd., Morrisville, NC 27560) was used to atomize the delivery solution containing the payload. Briefly, 7 μl of the delivery solution was pipetted directly into the nasal head. The spray head was directly connected to an air pressure source via a luer lock fitting. 1.5 bar of air pressure was delivered to the spray head for 330 ms to generate the spray. Current data indicates that the ultrasound spray emitter operating at 180 kHz is more efficient for delivering mRNA to T cells. The ultrasound spray emitter achieved 32.7% GFP uptake compared to 24.8% with the Ari Mist nebulizer (e.g., nebulizer 9304). Both the ultrasound emitter and the Ari Mist nebulizer resulted in higher GFP mRNA delivery compared to the NAD nasal spray emitter, which resulted in a delivery efficiency of 16.9%.

[0322] Figure 96 shows a series of results characterizing cell viability for the ultrasound emitter, Ari Mist nebulizer, and MAD nasal spray emitter. These results represent a minimum of three technical replicates for each spray head tested (e.g., ultrasound emitter, Ari Mist nebulizer, and MAD nasal spray emitter). Compared to the ultrasound head, which averaged 72.3% cell viability compared to untreated cells, the Ari Mist nebulizer improved cell viability with 85.3% compared to untreated cells. The MAD nasal spray emitter provided the highest cell viability with 99.7% cell viability compared to untreated cells.

[0323] Enclosure Atomization For example, during delivery of a solution using an atomizer, nebulizer, and / or ultrasound emitter, fine aerosols are generated. In some instances, the fine aerosols may contaminate adjacent wells of a multi-well filter plate. In some embodiments, an enclosure may be provided at the distal end portion of the atomizer, nebulizer, and / or ultrasound emitter, which may prevent contamination of adjacent wells of the filter plate.

[0324] Three rig platforms were constructed: Rig 1 (R1), Rig 3 (R3), and Rig 4 (R4) (Figures 126-128). The table below lists some of the rig features.

[0325] As illustrated in FIG. 126, R1 11700 includes a solution reservoir 9810 (e.g., an Elveflow sample reservoir) configured to provide permeabilization solution to an Ari Mist nebulizer 9804 and a pinch valve 9808 (e.g., an Elveflow pinch valve) configured to control delivery of the permeabilization solution to the nebulizer 9804. The nebulizer 9304 can be mounted to a spray head mount 11714 that can be configured to retain the nebulizer 9304 and facilitate alignment of the spray head. The pinch valve 9808 is configured to allow fluid control of the payload solution to the nebulizer 9304. The spray head mount 11714 can be mounted to a guide 11717 configured to facilitate up and down movement of the spray head mount 11714 along a vertical axis Z3. A spray head mount 11714 containing the nebulizer 9804 can be mounted on top of a plate holder 11718 which can be configured to receive a filter plate 11716 .

[0326] 127, the R3 11800 includes a nebulizer assembly 9301 having a sample reservoir syringe 9366 coupled to an Ari Mist nebulizer 9304 via a microvalve 9368 configured to control delivery of permeabilization solution to the nebulizer 9304. The nebulizer assembly 9301 can be mounted to a fluid head module 9308. The fluid head module 9308 can be mounted to a guide 11717 configured to facilitate up and down movement of the fluid head module 9308 along a vertical axis.

[0327] As illustrated in FIG. 128, the R4 9800 includes an LB-100 atomizer (not shown) coupled to a pinch valve 9808 (e.g., an Elveflow pinch valve) configured to control delivery of permeabilization solution from a sample reservoir (e.g., an Elveflow sample reservoir, not shown) to the atomizer. The atomizer can be located within a collar 9816 of a spray head mount 9814 that can be configured to retain the atomizer and facilitate alignment of the spray head. The collar 9816 containing the atomizer can be located over an opening of the stirred cell system 11900 such that the atomizer can deliver a payload to cells in the stirred cell system 11900.

[0328] (Table) Rig Features TIFF0007644048000014.tif60151

[0329] Details of the rig's features that allow fine control of spray parameters are provided below.

[0330] In some embodiments (e.g., R1 and R4), fluidic control of the delivery solution containing the payload can be achieved using an Elveflow pinch valve. Fluidic control can be achieved by a fluidic control system that can apply a constant pressure to an Elveflow fluid reservoir to force fluid through the pinch valve 9808. The volume of fluid that can be dispensed can be controlled by at least the amount of pressure applied, the length of time the valve 9808 is left open, and / or the diameter of the tubing used. The valve 9808 can be actuated by a metal oxide semiconductor field effect transistor (MOS FET), which can be controlled by a microprocessor.

[0331] The Elveflow-Pinch Valve 9808 described above had several limitations. For example, recalibration of R1 and R4 was required every time the system was refilled. Accuracy and precision were insufficient for dispensing volumes below 5 μl. For low volumes (<5 μl), the relative standard deviation for repeated dispenses (10) was approximately 9%. To address the observed limitations, the R3 was developed. As mentioned above, the R3 contains the Microvalve 9368 rather than the Pinch Valve 9808. Fluidic control of the delivery solution containing the payload can be achieved using the Microvalve 9368. When delivering volumes ranging from 1 μl to 100 μl, accuracy and precision were higher for the R3 using the Microvalve 9368.

[0332] Fluid control of the air delivered to the nebulizer / atomizer can be achieved using a solenoid valve. In some embodiments, electronics can be used to control the operation of the nebulizer / atomizer. To enable electronically controlled spray actuation, a system was designed using a microprocessor-based development board to allow easy development of time-controlled sequences. The development board used a microprocessor PIC16F1619. The spray actuation time and fluid delivery time can be manipulated by the development board's interface software. The microprocessor development board allows for pulsing of the nebulizer / atomizer spray. The system was then upgraded to include repeatable high-speed PLC (Programmable Logic Controller) technology to better align with industry standards and serve as a demonstration of automated delivery technology (which is based on ultra-high-speed PLC technology). The rig controller consisted of a PLC and Gyger controller and a program to facilitate communication between these two pieces of hardware. The hardware has operator interaction through momentary push buttons.

[0333] The parallel channel design of the Ari Mist Nebulizer 9304 inherently produces an off-center spray from the tip of the Nebulizer 9304. A custom spray head holder equipped with a goniometer allows for adjustment of the spray head alignment.

[0334] Several methods for delivering solutions using enclosed emitters were tested, and the results of GFP uptake using enclosed emitters were compared to the results of GFP uptake using unenclosed emitters.

[0335] Method 1: Using color to surround the emitter FIG. 97 shows an example of a nebulizer assembly 9501 that includes an enclosing collar 9502 that is positioned around the spray head 9504 of an Ari Mist nebulizer (e.g., nebulizer 9304). In the illustrated example, the spray head 9504 is positioned 27 mm above a well 9516a of a double height filter plate 9516. The collar 9502 was designed and manufactured for the Ari mist spray head 9504. The collar was fitted onto the spray head 9504. A double height walled 96 well filter plate 9516 was used.

[0336] Method 1A: Collar forms a seal with the well plate At a distance of 26 mm from the tip of the spray head 9504 to the bottom of the well 9516a, the collar 9502 engaged with the top edge of the well 9516a of the filter plate 9516 to form a seal. 6 The cells were seeded on a filter plate 9516 and centrifuged at 350×g for 5 min to remove the culture medium. The delivery solution (4 μl) containing GFP mRNA was sprayed onto the cells and incubated for 2 min. After the 2 min incubation, 50 μl of stop solution was added and incubated for 30 s. Finally, 100 μl of culture medium was added.

[0337] Method 1B: Leave a 1mm gap between the collar and the filter plate Collar 9502 was fitted with spray head 9504, with the tip of spray head 9504 positioned 27 mm from the bottom of well 9516a. The collar was therefore held 1 mm above the top surface of filter plate 9516. Note: A single hit protocol (e.g., one exposure to delivery solution and stop solution) was followed for the enclosing experiments.

[0338] Method 1: Results Figure 98 shows results characterizing the efficiency (GFP incorporation) corresponding to a spray head without collar 9502, a spray head with a collar that forms a seal with the filter plate, and a spray head with a collar where a 1 mm gap exists between the collar 9502 and the filter plate.

[0339] Tests corresponding to method 1B, with a 1 mm gap between collar 9502 and the top surface of filter plate 9516, show that collar 9502 did not affect GFP uptake. These results clearly demonstrated that in this setup, the data was comparable between wells with and without collar 9502. T cell viability was not affected by the use of the enclosing collar in either setup.

[0340] Method 1: Conclusion The results indicate that collar 9502 does not affect T cell viability. Spraying is affected by collar 9502 to varying degrees. This difference can be attributed to the degree to which the system is sealed. Sealing the system (e.g., method 1A) created a local pressure maximum that inhibited spraying. For method 1B, a gap was provided between collar 9502 and the top surface of filter plate 9516 to provide an outlet for the pressure. This gap allowed air to expand within the wells, allowing the spray to operate properly. Thus, a collar can be used to enclose the spray, provided it allows expansion space for the air to expand during the spraying process.

[0341] Method 2: Using X-pierce film to enclose the spray Rather than using a collar to surround the spray head, an X-pierce film (Sigma Aldrich, Catalog No. Z722529) was used to surround the spray head, which may be more beneficial than using a collar (e.g., collar 9502) to surround the spray but avoid forming a seal that may inhibit spraying.

[0342] FIG. 99 shows an example of a 96-well PCTE filter plate 9616 with an X-pierce film 9602 adhered to the top surface of the filter plate 9616. The filter base of the wells of the filter plate 9616 has a pore size of 0.4 μm. As shown in the illustrated example, the X-pierce film includes a pre-cut "X" that is positioned above the opening of each well of the filter plate 9616. In the experiment, 2.5×10 CD3+ T cells were cultured in 10 wells of the filter plate 9616. 5 1 was seeded into each well. The filter plate 9616 was centrifuged at 350×g for 1 min to remove the culture medium and generate a monolayer of cells. The air pressure delivered to the nebulizer was set at 1.65 bar, and the tip of the nebulizer spray head was positioned 12 mm above the top surface of the filter membrane located in the well and on which the monolayer of cells was formed. The cell monolayer was sprayed with a delivery solution (1 μl) containing GFP mRNA (2 μg). The cells were then incubated for 2 min. After the 2 min incubation, 50 μl of stop solution was added to each well and the wells were incubated for 30 s. Then, 100 μl of culture medium was added to each cell, as shown in Figure 99. The filter plate was incubated at 37°C for 17-24 h before analysis. A single hit protocol was followed for this enclosing experiment.

[0343] Method 2: Results Figure 100 shows results characterizing the efficiency (GFP incorporation) corresponding to tests performed in Rig 1 (R1) using a filter plate without an enclosure, tests performed in Rig 3 (R3) using a filter plate without an enclosure, and tests performed in R3 using a filter plate containing an X-pierce film enclosure on the wells of the filter plate. The rigs are described above. These results show that the efficiency was comparable in wells with and without film, thus indicating that the x-pierce film did not negatively affect the spray. The use of the X-pierce film did not affect cell viability.

[0344] An x-pierce film can be used to surround the spray and avoid cross contamination between wells without reducing efficiency (GFP uptake) or cell viability.

[0345] Fluid control of solutions to enable automation In some embodiments, the present high precision rig can provide an automated engineering solution for the payload delivery process. To enable this, microfluidic control of the delivery solution, cold stop solution, and culture media can be used.

[0346] Fluid control can be achieved by a fluid control system that can apply a constant pressure to the system to force fluid through pinch valves or microvalves. The volume of fluid that can be dispensed can be controlled by the amount of pressure applied, the length of time the valves are left open, and / or the diameter of the tubing used.

[0347] The valves can be actuated by metal oxide semiconductor field effect transistors (MOS FETs) which can be controlled by a microprocessor.

[0348] The fluid control system can enable delivery of volumes of fluid in the range of 2-10 μl using fine fluid control. Thus, the precision rig system 9000 can control the delivery of metered volume units in the range of 2-10 μl. In some embodiments, other volumes are possible.

[0349] Solution temperature control (temperature reservoir) In some embodiments, a fluid temperature control system can be implemented to control the temperature of the delivery solution, the stop solution, and the culture medium. The fluid temperature control system can include a heating system and a cooling system.

[0350] FIG. 101 shows an example of a heating system 11000 that can be used to heat the delivery solution, the stop solution, and the culture medium. The heating system can include a fluid reservoir 11002, a housing 11004, and a heating element 11006. The fluid reservoir 11002 can generally take the shape of a cylindrical tube that can be filled with a fluid, such as, for example, the culture medium. The housing 11004 can be, for example, an aluminum block having a passage for receiving the fluid reservoir 11002. The heating element 11006 can be located at a distal end of the housing 11004. The heating element 11006 can receive power from a power source and heat the housing 11004, thereby heating the fluid in the fluid reservoir. The heating element 11006 can heat the housing 11004 by conduction.

[0351] 76-77 show an example of a cooling system 11100 that can be used to heat the delivery solution, the stop solution, and the culture medium. The cooling system 11100 can include one or more fluid reservoirs 11102, e.g., 1.5 ml Eppendorf tubes, a housing 11104, a cooling element 11106, e.g., an electrothermal cooler, a heat sink 11108, and a fan 11110. The delivery and / or stop solutions can be filled into the one or more fluid reservoirs 11102. Power can be delivered to the cooling element 11106, which can generate a hot surface and a cold surface. The cold surface can be in contact with the housing 11104, and the hot surface can be in contact with the heat sink 11108. The fan 11110 can be coupled to or located on the heat sink 11108 to remove heat from the hot surface of the cooling element 11106.

[0352] In practice, the delivery and stop solutions can be kept at approximately 4°C, and the culture medium can be kept at approximately 20°C to 37°C.

[0353] Installing the spray head and temperature reservoir The needle emitter 9002 and the atomizer 9004 can be mounted to a support. FIG. 104 shows an example of a mounting assembly 11300 that can releasably retain the needle emitter 9002 and the atomizer 11304. The atomizer 11304 can be an ultrasonic atomizer that can operate at a frequency between 60 kHz and 120 kHz. The mounting assembly 11300 can include a support plate 11302 to which a connecting member 11305 is attached. The support plate can include a hole 11306 that can receive the atomizer 11304 and an opening 11308 that can receive a retaining element 11310 to which the needle emitter 9002 can be connected. The hole 11306 can have a slot 11312 extending therefrom. In some cases, the ultrasonic atomizer that can operate at 60 kHz can have the same design as the one that operates at 120 kHz. The mounting assembly 11300 can therefore accommodate an ultrasonic atomizer that can operate at either frequency.

[0354] FIG. 105 shows an exploded view of a portion of the mounting assembly 11300 with the retaining element 11310 and the needle emitter 9002. As shown in FIG. 105, the needle emitter 9002 can be inserted into the hole 11310a of the retaining element 11310, and the retaining element can be inserted into the opening 11308. The design of the retaining element 11310 as a sliding insert can allow for independent Z positioning, i.e. vertical positioning, of the needle emitter 9002 relative to the spray head emitter. This can accommodate a variety of atomizer heights ranging from 15-31 mm from the tip of the emitter to the bottom of the filter plate well while maintaining a consistent needle emitter height.

[0355] 106 illustrates an example of a mounting assembly 11500 that can releasably retain a needle emitter 9002 and an ultrasonic atomizer 11504. The mounting assembly 11500 can be generally similar to the mounting assembly 11300, but can be designed to function with an ultrasonic atomizer 11504 that can operate at 180 kHz. The mounting assembly 11500 can include a support plate 11502 to which a coupling member 11505 is attached. The support plate can include a hole (not shown) that can receive the atomizer 11504 and an opening 11508 that can receive a retention element 11510 to which the needle emitter 9002 can be coupled. The hole can have a slot 11512 extending therefrom.

[0356] In the illustrated embodiment, the collar 11514 can be concentrically mounted to the atomizer 11504 and the other two heads at the same height in the collar slots in the same head holder.

[0357] 107 illustrates an example of a mounting assembly 11600 that can releasably retain a needle emitter 9002 and a nebulizer 11604. The mounting assembly 11600 can be generally similar to the mounting assembly 11300 and can include a support plate 11602 to which a coupling member 11505 is attached. The nebulizer 11604 can be coupled to the support plate 11602 via a bracket 11606. The support plate 11602 can include an opening 11608 that can receive a retention element 11610 to which the needle emitter 9002 can be coupled.

[0358] Design templates similar to those shown in Figures 78-81 can be used to accommodate other emitter types.

[0359] The design of the mounting assemblies 11300, 11500, and 11600 allows the present precision rig system to serve as a platform for optimization of the delivery process. For example, four different types of spray heads can be accommodated.

[0360] Automation and Software Design The high precision rig system 9000 and / or the positive pressure system 9300 can include a software program and a user interface that can enable automation of the delivery process steps. For example, the software can be designed to control the translation stage 9010 and the valves 9011. As another example, the software program can be configured to control the movement of the mounting array 9302. The user interface can enable input of critical parameters. The sequence of the functional units can be selected by the user.

[0361] A system was designed that has the ability to ensure experimental repeatability but also provides the flexibility to adjust experiment-specific parameters. Such parameters can include the number of wells to be addressed or the volume dispensed into the wells. To achieve this synchronization, a traversing mechanism (e.g., actuator 9319) can be included that can be software controlled to position its carriage within the 200 mm location for the required time. The carriage includes six positions, each of which is coupled to an individually controlled high-precision fluid escapement chamber. Each position can be moved under the selection of the dispense location (e.g., payload, culture medium, and stop solution station). A device-specific software platform can be included. This software platform can include (1) a graphical user interface that allows the user to design the experiment and (2) local control by a controller (e.g., a programmable logic controller (PLC)), such as an Omron PLC. Figure 129 shows a schematic 12000 of the software platform design. The software can include a user-friendly experiment creation section and a background sequence generator. The user interface can include an experiment canvas that allows the user to vary the parameters of the experiment. User modifiable parameters include the location and number of wells to be addressed, vacuum and / or positive pressure, payload dispensing, sequence of steps including stop solution and culture medium, and corresponding volumes to be delivered. The user can also modify actuator speed and incubation time (FIG. 130). FIG. 130 shows a graphical user interface (GUI) 12100 of the software platform. In the illustrated example, the GUI includes an experiment canvas that allows the user to modify parameters.For example, user-modifiable parameters include the location and number of wells to be addressed, vacuum or positive pressure, payload, sequence of steps including dispensing of stop solution and / or culture media, and the corresponding volumes to be delivered. A background sequence generator outputs a program for the PLC, which provides local control of the mechanical and electronic systems. The software allows the operator (e.g., via an integrated display) to test different permutations of wells, media, and times for optimal experimental success without having to manually calculate dwell times between wells. These inputs allow the end user to perform multiple experiments.

[0362] In some embodiments, the system can address 96 locations within the filter plate substrate. This system can be faster and have multiple traversal mechanisms and a user-friendly HMI (human machine interface). The HMI can provide the operator with further automated utilities, such as automatic system purging or touch screen driven calibration sequences. This system can run as a standalone solution that still maintains interaction with the experiment planner to design the experiment. Further aspects of the experiment can be adjusted on a well-by-well basis, meaning that the possible permutations are significantly increased. For example, after dispensing fluids into several wells, the pressure applied to the fluid delivery system for the next experimental section can be automatically adjusted. This exemplary system has the ability to record the analysis from the experiment for further offline analysis.

[0363] FIG. 64 is a process flow diagram illustrating an exemplary process according to some aspects of the present subject matter. The exemplary process can be implemented, for example, by a controller of a delivery system. At 1610, a user input can be received, for example, by the controller. At 1620, a delivery solution application device can be operated to deliver atomized delivery solution to the cell monolayer in the well. At 1630, after application of the delivery solution, the cell monolayer can be incubated for a first time period. At 1640, a stop solution application device can be operated in response to expiration of the first incubation period. This operation can be performed to deliver a stop solution to the cell monolayer. At 1650, in response to application of the stop solution, the cell monolayer can be incubated for a second time period.

[0364] In some embodiments, the cycle of activating the delivery solution applying device, incubating for a first incubation period, activating the stop solution applying device, and incubating for a second incubation period can be repeated a predetermined number of times.

[0365] One or more aspects or features of the subject matter described herein may be implemented in digital electronic circuitry design, integrated circuit design, specially designed application specific integrated circuits (ASICs), field programmable gate array (FPGA) computer hardware, firmware, software, and / or combinations thereof. These various aspects or features may include implementation in one or more computer programs executable and / or interpretable by a programmable system including at least one programmable processor, which may be dedicated or general purpose, coupled to transmit and receive data and instructions to and from a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. Clients and servers are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0366] These computer programs may also be referred to as programs, software, software applications, applications, components, or codes, and include machine instructions for a programmable processor and may be implemented in high-level procedural languages, object-oriented programming languages, functional programming languages, logic programming languages, and / or assembly / machine languages. As used herein, the term "machine-readable medium" refers to any computer program product, apparatus, and / or device used to provide machine instructions and / or data to a programmable processor, such as, for example, magnetic disks, optical disks, memories, and programmable logic devices (PLDs), and encompasses machine-readable media that receive machine instructions as machine-readable signals. The term "machine-readable signal" refers to any signal used to provide machine instructions and / or data to a programmable processor. A machine-readable medium may store such machine instructions non-temporarily, such as, for example, a non-transitory solid-state memory or a magnetic hard drive or any equivalent storage medium. Alternatively or additionally, a machine-readable medium may store such machine instructions transiently, such as, for example, a processor cache or other random access memory associated with one or more physical processor cores.

[0367] To provide for user interaction, one or more aspects or features of the subject matter described herein can be implemented on a computer having a display device, such as a cathode ray tube (CRT) or liquid crystal display (LCD) or light emitting diode (LED) monitor, for displaying information to a user, a keyboard and a pointing device, such as a mouse or trackball, for providing input to the computer by the user. Other types of devices can be used to accommodate user interaction as well. For example, feedback provided to the user can be any form of sensory feedback, such as visual feedback, auditory feedback or tactile feedback, and input from the user can be received in any form, such as, but not limited to, acoustic input, voice input or tactile input. Other possible input devices include, but are not limited to, touch screens or other touch sensitive devices, such as single or multi-point resistive or capacitive track pads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software.

[0368] Equipment Enclosures The precision rig system 9000 and / or the positive pressure system 9300 can be kept within an enclosure to maintain stable ambient conditions. The enclosure can be custom made based on the parameters that govern the design of the enclosure.

[0369] Baseplate Temperature and Control In some embodiments, a temperature control system can be implemented to control the temperature of the base plate 9016. For example, cartridge heaters can be mounted at various locations on the base plate 9016 of the vacuum manifold assembly 9008. Thermodynamic analysis can be performed to study the heat transfer from the cartridge heaters to the well filter plate 9014. Temperature ramp experiments can be performed to study the effect of temperature on payload delivery.

[0370] Although some variations have been described above, other modifications are possible. For example, the filter plate can include any number of wells, and does not have to be a 96-well filter plate. In addition, the system can include any number of valves that can control vacuum pressure to any number of available wells. As another example, the system can include one or more needle emitters, atomizers, and / or nebulizers, each of which can be independently attached and controlled.

[0371] The present subject matter has many technical advantages. In general, the present subject matter provides a delivery system that can increase the consistency of the delivery process and the efficiency of delivery while maintaining cell health.

[0372] The delivery system allows for the provision of vacuum pressure to individual wells of a filter plate to remove culture medium, thereby generating a monolayer of cells. By applying vacuum pressure to individual wells on a filter plate, more precise control of vacuum pressure, consistency of vacuum pressure applied to each well can be achieved.

[0373] The delivery system allows for the dispensing of permeabilization solution in microliter volumes. Dispensing microliter volumes allows for better control of cell exposure to the solution, which may increase overall cell viability by reducing overexposure to the permeabilization solution. Furthermore, the system can be automated, which minimizes errors and improves precision.

[0374] The system allows for control of the temperature of the delivery solution, the temperature of the stop solution, and the temperature of the culture medium. Each solution can therefore be maintained at an optimal temperature to increase the efficiency of payload delivery and cell viability. The temperature of the baseplate can also be controlled, allowing for optimization of the temperature to maximize the efficiency of payload delivery.

[0375] The needle emitter and atomizer and / or nebulizer can be coupled to a mounting assembly that can accommodate a variety of atomizer / nebulizer heights ranging from 15-31 mm from the tip of the emitter to the bottom of the well in the filter plate while keeping the needle emitter height constant.

[0376] The system may comprise hardware, one or more software programs, and a user interface that may allow for automating the delivery process steps. The software may be designed to control the translation stages and valves. The user interface may allow for the input of critical parameters. The sequence of functional units may be selected by the user. This is beneficial for the following reasons:

[0377] The enclosure may function to maintain stable ambient conditions.

[0378] Further Exemplary Delivery System Aspects Scaling the delivery process involved designing a system to allow optimization, determining how to form cell monolayers on a larger scale, and optimizing atomization to allow intracellular delivery of mRNA to T cells.

[0379] Optimization work has to date achieved an efficiency of >50% mRNA delivery to T cells with >60% cell viability and up to 80% cell recovery.

[0380] A system was designed and constructed to facilitate scaling of the delivery process. The system is based on the commercially available Amicon stirred cell, a pressure-based sample concentration unit (sizes 50 ml and 200 ml, catalogs UFSC05001 and UFSC20001, respectively). Figure 108 shows an example of a stirred cell system 9700 configured to facilitate the formation of a monolayer of cells.

[0381] The stirred cell system 9700 can include a cap 9702, a body 9704, a membrane holder 9706, and a base 9708. The stirring bar 9710 can be located within the body 9704. The cap 9702 can be configured to couple to the body 9704 at a first end of the body 9704. A seal-forming element 9712 (e.g., a gasket) can be located between the cap 9702 and the first end of the body 9704 to form a seal between the cap 9702 and the first end of the body 9704. The membrane holder 9706 can receive the membrane 9714 and can be located adjacent to the second end of the body 9704 such that a coupling member 9716 of the membrane holder 9706 extends through an opening 9718 in the second end of the body 9704. The second end of the body 9704 can be coupled to the base 9708 and a seal-forming element 9720 (e.g., an o-ring) can be located therebetween to form a seal. A pressure conduit assembly 9722 can be coupled to the cap 9702 such that pressurized gas can be delivered to the body chamber formed by the cap 9702, the body 9704, and the base 9708. A filtrate tube assembly 9724 can be coupled to the coupling member 9716 of the membrane holder 9706 such that fluid can be evacuated from the chamber upon application of pressure.

[0382] In operation, culture medium containing cells can be delivered to the chamber and the cap 9702 can be coupled to the body 9704. A pressure line assembly 9722 can be coupled to the cap 9702 and a positive pressure can be applied to the chamber via the inlet line assembly 9722. In some embodiments, the pressure can be in the range of 50-1000 mbar and the pressure can be applied for 10-60 seconds. Thus, application of the positive pressure can force the culture medium out of the chamber through the coupling member 9716 and the filtrate line assembly 9724, across the membrane 9714. Once the culture medium is expelled, a monolayer of cells can remain on the membrane 9714. Thus, the stirred cell system 9700 can be used to form a cell monolayer on a filter membrane placed in the membrane holder of the stirred cell. Once the monolayer is formed, a nebulizer (e.g., LB-100 from Burgener Research) can be used to atomize the delivery solution onto the cells that are on the filter membrane.

[0383] The stirred cell system 9700 shown in FIG. 108 may have some limitations. For example, in some cases, the stirred cell system 9700 may be configured to detect a particular concentration (e.g., 10×10 6 109, the culture medium flows out through only one channel 9706a of the membrane holder 9706. The channel 9706a may become clogged when filtering culture medium with a cell suspension above a certain concentration. As another example, an even distribution of cells in the membrane 9714 was not achieved using the membrane holder 9706.

[0384] To address these limitations with the membrane holder 9706, a membrane holder 9906 was constructed as shown in FIG. 110. In this embodiment, the membrane holder 9906 is made of PTFE and can include a number of holes 9906a piercing the bottom surface in a pattern forming concentric circles. In use, when pressure is applied within the chamber of the stirred cell system, culture medium can flow out through the holes 9906a. The membrane holder 9906 was evaluated using dynabeads (microbeads used to mimic cells). FIG. 111 shows the membrane 9914 used with the membrane holder 9906 during the dynabead evaluation. As shown in FIG. 111, the results show that the dynabeads accumulated near the holes 9906a. Cells subjected to a delivery process using this holder resulted in poor uptake.

[0385] To prevent accumulation of cells near the holes and to generate a more even distribution of cells on the membrane, the membrane holder can include holes similar to the holes 9906a, but also include concentric channels that can facilitate radial flow between the holes. FIG. 112 shows another embodiment of a membrane holder 10006 that can be used with a stirred cell system (e.g., stirred cell system 9700). In the illustrated embodiment, the membrane holder 10006 includes holes 10006a, concentric channels 10006b, and straight, or linear, channels 10006c. The holes 10006a are formed along a pattern that includes a central circle, an outer circle, and linear spokes extending from the central circle toward the outer circle. The linear channels 10006c extend radially outward from a central point of the membrane holder 10006, and the concentric channels 10006b form concentric circles around the central point of the membrane holder 10006. The holes 10006a and channels 10006b, 10006c can facilitate even distribution of cells throughout the membrane and can facilitate rapid removal of culture medium. In some embodiments, the membrane holder 9706 illustrated in Figures 82-83 can be modified by drilling holes (e.g., hole 10006a) to form the membrane holder 10006.

[0386] Membrane holder 10006 was evaluated using dynabeads. Figure 113 shows membrane 10014 used with membrane holder 10006 in the dynabead evaluation. As shown in Figure 113, the results show that membrane 10014 used with membrane holder 10006 has a more even distribution of dynabeads than membrane 9914 used with membrane holder 9906.

[0387] In some embodiments, the membrane holder can include holes formed in the concentric channels. Figure 114 shows an example of a membrane holder 10106 including holes 10106a, concentric channels 10106b, and straight channels 10106c. In the illustrated example, holes 10106 are formed in the concentric channels 10106b and straight channels 10106c. This shape can facilitate an even outflow of culture medium so that a monolayer of cells can form on the membrane.

[0388] The R4 was assembled to allow atomization of the delivery solution using the LB-100 spray head. Figures 115-118 and 128 show an example of a solution delivery system, also referred to as the R4, that can be used to deliver permeabilization solution to a monolayer of cells. The solution delivery system can include a nebulizer assembly 9801 shown in Figure 115 and a mounting system 9802 shown in Figure 116.

[0389] The nebulizer assembly 9801 includes a nebulizer 9804 (e.g., an LB-100 spray head), a coupling element 9806 (e.g., an IDEX fitting) configured to facilitate delivery of air and liquid (e.g., permeabilization solution) to the nebulizer 9804, a solution reservoir 9810 (e.g., an Elveflow sample reservoir) configured to provide permeabilization solution to the nebulizer 9804, and a pinch valve 9808 configured to control delivery of the permeabilization solution to the nebulizer 9804. The mounting system 9802 can include a valve and reservoir mounting portion 9812, a spray head mounting portion 9814, and a nebulizer retaining collar 9816 for receiving the nebulizer 9804. The nebulizer assembly 9801 can be coupled to a mounting system 9082, as illustrated in FIGS. 91-92, to be fixed in place. The mounting system 9082 facilitates precise alignment of the nebulizer 9804 with the target area.

[0390] Further exemplary approaches to the formation of cell monolayers Two approaches for generating cell monolayers are described. In both methods, a stirred cell unit was assembled as shown in Figure 108. 6 A volume of 5-10 ml of cell suspension containing cells / ml was added to the stirred cell chamber, and the lid of the chamber was then closed.

[0391] Monolayers can be generated by applying vacuum to the bottom of the stirred cell: -50 to -1000 mbar is applied to the chamber for 10 to 60 seconds or until the filter membrane appears dry to the naked eye.

[0392] Alternatively, monolayers were formed by applying positive pressure through a tubing fitting on the lid of the stirred cell. Pressure was applied for a set time (10-60 seconds) and until the filter membrane appeared dry to the eye. In some cases, lower pressure was used to push >90% of the culture medium through the filter, and then the pressure was gradually increased over 10-15 seconds to finally achieve complete removal of the culture medium. Pressures of 100-200 mbar were applied to allow monolayer formation and complete removal of the cell culture medium.

[0393] The actual specific pressures and times varied depending on cell concentration, membrane type, pore size, and type of membrane holder. For example, in the original unmodified membrane holder, 50–100 × 10 6 T cells were used to form monolayers in 63.5 mm stirred cells. For PES (polyethylene sulfone) membranes, 50 × 10 6 For 60 × 10 cells, it was possible to remove the medium by applying 150 mbar for 20–30 s, whereas for 60 × 10 cells, 6 For cells, 30-60 s and 200 mbar were required to remove the medium. For PCTE (polycarbonate track etched) membranes, 250 mbar and 60 s were required for the lower cell concentration. 6For cells, after 2 minutes at 250 mbar, medium would still be present on the filter membrane. To remove this, the pressure was increased to 1 bar for 10 seconds and then decreased to normal levels (100 mbar). This was done several times to aid in medium removal.

[0394] Positive pressure was tested in unmodified filter holders (Figure 109) in 63.5mm and 44.5mm stirred cells with approximately 50x10^6 cells and a hydrophilic PCTE membrane with 2.0μm pores. The setup did not result in total removal of medium at any stage (0-600bar, 0-6min).

[0395] Using a modified filter holder (Figure 113) for the 44.5 mm membrane, a 1 μm PCTE hydrophobic membrane and a 20 × 10 6 For individual cells, a monolayer was achieved in 10 seconds at only 100 mbar, but could be achieved in even shorter times at higher pressures (500 mbar for 5 seconds).

[0396] The type of filter used to generate the monolayer was investigated. The filters varied in material, hydrophobicity, and pore size. Materials tested included PES, and polycarbonate track etched filters PCTE hydrophobic and hydrophilic membrane coatings, sizes included 13, 25 mm, 47 mm, and 63 mm diameter, with pore sizes ranging from 0.4 μm to 1.2 μm. PETE (polyester), silver, and gold membranes were also tested (see table below).

[0397] (table) TIFF0007644048000015.tif35128

[0398] Filters were evaluated for uniform monolayer formation, efficiency in removing culture medium, and cell recovery from the filter membrane. Dynabeads were used as cell representatives to assess monolayer formation. In addition, expanded T cells were used to assess monolayer formation, recovery, and viability after monolayer formation.

[0399] PES filters were investigated and found to allow for the formation of an even monolayer and efficient removal of the culture medium. However, the recovery of cells from the filter membrane was low (50%). PCTE track edge filters provided improved cell recovery from the filter membrane (50-90%) (Figure 121). However, the efficiency of culture medium removal was slower compared to PES filters (10 seconds for medium removal (at 150 mbar) vs. 45 seconds for PCTE filters). The best recovery achieved was with PCTE 0.4 Hydrophobic 20×10 6 At 100 mbar and 60 seconds, it was approximately 87% (Figure 122).

[0400] Micronization to enable intracellular delivery of mRNA to T cells Examination of the LB-100 spray head revealed a target area of ​​60 mm diameter. A force analysis of the LB-100 was performed (Figure 120). To optimize delivery with the LB-100 spray to a larger target area (25-65 mm filter membrane), the height, air pressure, volume delivered, number of hits, number of cells, and spray duration were adjusted.

[0401] To optimize LB-100 spray delivery, the height of the spray head to the target area was adjusted. Distances ranging from 30mm to 160mm from the tip of the spray head to the target area were investigated. The optimal distance range was found to be within 60-100mm from the tip of the spray head to the target area. Uptake increased with smaller distances and decreased with greater distances.

[0402] To optimize LB-100 spray delivery, air pressure was varied from 1 to 6 bar, and 2.5 to 3.0 bar was found to be the optimal range for intracellular delivery. GFP mRNA delivery was reduced at pressures below 2.5 bar, and cell viability was reduced at pressures above 3.0 bar. Increasing air pressure increased the effective target area.

[0403] To optimize LB-100 spray delivery, the delivered volume was varied from 10 to 300 μl, and a volume of 80 to 100 μl was found to be optimal for intracellular delivery. 6 5–10 ml of cell suspension of human primary T cells ranging from 5–10 cells / ml were added to a stirred cell unit (Merck Millipore; PCTE filter, pore size 0.4 μm or 1 μm). A cell monolayer was formed by applying a positive pressure ranging from 100–150 mbar for 20–50 s. The cell monolayer was sprayed with 10–300 μl of delivery solution containing 0.1 μg / μl GFP mRNA and incubated for 2 min. Stop solution (1 ml) was added, followed by a 30 s incubation and the addition of culture medium (4 ml) to the filter membrane. The cells were incubated in a humidified incubator at 37 °C and 5% CO. 2 The cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry after 17–24 h.

[0404] To increase delivery efficiency, an evaluation of the optimal number of hits was performed comparing a one-hit strategy with a two-hit strategy. The data showed an increase in delivery efficiency when cells received a second hit. 65–10 ml of cell suspension of human primary T cells ranging from 50–100 cells / ml were added to a stirred cell unit (Merck Millipore; PCTE filter, pore size 0.4 μm or 1 μm). A cell monolayer was formed by applying a positive pressure ranging from 100–150 mbar for 20–50 s. The cell monolayer was sprayed with 80–100 μl of delivery solution containing 0.1 μg / μl GFP mRNA and incubated for 2 min. Stop solution (1 ml) was added, followed by a 30 s incubation, and culture medium (4 ml) was added to the filter membrane. For the two-hit strategy, the cells were incubated for 2 h before the spraying process was repeated (as described above). The cells were incubated at 37 °C and 5% CO in a humidified incubator. 2 The cells were incubated overnight at RT and assessed for GFP fluorescence by flow cytometry after 17-24 hours. The double-hit process resulted in increased delivery efficiency (Figure 123).

[0405] An exemplary embodiment 12300 of the Midi system is shown in Fig. 132. In the illustrated example, the Midi system 12300 comprises a stirred cell system 11900 (e.g., a 63 mm stirred cell system) with a membrane holder 10006 (e.g., a 44 mm membrane holder). An enclosing film 12303 is attached to the opening of the stirred cell unit. The collar of the spray head holder 9814 is inserted into the stirred cell system 11900 through a "slit" in the enclosing film 12303. In the illustrated example, an LB-100 atomizer is held in the collar of the spray head holder 9814 and positioned so that the tip of the spray head is 82 mm from the top surface of the membrane holder 10006.

[0406] Figure 133 is a plot representing data characterizing efficiency (GFP incorporation) and viability corresponding to studies performed on the Midi system 12300. The data show an average delivery efficiency of 59.63% ± 1.2 and an average viability data of 74.6% ± 5.3 across three technical replicates.

[0407] To increase the delivery efficiency, the number of cells seeded on the filter membrane was examined. Cells 13 × 10 3 pieces / mm 2 A cell density of 1000 μl was seeded onto a stirred cell unit (Merck Millipore; PCTE filter, pore size 0.4 μm or 1 μm). A cell monolayer was formed by applying a positive pressure ranging from 100 to 200 mbar for 10 to 50 seconds. The cell monolayer was sprayed with 80 to 100 μl of delivery solution containing 0.1 μg / μl of GFP mRNA and incubated for 2 min. Stop solution (1 ml) was added and incubated for 30 seconds, after which culture medium (4 ml) was added to the filter membrane. The cells were incubated at 37 °C and 5% CO in a humidified incubator. 2 The cells were incubated overnight at 4°C and assessed for GFP fluorescence by flow cytometry after 17–24 h.

[0408] The duration of the spray was investigated. This was achieved by adjusting the open time of the valves that control the flow of air and payload to the atomizer. Spray durations from 280 to 700 ms were tested.

[0409] FIG. 134 illustrates one exemplary embodiment of a delivery system configured to facilitate the generation of a monolayer of cells and the delivery of a payload to the cells.

[0410] In some embodiments, the filter membrane can be vibrated before, after, and / or during delivery of the permeabilization solution to facilitate and enhance exposure of the cells to the permeabilization solution. To aid in the formation of a monolayer of cells on the filter membrane, the membrane can be vibrated before, after, and / or during the formation of the monolayer.

[0411] The vibration can be provided by an eccentric rotating mass (ERM) system or a linear resonant actuator (LRA) system. For example, in a preferred embodiment, one, two, or three actuators (LRAs) corresponding to the X-axis, Y-axis, and Z-axis can be attached to the membrane or the corresponding membrane holder (e.g., membrane holder 10006, 10106) such that actuation of the actuators causes the membrane to vibrate. The advantage of the LRA is that each vibration axis can be driven independently. Thus, a controllable vibration pattern can be generated in the membrane. In addition, identifying mechanical resonance points due to the physical characteristics of the membrane can improve the degree of control that can be exhibited over the membrane. In some embodiments, a three-axis accelerometer device can be mechanically coupled to the membrane and / or membrane holder to obtain data characterizing the movement and / or deflection of the membrane and / or membrane holder. Data from the accelerometer can be used in a feedback control system to control the actuation of the LRA. For example, the accelerometer can be used to monitor the vibration of the membrane and / or membrane holder. In some embodiments, the data from the accelerometer can be used as a control feedback signal to adjust the vibration generated by the LRA. For example, the data from the accelerometer can be used to generate an error signal between the desired vibration pattern and the achieved vibration pattern. As an example, the driving vibration frequency can be determined based on the stiffness of the membrane and / or the size of the cells on the membrane. An exemplary vibration pattern can be provided with a 3000Hz sinusoidal signal on the x-axis and y-axis and no signal on the z-axis. The deviation is 1mm peak to peak, there is no phase difference between the x-drive waveform and the y-drive waveform, and both can be coherent. Many other patterns are possible, such as those that provide rotation and / or shaking on the x-, y-, and / or z-axis.

[0412] The subject matter can include a non-viral, vector-free method of achieving intracellular delivery by mild, reversible permeabilization. The subject matter can include (1) a permeabilization solution containing a low dose of ethanol as a permeabilization agent, and (2) a means of applying the delivery solution to target cells in a dropletized form. The technique provides tight control over the volume, time, and pressure at which the permeabilization solution is applied to the cells, thereby achieving high levels of delivery efficiency and cell viability.

[0413] The steps of this process may include: generating a monolayer of target cells; removing the supernatant from the cells; mixing the cargo with the delivery solution and applying it to the cells in droplet form and incubating for 2 minutes; during this period, the ethanol permeabilizes the cell membrane and the cargo diffuses into the cell; the cargo enters directly into the cytoplasm in an endocytosis-independent manner; then applying a "stop" solution and incubating for 30 seconds - this acts to dilute the permeabilized delivery solution and allows the cell membrane to begin to reseal; then adding culture medium to complete the process.

[0414] Other Aspects Although the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative of the invention and not to limit its scope, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0415] The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. The U.S. patents and published or unpublished U.S. patent applications referred to herein are incorporated by reference. All published foreign patents and patent applications referred to herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature referred to herein are incorporated by reference.

[0416] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. Systems including: a housing configured to receive a plate containing a well; wherein the plate contains a population of cells; a pressure differential application device configured to apply a pressure differential to the well; a delivery solution application device configured to deliver atomized delivery solution to said well; a reservoir in fluid communication with the delivery solution application device; wherein the reservoir comprises a vector-free delivery solution, the delivery solution comprising an aqueous solution comprising a payload and an alcohol at a concentration greater than 5% (v / v), wherein the aqueous solution is isotonic, and wherein the population of cells is contacted with the aqueous solution by propelling the aqueous solution with a gas to form a spray, wherein the payload comprises a gene editing composition; a stop solution application device configured to deliver a stop solution to the well; and A culture medium application device configured to deliver culture medium to said well.

2. Addressable Well Assembly Further equipped with the addressable well assembly comprising: aligning the pressure differential application device adjacent to the well for applying the pressure differential to the well; aligning the delivery solution application device adjacent to the well for delivering the atomized delivery solution to the well; aligning the stop solution application device adjacent to the well for delivering the stop solution to the well; and / or Aligning the culture medium application device adjacent to the well for delivering the culture medium to the well. The system of claim 1 , configured to:

3. 3. The system of claim 2, wherein the addressable well assembly comprises a movable base plate configured to receive the plate containing the wells and move the plate in at least one direction.

4. 3. The system of claim 2, wherein the addressable well assembly comprises a mounting assembly configured to couple to the delivery solution application device, to the stop solution application device, and to the culture medium application device.

5. The system of claim 1 , wherein the delivery solution application device comprises a nebulizer.

6. The system of claim 1 , wherein the delivery solution application device is configured to deliver between 10 and 300 microliters of the delivery solution per actuation.

7. The system of claim 1 , further comprising a temperature control system configured to control a temperature of the delivery solution and / or a temperature of the plate containing the wells.

8. 10. The system of claim 1, further comprising an enclosure configured to control an environment of the plate containing the wells.

9. 2. The system of claim 1, wherein the pressure differential application device includes a nozzle assembly configured to form a seal with an opening of the well and configured to deliver steam to the well to increase or decrease pressure within the well, thereby pushing a liquid portion of the culture medium out of the well such that a layer of cells remains within the well.

10. The system of claim 1 , wherein the stop solution application device comprises a needle emitter configured to couple to a stop solution reservoir.

11. 10. The system of claim 1, wherein the culture medium application device comprises a needle emitter configured to couple to a culture medium reservoir.

12. Receive user input, activating the delivery solution application device to deliver the atomized delivery solution to the cell monolayer in the well; incubating the cell monolayer for a first incubation period after application of the delivery solution; activating the stop solution application device in response to expiration of the first incubation period to deliver the stop solution to the cell monolayer; and The cell monolayer is incubated for a second incubation period and in response to application of the stop solution. The system of claim 1 , further comprising a controller configured to:

13. The control device, repeating the actuation of the delivery solution applying device, the incubation for the first incubation period, the actuation of the stop solution applying device, and the incubation for the second incubation period a predetermined number of iterations. The system of claim 12, further configured to:

14. Activating a positive pressure system to remove the supernatant from the well and generate a cell monolayer within the well. The system of claim 1 , further comprising a controller configured to:

15. 2. The system of claim 1, wherein the delivery solution application device includes a spray head and a collar surrounding a distal end of the spray head, wherein the collar is configured to prevent contamination between wells in a multi-well plate, and wherein the collar is configured to provide a gap between the plate and the collar.

16. 16. The system of claim 15, wherein the delivery solution application device further comprises a film surrounding a distal end of the spray head.

17. The system of claim 1 , further comprising a vibration system coupled to the membrane holder and configured to vibrate the membrane.

18. The system of claim 1 , wherein the well contains a population of non-adherent cells.

19. The system of claim 1 , wherein the alcohol comprises ethanol.

20. The system of claim 1 , wherein the aqueous solution comprises greater than 10% ethanol.

21. 2. The system of claim 1, wherein the aqueous solution comprises 20-30% ethanol.

22. The system of claim 1 , wherein the aqueous solution comprises 27% ethanol.

23. 2. The system of claim 1, wherein the aqueous solution comprises 100 to 125 mM KCl.

24. 2. The system of claim 1, wherein the aqueous solution comprises 106 mM KCl.

25. The system of claim 18 , wherein the non-adherent cells comprise peripheral blood mononuclear cells.

26. The system of claim 18 , wherein the non-adherent cells comprise immune cells.

27. The system of claim 18 , wherein the non-adherent cells comprise T lymphocytes.

28. The system of claim 1 , wherein the payload comprises messenger ribonucleic acid (mRNA).

29. The system of claim 1, wherein the gene editing composition reduces expression of PD-1.

30. The system of claim 28, wherein the mRNA encodes a chimeric antigen receptor.

31. The system of claim 18, wherein the population of non-adherent cells constitutes a monolayer.

32. The system of claim 1 , wherein the delivery solution is non-viral.

33. The system of claim 1 , wherein the payload comprises messenger ribonucleic acid (mRNA).

34. The system of claim 1, wherein the gene editing composition reduces expression of PD-1.

35. The system of claim 33, wherein the mRNA encodes a chimeric antigen receptor.

36. 2. The system of claim 1, wherein the gene editing composition comprises one or more of: (a) a gene editing protein, (b) an RNA molecule, and / or (c) a ribonucleoprotein (RNP).

37. The system of claim 1, wherein the gene editing composition comprises a compound or complex that cuts, nicks, splices, rearranges, translocates, recombines, or modifies genomic DNA.

38. The system of claim 1, wherein the gene editing composition comprises: (i) a compound that can be included in a gene editing complex that cuts, nicks, splices, rearranges, moves, recombines, or modifies genomic DNA; or (ii) a compound that can be processed or modified into a compound that can be included in a gene editing complex that cuts, nicks, splices, rearranges, moves, recombines, or modifies genomic DNA.

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