Vector-free intracellular delivery by reversible permeabilization

Abstract

To provide compositions and methods to facilitate transfection of suspension cells, such as non-adherent cells.SOLUTION: Provided is a method of delivering a payload across a plasma membrane of a non-adherent cell, 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 including the payload and an alcohol at greater than 5% (v / v) concentration.SELECTED DRAWING: None

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 hereby incorporated by reference in its entirety.

[0002] Field of the Invention The present invention relates to the delivery of agents into mammalian cells.

Background Art

[0003] Background of the Invention Cell transfection efficiency varies among different cell types. Transfection of floating cells, such as non - adherent cells, has been found to be very difficult with conventional methods. Therefore, there is a need for compositions and methods to facilitate 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 comprises providing a population of non - adherent cells and contacting the population of cells with an isotonic aqueous solution having a volume, the aqueous solution comprising the payload and an alcohol at a concentration greater than 5% (v / v). For example, the alcohol is ethanol, for example, ethanol in an amount greater than 10%. In some examples, the aqueous solution comprises 20 - 30% ethanol, for example, 27% ethanol.

[0005] The aqueous solution for delivering cargo to cells contains salts, such as 12.5 - 500 mM potassium chloride (KCl). For example, the solution is isotonic with respect to the cytoplasm of mammalian cells such as human T cells. An example of such an isotonic delivery solution is 106 mM KCl.

[0006] This method can be used to deliver one or more types of any cargo molecule to adherent or non - adherent mammalian cells and is particularly useful for delivering cargo to non - adherent cells. This is because prior to the present invention, it was difficult to do so. In some examples, non - adherent cells include peripheral blood mononuclear cells, and for example, non - adherent cells include immune cells such as T cells (T lymphocytes). 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, the cargo in the delivery solution is delivered to a population of non - adherent cells that form a monolayer. For example, the monolayer is contacted with a spray of the aqueous delivery solution. This method delivers the payload / cargo (compound or composition) into the cytoplasm of the cells, where the population of cells has a higher percentage of survival compared to delivery of the 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 includes messenger ribonucleic acid (mRNA), such as mRNA encoding a gene - editing composition. For example, the gene - editing composition reduces the expression of immune checkpoint inhibitors 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 / floating cells is present on a membrane filter. In some embodiments, after contacting the cell monolayer with a spray of delivery solution, the membrane filter is vibrated. The membrane filter can be vibrated or shaken before, during, and / or after spraying the delivery solution onto the cells.

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

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

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

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

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

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

[0016] The stop solution applying device can include a needle emitter configured to be connected to a stop solution reservoir.

[0017] The culture medium applying device can include a needle emitter configured to be connected to a culture medium reservoir.

[0018] The system can further comprise a control device configured to receive user input, operate a delivery solution applying device to deliver an atomized delivery solution to a monolayer of cells within a well, incubate the monolayer of cells over a first incubation period after application of the delivery solution, operate a stop solution applying device in response to expiration of the first incubation period to deliver a stop solution to the monolayer of cells, and incubate the monolayer of cells over a second incubation period and in response to application of the stop solution. The control device can be further configured to repeat the operation of the delivery solution applying device, the incubation over the first incubation period, the operation of the stop solution applying device, and the incubation over the second incubation period a predetermined number of times.

[0019] The system can further include a control device configured to operate a positive pressure system to remove supernatant from the wells and generate a cell monolayer within the wells.

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

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

[0022] The system can further include a vibration system that is connected to a membrane holder and is configured to vibrate the membrane.

[0023] The system can further include a plate configured such that the wells contain a population of non-adherent cells.

[0024] The delivery solution includes an isotonic aqueous solution that includes a payload and an alcohol at a concentration greater than 5 percent (v / v). The alcohol can include ethanol. The aqueous solution can include more 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 include peripheral blood mononuclear cells. The non-adherent cells can include immune cells. The non-adherent cells can include T lymphocytes. The population of non-adherent cells can form 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 the expression of PD-1. The mRNA can encode a chimeric antigen receptor.

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

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

[0029] The compounds loaded into the MPS composition are processed or purified. For example, polynucleotides, polypeptides or other agents are purified and / or isolated. Specifically, as used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, when produced by recombinant techniques, is substantially free of other cellular material or culture medium, and when chemically synthesized, is substantially free of chemical precursors or other chemicals. 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 in which the desired compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) by weight. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain the genes or sequences that flank it in its natural state. Purified also indicates sterility safe for administration to a human subject, e.g., lacking infectious agents 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" means a nucleotide or polypeptide separated from the components that are naturally associated with it. Typically, nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99% by weight and are free of the proteins and natural organic molecules that are naturally associated with them.

[0031] A small molecule is a compound with a mass of less than 2000 Daltons. The molecular mass of the 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 term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or non-limiting 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 claim to the specified materials or steps and those that do not materially affect the "basic and novel characteristics" of the claimed invention.

[0033] [Invention 1001] The step of providing a population of non-adherent cells, and The step of contacting the population of cells with an isotonic aqueous solution of a certain volume, wherein the aqueous solution contains a payload and an alcohol at a concentration of more than 5% (v / v), A method for delivering the payload across the plasma membrane of non-adherent cells, comprising the above steps. [Invention 1002] The method of Invention 1001, wherein the alcohol comprises ethanol. [Invention 1003] The method of Invention 1002, wherein the aqueous solution contains more than 10% ethanol. [Invention 1004] The method of Invention 1002, wherein the aqueous solution contains 20 - 30% ethanol. [Invention 1005] The method of Invention 1001, wherein the aqueous solution contains 27% ethanol. [Invention 1006] The method of Invention 1001, wherein the aqueous solution contains 12.5 - 500 mM KCl. [Invention 1007] The method of the present invention 1001, wherein the aqueous solution contains 106 mM of KCl. [The present invention 1008] The method of the present invention 1001, wherein the non-adherent cells contain peripheral blood mononuclear cells. [The present invention 1009] The method of the present invention 1001, wherein the non-adherent cells contain immune cells. [The present invention 1010] The method of the present invention 1001, wherein the non-adherent cells contain T lymphocytes. [The present invention 1011] The method of the present invention 1009, wherein the immune cells are activated by a ligand of CD3, a ligand of CD28, or a combination thereof. [The present invention 1012] The method of the present invention 1001, wherein the population of non-adherent cells forms a monolayer. [The present invention 1013] The method of the present invention 1001, wherein the monolayer is contacted with a spray of the aqueous solution. [The present invention 1014] The method of the present invention 1001, wherein the method delivers the payload into the cytoplasm of the cells, and the population of the cells has a higher survival percentage compared to the delivery of the payload by electroporation. [The present invention 1015] The method of the present invention 1001, wherein the payload contains messenger ribonucleic acid (mRNA). [The present invention 1016] The method of the present invention 1005, wherein the mRNA encodes a gene editing composition. [The present invention 1017] The method of the present invention 1016, wherein the gene editing composition reduces the expression of PD-1. [The present invention 1018] The method of the present invention 1013, wherein the monolayer is present on a membrane filter. [The present invention 1019] The method of the present invention 1013, wherein the membrane filter is vibrated after contact with the spray. [The present invention 1020] The method of the present invention 1015, wherein the mRNA encodes a chimeric antigen receptor. [The present invention 1021] A housing configured to receive a plate containing wells, A pressure difference applying device configured to apply a pressure difference to the wells, A delivery solution applying device configured to deliver an atomized delivery solution to the wells, A stop solution applying device configured to deliver a stop solution to the wells, and A culture medium applying device configured to deliver a culture medium for culturing to the wells A system comprising. [The present invention 1022] An addressable well assembly Further comprising, The addressable well assembly is Aligning the pressure difference applying device for applying the pressure difference to the well adjacent to the well, Aligning the delivery solution applying device for delivering the atomized delivery solution to the well adjacent to the well, Aligning the stop solution applying device for delivering the stop solution to the well adjacent to the well, and / or Aligning the culture medium applying device for delivering the culture medium for culturing to the well adjacent to the well The system of the present invention 1021, which is configured as follows. [The present invention 1023] The system of the present invention 1022, wherein the addressable well assembly includes 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 the present invention 1022, wherein the addressable well assembly includes a mounting assembly configured to connect to the delivery solution applying device, the stop solution applying device, and the culture medium applying device. [The present invention 1025] The system of the present invention 1021, wherein the delivery solution applying device includes a nebulizer. [The present invention 1026] The system of the present invention 1021, wherein the delivery solution application device is configured to deliver 10 to 300 microliters of the delivery solution per operation. [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 including the well. [The present invention 1028] The system of the present invention 1021, further comprising an enclosure configured to control the environment of the plate including the well. [The present invention 1029] The system of the present invention 1021, wherein the pressure difference application device includes a nozzle assembly configured to form a seal with the opening of the well and to deliver vapor to the well to increase or decrease the pressure therein, thereby pushing out the liquid portion of the culture medium from the well so that a layer of cells remains in the well. [The present invention 1030] The system of the present invention 1021, wherein the stop solution application device includes a needle emitter configured to connect to a stop solution reservoir. [The present invention 1031] The system of the present invention 1021, wherein the culture medium application device includes a needle emitter configured to connect to a culture medium reservoir. [The present invention 1032] Receiving user input, Operating 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, Operating the stop solution application device in response to the end of the first incubation period to deliver the stop solution to the cell monolayer, and Incubating the cell monolayer for a second incubation period and in response to the application of the stop solution The system of the present invention 1021, further comprising a control device configured as described above. [The present invention 1033] The control device operates the delivery solution applicator, incubates for the first incubation period, operates the stop solution applicator, and incubates for the second incubation period, repeating the above for a predetermined number of times. The system of the present invention 1022, further configured as described above. [The present invention 1034] Operate a positive pressure system to remove the supernatant from the well and generate a cell monolayer in the well. The system of the present invention 1021, further comprising a control device configured as described above. [The present invention 1035] The delivery solution applicator of the system of the present invention 1021 includes a spray head and a collar surrounding the distal end of the spray head, where the collar is configured to prevent contamination between wells in a single 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 the present invention 1035, where the delivery solution applicator includes a spray head and a film surrounding the distal end of the spray head. [The present invention 1037] The system of the present invention 1021, further comprising a vibration system, where the vibration system is connected to a membrane holder and is configured to vibrate the membrane. [The present invention 1038] A plate, where the well is configured to contain a population of non-adherent cells. The system of the present invention 1021, further comprising the above. [The present invention 1039] The system of the present invention 1021, where the delivery solution contains an isotonic aqueous solution, and the aqueous solution contains a payload and alcohol at a concentration of more than 5% (v / v). [The present invention 1040] The system of the present invention 1038, wherein the alcohol contains ethanol. [The present invention 1041] The system of the present invention 1039, wherein the aqueous solution contains more than 10% ethanol. [The present invention 1042] The system of the present invention 1039, wherein the aqueous solution contains 20 - 30% ethanol. [The present invention 1043] The system of the present invention 1038, wherein the aqueous solution contains 27% ethanol. [The present invention 1044] The system of the present invention 1038, wherein the aqueous solution contains 12.5 - 500 mM KCl. [The present invention 1045] The system of the present invention 1038, wherein the aqueous solution contains 106 mM KCl. [The present invention 1046] The system of the present invention 1037, wherein the non - adherent cells contain peripheral blood mononuclear cells. [The present invention 1047] The system of the present invention 1037, wherein the non - adherent cells contain immune cells. [The present invention 1048] The system of the present invention 1037, wherein the non - adherent cells contain T lymphocytes. [The present invention 1049] The system of the present invention 1038, wherein the payload contains messenger ribonucleic acid (mRNA). [The present invention 1050] The system of the present invention 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 the present invention 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 mammalian cells. [The present invention 1054] The system of the present invention 1037, wherein the population of non-adherent cells forms a monolayer. [The present invention 1055] A composition comprising an isotonic aqueous solution containing KCl at a concentration of 10-500 mM and ethanol at a concentration of more than 5% (v / v) for use in delivering a cargo compound or cargo composition to mammalian cells. [The present invention 1056] The composition of the present invention 1055, wherein the KCl concentration is 106 mM and the alcohol concentration is 27%. [The present invention 1057] Devices, systems, techniques, compositions, and articles described or illustrated herein. Other features and advantages of the present invention will become 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 one of ordinary skill in the art to which this invention belongs. In the practice or testing of 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 referred to herein are incorporated herein by reference. Genbank and NCBI registrations indicated by accession numbers referred to herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature referred to herein are incorporated herein by reference. In case of conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Brief Description of the Drawings

[0034]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

Figure 56

Figure 57

Figure 58

Figure 59

Figure 60

Figure 61

Figure 62

Figure 63

Figure 64

Figure 65

Figure 66

Figure 67

Figure 68

Figure 69

Figure 70

Figure 71

Figure 72

Figure 73

Figure 74

Figure 75

Figure 76

Figure 77

Figure 78

Figure 79

Figure 80

Figure 81

Figure 82

Figure 83

Figure 84

Figure 85

Figure 86

Figure 87

Figure 88

Figure 89

Figure 90

Figure 91

Figure 92

Figure 93

Figure 94

Figure 95

Figure 96

Figure 97

Figure 98

Figure 99

Figure 100

Figure 101

Figure 102

Figure 103

Figure 104

Figure 105

Figure 106

Figure 107

Figure 108

Figure 109

Figure 110

Figure 111

Figure 112

Figure 113

Figure 114

Figure 115

Figure 116

Figure 117

Figure 118

Figure 119

Figure 120

Figure 121

Figure 122

Figure 123

Figure 124

Figure 125

Figure 126

Figure 127

Figure 128

Figure 129

Figure 130

Figure 131

Figure 132

Figure 133

Figure 134

Mode for Carrying Out the Invention

[0035] Detailed Description The difficulty of transfecting molecules into non - adherent cells has plagued research and therapies, such as cell therapy, gene therapy, and genetic modification, for decades. As a reason for the difficulty of transfecting such cells, it can be mentioned that non - adherent cells lack cell - surface heparan sulfate proteoglycans, which are molecules responsible for cell adhesion to the extracellular matrix. Transfection methods such as electroporation and / or nucleofection have the drawback that they impair the cell viability, the ability of the cells to resume growth after treatment, and the function of the cells, such as the immune activity of lymphocytes. The transfection compositions and transfection methods (solporation) described herein are considered to have significant advantages compared to previous methods of introducing cargo molecules into mammalian cells, such as non - adherent / suspension cells that 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 mixture of compounds (composition) is delivered into the cytoplasm of a eukaryotic cell by contacting the cell with a solution containing the compound to be delivered (e.g., payload) and an agent that reversibly permeates or lyses the cell membrane. Preferably, the solution is delivered to the cell in the form of a spray, e.g., in the form of aqueous particles (see, e.g., 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 lyse the eukaryotic cell membrane include alcohols and detergents, such as ethanol and Triton X-100, respectively. Other exemplary detergents, such as 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 octyl glucoside.

[0037] An example of conditions for achieving coating of a population of coated cells involves delivery of a microparticle spray, e.g., dropping or pipetting a bolus volume of solution onto the cells such that a significant number of cells are not immersed or submerged by the bolus volume of fluid is excluded from this condition. Thus, the mist or spray contains a ratio of fluid volume to cell volume. Alternatively, the conditions involve a ratio of mist volume or spray volume to the exposed cell membrane area, e.g., when the cells are present as a confluent layer or substantially confluent layer on the bottom of a tissue culture vessel, such as in a well of a tissue culture plate, e.g., a microtiter tissue culture plate, where the cells are on a substantially flat surface.

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

[0039] In one aspect, delivery of a payload across a cell's plasma membrane involves providing a population of cells and contacting that population of cells with a volume of aqueous solution. The aqueous solution includes the payload and an alcohol content of greater than 5 percent. 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 a cell's plasma membrane includes an aqueous solution that includes the payload, an alcohol greater than 5 percent in concentration, a salt greater than 46 mM, a sugar less than 121 mM, and a buffer less than 19 mM. The concentration of the alcohol, such as ethanol, does not exceed 50%.

[0041] One or more of the following features can be included in any combination that is conceivable. The volume of the solution delivered to the cells is in multiple units, such as a spray, such as multiple droplets on an aqueous particle. The volume is described in comparison to individual cells or in comparison to the exposed surface area of a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, such as 85%, 90%, 95%, 97%, 98%, 100% confluent) population of cells. For example, the volume can be 6.0×10 -7 microliters per cell to 7.4×10 -4 microliters per cell. The volume can be 4.9×10 -6 microliters per cell to 2.2×10 -3 microliters per cell. The volume can be 9.3×10 -6 microliters per cell to 2.8×10 -5 microliters per cell. The volume can be about 1.9×10 -5 microliters per cell, where "about" is within 10%. The volume can be 6.0×10 -7 microliters per cell to 2.2×10 -3It is in microliters. The volume can be from 2.6×10 -9 microliters per square micrometer of exposed surface area to 1.1×10 -6 microliters per square micrometer of exposed surface area. The volume can be from 5.3×10-8 microliters per square micrometer of exposed surface area to 1.6×10 -7 microliters per square micrometer of exposed surface area. The volume can be about 1.1×10 -7 microliters per square micrometer of exposed surface area. The "about" can be within 10%.

[0042] Cell confluence refers to cells that are in contact with each other on the surface. For example, confluence can be expressed as an estimated (or counted) percentage. For example, 10% confluent means that 10% of the surface, such as the surface of a tissue culture flask, 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, sedimentation by vacuum, aspiration of the tissue culture medium from the top of the cell population, or aspiration or vacuum removal from the bottom of the vessel.

[0043] The contact between the cell population and the aqueous solution of the said volume can be carried out by spraying the aqueous solution with a gas to form a spray. This gas can contain nitrogen, ambient air or an inert gas. The spray can contain discrete volume units with diameters in the range of 1 nm to 100 μm, such as 30 to 100 μm. The spray contains discrete volume units with a diameter of about 30 to 50 μm. An aqueous solution with a total volume of 20 μl can be delivered as a spray to a cell occupancy area of about 1.9 cm 2 For example, to one well of a 24-well culture plate. An aqueous solution with a total volume of 10 μl can be delivered to about 0.95 cm 2The cell occupancy area is delivered to, for example, 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 alcohol, and the second volume does not contain alcohol (and optionally does not contain the payload). The cell population can be contacted with the aqueous solution for 0.1 to 10 minutes before adding the second volume of buffer or culture medium to sediment or suspend the cell population. The buffer or culture medium can be phosphate buffered saline (PBS). The cell population can be contacted with the aqueous solution for 2 seconds to 5 minutes before adding the second volume of buffer or culture medium to sediment or suspend the cell population. The cell population can be contacted with the aqueous solution, for example, an aqueous solution containing the payload, for 30 seconds to 2 minutes before adding the second volume of buffer or culture medium, for example, one that does not contain the payload, to sediment or suspend the cell population. The cell population can be contacted with the spray for about 1 to 2 minutes before adding the second volume of buffer or culture medium to sediment or suspend the cell population. During the time between the spraying of the cells and the addition of the buffer or culture medium, the cells remain hydrated by the layer of water from the spray volume.

[0044] The aqueous solution can contain an ethanol concentration of 5 to 30%. The aqueous solution can contain one or more of 75 to 98% H 2 O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 500 mM KCl, 2 to 35 mM ammonium acetate, and 1 to 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. For example, 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 are primary cells as well as immortalized cells (such as cells of cell lines). Exemplary non - adherent / suspension cells include primary hematopoietic stem cells (HSC), T cells (such as CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine - induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as Jurkat T cells.

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

[0047] Antibodies generally have a molecular mass of about 150,000 Da. Antibodies can include anti-actin antibodies, anti-GAPDH antibodies, anti-Src antibodies, anti-Myc ab, and / or anti-Raf antibodies. Antibodies can include green fluorescent protein (GFP) plasmids, GLuc plasmids, and BATEM plasmids. The DNA molecule can be greater than 5,000,000 Da. In some examples, the antibody can be a monoclonal antibody derived from a mouse, such as ibritumomab tiuxetin, muromomab-CD3, tositumomab, a human antibody, or a humanized mouse (or derived from another species) 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, gemtuzumab ozogamicin, trastuzumab, tocilizumab, ipilimumamb, or panitumumab. Antibodies can include antibody fragments, such as abatecept, aflibercept, alefacept, or etanercept. The present invention includes 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 containing the binding specificity of an antibody derived from, for example, a mouse, and the remaining portion of another antibody derived from, for example, a human.

[0048] The payload can include a therapeutic agent. A therapeutic agent, such as a drug or an active agent, can mean any compound useful for a therapeutic or diagnostic purpose, and this 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. In the methods described herein, the therapeutic agents described in U.S. Patent No. 7,667,004 (incorporated herein by reference) can be used. The therapeutic agent can include at least one of cisplatin, aspirin, statins (such as pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chlorpromazine HCl, thioridazine HCl, polymyxin B sulfate, chloroquine, benfluorex HCl, and phenazopyridine HCl), and fluoxetine. The payload can include a diagnostic agent. The diagnostic agent can include at least one of a detectable label or marker, such as methylene blue, patent blue V, and indocyanine green. The payload can include a fluorescent molecule. The payload can include detectable nanoparticles. The nanoparticles can include quantum dots.

[0049] The population of non-adherent cells can be substantially confluent, for example, exceeding 75% confluence. Cell confluence refers to cells that are in contact with each other on the surface. For example, confluence can be expressed as an estimated (or counted) percentage. For example, 10% confluence means that 10% of the surface, such as the surface of a tissue culture flask, 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, sedimentation by vacuum, aspiration of the tissue culture medium from the top of the cell population, or aspiration or vacuum removal from the bottom of the flask. The population of cells can form a monolayer of cells.

[0050] The alcohol can be selected from methanol, ethanol, isopropyl alcohol, butanol, and benzyl alcohol. The salt can be selected from NaCl, KCl, Na 2 HPO 4 、KH 2 PO 4 、and C 2 H 3 O 2 NH. Preferably, 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 a method for delivering molecules across a plasma membrane. The present subject matter is useful in the field of intracellular delivery and has applications in the delivery of molecular biology and pharmacological therapeutic agents to target sites such as cells, tissues, or organs. The method of the present subject matter includes the steps of introducing a molecule into an aqueous composition to form a matrix, atomizing the matrix into fine particles by spraying, and contacting the matrix with the plasma membrane.

[0052] The present subject matter relates to a composition used for delivering molecules across a plasma membrane. The present subject matter is useful in the field of intracellular delivery and has applications in the delivery of molecular biology and pharmacological therapeutic agents to target sites such as cells, tissues, or organs. The composition of the present subject matter includes an alcohol, a salt, a sugar, and / or a buffer.

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

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

[0055] Most preferably, the payload delivery solution contains 25% (v / v) alcohol. Alternatively, the payload can contain 2 - 8% (v / v) alcohol or 2% alcohol. The alcohol can include ethanol, and the payload contains 5%, 10%, 20%, 25%, 30%, and up to 40% or 50% (v / v) ethanol, e.g., 27% ethanol. The exemplary method can include methanol as the alcohol, and the payload can contain 5, 10, 20, 25, 30, or 40% (v / v) methanol. The payload can contain 2 - 45% (v / v) methanol, 20 - 30% (v / v) or 25% (v / v) methanol. Preferably, the payload contains 20 - 30% (v / v) methanol. Further alternatively, the alcohol is butanol, and the payload contains 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.

[0057] According to the present subject matter, the payload can contain at least one salt. The salt is NaCl, KCl, Na2 HPO 4 、 C 2 H 3 O 2 NH 4 、 and KH 2 PO 4 may be selected from. For example, the KCl concentration ranges from 2 mM to 500 mM. In some preferred embodiments, the concentration is above 100 mM, for example 106 mM.

[0058] According to the exemplary methods of the present subject matter, the payload may include a sugar (e.g., sucrose or a disaccharide). According to the exemplary methods, the payload includes less than 121 mM of sugar, 6 - 91 mM or 26 - 39 mM of sugar. Further, the payload includes 32 mM of 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 of sucrose.

[0059] According to the exemplary methods of the present subject matter, the payload may include a buffer (e.g., a weak acid or a weak base). The buffer may include zwitterions. According to the exemplary methods, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The payload may include less than 19 mM of buffer (e.g., 1 - 15 mM or 4 - 6 mM or 5 mM of buffer). According to the exemplary methods, the buffer 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 the exemplary methods of the present subject matter, the payload includes ammonium acetate. The payload may include less than 46 mM of ammonium acetate (e.g., 2 - 35 mM, 10 - 15 mM, or 12 mM of ammonium acetate). The payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM of ammonium acetate.

[0061] The volume of the aqueous solution effected by the gas injecting the aqueous solution may include compressed air (e.g., ambient air). Other embodiments may include inert gases such as helium, neon, and argon.

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

[0063] In certain aspects of the present subject matter, the population of cells can be substantially confluent and can exceed substantially 75 percent confluent. In a preferred embodiment, 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 examples, 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 cell plasma membrane can include small chemical molecules, peptides or proteins, polysaccharides or nucleic acids or nanoparticles. The small chemical molecules can be less than 1,000 Da, the peptides can have a molecular weight of about 5,000 Da, the siRNA can have a molecular weight of around 15,000 Da, the antibodies can have a molecular weight of about 150,000 Da, and the DNA can have a molecular weight greater than 5,000,000 Da or 5,000,000 Da. In a preferred aspect, the payload includes mRNA.

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

[0067] According to the 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 subject matter, a method for delivering molecules of multiple molecular weights across a plasma membrane is provided, the method comprising 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 comprises introducing a first molecule having a first molecular weight and a second molecule having a second molecular weight into the payload, wherein the first molecule and the second molecule may have different molecular weights or the first molecule and the second molecule may have the same molecular weight. According to the exemplary method, the first molecule and the second molecule may be different molecules.

[0070] In some embodiments, the payload to be delivered can include a therapeutic or diagnostic agent, such as cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chlorpromazine HCl, thioridazine HCl, polymyxin B sulfate, chloroquine, benfluorex HCl, and phenazopyridine HCl), and fluoxetine. Other therapeutic agents can include antimicrobial agents (aminoclycloside (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., virginiamycin, quinupristin-dalfopristin), carbapenems, lipopeptides, oxazolidinones, cycloserine, ethambutol, ethionamide, isoniazrid, para-aminosalicylic acid, and pyrazinamide). In some examples, antiviral agents (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] Additional exemplary payloads can also include a detectable marker or label, such as methylene blue, patent blue V, and indocyanine green.

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

[0073] In a further aspect, the payload to be delivered can include a composition that edits genomic DNA (i.e., a gene editing tool). For example, a gene editing composition can include a compound or complex that cuts genomic DNA, nicks genomic DNA, splices genomic DNA, rearranges it, moves it, recombines it, or otherwise modifies it. Alternatively or in addition to the above, a gene editing composition can include (i) a compound that can be included in a gene editing complex that cuts genomic DNA, nicks genomic DNA, splices genomic DNA, rearranges it, moves it, recombines it, or otherwise modifies it, or (ii) a compound that can be processed or modified by a compound included in a gene editing complex that cuts genomic DNA, nicks genomic DNA, splices genomic DNA, rearranges it, moves it, recombines it, or otherwise modifies it. In various aspects, a 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 aspects, the gene editing composition includes 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 a Flp recombinase. In a further aspect, the gene editing protein can be a fusion protein (megaTAL) that combines a homing endonuclease with a modular DNA binding domain of a TALEN. For example, megaTAL can be delivered as a protein or an mRNA encoding the megaTAL protein can be delivered to cells.

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

[0076] In certain embodiments, the gene editing composition comprises an RNP, which comprises a Cas protein and an sgRNA or a crRNA and a tracrRNA. Aspects of the subject matter are particularly useful for controlling when or for 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 a population of cells or progeny thereof, (a) after contacting the population of cells with an aqueous solution 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, or (b) after contacting the population of cells with an aqueous solution 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.

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

[0079] Aspects of the invention relate to a cell comprising one gene editing compound and inserting another gene editing compound into the cell. For example, one component of the RNP can be introduced into a cell that expresses or otherwise already contains another component of the RNP. For example, a cell in a population of cells or progeny thereof can comprise an sgRNA, a crRNA, and / or a tracrRNA. In some embodiments, a population of cells or progeny thereof expresses an sgRNA, a crRNA, and / or a tracrRNA. Alternatively or in addition to the above, a cell in a population of cells or progeny thereof expresses a Cas protein.

[0080] Various embodiments of the subject matter herein include a Cas protein. In some aspects, 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 the 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, such as 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, (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 crRNA molecule, (d) 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 crRNA molecule and further comprises a tracrRNA molecule, or (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

[0083] In various 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 various aspects, increasing the number of times a cell undergoes the delivery process (or increasing the number of administrations) may increase the editing rate. In some aspects, the number of administrations may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations.

[0085] In various aspects, the first and second sgRNAs or the first and second crRNA molecules as a whole include nucleic acid sequences complementary to target sequences 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 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 aspects, the use of pairs of RNPs containing the first and second sgRNAs or the first and second crRNA molecules can be used to generate polynucleotide molecules containing a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.

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

[0087] In various aspects, the first and second sgRNAs or the first and second crRNA molecules are complementary to sequences adjacent to an extrachromosomal sequence within an expression vector.

[0088] Aspects of the present subject matter relate to the delivery of multiple components of a gene editing complex, where the multiple components do not form a complex 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 present subject matter enables 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, or progeny thereof, of a population of cells become gene variants after contact with an aqueous solution. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more, or progeny thereof, of a population of cells remain viable after contact with an aqueous solution.

[0090] In certain embodiments, the gene editing composition induces single-stranded or double-stranded breaks in intracellular DNA. 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 (including repair templates) using the CRISPR-Cas system 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 subject matter, the 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 -7 microliters per cell to 7.4×10 -4 microliters per cell. In certain embodiments, the spray comprises colloidal particles or sub-particles having diameters ranging from 10 nm to 100 μm. In various embodiments, the volume is 2.6×10 -9 microliters per square micrometer of exposed surface to 1.1×10 -6 microliters per square micrometer of exposed surface area.

[0092] In some embodiments, the RNP has 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 a population of cells with the aqueous solution of the above volume can be performed by a gas that sprays 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 immerse or suspend the population of cells.

[0094] In various embodiments, the population of cells includes at least one of primary cells or immortalized cells. For example, the population of cells can include mesenchymal stem cells, lung cells, nerve 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+ subset of T cells is used. CD8 + 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 GFP mRNA can be delivered by platform delivery technology (i.e., 3% expression and 96% viability in 24 hours). In a further aspect, NK cell lines such as NK92 can be used, for example.

[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 and CAR NK cells, respectively); T cells expressing modified T cell receptors (TCRs); MSCs modified using viruses or non-virally to overexpress therapeutic proteins that complement innate properties (e.g., delivery of Epo using a lentiviral vector or delivery of 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 externally regulate targeting (Park et al., 2015); MSCs functionalized with targeting moieties to enhance homing to the site of treatment using 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., engineering of the cell surface with palmitated protein that acts as a hydrophobic anchor for subsequent antibody conjugation) (Park et al., 2015). For example, T cells modified to express a chimeric antigen receptor, such as primary T cells or T cell lines (CAR T cells), can be further treated according to the present invention with a complex containing a gene editing protein and / or a guide nucleic acid specific for the CAR coding sequence in order to edit the gene encoding the CAR and thereby reduce or stop the expression of the CAR in the modified T cells.

[0096] Aspects of the invention relate to expression vector-free delivery of gene editing compounds and gene editing complexes to cells and tissues, such as delivery of Cas-gRNA ribonucleoprotein for genome editing in primary human T cells, hematopoietic stem cells (HSCs), and mesenchymal stromal cells (MSCs). In some examples, mRNA encoding the aforementioned proteins is delivered to the 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 content of each of these documents being 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, Csm3, 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 the Cas9 protein of Streptococcus pyogenes can be found in the SwissProt database under accession number Q99ZW2 and in the NCBI database under accession number Q99ZW2.1. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 are other examples of the Cas9 protein amino acid sequence. Another non-limiting example is the Cas9 protein of Streptococcus thermophilus, whose amino acid sequence can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, unmodified CRISPR enzymes such as Cas9 have DNA cleavage activity. In certain embodiments, the CRISPR enzyme is Cas9 and can be Cas9 derived from Streptococcus pyogenes or Streptococcus pneumoniae. In various embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, e.g., 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 CRISPR enzyme that has been mutated such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence as compared to the corresponding wild-type enzyme. For example, a substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves one strand). Other examples of mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. In aspects of the invention, the nickase can be used for genome editing by homologous recombination.

[0099] In certain embodiments, Cas9 nickase can be used in combination with a guide sequence, for example, in combination with two guide sequences that each target the sense and antisense strands of a DNA target, respectively. This combination allows for nicking both strands and using it to induce NHEJ.

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

[0101] In certain embodiments, the protein to be delivered (e.g., a Cas protein or a variant thereof) can include an intracellular localization signal. For example, the Cas protein within the RNP can include an intracellular localization signal. Depending on the context, for example, a fusion protein comprising Cas9 and a nuclear localization signal can be referred to herein as "Cas9" without specifying that the 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 can 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 these references being incorporated herein by reference. In various embodiments, the Cas protein can include multiple localization signals, such as two, three, four, five, or more nuclear localization signals. In some embodiments, the localization signal is at the N-terminus of the Cas protein, and in other embodiments, the localization signal is 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 can be from a particular organism, such as, but not limited to, a mammal including a human, mouse, rat, rabbit, dog, or non-human primate, or can be derived from those particular organisms. Generally, codon optimization refers to the process of altering a nucleic acid sequence by replacing at least one codon of the native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons, or a number greater than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more) with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage frequency among organisms) often correlates with the translational efficiency of messenger RNA (mRNA), and it is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons that are most frequently used for peptide synthesis.

[0103] Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization. Codon usage tables can be readily obtained, for example, from 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 for codon-optimizing specific sequences for expression in specific host cells, such as Gene Forge (Aptagen, Jacobus, Pennsylvania), can also be used. 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 codons that are most frequently used for a particular amino acid.

[0104] Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence that is sufficient to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using an appropriate alignment algorithm. In some embodiments, the degree of complementarity is 100%. Optimal alignment can be determined using any appropriate algorithm for aligning sequences, and non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is of a length of 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, 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. In certain embodiments, the guide sequence is of a length less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides, or even less than those.The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be evaluated by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex comprising the guide sequence to be tested are provided to a host cell having the corresponding target sequence, for example by transfection with a vector encoding components of the CRISPR sequence, and then evaluation of preferential cleavage within the target sequence can be performed, for example, by the Surveyor assay described herein. Similarly, cleavage of a target polynucleotide sequence can be evaluated in vitro by providing the target sequence, components of a CRISPR complex comprising the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction.

[0105] CRISPR-Cas technologies that facilitate genome engineering in a wide range of cell types are rapidly evolving. It has recently been revealed that delivery of the Cas9-gRNA editing tool in the form of ribonucleoprotein (RNP) offers several advantages compared to delivery of plasmids encoding Cas9 and gRNA. Advantages 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, particularly with respect to certain clinically important cell types. Therefore, there is a need to provide a vector-free, e.g., virus vector-free, approach for delivering biologically important payloads, e.g., RNPs, across the plasma membrane into cells. The term "cargo" or "payload" is used to describe a compound or composition that is delivered as an aqueous solution across the cell plasma membrane into the interior of the cell.

[0106] The present subject matter relates to delivery techniques that facilitate delivery of a wide range of payloads to cells with low toxicity. Genome editing can be achieved by delivering RNPs to cells using some aspects of the present subject matter. Thereafter, the levels decline until Cas9 is no longer detectable. The delivery technique itself does not have a detrimental effect on the viability or functionality of Jurkat T cells and primary T cells. The present subject matter enables gene editing with Cas9 RNPs 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 the use of expression vectors, a certain amount of Cas, gRNA, or RNP can be added at a more precise timing over a limited length of time. Components expressed from vectors can be produced in various amounts over various lengths of time, which makes it difficult to achieve consistent gene editing without off-target editing. In addition, pre-formed complexes (RNPs) of Cas and gRNA cannot be delivered with expression vectors.

[0108] In one aspect, the present subject matter describes cells attached to a solid support (e.g., a strip, polymer, bead, or 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, amylases, natural and modified celluloses, polyacrylamide, rhyolite, and magnetite. For the purposes of the present subject matter, the carrier can be somewhat soluble or insoluble to some extent. 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, e.g., a sheet or a test strip. Preferred supports include polystyrene beads.

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

[0110] The present subject matter further relates to devices, systems, techniques, and articles for delivering a payload across a plasma membrane. The present subject matter also relates to devices for delivering a payload such as a protein or protein complex across a plasma membrane. The present subject matter can be used in the field of intracellular delivery and has applications, for example, in the delivery of molecular biology and pharmacological therapeutic agents to target sites such as cells, tissues, or organs.

[0111] In some embodiments, a device for delivering a payload across a plasma membrane can include an atomizer having at least one atomizer emitter and a support adapted to the atomizer. The method can further include the step of atomizing the payload before 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 tube. 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 commercially available from LMA Teleflex of North Carolina, USA. The atomizer can be an intranasal mucosal atomization device commercially available from LMA Teleflex of North Carolina, USA under catalog number MAD300.

[0113] The atomizer can be adapted to provide a colloidal suspension of particles having a diameter of 30 to 100 μm before bringing the plasma membrane into contact with the payload. The atomizer can be adapted to provide a colloidal suspension of particles having a diameter of 30 to 80 μm. The atomizer can be adapted to provide a colloidal suspension of particles having a diameter of 50 to 80 μm.

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

[0115] The apparatus can include a sample reservoir. The sample reservoir can be in fluid communication with the atomizer. The sample reservoir can be in fluid communication with the atomizer emitter. The gas reservoir and the sample reservoir can both be in fluid communication with the atomizer emitter.

[0116] The device can comprise a sample valve positioned between the sample reservoir and the gas reservoir. The device can comprise a sample valve positioned between the sample reservoir and the gas guide. The sample valve can be adapted to regulate the sample flow from the sample reservoir. The sample valve can be adapted to enable a continuous or semi - continuous sample flow. The sample valve can be adapted to enable a semi - continuous sample flow. The sample valve can be adapted to enable a semi - continuous sample flow of a specified quantity. The sample valve is adapted to enable a semi - continuous sample flow of 0.5 to 100 μL. The sample valve can be adapted to enable a semi - continuous sample flow of 10 μL. The sample valve can be adapted to enable a semi - continuous sample flow of 1 μL to an area of 0.065 to 0.085 cm 2 ².

[0117] The atomizer and the support can be placed at a distance from each other. The support can include a solid support. The support can include a plate containing sample wells. The support can include a plate containing sample wells selected from 1, 6, 9, 12, 24, 48, 384, 1536, or more wells. Alternatively, the support can include a scaled-up configuration capable of accommodating a monolayer of more cells than a plate, such as 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 a combination thereof. The support can include a heating element. The support can include a resistive element. The support can be reciprocally attachable to the device. The support can be reciprocally movable with respect to the device. The support can be reciprocally movable with respect to the atomizer. The support can be reciprocally movable with respect to the atomizer emitter. The support can include a support actuator for reciprocally moving the support with respect to the atomizer. The support can include a support actuator for reciprocally moving the support with respect to the atomizer emitter. The support can include a support actuator for reciprocally moving the support in a direction transverse to the longitudinal axis of the atomizer emitter. The support can include a support actuator for reciprocally moving the support in a direction transverse to the longitudinal axis of the atomizer emitter.

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

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

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

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

[0122] The atomizer emitter can be adapted to provide a conical spray zone (e.g., generally a conical spray zone). The atomizer emitter can be adapted to provide a 30° conical spray zone. The apparatus can further include a microprocessor for controlling any or all of the parts of the apparatus. The microprocessor can be arranged to control any or all of the sample valve, the support actuator, the valve, and the regulator. The apparatus can include an atomizer having at least one atomizer emitter and a support adapted to the atomizer, and 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 - 100 μm. The apparatus can include a sample reservoir, 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 - 100 μL. The atomizer and the support can be spaced apart and can generally define a conical spray zone therebetween. Also, the distance between the atomizer and the support can be about twice the diameter of the circular bottom surface of the area of the cell 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 bottom surface of the area of the cell to which the molecules are to be delivered can be 15.5 mm. The apparatus can include a gas guide, and the pressure within the gas guide can be 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 can be saponin - free.

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

[0125] Optimization of T Cell Preparations for Solporation The 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 methods for intracellular delivery of polymers and nucleic acids described herein have successfully facilitated 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 for efficient introduction of mRNA into human T cells using a vector-free delivery platform.

[0126] There are significant differences among clinical groups and T cell engineering companies in the ex vivo activation of human lymphocytes. There is no single standardized protocol for the expansion of primary human T cells. Therefore, an extensive evaluation of culture media, additives, activation methods, and timing schedules was initiated to identify the optimal ex vivo culture conditions that promote the most efficient introduction of mRNA using the vector-free reversible permeabilization method technology. Here, the cell isolation protocols and the results / conclusions of each experiment initiated are described.

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

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

[0129] Delivery method. Activated T cells were seeded at 1.5 × 10 6 cells per well in a 96-well filter plate (Acroprep, 1.2 μm Supor membrane, Pall, USA). The medium was removed from the wells by centrifugation at 300 × g for 5 minutes. Next, 7 μl of the delivery solution containing 4 μg of GFP mRNA (32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 27% ethanol, all from Sigma-Aldrich, in molecular grade water) was sprayed into each well using a vector-free delivery spray device. The atomizer used for this device was the MAD Nasal™ micronization device for nasal mucosa (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, Vannes Cedex, France) via a polyurethane tube (outer diameter 6 mm, inner diameter 4 mm, 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 the spray actuator button (SMC, Tokyo, Japan). After delivery, the cells were incubated in this solution for 2 minutes and then 50 μl of the stop solution (0.5× PBS) was added. After 30 seconds, T cell medium was added (100 μl), and the cells were allowed to recover overnight at 37 °C and 5% CO 2 . Uptake and viability were evaluated 24 hours after delivery.

[0130] Cell viability, FACS sample preparation, and analysis. To evaluate cell viability after the vector-free delivery method, cells were stained with 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, protected from light, 5 - 10 minutes at room temperature), 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 whole cells using forward scatter 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 types of media for T cell culture and expansion were evaluated using mRNA as a cargo, for example, model cargo GFP mRNA, and GFP expression was used as an indicator of a favorable culture method. cRPMI, a serum-containing medium typically used for culturing primary immune cells, was tested. However, since serum is a highly variable supplement in cell culture media, three types of serum-free and xenofree expansion media optimized for 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, and 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 proliferation protocols. The first proliferation protocol tested was the 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 together with an alternative activation method using "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 a 2-hour incubation, the supernatant was removed, leaving adherent monocytes. T cells cultured in cRPMI were added to the plates and co-incubated with monocytes for several days. In a similar protocol, the A549 cell line was allowed to adhere for up to 2 hours. The medium was removed, T cells were added to the plates, and after co-incubation, mRNA was delivered using a vector-free delivery technique. Since the uptake efficiency varied within and between experiments when feeder cells were used (Figure 1), the ImmunoCult™ Human CD3 / CD28 T Cell Activator reagent was used in further evaluations.

[0134] T Cell Activation Using Dynabeads® T cells are activated using methods known in the art, such as antibodies that bind to cell surface proteins such as CD3 and / or CD28, feeder cells, and / or magnetic beads containing an immune activating molecule. For example, Dynabeads® were considered as an alternative to the ImmunoCult™ Human CD3 / CD28 T Cell Activator. Dynabeads® are superparamagnetic beads coated with antibodies against human CD3 and CD28 that provide the primary and co-stimulatory signals necessary for T cell activation and expansion. The recommended bead-to-cell ratio is 1:1, but another condition using three beads per cell was also used to evaluate whether a more efficient and rapid expansion would positively affect the uptake of mRNA into T cells using the electroporation delivery method. Increasing the bead-to-cell ratio significantly improved the uptake rate (Figure 2A). Next, repeated experiments were performed with the ImmunoCult™ Human CD3 / CD28 T Cell Activator included at three times the manufacturer's recommended concentration, but this concentration did not result in the same efficiency improvement as that observed using Dynabeads (Figure 2B).

[0135] Culture medium: Prime XV A culture medium compatible with the cells was used in this delivery method. For example, Prime-XV (Irvine Scientific) is the first serum-free and animal component-free medium tested with 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), this activation method was used to continue testing alternative culture media. This medium was tested using Dynabeads to induce proliferation, along with an alternative activation method that stimulates T cell proliferation by binding anti-human CD3 antibody to the cell culture plate and then adding soluble anti-CD28 to the medium. Dynabead-activated T cells cultured in Prime XV showed a significantly improved uptake efficiency (up to 50%) compared to cells stimulated with soluble α-CD3 / CD28 (<20%) (Figure 3).

[0136] Delivery to T cells cultured in Prime XV improved mRNA uptake using vector-free technology, but this was associated with cell handling issues such as removing cells from culture 24 hours after initial seeding and cell recovery rates from Dynabeads during washing (Figures 4A and 4B).

[0137] Culture medium: Supplementary Cytokines In some examples, high concentrations of IL-2 (200 U / ml instead of 100 U / ml) were supplemented to the cell culture medium to enhance the proliferation rate (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, this is also a serum-free xeno-free T cell culture medium. In this example, T cells were cultured in Immunocult Expansion Medium and activated using Dynabeads® at a bead-to-cell ratio of 3 to 1. GFP mRNA was delivered to the cells on day 1 and day 2 after activation and evaluated 24 hours later (Figure 5).

[0139] Timing of Delivery after T Cell Activation The cells were activated for 15 - 21 hours, with 19 hours being preferred.

[0140] An optimal “window” for post-activation delivery after addition of Dynabeads was identified. Using a vector-free technology, mRNA was delivered at several time points. Optimal GFP expression was observed when the mRNA payload, for example the model payload GFP mRNA, was delivered 19 hours after activation as compared to 17 and 21 hours (Figure 6). A study was initiated to determine whether there is a correlation between increased cell size and time after activation. Cell size was estimated using forward scatter (FSC) data obtained from flow cytometry analysis. It was observed that the maximum transfection efficiency correlates with the time when the cell size is actively increasing. Exemplary results are illustrated in the table below.

[0141] (Table) Timing of delivery after T cell activation TIFF2025084859000001.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 beneficial compared to Dynabeads® as excess reagent can be removed by centrifugation and no magnetic separation of beads from the cells and subsequent washing are required. Therefore, by using Immunocult as a medium and evaluating this reagent over a period of 3 days, the preferred day for mRNA delivery by the vector-free delivery technology described herein was determined. The initiation of T cell proliferation by TransAct was not as active as that by Dynabeads. Therefore, optimal delivery of mRNA to T cells was observed 24 hours later than that shown when bead activation was used, but this was accompanied by enhanced transfection efficiency (Figure 7).

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

[0144] Effect of Recovery Period after Thawing before Activation The benefit to delivery of allowing cells to recover overnight prior to addition of the activation reagent was evaluated compared to adding the recovery agent immediately after thawing without a recovery period. When T cells were thawed from liquid nitrogen storage, they were allowed to recover overnight in culture medium only prior to addition of the activation reagent. This step resulted in a 30% improvement in transfection efficiency. In some examples, fresh primary non-adherent cells are used in this method, and in another example, primary non-adherent cells are frozen, e.g., for storage, and then thawed prior to the payload delivery method. Thus, this method can optionally include a freeze / thaw step of primary non-adherent cells. These data indicate that a recovery period prior to activation (after thawing) is useful.

[0145] Cell Culture Density Prior to vector-free delivery, cells were cultured in Immunocult expansion medium at various seeding densities (1×10 6 cells / ml and 5×10 6 cells / ml). The cell density during culture prior to electroporation was 1 - 5×10 6 cells / mL. An increase in seeding number led to an improvement in uptake efficiency (Figure 10).

[0146] T Cell Activation Using Zinc Zinc influx can assist 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 tendency for transfection efficiency to improve. Thus, zinc is an optional component of the cell culture medium in the activation step. The range of zinc concentration is from 0.03 mM to 3 mM.

[0147] Optimization of T cell culture conditions to maximize mRNA delivery efficiency Through the evaluation of multiple culture media, activation methods, supplements, and time course, a preconditioning protocol was developed to maximize the transfection efficiency of mRNA, such as model payload GFP mRNA, into human primary T cells using vector-free delivery technology. In this evaluation, cells were cultured at a density of 5×10 6 cells / ml in Immunocult™ T cell expansion medium supplemented with 200 U / ml of IL-2. After thawing the cells and allowing them to recover overnight, 1× T cell TransAct™ (Miltenyi) was added for 48 hours prior to vector-free delivery of nucleic acids.

[0148] Human peripheral blood mononuclear cells (PBMCs) were recovered from Leuko Pak (AllCells, Alameda, CA) by centrifugation on a Percoll gradient. CD4-enriched T cells were isolated by negative selection to collect the purified population using anti-CD8 microbeads, and the flow-through fraction was collected from an LD column (Miltenyi). Cells were cultured in complete RPMI supplemented with IL-2 (200 U / ml) using standard cell culture medium, such as RPMI basal medium, heat-inactivated fetal bovine serum (FBS) (10% v / v), penicillin-streptomycin, and L-glutamine. After allowing the cells to recover for 4 hours, Dynabeads® were added at a bead-to-cell ratio of 3 to 1. mRNA was delivered to the cells on day 1 after activation, and uptake was evaluated 24 hours later. In this cell type, an improvement in uptake efficiency was achieved by multiple hits, >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 as well as 250 IU / ml of IL-2. Cells were at 1×10 6Seeded at a density of cells / mL and supplemented with anti-CD3 and anti-CD28 antibodies (Miltenyi) prior to culture. mRNA, such as test payload GFP mRNA, was delivered to the cells by electroporation either on the second or third day after initiation, and uptake was evaluated 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 generally 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 that are substantially aligned on the same surface and are often in contact with each other. The cells can be adherent cells (cells that attach to the substrate) or non-adherent cells (cells that float or are suspended in the culture medium). Adherent cells grow and attach on the substrate, thereby forming a monolayer. A monolayer can also be generated from non-adherent cells or "floating cells". The terms "non-adherent cells" and "floating cells" are used interchangeably herein.

[0151] Several techniques can be used to generate a cell monolayer from non-adherent or floating cells prior to delivery of the payload to the cells. Such techniques include sedimenting the cultured floating 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 (such as use of the Transwell insert system described below).

[0152] Transwell insert To form a monolayer of floating cells that allows the cells to be optimally presented to the spray, a Transwell insert system was used. 1×10 in 400 μl per insert 6Cells were seeded at [quantity] and the Transwell insert (Greiner bio-one; catalog number 655640; 12-well ThinCert; PET 0.4 μm) was placed in a device that allows application of a vacuum (-0.5 bar to -0.65 bar) to the bottom of the insert to remove the medium (see Figures 14A, B, and C). When the medium was removed, the remaining cells formed a monolayer to 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 a delivery solution (10 μl) containing a test payload such as fluorescently labeled β-lactoglobulin (BLG), bovine serum albumin (BSA), or ovalbumin (OVA) was sprayed. After a 2-minute incubation, a stop solution (50 μl) was applied, and after 30 seconds, normal medium (100 μl) was applied. Expression levels of 55.6, 28.5, and 15.3% were achieved (Figure 15). The insert system was found to be useful as an exemplary technique for generating monolayers using non-adherent cells.

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

[0154] To generate floating cell monolayers, such membrane-based systems were evaluated. A 96-well filter-bottom plate provides a 96-well format with a filter bottom in the well. A Pall Supor filter plate (AcroPrep Advance; PES 1.2 μm, catalog number 8039) was evaluated. 1×10 6Individual human primary T cells were seeded at 100 μl per well and the plates were centrifuged at 300 × g for 5 minutes. The medium was removed by centrifugation, and once the remaining cells formed a monolayer, the plates were placed inside a Solupore device and the delivery solution containing mRNA was sprayed onto the cells. After a 2-minute incubation, the stop solution (50 μl) was applied, and 30 seconds later, normal medium (100 μl) was applied. The cells were incubated for 2 hours and then this process was repeated. At the end of this spray, the cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. GFP expression levels of 52% ± 3.6 were achieved in 5 donors and 5 experiments, and the viability was 97% ± 3.3 (Figures 16A, B, C). PES plates contain a mesh-like filter where cells can potentially become non-recoverable. Other filter types, such as track-etched filters, were evaluated. A track-etched membrane is a thin (about 5 - 25 micrometers) polymeric membrane, and its small pores are formed by irradiating the initial non-porous material with high-energy particles and then etching the latent tracks (usually with a caustic etching solution (e.g., NaOH)) to form small pores of a given diameter through the membrane.

[0155] 96-well polycarbonate track-etched (PCTE) plates An alternative membrane filter system can be used for the generation of a cell monolayer 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 5Individual cells were seeded with human primary T cells and centrifuged at 350×g for 2 minutes. Once the medium was removed and a cell monolayer was formed, the plate was placed inside the Solupore device, and a delivery solution containing a test payload such as GFP mRNA was sprayed onto the cells. After a 2-minute incubation, a stop solution (50 μl) was applied, and 30 seconds later, normal medium (100 μl) was applied. The cells were incubated for 2 hours, and then this process was repeated. At the end of this spray, the cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. A GFP expression level of 72% ± 5 was achieved, and the viability was 75.0% ± 3.5 (Figure 17). The results of comparing the PES plate and the PCTE plate are shown in Figure 18. This result indicates that uptake is enhanced when using the PCTE plate. Thus, hydrophilic membrane filters, optionally track-etched filters, are useful exemplary membranes and are preferred in some embodiments.

[0156] Solvent removal We also addressed methods for removing the medium from the cells. Using a filter plate, centrifugation, vacuum pressure, and positive pressure were evaluated. A 96-well filter plate (Pall; Supor, 1.2 μm; catalog number 8039) was seeded with 1×10 6 individual human primary T cells per well. The medium was removed by centrifugation at 300×g for 5 minutes or by vacuum pressure (-20 mBar, 30 seconds; see Figures 105 - 107). A 4 μl delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. After a 2-minute incubation, a stop solution (50 μl) was applied, and 30 seconds later, normal medium (100 μl) was applied. The cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry (Figures 19A, B). In another experiment, an Agilent PCTE filter plate (Agilent; PCTE 0.4 μm) was used with 2.5×10 5Individual cells were seeded with human primary T cells. The medium was removed either by centrifugation (350×g for 2 minutes) or by positive pressure (200 mBar for 1 minute). Once the medium was removed and a cell monolayer was formed, the plate was placed inside the Solupore device, and the delivery solution containing GFP mRNA was sprayed onto the cells. After a 2-minute incubation, the stop solution (50 μl) was applied, and 30 seconds later, the normal medium (100 μl) was applied. The cells were incubated for 2 hours, and then this process was repeated. At the end of this spray, the cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry (Figures 19A, B). A cell monolayer was 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 In an alternative method, the use of T cell activation beads (e.g., DynaBeads at a 3:1 ratio) is combined with a magnet. After overnight activation, the T cells (those 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. Next, the mRNA was delivered by solporation. GFP expression was detected by light microscopy after 24 hours.

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

[0159] The delivery of mRNA, such as the test payload / cargo GFP mRNA, to BM-MSC and iPSC-MSC was evaluated. The delivery of various cargo compounds, such as 10 kDa dextran, to primary BM-MSC using a vector-free intracellular delivery method involving reversible permeabilization has been reported previously (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 mRNA delivery efficiency to BM-derived MSC and iPSC-derived MSC.

[0160] Multiple treatments, such as 3 times of GFP mRNA, were delivered to BM-MSC and iPSC-MSC over a period of 2 days. The expression of GFP protein in the cells was confirmed 24 hours after the last mRNA delivery by fluorescence microscopy (Figure 72A). Flow cytometry analysis showed delivery efficiencies of 29.5±10.4% and 31.0±2.2% (n = 3) in BM-MSC and iPSC-MSC, respectively (Figures 21A, B).

[0161] Optimization of Micronization of Delivery Solution The mucosal atomization device (MAD Nasal (trademark)) spray head used to atomize the payload solution dispensed volumes in milliliters, while solporation operates in microliter volumes. The use of a microliter volume atomizer / droplet delivery system is preferred.

[0162] Alternative spray heads were investigated to increase uptake and improve reproducibility across multiple repeats (within experiments) and multiple experiments (between experiments), two main objectives. To find out which is optimal for mRNA delivery to T cells, we began evaluating alternative atomizer devices. An atomizer enables the application of the delivery solution in droplet form to a cell monolayer. In addition to the identification of the atomizer, a control device was designed and assembled that enables fine control of the atomization process. Various parameter sets were tested to find the optimal parameters for mRNA delivery to T cells.

[0163] The results showed that microliter volume delivery devices such as the Ari Mist nebulizer and the 180 kHz ultrasonic nebulizer gave comparable uptake and reproducibility, and that cell viability was slightly higher with the Ari Mist. Such microliter volume delivery devices, such as the Ari Mist head, are preferred because they are small in size, easy to handle, and do not require a power box to operate, and can be incorporated into an automated solution. Other units, such as the Burgener atomization technique (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] Micronization of Delivery Solution to Generate Monodisperse Droplets The cell membrane permeabilization treatment solution was delivered onto the cell monolayer using various methods. For example, the permeabilization solution can be atomized using ultrasound or sprayed using a pneumatic nebulizer.

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

[0166] The ultrasonic treatment tests were conducted at 60kHz, 130kHz, and 180kHz. The liquid can be extruded to the ultrasonic nozzle by a pumping system and atomized into fine mist sprays using high-frequency sound wave vibrations.

[0167] A gas (air) curtain can assist this process. An auxiliary piece called a shaper is attached around the ultrasonic head, and the function of the air is to shape the mist of the ultrasonically treated liquid. For example, air has a dual function of not only shaping the spray but also promoting the entry of the payload into the cells. This was made possible because the air supplied through the shaper was at a pressure exceeding the pressure used for the shaping function.

[0168] Tests with the ultrasonic nebulizer gave useful results for the delivery of cargo / payload to mammalian cells, such as non-adherent cells. Up to 60% of dextran-Alexa488 (model cargo) was delivered to U2OS cells using the 180kHz ultrasonic head (Figures 22A, B).

[0169] An electrical input can be transmitted through a piezoelectric transducer into mechanical energy in the form of vibrations. When introduced into a nozzle, these vibrations create surface tension waves in a liquid, resulting in atomization of the liquid. Each ultrasonic probe operated at a given resonance frequency. The operating frequency can determine the size of the generated droplets. The size of the droplets can also be affected by the power driving the ultrasonic probe, although not as much as by the operating frequency. An auxiliary air flow can be used to assist in spray control and shaping.

[0170] Exemplary ultrasonic spray emitters produce a fine spray with a narrow droplet size distribution, and that narrow size distribution results in an even deposition of delivery solution and payload onto cells. For example, for the preferred parameters evaluated for the MAD nasal spray head, reducing the delivered volume and reducing the ethanol concentration improved the delivery efficiency and survival rate with the ultrasonic spray head.

[0171] Additional nebulizers for generating droplets (microliter volumes) were tested, such as the Ari Mist, Peek Mira Mist, T2100, X175, and PFA250 nebulizers (Burgener Research). These exemplary nebulizers operate with compressed gas and require a pump to supply the sample solution. These exemplary atomizers have two parallel channels, one for gas (air) and the other for the liquid to be atomized. Both paths end 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 stream. Collisions with gas molecules can break up the liquid into small droplets, resulting in atomization.

[0172] The Burgener nebulizers tested (U.S. Patent No. 6,634,572) have different inner diameters, materials, and optimal flow rates. In preliminary tests, the nebulizers gave equivalent cargo mRNA expression. Characterization of the droplet size revealed that the droplets ranged from 1 - 20 μm, with the peak number of droplets in the range of 5 - 7 μm. The Sauter mean (D32 ) The average particle size defined as diameter is 13 μm (see http: / / www.burgener.com / EnhancedData.html). Among a set of Burgener nebulizers tested, Ari Mist was chosen as the preferred spray head based on several factors listed below: it had good uptake and survival rates, its specifications (optimal flow rate, inner diameter) were compatible with the characteristics of the pumping system, and its inner diameter (225 μm) was small enough to handle low volumes of liquid without the inconvenience of clogging.

[0173] Certus Digital Dispensing Technology An 8-channel dispensing head (catalog number D196057) and two valve sizes (nozzle diameter 0.10, travel 0.03 (catalog number 21765) and nozzle diameter 0.15, travel 0.03 (catalog number 21766)) were attached to the Certus Flex liquid dispensing instrument. Each channel is individually controlled using Certus' proprietary software and electronics. Certus Flex enables contactless dispensing of liquids and macromolecules using Gyger microvalve technology and pneumatic control. Volumes in the nanoliter (nl) range can be delivered with high precision (CV 5% at 100 nl; CV represents the coefficient of variation or relative standard deviation).

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

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

[0176] The results indicate that the cell viability was equivalent to that of untreated cells when using this system. When using this system, delivery of GFP mRNA was not observed. This was seen for all parameters tested (Figure 61). Thus, even when using the Certus digital dispensing technology to deliver droplets in the nl to μl range, uptake of GFP mRNA into T cells did not occur.

[0177] (Table 1) Plate Delivery Template Parameters TIFF2025084859000002.tif55146

[0178] (Table 2) Dispensing Head Channel Configuration TIFF2025084859000003.tif62147

[0179] Use of equipment to enable fine control of spraying A test rig was assembled to control important spray parameters and enable spray mechanization. The plates containing the cell suspension were centrifuged before being placed in the test rig. The delivery solution containing the payload was filled into an elveflow fluid reservoir or a syringe system. The flow control of the delivery solution containing the payload was achieved using either a pinch valve or a micro valve. The addition of the stop and culture medium was done manually.

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

[0181] (a) Flow control of the delivery solution containing the payload (i) Elveflow-pinch valve Elveflow refers to a microfluidic reservoir used with a 1.5 ml Eppendorf tube or a 50 ml Falcon tube depending on the required sample reservoir size (Elvesys, Paris, France, 75011, Avenue Philippe Auguste 83, Innovation Center). The pinch valve can refer to any pinch valve, and one example is the electronic Clippard pinch valve (Clippard, Cincinnati, Ohio 45239, USA, 7390 Colerain Avenue). The flow control can be achieved by a flow 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 tube used.

[0182] The valve can be actuated by a metal-oxide semiconductor field-effect transistor (MOS FET) that can be controlled by a microprocessor.

[0183] (ii) Syringe - Microvalve The above - mentioned Elveflow - pinch valve system had the following constraints. - When the elveflow sample reservoir was refilled, the calibration of the system was not maintained. - The accuracy and precision when dispensing volumes less than 5 μl were insufficient (for low volumes (<5 μl), the relative standard deviation in repeated dispensing was approximately 9%. These data were generated at Avectas and are summarized in Figure 54). Calibration data for the delivery solution used the Elveflow - pinch valve system.

[0184] To address these constraints, a new fluid system was used.

[0185] This may involve the use of a microvalve fluid system such as the Gyger microvalve (SMLD300, Fritz Gyger AG, Switzerland, Guvatt (Thun) 3645, Bodmersstrasse 12). 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 microvalves and pinch valves, there was no difference in delivery efficiency (Figure 63A, B).

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

[0187] (c) Electronics for controlling spray operation To enable electronically controlled spray operation, the system was designed using a microprocessor-based development board to facilitate the easy development of a time control sequence. A microprocessor, such as the PIC16F1619, was used on the development board. The spray operation time and fluid delivery time can be manipulated by the interface software of the development board. This microprocessor development board enables the pulsing of the nebulizer spray.

[0188] Next, by leveraging repeatable high-speed PLC technology (programmable logic controller), the system was upgraded to better align with industry standards and serve as a proof-of-concept for automated Solupore (trademark) technology, which is based on ultra-high-speed programmable logic controller (PLC) technology. The test rig controller consisted of a PLC, a Gyger controller, and a program that communicated between these two pieces of hardware. The hardware included an operator interface via momentary push buttons.

[0189] (d) Alignment of the spray head Spray heads, such as the Ari Mist nebulizer parallel path design, produce an off-center spray from the tip of the nebulizer. The alignment of the spray head can be adjusted using a custom spray head holder equipped with a goniometer.

[0190] Identification of optimal parameters for delivery of mRNA to T cells Work was done to characterize and optimize the sprays produced by three types of ultrasonic heads and the Ari Mist head. The characteristics of the various sprays were evaluated using high-speed camera recordings. The force of the spray received by the cell monolayer was measured by force sensor analysis. In some examples, the volume delivered to the wells of a 96-well plate was evaluated using a colorimetric assay.

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

[0192] Using EGFP mRNA as the payload, a large number of sets of parameter combinations were tested using three types of ultrasonic probes and an Ari Mist nebulizer.

[0193] All sets of parameters resulted in GFP expression, and the uptake varied from 5% to 30%.

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

[0195] The uptake and reproducibility obtained with Ari Mist, 180 kHz ultrasound, and the Mad Nasal spray head were compared. The results are presented in vertical data plots for Ari Mist, 180 kHz ultrasound, and the Mad Nasal spray head (Figures 24, 25, and 26 respectively). The vertical data also shows the transition of the optimization of delivery parameters over several weeks. The uptake reached 20 - 30% positive cells with 180 kHz ultrasound (Figure 25) and Ari Mist (Figure 24), but fluctuated below 20% when cells were sonoporated using the MAD nasal spray head (Figure 26).

[0196] When using an ultrasonic nebulizer or a Burgener nebulizer (Figs. 24, 25), an improvement in reproducibility was seen compared to the MAD nasal spray head (Fig. 26). The reproducibility of uptake was represented by the standard deviation (StDev). The main purpose of evaluating other spray heads was to identify a nebulizer that gives the narrowest possible standard deviation of uptake within multiple repetitions of one experiment. By averaging the standard deviation of uptake across all experiments, longitudinal data were examined, and the average standard deviation of uptake was 2.4% for the 180 kHz ultrasonic spray head, 2.1% for Ari Mist, while it was 4.5% for MAD nasal. In both cases, it was improved compared to the MAD nasal nebulizer.

[0197] The results showed that the Ari Mist and 180 kHz ultrasonic nebulizers gave equivalent levels of delivery efficiency, with 20 - 30% GFP-positive cells detected. The cell viability was higher for the Ari Mist head. Furthermore, this nebulizer is smaller, easier to handle, and does not require power to operate. All these reasons contributed to the selection of the nebulizer as a spray head for sonoporation, e.g., the Ari Mist nebulizer, and to the optimized delivery parameters evaluated for mRNA delivery, as shown, for example, in Table 4.1. This set of parameters is the result of an extensive screening and serves as a starting point for further refined optimization studies that fine-tune other parameters such as the composition of the delivery solution, the number of cells, and the filter plate, in addition to volume, spray distance, and length, to further improve mRNA delivery. Table 4.1 shows a list of the parameters and ranges tested and includes the parameters favorable for the delivery of mRNA (e.g., model cargo GFP mRNA) to T cells. Table 4.1 includes the ranges tested and the favorable parameters for both the benchtop Flexi (benchtop) system and the Midi (scale-up) system.

[0198] Optimization of the process for applying the delivery solution to cells Atomizer height To increase the mRNA uptake efficiency and expression in human primary T cells, several parameters were evaluated. To observe whether there is an effect on the delivery of GFP mRNA to T cells, the height of the AriMist atomizer was evaluated. 96-well filter plates (Pall; Supor, 1.2 μm; catalog number 8039) were seeded with 1×10 6 human primary T cells per well. The plates were centrifuged at 300×g for 5 minutes, and 4 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. The atomizer height was evaluated at 31, 26, and 11 mm above the bottom of the well (in another experiment, the comparison between 26 mm and 12 mm was evaluated). After a 2-minute incubation, the stop solution (50 μl) was applied, and after 30 seconds, the normal medium (100 μl) was applied. The cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. mRNA uptake by T cells was achieved when the atomizer was placed 31 mm and 26 mm above the bottom of the well. In some cases, part of the 4 μl delivered did not enter the well. A preferred height of 12 mm above the bottom of the well was selected (Figures 27A, B). This enabled accurate dosing of the payload and prevented overspray that could contaminate other wells. The reduction in height also allowed for a reduction in the volume delivered. The height range is from 11 mm to 31 mm above the bottom of the well (filter).

[0199] Volume delivered To determine the optimal volume delivered that could maximize mRNA uptake by T cells, a comparison of volumes was initiated. 96-well filter plates (Pall; Supor, 1.2 μm; catalog number 8039) were seeded with 1×10 6Individual human primary T cells were seeded. The plates were centrifuged at 300×g for 5 minutes, and 4, 1, or 0.5 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. After a 2-minute incubation, the stop solution (50 μl) was applied, and after 30 seconds, the normal medium (100 μl) was applied. The cells were incubated overnight in a humidified incubator at 37 °C and 5% CO 2 and evaluated for GFP fluorescence by flow cytometry. An optimal volume of 1 μl per well was determined (Figure 28). In the Solupore test rig system, the volume sprayed can be adjusted by changing the pressure applied to the ElveFlow or by the duration of keeping the valve open. In the first condition, the duration of valve opening was set to 280 ms and the pressure to 70 mBar. In the second condition, the valve opening time was reduced to 140 ms and the pressure was set to 140 mBar. The optimal method was to reduce the valve opening time to 140 ms.

[0200] Tonicity of the delivery solution In the previous experiment, a delivery solution that was hypotonic compared to the cells was used. Delivery solutions with their tonicity changed by further addition of KCl were evaluated. A 96-well filter plate (Pall; Supor, 1.2 μm; catalog number 8039) was seeded with 1×10 6 individuals per well with human primary T cells. The plates were centrifuged at 300×g for 5 minutes, and 4 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. In the first condition, the delivery solution contained 12.5 mM KCl, which resulted in a solution hypotonic to the cells. The second condition contained 106 mM KCl, which resulted in a solution isotonic to the cytoplasm of the cells. Other concentrations between 10 mM and 500 mM, such as 12.5 mM KCl, 328 mM, and 500 mM KCl, were also tested. At high tonicities, 328 mM and 500 mM KCl, a decreased level of GFP expression was shown. Thus, the useful range is 12.5 - 500 mM, such as 50 - 150 mM, such as 100 - 125 mM, such as 100 - 110 mM, and 106 mM is the preferred KCl concentration.

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

[0202] Multiple "hits" Due to the gentle nature of the Solupore technology, cells can be treated multiple times without a decline in cell viability or functionality. For example, we initiated an evaluation of the preferred number of "hits" (treatments) with 1-hit, 2-hit, and 3-hit strategies. A 96-well filter plate (Pall; PES, 1.2 μm) was seeded with human primary T cells at 1 × 10 6 cells per well. The plate was centrifuged at 300 × g for 5 minutes, and 1 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. Once the cells were sprayed, the stop solution (50 μl) was applied after 2 minutes of incubation, and the normal medium (100 μl) was applied 30 seconds later. For the 2-hit strategy, the cells were incubated for 2 hours before repeating the spray process, and for the 3-hit strategy, it was repeated once more after a 2-hour incubation. Before each additional hit and at the end of the 2-hour incubation, the wells containing the cell suspension were sealed with a film (e.g., Parafilm M), and the plate was placed on a stirrer such as a vortex mixer and held for 15 seconds. Then the cell suspension was mixed 3 times with a pipette. Vortexing and mixing enabled the "shuffling" of the cell orientation before the next hit. The cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. Considering the uptake, viability, and cell yield, the 3-hit strategy was considered optimal (Figure 30).

[0203] Cell seeding density We began to evaluate the optimal T cell seeding density using an Agilent PCTE plate. Human primary T cells were seeded in a 96-well filter plate (Agilent; PCTE, 0.4 μm) at 1.25, 2.5, 3.5, 5, and 7.5×10 5 cells per well. The plate was centrifuged at 350×g for 2 minutes, and 1 μl of delivery solution containing 0.57 μg / μl of mRNA was sprayed onto the cell monolayer. After the cells were sprayed, a stop solution (50 μl) was applied after a 2-minute incubation, and then normal medium (100 μl) was applied 30 seconds later. The cells were incubated for 2 hours, and then this process was repeated. At the end of this spray, the cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. A seeding density of 3.5×10 5 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 ; Figure 32). To confirm the seeding density, the density was calculated based on the average diameter of the activated T cells and the area of the addressable region 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 cells.

[0204] Masking We had previously noticed that there was a region of negative cells near the edge of the well. The reason for this edge effect is thought to be any (or a combination) of insufficient sprayer targeting, an increase in volume near the edge due to the meniscus, non-effective droplet collisions at the edge, or pressure disturbances at the edge. A strategy to overcome the edge effect is thought to be to generate a seeding mask that is present during seeding and removed after centrifugation to prevent cells from being seeded near the well edge. To test this theory, a mask that reduced the well diameter from 5.2 mm to 4 mm was placed in the PCTE plate wells. 2.5×10 5Human T cells of individual cells were seeded into the mask, and 3.5×10 5 individual cells were seeded into wells without a mask as a control. The plates were centrifuged at 350×g for 2 minutes, and the masks were removed before the cells were spray-treated. This means that the cells were seeded only up to approximately 0.5 mm from the walls of the wells. A delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cells using a one-hit strategy. At the end of this process, the cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator and evaluated for GFP fluorescence by flow cytometry. The results showed that samples from masked wells gave 67.7% uptake, while samples without a mask gave 53.1% uptake. This suggests that there is an edge effect and that it is canceled out by the presence of a mask for seeding (Figures 60A, B). Thus, optionally, by preventing cells from being seeded to the edges of the wells, an increase in transfection efficiency is brought about.

[0205] force To examine the correlation between the force exerted by the spray and the uptake, force sensor analyses were performed at different heights and pressures to establish a baseline for the results. The pressure of pushing air through the atomizer was adjusted to 0.5 - 2 bar, and the force received by the bottom of the well was measured using a force sensor. The height was also adjusted to 31, 26, and 11 mm. As a result, air pressure was shown to be the single largest factor affecting the force exerted by the spray, and a slight drop in force occurred at the low height of 11 mm. For a force of 2.0 bar at 11 mm, the same force occurred at higher heights and a normal pressure of 1.65 bar (Figure 55). This experiment was repeated for a more robust analysis of the factors affecting the force exerted by the spray. Again, air pressure was the largest factor for the force exerted by the spray (Figure 56). At low pressure, the force experienced at different heights became negligible, and the forces at 1.15 bar and heights of 11, 26, and 31 mm were indistinguishable. At 2.15 bar, the forces at heights of 26 mm and 31 mm were greater than the force at 11 mm. This can be explained by the fact that according to Force = Mass × Acceleration, at high pressure, a higher height drags more air from the atmosphere into the spray path, and thus the mass of the spray increases. Next, the same parameters as in Figure 4.11 were tested to correlate the force profile with the uptake of GFP mRNA in CD3+ T cells. 96-well filter plates (Pall; Supor, 1.2 μm; catalog number 8039) were seeded with 1×10 6 human primary T cells per well. The plates were centrifuged at 300×g for 5 minutes, and 4, 2, 1, or 0.67 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. After a 2-minute incubation, a stop solution (50 μl) was applied, and after 30 seconds, normal medium (100 μl) was applied. The cells were placed in a humidified incubator at 37°C and 5% CO 2Incubate overnight and evaluate GFP fluorescence by flow cytometry. The 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 received from the spray is not directly correlated with uptake. There may be a minimum force required to achieve uptake and a maximum force at which no decrease in viability is seen, but this range is wide (1 - 2 bar).

[0206] The range of conditions suitable for mRNA delivery to non - adherent cells, such as T cells, for example primary human T cells, is summarized below. TIFF2025084859000004.tif125128

[0207] The preferred, for example optimal, conditions for mRNA delivery to T cells are outlined in Table 4.1.

[0208] (Table 4.1) Conditions for mRNA delivery to T cells over time TIFF2025084859000005.tif157146

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

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

[0211] In most delivery methods, a balance must be struck between effective delivery and maintenance of cell viability. To examine this balance, integrated characteristics of cell loss, cell viability, and transfection efficiency were obtained for the delivery technology of the present subject matter compared to electroporation, using the transfection score ((transfected cells / total cells) × (live cells / total cells)). A score of 1.0 indicates 100% transfection efficiency and 100% cell viability, indicating that no cells are lost during this procedure. The transfection score was 0.33 (±0.05) for the technology of the present subject matter and 0.51 (±0.13) for electroporation, and there was no significant difference between the scores (Figure 33C).

[0212] To benchmark this technology, a comparison of Solupore delivery and nucleofection (4D; Lonza) delivery of mRNA to human T cells was initiated. The amount of mRNA delivered per cell (μg) was matched. For soluporation, human primary T cells were seeded at 1×10 6 cells per well in a 96-well filter plate (Pall; PES, 1.2 μm). The plate was centrifuged at 300×g for 5 minutes, and 1 μl of the delivery solution containing 0.57 μg / μl of GFP mRNA was sprayed onto the cell monolayer. Once the cells were sprayed, a stop solution (50 μl) was applied after 2 minutes of incubation, and normal medium (100 μl) was applied 30 seconds later. The cells were incubated overnight at 37 °C and 5% CO 2 in a humidified incubator 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). Next, the cells were added to a nucleocuvette strip and nucleofected according to the instructions. 100 μl of medium was added to each well and transferred to a recovery flask containing 10 ml of medium. The cells were incubated overnight at 37 °C and 5% CO 2Incubate overnight and evaluate GFP fluorescence by flow cytometry. The GFP mRNA expression levels were 40.3% and 89.3%, respectively (Figure 34). The mean median fluorescence intensities from 5 experiments and 2 donors were 203,059 and 113,895, respectively (Figures 35A, B, C, D). The dose responses of mRNA delivered by each technique are shown in Figure 36.

[0213] Endocytosis-independent Diffusion of cargo into cells and re-sealing of the plasma membrane. Since the ability of this method to deliver a wide range of cargo to a series of cell types was demonstrated, the mechanism of cargo uptake into cells and the reversal of cell permeability were investigated. The limitations of other delivery techniques are that they rely on active uptake pathways such as endocytosis, which can lead to the sequestration of cargo so that it cannot be used for its function in cells. For example, liposome-mediated delivery requires both clathrin-mediated endocytosis 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), while 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 solporation, immediate uptake of the card into cells was observed. When 10 kDa dextran-FITC was used as a model cargo, the cargo could be seen inside the cells within 30 seconds from the application of the delivery solution, before adding the stop solution (Figs. 37A, B, C, C). The rapid influx of the cargo into the cells indicates that endocytosis is unlikely to be involved in its delivery. Results showing loading of a wide range of molecular species into a series of cell types indicate that a diffusion mechanism through the cell membrane is the mechanism of entry of macromolecules into cells. For the purpose of investigating 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 caveola-mediated endocytosis and micropinocytosis, respectively. Since the expression of EGFP mRNA remained unchanged in the presence of these inhibitors, this method was shown to result in direct delivery into the cytoplasm of the cells and to be independent of endocytosis (Fig. 37C). Furthermore, in addition to following the method reported by D'Astolfo et al. (2015), Lipofectamine 2000 was included as a positive control to confirm the Dynasore-mediated inhibition of clathrin-mediated endocytosis (Fig. 37C).

[0215] This delivery method is extremely gentle on cells, and it has been found that visible cell death or cell damage, if any, is minimal. In this method, the permeabilized plasma membrane can rapidly reform reseals, thus maintaining a high level of cell viability. To examine the rate of recovery of the cell membrane after permeabilization, the delivery solution was applied to A549 cells without cargo. At subsequent time points (0 - 182.5 minutes), this delivery solution was removed, and 50 μl of PBS (100 μg / ml) containing propidium iodide (PI) was added. After a 2-minute incubation, the PI solution was removed and the cells were harvested. PI uptake was analyzed by flow cytometry. For the basal level of PI uptake, untreated cells were given 50 μl of PI in PBS. The results show that cells remain permeable to PI for several minutes but reform reseals over 6 minutes after treatment (Figure 37C). There is no further uptake after 6 minutes. Thus, not only does a load occur in cells within 2 minutes of exposure to the delivery solution, but the membrane effectively recovers its integrity within 6 minutes of the start of this procedure. These data indicate that endocytosis is not involved in the delivery of agents using sonoporation.

[0216] Gene Editing in T Cells To demonstrate further functional output of T cells after delivering cargo, gene editing of T cells was evaluated using CRISPR / Cas9 RNP delivery.

[0217] CRISPR / Cas9 RNP delivery. The PDCD1 gene encoding the PD-1 protein A 2-guide RNA strategy was used to knockdown TIFF2025084859000006.tif105150.

[0218] TIFF2025084859000007.tif30150 (PD1-1 protein amino acid sequence, SEQ ID NO:2). An equimolar amount of crisprRNA (similarly an equimolar amount of TIFF2025084859000008.tif4128 crisprRNA and TIFF2025084859000009.tif was incubated with the mixture with 4128 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))) and tracrRNA at room temperature for 10 minutes. Next, 5 μg of Cas9 (IDT) was added so that the final molar ratio of Cas9 to guide RNA was 1:3, and the mixture was incubated at room temperature for an additional 10 minutes. Cas9 RNP (in buffer, containing α-crystallin (220 μM) and ethanol (25% v / v)) was delivered to T cells using the described vector-free intracellular delivery method. Using the vector-free delivery method described herein, 1.5×10 6 cells per treatment were delivered with RNP, and PD-1 expression was analyzed 72 hours after transfection.

[0219] Cell viability. Preparation and analysis of FACS samples: The cell viability after the vector-free intracellular delivery method was evaluated using a 7-AAD viability staining solution (Sigma). Cells were washed in PBS + 1% fetal bovine serum (FACS buffer) and then incubated with 7-AAD (1:40, in the dark, at room temperature for 5 - 10 minutes), 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 whole cells using forward scatter parameters and side scatter parameters. Single cells were selected by excluding doublets in a plot of FSC height versus FSC area. GFP expression was analyzed in gated live cells.

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

[0221] Delivery of the CRISPR / Cas9 gene editing tool into activated T cells resulted in a 28% reduction in PD-1 expression with the vector-free intracellular delivery described herein, while it was 53% inhibition in electroporated cells (Figures 38A, B).

[0222] The proliferative capacity of the gene-edited cells was evaluated over 4 days by observing the formation of T cell aggregates in culture. Cells were returned to culture after RNP delivery. Cells were observed using a microscope 4 days later. Since cell aggregation in cells transfected by the vector-free intracellular method was similar to that of untreated control activated cells, it was shown that they were not affected by the described vector-free intracellular delivery method. In contrast, the growth rate of electroporated cells was significantly affected, as indicated by significantly less cell aggregation (Figure 39).

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

[0224] Materials and Methods Cell culture. Human peripheral blood mononuclear cells (PBMCs) were collected 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 the stored aliquots, CD3 + T cells were cultured in human recombinant interleukin-2 (IL-2) in a humidified tissue culture incubator at 37 °C and 5% CO 2 and primary stimulation and costimulatory antibody activation were performed using various protocols (see below).

[0225] Construction of CD19 CAR plasmid The CD19 CAR plasmid was obtained commercially (Creative Biolabs, New York, USA), and mRNA was generated from the plasmid. The full length of the chimeric antigen receptor (CAR) was synthesized and subcloned into a lentiviral vector. The insert was confirmed by Sanger sequencing. The structure of the CAR vector is illustrated in Fig. 86. The amino acid sequence of the scFv (anti-CD19 scFv VL-linker-VH) is shown in Fig. 87A. The nucleotide sequence of the CAR cassette (codons optimized) is shown in Fig. 87B. The amino acid sequence of the CAR cassette is shown in Fig. 87C. The restriction digestion map is shown in Fig. 88. The quality control results of the vector design are shown in Fig. 89. The CAR sequence alignment validation is shown in Fig. 90. The sequence alignment results showed that the sequence of the 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, and ethanol (27% v / v) was also added, and delivered to 1.5×10 6 T cells using the technique described herein. Cells were harvested 24 hours later and analyzed for mRNA expression using flow cytometry.

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

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

[0229] Evaluation of the functionality of T cells after sonoporation To demonstrate that the functionality of T cells transfected using the Solupore technology is equivalent to or greater than that of cells transfected using, for example, electroporation with a Neon electroporator and nucleofection using, for example, 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) Analysis of T cell membrane protein expression The aim of this study was to determine whether the expression of cell surface proteins in T cells is affected by the sonoporation process and to compare it with electroporation and nucleofection.

[0231] Methods: Activated T cells were seeded at 1.5×10 6 cells per well in 96-well filter plates (Acroprep, 1.2 μm Supor membrane; Pall, USA). The medium was removed from the wells by centrifugation at 300×g for 5 minutes. Next, 7 μl of delivery solution containing 4 μg of GFP mRNA (32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 27% ethanol (all from Sigma-Aldrich) in molecular grade water) was sprayed into each well using a vector-free delivery spray device. After delivery, the cells were incubated in this solution for 2 minutes, and then 50 μl of stop solution (0.5×PBS) was added. After 30 seconds, T cell medium was added (100 μl), and the cells were allowed to recover overnight at 37 °C and 5% CO 2 . For electroporation and nucleofection, 5×10 6 cells and 2.5×10 6 cells were used per transfection, respectively. GFP expression and viability were evaluated 6 hours and 24 hours after delivery.

[0232] The expression of cell surface CD4 and CD8 was examined 6 hours and 24 hours after any one of nucleofection, electroporation, or sonoporation. Six hours after transfection, the percentage of T cells expressing CD4 and CD8 did not change for each transfection method compared to untreated control cells. However, at 24 hours, expression was significantly reduced in electroporated cells, while expression in sonoporated cells and nucleofected cells was similar to that of untreated control cells (Figures 47A, B).

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

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

[0235] The highest level of gene expression changes occurred with Neon electroporation. However, the drawback of electroporation is the reduction in viability and functionality of the treated cells. Of the 20,893 mRNAs analyzed, with Neon electroporation, a total of 317 changed at all time points (6 hours and 24 hours) and in both donors, 32 changed with Solupore (Tables 5.1 and 5.2), and 24 changed with the nucleofector (Tables 5.3 and 5.4; Figures 48A, B, C). Notably, the false positive background level for this microarray analysis was approximately 10%. In the case of Solupore and nucleofection, the number of genes that changed was only slightly higher than this threshold, demonstrating that the level of perturbation of cell gene expression caused by Solupore and the nucleofector is minimal. The high level of gene expression changes indicates that the Neon electroporation process causes more perturbation to the cells than solporation or nucleofection. Since cell perturbation is undesirable, this data indicates that Neon electroporation is not as desirable a transfection method as solporation and nucleofection. Soluporation has several major advantages compared to electroporation and nucleofection. These advantages include the high-level and reliable delivery of cargo such as mRNA to primary human cells while maintaining the integrity, function, and proliferative capacity of the treated cells.

[0236] (Table 5.1) List of gene expression changes in sonoporated cells compared to untreated control cells 6 hours after transfection (Numbers indicate fold change compared to untreated control cells) TIFF2025084859000010.tif112150

[0237] This data indicates that in solporated cells, only a small number (e.g., negligible) of gene expression changes occur 6 hours after transfection.

[0238] (Table 5.2) List of gene expression changes in sonoporated cells compared to untreated control cells 24 hours after transfection The numbers indicate the fold change compared to the untreated control cells. TIFF2025084859000011.tif155150

[0239] This data indicates that in the cells transfected by soloporation, only a small number (e.g., negligible) of gene expression changes occur 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 The numbers indicate the fold change compared to the untreated control cells. TIFF2025084859000012.tif61148

[0241] This data indicates that in the cells transfected by nucleofection, only a small number (e.g., negligible) of gene expression changes occur 6 hours after transfection.

[0242] (Table 5.4) List of gene expression changes in nucleofected cells compared to untreated control cells 24 hours after transfection The numbers indicate the fold change compared to the untreated control cells. TIFF2025084859000013.tif96151

[0243] This data indicates that in the cells transfected by nucleofection, only a small number (e.g., negligible) of gene expression changes occur 24 hours after transfection.

[0244] (iii) Cell proliferation analysis For therapeutic applications, T cells need to be able to proliferate after modification. Therefore, the proliferation ability of T cells after soloporation, electroporation, and nucleofection was examined. The proliferation ability of cells after cryopreservation and thawing was also tested.

[0245] Method: A 96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA) was seeded with 1.5×10 6 cells per well of activated T cells. Next, 1 μl of delivery solution containing 0.2 μg of GFP mRNA was sprayed into each well. A second spray was performed 2 hours later. For electroporation and nucleofection, 5×10 6 cells and 2.5×10 6 cells were used per transfection. The cells were transferred to an incubator at 37 °C and 5% CO 2 . The next day, the cells were harvested and counted. Next, the cells were re-seeded at 0.5×10 6 cells / ml by adding medium + IL-2 daily for 7 days. In another experiment, 24 hours after transfection, the cells were cryopreserved in 10% DMSO and fetal bovine serum. The cells were thawed and seeded on day 0 at 0.5×10 6 cells / ml in Immunocult medium + IL-2. The cells were counted and re-seeded daily for 5 days by adding medium.

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

[0247] (iv) Interferon-γ (IFNg) secretion analysis For therapeutic application, T cells need to be able to produce IFNγ after modification. Therefore, the IFNγ production ability of T cells after sonoporation, electroporation, and nucleofection was examined. Two different activation methods, phorbol myristate acetate / ionomycin (PMA / I) and Dynabeads, were examined. The IFN-γ secretion ability of the cells was also tested after cryopreservation and thawing following sonoporation.

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

[0249] In T cells after soluporation, nucleofection, or electroporation, IFNγ production was not reduced compared to control cells (Figure 51). Freshly thawed soluporated cells had not lost their ability to secrete IFN-γ 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 an NSG mouse model was studied. NOD scidγ mice (NSG mice) are an immunodeficient experimental mouse strain approved by The Jackson Laboratory in the art.

[0251] If transfected cells are adversely affected by the Solupore delivery technology, their ability to engraft and induce graft-versus-host disease (GvHD) will be impaired. A comparison with nucleofection was also performed. It was not realistic to include Neon electroporation as a comparator because this process results in a large loss of cells, making the number of cells that need to be electroporated infeasibly high.

[0252] Method: 3 kDa dextran-Alexa488 at 20×10 6 cells and 5×10 6Individual PBMCs were delivered by electroporation and nucleofection, respectively. Electroporation was performed using a 44.45 mm stirred cell system. A monolayer of cells was formed on a 1 μm PCTE hydrophilic membrane (Sterlitech) by applying a pressure of 100 mbar for 15 - 25 seconds until all the medium was removed. 5 ml of delivery solution containing 3 μM dextran 3000 was prepared and filled into an LP100 atomizer. The cells were electroporated and after 1 minute and 30 seconds, the membrane was gently transferred to a 60 mm cell culture Petri dish. After 2 minutes, 1 ml of stop solution was added to the membrane, incubated for 30 seconds, and then 4 ml of medium was added to the cells. The Petri dish was transferred to an incubator at 37°C with 5% CO 2 for 30 minutes. For nucleofection, 5×10 6 cells were used per transfection. The cells were harvested, washed twice with 1×PBS, resuspended according to the mouse body weight, i.e., 1×10 6 per gram, and intravenously injected via the tail vein based on the body weight per mouse (1×10 6 per gram). The 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. Also, blood and spleen were collected at the end of the study and prepared for analysis.

[0253] PBMCs were isolated from three different donors (D119, D120, D121), and the cells from each donor were subjected to either electroporation or nucleofection. For this study, there were four groups of NOD scidγ (NSG) mice at five mice per group. Each group was: 1. no cells, 2. UT, 3. electroporation, 4. nucleofection (4 groups × 5 mice per group × 3 donors = 60 mice). The cells were injected into the mice on day 0. When the animals were monitored daily, GvHD symptoms were present in all animals that received cells by day 14 post-injection, indicating that the cells were viable and functional. Blood samples were collected from the mice on day 14 and analyzed by flow cytometry for the presence of human PBMCs and T cell subsets as indicated by CD45 expression. The results confirmed that the cells were viable and functional as CD45+ cells were present in the untreated and electroporated groups at this time point (Figures 53A, B, C). At this time point, fewer CD45+ cells were detected in the nucleofected cells. These data indicate that electroporated human PBMCs retain viability and functionality in vivo and have the ability to induce GvHD in NSG mice. These results demonstrate the successful engraftment of electroporated cells into NSG mice and show that their viability and functionality are maintained after electroporation.

[0254] Filter membrane conditions for delivering a payload composition to cells To facilitate and enhance the exposure of cells (non - adherent or adherent cells) to the treatment solution, the filter membrane is optionally vibrated before, after, during delivery, or in any permutation thereof. The membrane can be vibrated before, after, during delivery, or in any permutation thereof to assist in the formation of a monolayer of cells on the filter membrane. The vibration of the filter membrane can be carried out using readily available devices, such as piezoelectric accelerometers like the Miniature Triaxial DeltaTron® accelerometer (available from Bruel and Kjaer, www.bksv.com). Several suitable vibration motors can also be obtained 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 connection 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. Additionally, if the mechanical resonance points due to the physical characteristics of the membrane are identified, the degree of control that can be exerted on the membrane will be improved. To feedback the displacement caused by the membrane, a 3-axis accelerometer device will be mechanically coupled to the filter membrane or the holder. By using the accelerometer system for monitoring or as a control feedback signal to the vibration system, an error signal between the desired vibration pattern and the achieved vibration pattern can be generated. The selection of the driving vibration frequency is made based on the stiffness of the membrane and the size of the cells on the membrane. An exemplary vibration pattern is provided by a 3000 Hz sine wave signal on the x-axis and y-axis and no signal on the z-axis. The displacement is 1 mm between peaks, there is no phase difference between the x-driving waveform and the y-driving waveform, and both are coherent. Additionally, many patterns are possible, such as those that provide rotation and oscillation to the x, y, and / or z axes.

[0257] Device for delivering cargo molecules to mammalian cells This subject generally relates to the delivery of biological payloads to cells. The delivery of biological payloads to cells can involve the atomization and delivery of a permeabilization treatment solution to a monolayer of cells. Current techniques may be insufficient in some respects. These will be detailed below.

[0258] Prior to being given the payload, the cells can be in a state of being submerged or suspended in a culture medium. To achieve effective payload uptake, it can be beneficial to remove the culture medium so that the cells can be exposed to the permeabilization treatment solution and to organize the cells into a monolayer form. In some of the current techniques, the medium is removed by centrifugation. This technique is effective for removing the medium but typically does not result in the formation of a uniform monolayer of cells.

[0259] The cells can be aligned under a solution atomizer or solution nebulizer so as to ensure proper distribution of the permeation treatment solution to the monolayer of cells. In current techniques, generally a manual system is used to align the cells under the atomizer, which is prone to operator variability. The atomizer / nebulizer can be used to dispense the permeation treatment solution onto the monolayer of cells. However, while a particular atomizer may be designed to dispense milliliter amounts of solution, the dispensed volume is preferably on the order of microliters, and may not even be necessary. In addition, transfection protocols for payload delivery may involve some steps where time is critical. Currently, fluid handling is generally manually controlled and thus inherently unstable.

[0260] Due to these drawbacks, the data can be inherently inconsistent. The lack of reproducibility of the data can impede further development of the payload delivery process. To address the above problems, some aspects of the present subject matter provide a delivery system that can enhance the consistency and delivery efficiency of the delivery process while maintaining the integrity of the cells.

[0261] Exemplary delivery system FIG. 65 shows an example 8800 of a delivery system configured to deliver a payload to cells. The delivery system 8800 can include a housing 8802 configured to receive a plate 8804 that includes wells. The delivery system 8800 can include a pressure difference application device 8806 configured to apply a pressure difference to the wells, a delivery solution application device 8808 configured to deliver an atomized delivery solution to the wells, a stop solution application device 8810 configured to deliver a stop solution to the wells, and a culture medium application device 8812 configured to deliver a culture medium to the wells.

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

[0263] Delivery system 8800 may also include a control system 8814, an operating system 8816, a support frame 8818, and a sensing and management system 8820. In some embodiments, a pressure differential application device 8806, a delivery solution application device 8808, a stop solution application device 8810, and / or a culture medium application device 8812 can be connected to the support frame 8818. The operating system 8816 can be connected 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 operating system 8816 can be connected to the pressure differential application device 8806, the delivery solution application device 8808, the stop solution application device 8810, and / or the culture medium application device 8812 and configured to move them. For example, the support frame 8818 can be or include a fluid head module 9308 described below with respect to FIGS. 84-89. The operating system can be or include an actuator 9319 described below with respect to FIGS. 84 and 85. In some embodiments, a vibration system can 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 a thermal management system. As an example, the sensors and / or the thermal management system can be connected to the pressure differential application device 8806, the delivery solution application device 8808, the stop solution application device 8810, and / or the culture medium application device 8812 and 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 connected to an operating system 8816 as well as a sensing and management system 8820. The control system can be configured to control the operating system 8816 as well as the sensing and management system 8820. As an example, the control system 8814 can be or can include the control system 9306 described later with respect to FIG. 84.

[0266] Exemplary vacuum pressure system FIG. 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, temperature control of the base plate.

[0267] This delivery system can address the following parts that cause variability in relation to payload delivery to cells: monolayer formation, atomization, automation of payload delivery, and temperature control of the solution and culture vessel.

[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 the medium using vacuum and generation of a monolayer of cells, atomization of the permeation treatment solution to generate monodisperse droplets, fluid control of the solution to enable automation, temperature control of the solution, mounting of the spray head and temperature reservoir, automation and software design, enclosure of the device, and temperature control of the base plate.

[0269] Figures 67 to 69 illustrate an exemplary embodiment 9000 of a high-precision rig system. 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, valves 9011 (shown in Figures 68 to 69), and a manifold 9024. In some embodiments, the vacuum manifold assembly 9008 can be connected to the translation stage 9010 via a connecting 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 (shown in Figures 68 to 69). As shown in Figures 68 to 69, the filter plate can be a 96-well filter plate. The filter plate 9014 can seat within a recess of the base plate 9016, and the top plate can be positioned over the filter plate 9014 and connected to the base plate 9016 to fix the filter plate 9014 in the correct position.

[0270] The needle emitter 9002 can function to deliver a culture medium capable of containing 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 generally be in the form of a cylindrical tube and can be capable of applying a vacuum pressure to the corresponding wells of the filter plate 9014. The vacuum pressure can be sent to each valve 9011 via a port 9024a of the manifold 9024 and can be sent to the vacuum fitting 9020. The atomizer 9004 can atomize a permeation treatment solution and deliver it to the cells within the wells of the filter plate 9014. Systems, devices, and methods related to the delivery of a permeation treatment solution to a monolayer of cells are discussed in further detail below.

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

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

[0273] Referring to FIGS. 70-73, the base plate 9016 can include a first set and a second set of connecting holes 9016b, 9016c. The first set of connecting holes 9016b can be aligned with the connecting holes 9018b in the top plate 9018 such that the base plate 9016 can be connected to the top plate 9018 by a connecting member, such as a bolt or a screw, that can extend through the connecting holes 9016b, 9018b. The connecting member 9012 can be connected to the base plate 9016 via a connecting element that can extend into the second set of connections 9016c.

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

[0275] The filter plate 9014 can have wells with active openings 9014b in addition to having wells with inactive openings 9014c. The active openings 9014b can be located over and in fluid communication with the openings 9030b in the base plate 9016. In other words, certain wells of the filter plate 9014 can be active and other wells of the filter plate 9014 can be inactive.

[0276] The gasket 9026 can have holes 9026b that can align with the active 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 fluid communication between the active openings 9014b of the wells and the corresponding vacuum fittings 9020 while isolating each active opening 9014b from other active openings 9014b and inactive openings 9014c.

[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 over the filter plate 9014 and connected to the base plate 9016 as described above.

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

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

[0280] As described above, the vacuum pressure can be sent to each valve 9011 via the manifold 9024 and can be sent to the vacuum joint 9020. FIGS. 79-81 show various views of a portion of the high-precision rig system 9000. As shown in FIGS. 79-81, the manifold can include eight ports 9024a, and the tube 9034 can extend from six ports 9024a on the manifold to six valves 9011. The tube can also connect the ports on each valve 9011 to the vacuum joint 9020 connected to the base plate 9016. A vacuum line (not shown) can be connected to one of the remaining two open ports 9024a on the manifold 9024 to seal the remaining port 9024a.

[0281] The vacuum manifold system 9006 can be used to enable the formation of a monolayer of floating cells. As described above, the needle emitter 9002 can deliver a culture medium containing cells into the wells of the filter plate 9014. The vacuum pressure can be applied to the 8-channel manifold via a vacuum line. The valves 9011 can be opened and closed individually. Therefore, access to each active well can be controlled individually. When the valve 9011 is opened, the applied vacuum pressure can be sufficient for the effective removal of the culture medium in which the cells are suspended without causing shear stress that could lead to damage. The extracted medium exits 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 the cell viability can be maintained. The formation of the cell monolayer can be achieved without damaging the cells.

[0282] The vacuum manifold system 9006 was assembled and tested. During the test, a culture medium containing cells was added to the wells of a 96-well filter plate 9014. A vacuum pressure was applied via the manifold 9024. A vacuum pressure in the range of 10 bar to 100 mbar was applied to individual wells by opening the 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 so that it did not return to the wells by "wicking" when the filter plate 9014 was removed.

[0284] At pressures lower than -50 to -10 mBar, the medium remained close enough to the wells such that when the filter plate 9014 was removed from the vacuum manifold assembly 9008, some of the medium would return to the wells by "wicking". The amount of liquid returning to the wells by wicking was approximately 5 - 10 μl. This is not a problem when the vacuum manifold assembly 9008 is part of the high-precision rig system 9000, as the wells are sprayed and the culture medium is replaced before the filter plate 9014 is removed from the manifold.

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

[0286] At a low pressure of about -10 mBar, the culture medium was removed at a gentler, more controlled rate, which was more gentle on the cells. This occurred both in the presence and absence of cells and could lead to an improvement in cell viability and recovery from the wells.

[0287] It was considered that pulsing the valve 9011 on - off during testing did not promote the removal of the culture medium.

[0288] Centrifugation was also investigated as a method for forming a cell monolayer. However, this was found to be ineffective as it formed a non - uniform cell layer. Figure 82 shows the distribution of Glosarium particles observed after centrifugation and after vacuum extraction. Glosarium particles are included. The top three images represent the distribution of Glosarium particles obtained when the culture medium was removed using vacuum pressure, and the bottom three images represent the distribution of Glosarium particles obtained when the culture medium was removed using centrifugation. The centrifugation samples were centrifuged at 350×g for 5 minutes, and the vacuum pressure samples were depressurized at -800 mbar for 15 seconds.

[0289] As shown in FIG. 82, the vacuum pressure sample showed a uniform distribution of Glosamer particles, while after centrifugation, a non-uniform crescent-shaped distribution of Glosamer particles was observed.

[0290] Even at -10 mBar, equivalent results were achieved for mRNA uptake using the vacuum method and the centrifugation method. It was found that the GFP MFI was higher for the samples prepared by the vacuum method compared to the centrifugation method (about 300,000 units and 50,000 units). This indicates 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 seconds 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. By performing more work, the length of time required to remove the medium at a lower vacuum pressure can be investigated.

[0292] In some embodiments, it is contemplated that the residual medium can be drained simultaneously from all wells at a low pressure to remove it from the discharge port (no plate on the manifold).

[0293] The vacuum manifold system 9006 enables the application of vacuum pressure to the individual wells of the filter plate 9014. By applying vacuum pressure to the individual wells on the filter plate 9014, more precise control of the vacuum pressure and higher consistency of the vacuum pressure applied to each well can be achieved. Existing vacuum manifolds apply vacuum to the entire 96-well filter plate. During the tests, a low vacuum pressure was effective for the removal of the culture medium and the formation of a uniform monolayer of cells within the wells.

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

[0295] The positive pressure delivery system can address the following sources of variability related to 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 removal of media using positive pressure and generation of a monolayer of cells, atomization of the permeation treatment solution to produce monodisperse droplets, fluid control of solutions to enable automation, temperature control of solutions, attachment of emitters, atomizers, nebulizers, and temperature reservoirs, automation and software design, enclosure of the instrument, and temperature control of a base plate configured to hold a well plate.

[0297] Examples of a positive pressure system 9300 are shown in FIGS. 84 - 85. This positive pressure system 9300 uses positive pressure as an alternative to vacuum for removing culture media. The positive pressure enhances the accuracy and precision of delivery of a small volume (1 μl - 100 μl) of fluid for extruding the culture media from 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, which 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 a culture medium and a stop solution that can contain cells to the wells of a filter plate 9316. The nebulizer 9304 can be configured to atomize a permeabilization treatment solution and deliver it to the cells within the wells of the filter plate 9316. The positive pressure nozzle assembly 9310 can be configured to apply positive pressure to the wells of a well filter plate 9316 to remove the liquid portion of the culture medium from the wells, thereby creating a monolayer of cells. Systems, devices, and methods related to the delivery of a permeabilization treatment solution to a monolayer of cells are discussed in further detail below.

[0300] In some embodiments, the positive pressure system 9300 can include a guide rail 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 rail 9318, thereby enabling the mounting array 9302 to be translated along an axis X2 defined by the guide rail 9318. For example, an actuator 9319 can move the mounting array 9302 along the guide rail 9318. The guide rail 9318 can also be moved along an axis Y2 that can be perpendicular to the axis X2. The actuator 9319 can also move the mounting array 9302 along the axis Y2. Therefore, the mounting array 9302, which includes the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 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, the user can insert the filter plate 9316 into the base 9312 and then position it within the housing 9314. The filter plate 9316 can be kept stationary after being received within the housing 9314. The filter plate 9316 can be selectively addressed by a needle emitter 9303, a nebulizer 9304, and / or a positive pressure nozzle assembly 9310 that are coupled to the fluid head module 9308 of the mounting array 9302. In the illustrated example, the mounting array 9302 includes six fluid head modules 9308. Two of the fluid head modules 9308 include needle emitters 9303, three of the fluid head modules 9308 include nebulizers 9304, and one of the fluid head modules 9308 includes a positive pressure nozzle assembly 9310. The mounting array 9302 can be moved along axes X2, Y2 such that each of the fluid head modules 9308 including a needle emitter 9303, a nebulizer 9304, and / or a positive pressure nozzle assembly 9310 can address 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 vertically parallel to the axis Z2, thereby enabling independent operation of the fluid head modules 9308. In some embodiments, the movement of the fluid head modules 9308 as well as the operation of the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310 are controlled independently. One or more movements of the fluid head module 9308, as well as the operation of the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310 attached thereto, can occur simultaneously. In some embodiments, the fluid head modules 9308 activate the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310 as they move towards the filter plate 9316. The fluid head modules 9308 can accommodate various fluid dispensing assemblies, including, but not limited to, needle assemblies, Ari Mist nebulizers, and positive pressure nozzle assemblies, as shown in FIGS. 84-85.

[0303] The actuation and control system (ACS) 9306 can include at least one data processor and can control the movement of the mounting array 9302 and / or the fluid head modules 9308. The ACS 9306 can also control the operation 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 the data processor of the ACS 9306. The data processor can receive instructions, process the instructions, and execute the instructions. For example, the data processor can deliver control signals to the actuators and / or motors of the ACS 9306 to adjust the position of the mounting array 9302 and / or to activate 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 serves as a mounting point for various fluid dispensing assemblies (such as the needle emitter 9303, the nebulizer 9304, and / or the positive pressure nozzle assembly 9310) so that they can be connected to the mounting array 9302. The fluid head module 9308 also provides actuation along the axis Z2 so that the dispensing assemblies can be selectively actuated.

[0305] An enlarged view of the fluid head module 9308 is shown in FIG. 86. 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 enable high tolerance locating of the fluid head module 9308.

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

[0307] Removal of medium using positive pressure and generation of cell monolayer As described above, the positive pressure nozzle can be configured to apply positive pressure to the wells of a well filter plate to remove the culture medium and create a monolayer of cells. An enlarged view of the 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 can include an internal lumen formed from a tube 9320 positioned within a housing 9322. The tube 9320 can have openings 9321, 9323 adjacent to a first end and a second end 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 connecting member 9324 connected to the first end 9326 of the tube 9320. The connecting member 9324 can be configured to form a seal with an opening 9321 adjacent to 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 the internal lumen 9325 of the connecting member 9324. The internal lumen 9325 of the connecting member 9324 can be in fluid communication with the lumen of the tube 9320 via the opening 9321, and the lumen of the tube 9320 can be in fluid communication with the well 9316a via the opening 9323. The compressed air can increase the pressure within the tube 9320, thereby increasing the pressure within the well 9316a. Thus, the positive pressure nozzle assembly 9310 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 outside the tube 9320 adjacent to the opening 9323. The seal-forming element 9328 encompasses the well 9316a and is configured to form a seal with the upper surface 9330 of the filter plate 9316, thereby isolating the well 9316a. The seal-forming element 9328 is configured to prevent air leakage between the second end 9327 of the nozzle assembly 9310 and the upper surface 9330 of the filter plate 9316, thereby improving the accuracy and precision of the pressure control within the well 9316a. For example, when the nozzle assembly 9310 is engaged with the filter plate 9316, upon application of compressed air, liquid will 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 for controlling 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 that includes a valve 9432. In the illustrated example, the nozzle assembly 9410 includes a first tube portion 9420a and a second tube portion 9420b connected to the valve 9432. The second tube portion 9420b can include a seal-forming element located adjacent to the distal opening 9423 of the second tube portion 9420b. This nozzle can generally function in the same manner as 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 upper surface of the filter plate such that the seal-forming element 9428 encompasses the opening of the well and forms a seal with the upper surface of the filter plate. Compressed air can be delivered to the internal lumen of the first tube portion 9420a. When the valve is in the open position, air can pass through the valve 9432 and reach the wells of the filter plate via the second tube portion 9420b. Alternatively, the valve 9432 can be adjusted to the off position to prevent airflow to the wells. The valve 9432 can be adjusted to control the air pressure within the wells.

[0311] Atomization of the delivery solution to generate monodisperse droplets FIG. 93 shows an enlarged view of a nebulizer assembly 9301 that can be connected 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 connected to the microvalve 9368 via a connecting member 9370 (e.g., a pre-column connector). The microvalve 9368 can be held and / or connected within a valve holder 9372 that can be connected to the syringe 9366 via an adapter 9374. The nebulizer assembly 9301 enables the delivery of a payload solution to the nebulizer 9304 with high accuracy and precision.

[0312] There are several different ways to deliver a permeabilization treatment solution to a monolayer of cells. For example, the permeabilization treatment solution can be atomized using ultrasound or nebulized using a nebulizer.

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

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

[0315] High-speed camera recordings were used to evaluate the effect on spray characteristics. The force experienced by the cells was measured by force sensor analysis. The volume delivered into the wells was evaluated using a colorimetric assay.

[0316] The sonication tests were performed at 60 kHz, 130 kHz, and 180 kHz. The liquid can be extruded into the ultrasonic nozzle by a pumping system and atomized into a fine mist spray using high-frequency sound wave vibrations.

[0317] A piezoelectric transducer was used to convert an electrical input into mechanical energy in the form of vibrations that create surface tension waves in the liquid and result in atomization of the liquid when introduced into the nozzle. Each ultrasonic probe operated at a given resonance frequency. The operating frequency can determine the size of the generated droplets. The size of the droplets can also be affected by the power at which the ultrasonic probe is operated, although not as much as the operating frequency. An auxiliary air flow can be used to assist in spray control and shaping.

[0318] The atomization test was performed using an Ari Mist nebulizer 9304 (Figure 125). As shown in Figure 125, the nebulizer 9304 includes a connector 9307 for liquid and a connector 9309 for air. The Ari Mist nebulizer operates with compressed gas and requires a pump to supply the sample solution. This atomizer has two channels. One is for gas (air), and the other is for the liquid to be atomized, and both flow along parallel paths. Both paths end at the tip of the nebulizer with an orifice for gas and an outlet for 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 atomization.

[0319] An ultrasonic emitter operating at 180 kHz was found to be more effective in the delivery of payload to T cells compared to 130 kHz, 60 kHz ultrasonic heads, and the Ari Mist nebulizer. Figure 94 shows a series of results characterizing the efficiency of payload delivery for an ultrasonic 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 the consistency between multiple repetitions was at a high level (±1%). The integrity of the cells was maintained after delivery (survival rate 85%).

[0320] These results indicate that the ultrasonic spray emitter produces a monodisperse spray that results in an even deposition of the delivery solution and payload onto the cells. Furthermore, reducing the volume to be delivered and lowering the ethanol concentration improved the delivery efficiency by the ultrasonic spray head.

[0321] Figure 95 shows another set of results characterizing GFP uptake for ultrasonic emitters, Ari Mist nebulizers, and MAD nasal spray emitters. A micronization device for intranasal mucosa, MAD Nasal (trademark) (Teleflex, Morrisville, North Carolina 27560, Carrington Mill Boulevard 3015), was used to micronize a 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 a pneumatic source via a luer lock connector. A pneumatic pressure of 1.5 bar was supplied to the spray head over 330 ms to generate a spray. According to current data, an ultrasonic spray emitter operating at 180 kHz is more efficient for delivering mRNA to T cells. GFP uptake of 32.7% was obtained with the ultrasonic spray emitter, compared with 24.8% with the Ari Mist nebulizer (e.g., nebulizer 9304). Both the ultrasonic 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 set of results characterizing cell viability for ultrasonic emitters, Ari Mist nebulizers, and MAD nasal spray emitters. These results represent at least three technical replicate experiments for each spray head tested (e.g., ultrasonic emitter, Ari Mist nebulizer, and MAD nasal spray emitter). The Ari Mist nebulizer improved cell viability, with 85.3% compared to untreated cells, compared to the ultrasonic head, which had an average cell viability of 72.3% compared to untreated cells. The MAD nasal spray emitter resulted in the highest cell viability, with a cell viability of 99.7% compared to untreated cells.

[0323] Enclosure atomization During delivery of a solution using, for example, an atomizer, a nebulizer, and / or an ultrasonic emitter, fine aerosols are generated. In some examples, the fine aerosols can contaminate adjacent wells of a multi-well filter plate. In some aspects, an enclosure can be provided at a distal end portion of the atomizer, the nebulizer, and / or the ultrasonic emitter, thereby preventing contamination of adjacent wells of the filter plate.

[0324] Three lig platforms: Lig 1 (R1), Lig 3 (R3), and Lig 4 (R4) were assembled (Figs. 126 - 128). Some of the characteristics of the ligs are shown in the table below.

[0325] As illustrated in Fig. 126, R1 11700 includes a solution reservoir 9810 (e.g., an Elveflow sample reservoir) configured to provide a permeate treatment solution to an Ari Mist nebulizer 9804 and a pinch valve 9808 (e.g., an Elveflow pinch valve) configured to control delivery of the permeate treatment solution to the nebulizer 9804. The nebulizer 9304 can be mounted to a spray head mounting portion 11714 configured to hold the nebulizer 9304 and facilitate alignment of the spray head. The pinch valve 9808 is configured to enable fluid control of the payload solution to the nebulizer 9304. The spray head mounting portion 11714 can be mounted to a guide 11717 configured to facilitate vertical movement of the spray head mounting portion 11714 along a vertical axis Z3. The spray head mounting portion 11714 including the nebulizer 9804 can be mounted on top of a plate holder 11718 configured to receive a filter plate 11716.

[0326] As illustrated in FIG. 127, R3 11800 includes a nebulizer assembly 9301 having a sample reservoir syringe 9366 connected to an Ari Mist nebulizer 9304 via a microvalve 9368 configured to control the delivery of the permeation treatment 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 vertical movement of the fluid head module 9308 along a vertical axis.

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

[0328] (Table) Characteristics of the rig TIFF2025084859000014.tif60151

[0329] The characteristics of the rig that enable fine control of the spray parameters are described in detail below.

[0330] In some embodiments (e.g., R1 and R4), fluid control of the delivery solution containing the payload can be achieved using an Elveflow pinch valve. The fluid control can be achieved by a fluid control system that can apply a constant pressure to an Elveflow fluid reservoir to push the 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 tube used. The valve 9808 can be actuated by a metal-oxide semiconductor field-effect transistor (MOS FET) that can be controlled by a microprocessor.

[0331] The above Elveflow-pinch valve 9808 had several limitations. For example, recalibration of R1 and R4 was required each time the system was refilled. Accuracy and precision were insufficient for dispensing volumes less than 5 μl. For low volumes (<5 μl), the relative standard deviation in repeated dispensing (10) was approximately 9%. To address the observed limitations, R3 was developed. As described above, R3 includes a microvalve 9368 instead of the pinch valve 9808. Fluid control of the delivery solution containing the payload can be achieved using the microvalve 9368. When delivering volumes in the range of 1 μl to 100 μl, the accuracy and precision were higher for R3 using the microvalve 9368.

[0332] The flow 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. The system was designed using a microprocessor-based development board to enable the easy development of a time control sequence to allow for electronically controlled spray operation. This development board used the microprocessor PIC16F1619. The spray operation time and the fluid delivery time can be manipulated by the interface software of the development board. This microprocessor development board enables the pulsing of the nebulizer / atomizer spray. Next, the system was upgraded by including repeatable high-speed PLC (programmable logic controller) technology to better align with industry standards and serve as a proof-of-experiment for automated delivery technology (which is based on ultra-high-speed PLC technology). This rig controller consisted of a PLC and a Gyger controller, as well as a program to facilitate communication between these two pieces of hardware. The hardware included interaction with the operator via momentary push buttons.

[0333] The parallel path design of the Ari Mist nebulizer 9304 inherently produces a spray that is eccentric from the tip of the nebulizer 9304. The alignment of the spray head can be adjusted using a custom spray head holder fitted with a goniometer.

[0334] Several methods for delivering the solution using an enclosed emitter were tested. The results of GFP uptake using an enclosed emitter were compared to the results of GFP uptake using an unenclosed emitter.

[0335] Method 1: Use of color to surround the emitter Figure 97 shows an example 9501 of a nebulizer assembly including an enclosing color 9502 located around the spray head 9504 of an Ari Mist nebulizer (for example, nebulizer 9304). In the illustrated example, the spray head 9504 is located 27 mm above the well 9516a of the double-height filter plate 9516. The color 9502 is designed and manufactured for the Ari mist spray head 9504. This color was equipped on the spray head 9504. A 96-well filter plate 9516 with double-height walls was used.

[0336] Method 1A: The color forms a seal with the well plate At a distance of 26 mm from the tip of the spray head 9504 to the bottom surface of the well 9516a, the color 9502 engaged with the upper end of the well 9516a of the filter plate 9516 to form a seal. 1.5×10 6 CD3+ T cells were seeded on the filter plate 9516 and centrifuged at 350×g for 5 minutes to remove the culture medium. A delivery solution (4 μl) containing GFP mRNA was sprayed onto the cells and incubated for 2 minutes. After a 2-minute incubation, 50 μl of stop solution was added and incubated for 30 seconds. Finally, 100 μl of culture medium was added.

[0337] Method 1B: Provide a 1-mm gap between the color and the filter plate The color 9502 was equipped on the spray head 9504, and the tip of the spray head 9504 was positioned at a distance of 27 mm from the bottom surface of the well 9516a. Thus, the color was held 1 mm above the upper surface of the filter plate 9516. Note: In the enclosure experiment, a one-hit protocol (for example, one exposure to the delivery solution and the stop solution) was followed.

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

[0339] Tests corresponding to Method 1B, where there is a 1 mm gap between color 9502 and the upper surface of filter plate 9516, showed that color 9502 had no effect on GFP uptake. These results clearly showed that the data was equivalent between wells with color 9502 and wells without color 9502 in this mechanism. The viability of T cells was not affected regardless of which mechanism used an enclosing color.

[0340] Method 1: Conclusion This result shows that color 9502 has no effect on the viability of T cells. Color 9502 affects the spray to varying degrees. This difference can be attributed to the degree to which the system is sealed. By sealing the system (e.g., Method 1A), local pressure maxima that inhibit the spray were formed. For Method 1B, a pressure outlet was provided by creating a gap between color 9502 and the upper surface of filter plate 9516. This gap allowed for the expansion of air within the well and enabled the spray to operate correctly. Therefore, if an expansion space for air to expand during the spray process is allowed, a color can be used to surround the spray.

[0341] Method 2: Use of X-pierce film to surround the spray Rather than using a color to surround the spray head, the spray was surrounded using an X-pierce film (Sigma Aldrich, catalog number Z722529). To surround the spray while avoiding the formation of a seal that could inhibit the spray, it may be more beneficial to use an X-pierce film to surround the spray head than to use a color (e.g., color 9502).

[0342] Figure 99 shows an example of a 96-well PCTE filter plate 9616 in which an X-pierce film 9602 is adhered to the upper surface of a 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" located above the opening of each well of the filter plate 9616. In the experiment, 2.5×10 5 cells were seeded in each well. The filter plate 9616 was centrifuged at 350×g for 1 minute to remove the culture medium and generate a monolayer of cells. The air pressure delivered to the nebulizer was set to 1.65 bar, and the tip of the spray head of the nebulizer was positioned 12 mm above the upper surface of the filter membrane located within the well and on which a monolayer of cells was formed. A delivery solution (1 μl) containing GFP mRNA (2 μg) was sprayed onto the cell monolayer. The cells were then incubated for 2 minutes. After a 2-minute incubation, 50 μl of stop solution was added to each well and the wells were incubated for 30 seconds. Thereafter, as shown in Figure 99, 100 μl of culture medium was added to each cell. The filter plate was incubated at 37°C for 17 - 24 hours and then analyzed. This encapsulation experiment followed a one-hit protocol.

[0343] Method 2: Results Figure 100 shows the results of characterizing the efficiency (GFP uptake) corresponding to a test performed with Lig 1 (R1) using a filter plate without an enclosure, a test performed with Lig 3 (R3) using a filter plate without an enclosure, and a test performed with R3 using a filter plate including an X-pierce film enclosure over the wells of the filter plate. The ligs were described above. These results show that the efficiency is the same whether the well has a film or not, and thus indicate that the x-pierce film did not have a negative impact on the spray. The use of the X-pierce film did not affect cell viability.

[0344] Using the x-pierce film, cross-well cross-contamination can be avoided around the spray without reducing either the efficiency (GFP uptake) or the cell viability.

[0345] Fluid control of the solution to enable automation In some embodiments, the 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 medium can be used.

[0346] Fluid control can be achieved by a fluid control system that can apply a constant pressure to the system to extrude fluid through a pinch valve or a microvalve. The volume of fluid that can be dispensed can be controlled by the amount of pressure applied, the length of time the valve is open, and / or the diameter of the tube used.

[0347] The valve can be actuated by a metal-oxide semiconductor field-effect transistor (MOS FET) that can be controlled by a microprocessor.

[0348] The fluid control system can be enabled to deliver fluid in volumes ranging from 2 to 10 μl using microfluidic control. Therefore, the high-precision rig system 9000 can control the delivery of metered volume units in the range of 2 to 10 μl. In some embodiments, other volumes are also possible.

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

[0350] Figure 101 shows an example 11000 of a heating system that can be used to heat a delivery solution, a stop solution, and a 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 be in the shape of a cylindrical tube that can be filled with a fluid such as, for example, a 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 the distal end of the housing 11004. The heating element 11006 can receive power from a power source and can heat the fluid in the fluid reservoir by heating the housing 11004. The heating element 11006 can heat the housing 11004 by conduction.

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

[0352] In practice, the delivery solution and the stop solution can be maintained at approximately 4°C, and the culture medium can be maintained at approximately 20°C - 37°C.

[0353] Mounting of the spray head and temperature reservoir The needle emitter 9002 and the atomizer 9004 can be mounted on a support. FIG. 104 shows an example 11300 of a mounting assembly that can releasably hold the needle emitter 9002 and the atomizer 11304. The atomizer 11304 can be an ultrasonic atomizer that can operate at a frequency of 60 kHz to 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 may be connected. The hole 11306 can have a slot 11312 extending therefrom. Optionally, an ultrasonic atomizer that can operate at 60 kHz can have the same design as one that operates at 120 kHz. Therefore, the mounting assembly 11300 can 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, and the retaining element can be inserted into the opening 11308. The design of the retaining element 11310 as a slide-in 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 various atomizer heights in the range of 15 to 31 mm from the tip of the emitter to the bottom of the well of the filter plate while maintaining a consistent needle emitter height.

[0355] Figure 106 shows an example 11500 of a mounting assembly that can releasably hold a needle emitter 9002 and an ultrasonic atomizer 11504. The mounting assembly 11500 can generally be 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 connecting 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 retaining element 11510 to which the needle emitter 9002 may be connected. The hole can have a slot 11512 extending therefrom.

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

[0357] Figure 107 shows an example 11600 of a mounting assembly that can releasably hold a needle emitter 9002 and a nebulizer 11604. The mounting assembly 11600 can generally be similar to the mounting assembly 11300 and can include a support plate 11602 to which a connecting member 11505 is attached. The nebulizer 11604 can be connected to the support plate 11602 via a bracket 11606. The support plate 11602 can include an opening 11608 that can receive a retaining element 11610 to which the needle emitter 9002 may be connected.

[0358] A design template similar to that shown in FIGS. 78 - 81 can be used to accommodate other emitter types.

[0359] The designs of the mounting assemblies 11300, 11500, and 11600 enable this high-precision rig system to function as a platform for optimizing the delivery process. For example, it can accommodate four different spray heads.

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

[0361] A system has been designed that has the ability to ensure the repeatability accuracy of experiments and can also provide 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, including a traversing mechanism (e.g., actuator 9319), it can be software-controlled to position its carriage within the required time and within a 200 mm location. The carriage includes 6 positions, and each of these positions is connected to an individually controlled high-precision fluid escapement chamber. It is possible to move each position under the selection of dispensing locations (e.g., payload, culture medium, and stop solution stations). A device-specific software platform can be included. This software platform can include (1) a graphical user interface that enables the user to design experiments and (2) local control by a control device (e.g., a programmable logic controller (PLC)), such as an Omron PLC. Figure 129 shows a schematic diagram 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 enables the user to change the parameters of the experiment. Parameters that can be changed by the user include the location and number of wells to be addressed, vacuum and / or positive pressure, payload dispensing, the sequence of steps including stop solution and culture medium, and the corresponding volumes to be delivered. The user can also change the actuator speed and incubation time (Figure 130). Figure 130 shows the graphical user interface (GUI) 12100 of the software platform. In the illustrated example, the GUI includes an experiment canvas that enables the user to change parameters.For example, parameters that can be changed by the user include the location and number of wells to be addressed, vacuum or positive pressure, payload, stop solution and / or sequence of steps including dispensing of culture medium, and the corresponding volumes to be delivered. The background sequence generator outputs a program for the PLC, which results in local control of the mechanical and electronic systems. With this software, the operator can (e.g., via an integrated display) test various rearrangements of wells, media, and times without having to manually calculate the dwell time between wells for the success of an optimal experiment. With these inputs, the end user can perform multiple experiments.

[0362] In some embodiments, the system can address 96 positions within a filter plate body. This system can be faster and can have multiple traversing mechanisms and a user-friendly HMI (Human Machine Interface). The HMI can provide the operator with additional automation utilities such as an automatic system purge or a touch screen-driven calibration sequence. This system can operate as a stand-alone solution that still maintains contact with the experimenter for planning experiments. Further aspects of the experiment can be adjusted for each well, which means that the possible rearrangements increase significantly. For example, after dispensing fluid into some 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 analysis from experiments for further offline analysis.

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

[0364] In some embodiments, the operation of the delivery solution application device, the incubation over the first incubation period, the operation of the stop solution application device, and the incubation over the second incubation period can be repeated a predetermined number of times.

[0365] One or more aspects or features described in this specification can be realized in digital electronic circuit design, integrated circuit design, specially designed application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs) computer hardware, firmware, software, and / or combinations thereof. These various aspects or features can include an implementation in one or more computer programs that are executable and / or interpretable in a programmable system including at least one programmable processor, which may be special purpose or general purpose, coupled to communicate data and instructions between a memory system, at least one input device, and at least one output device. A programmable system or computing system can include clients and servers. Clients and servers are generally remotely located from each other and typically interact via a communication network. The relationship of client and server arises by computer programs operating on respective computers and having a client-server relationship to each other.

[0366] These computer programs can also be referred to as programs, software, software applications, applications, components, or code, and include machine instructions for programmable processors and can 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, memory, and programmable logic devices (PLDs), and includes a machine-readable medium that receives the machine instructions as a machine-readable signal. 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 can store such machine instructions, not transitorily, such as, for example, a non-transitory solid state memory or a magnetic hard drive or any equivalent storage medium. Instead of or in addition to the foregoing, a machine-readable medium can also store such machine instructions transitorily, such as, for example, a processor cache or other random access memory associated with one or more physical processor cores.

[0367] To provide interaction with a user, 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), liquid crystal display (LCD), or light emitting diode (LED) monitor for displaying information to the user, a keyboard as an input means to the computer by the user, and a pointing device such as a mouse or trackball. Other types of devices can be used as well to accommodate interaction with the user. For example, the feedback provided to the user can be any form of sensory feedback such as, for example, visual feedback, auditory feedback, or tactile feedback, and the input received from the user can be in any form, including but not limited to acoustic input, voice input, or tactile input. Other input devices that can be considered include, but are not limited to, touchscreens or other touch sensor-based devices, such as single-point or multi-point resistive or capacitive trackpads, speech recognition hardware and software, optical scanners, optical pointers, digital image capture devices, and associated interpretation software.

[0368] Enclosure for the equipment The high-precision rig system 9000 and / or the positive pressure system 9300 can be housed within an enclosure to maintain stable ambient conditions. The enclosure can be customized based on parameters that influence the design of the enclosure.

[0369] Temperature and control of the base plate 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. A thermodynamic analysis can be performed to examine the heat transfer from the cartridge heaters to the well filter plate 9014. Temperature ramp experiments can be conducted to examine the effect of temperature on payload delivery.

[0370] Some variations have been described above, but other modifications are possible. For example, the filter plate can include any number of wells and need not be a 96-well filter plate. Additionally, the system can include any number of valves that can control the vacuum pressure to any number of active wells. As another example, the system can include one or more needle emitters, atomizers, and / or nebulizers that can each be independently mounted and controlled.

[0371] The present subject matter has many technical advantages. Generally, the present subject matter provides a delivery system that can enhance the consistency and delivery efficiency of the delivery process while maintaining the integrity of the cells.

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

[0373] The present delivery system enables the dispensing of a permeabilization treatment solution in volumes on the order of microliters. The microliter dispensing volume allows for better control of the exposure of the cells to the solution, which can increase the overall cell viability by reducing overexposure to the permeabilization treatment solution. Additionally, the system can be automated, which minimizes errors and improves accuracy.

[0374] This system enables the control of the temperature of the delivery solution, the stop solution, and the culture medium. Therefore, each solution can be maintained at an optimal temperature to increase the efficiency of payload delivery and cell viability. The temperature of the base plate can also be controlled. This enables the optimization of the temperature for maximizing the efficiency of payload delivery.

[0375] The needle emitter and the atomizer and / or nebulizer can be connected to a mounting assembly that can accommodate various atomizer / nebulizer heights in the range of 15 - 31 mm from the tip of the emitter to the bottom of the well of the filter plate while keeping the needle emitter height constant. This makes it possible.

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

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

[0378] Further exemplary aspects of the delivery system Scaling of the delivery process involved designing a system for optimization, determining methods for forming cell monolayers on a larger scale, and optimizing atomization to enable intracellular delivery of mRNA to T cells.

[0379] Through optimization efforts, mRNA delivery efficiency to >50% of T cells has been achieved to date with >60% cell viability and a maximum 80% cell recovery rate.

[0380] The system was designed and constructed to facilitate the scaling of the delivery process. This system is based on commercially available Amicon stirred cells, pressure-based sample concentration units (sizes 50 ml and 200 ml, catalog numbers UFSC05001 and UFSC20001, respectively). Figure 108 shows an example 9700 of a stirred cell system 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. A stir bar 9710 can be located within the body 9704. The cap 9702 can be configured to connect 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 therebetween. The membrane holder 9706 can receive a membrane 9714 and can be located adjacent to a second end of the body 9704 such that a connecting member 9716 of the membrane holder 9706 extends through an opening 9718 at the second end of the body 9704. The second end of the body 9704 can be connected 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-conducting tube assembly 9722 can be connected to the cap 9702 such that a pressurized gas can be delivered to a body chamber formed by the cap 9702, the body 9704, and the base 9708. A filtrate tube assembly 9724 can be connected to the connecting member 9716 of the membrane holder 9706 such that fluid can be discharged from the chamber when pressure is applied.

[0382] During operation, a culture medium containing cells can be delivered to the chamber, and the cap 9702 can be connected to the main body 9704. The pressure conduit assembly 9722 can be connected to the cap 9702, and a positive pressure can be applied to the chamber via the inlet conduit assembly 9722. In some embodiments, the pressure can be in the range of 50 to 1000 mbar, and the pressure can be applied for 10 to 60 seconds. Thus, when the positive pressure is applied, the culture medium can be pushed out of the chamber through the connecting member 9716 and the filtrate conduit assembly 9724 across the membrane 9714. When the culture medium is removed, a monolayer of cells can remain on the membrane 9714. Thus, the stirred cell system 9700 can be used to form a monolayer of cells on a filter membrane installed in the membrane holder of the stirred cell. Once the monolayer is formed, a delivery solution can be atomized onto the cells on the filter membrane using a nebulizer (e.g., the LB-100 of Burgener Research).

[0383] The stirred cell system 9700 shown in FIG. 108 can have some limitations. For example, in some cases, the stirred cell system 9700 cannot filter a culture medium having a cell suspension above a certain concentration (e.g., a concentration above 10×10 6 / mL when using a PCTE membrane). This can be, in part, due to the shape of the membrane holder 9706. As shown in FIG. 109, the culture medium flows out through the only channel 9706a of the membrane holder 9706. The channel 9706a can become clogged when filtering a culture medium having 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 constraints associated with membrane holder 9706, membrane holder 9906 shown in FIG. 110 was constructed. In this embodiment, membrane holder 9906 is made of PTFE and may include a plurality of holes 9906a that penetrate the bottom surface in a pattern forming concentric circles. In use, when pressure is applied within the chamber of the stirred cell system, the culture medium can flow out through holes 9906a. Membrane holder 9906 was evaluated using dynabeads (microbeads used to mimic cells). FIG. 111 shows membrane 9914 used with membrane holder 9906 during dynabead evaluation. As shown in FIG. 111, the results indicate that dynabeads accumulated near holes 9906a. Cells subjected to the delivery process using this holder resulted in insufficient uptake.

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

[0386] The membrane holder 10006 was evaluated using dynabeads. Figure 113 shows the membrane 10014 used with the membrane holder 10006 in the dynabead evaluation. As shown in Figure 113, the results indicate that the dynabeads are more evenly distributed on the membrane 10014 used with the membrane holder 10006 than on the membrane 9914 used with the membrane holder 9906.

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

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

[0389] The nebulizer assembly 9801 includes a nebulizer 9804 (e.g., an LB-100 spray head), a connection element 9806 (e.g., an IDEX connector) configured to facilitate the delivery of air and liquid (e.g., the permeabilization treatment solution) to the nebulizer 9804, a solution reservoir 9810 (e.g., an Elveflow sample reservoir) configured to provide the permeabilization treatment solution to the nebulizer 9804, and a pinch valve 9808 configured to control the delivery of the permeabilization treatment 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 housing the nebulizer 9804. The nebulizer assembly 9801 can be connected to the mounting system 9082 as illustrated in FIGS. 91-92 so as to be fixed in a stationary position. The mounting system 9082 facilitates the accurate alignment of the nebulizer 9804 with the target area.

[0390] Further exemplary approach to cell monolayer formation Two approaches for generating cell monolayers are described. In either method, the stirred cell unit was assembled as shown in Figure 108. A cell suspension with a volume of 5 - 10 ml containing 0.4 - 10×10 6 cells / ml was added to the stirred cell chamber, and then the chamber lid was closed.

[0391] The monolayer can be generated by applying a vacuum pressure to the bottom of the stirred cell. In this method, -50 to -1000 mbar was applied to the chamber for 10 - 60 seconds or until the filter membrane appeared dry visually.

[0392] Alternatively, the monolayer was formed by applying a positive pressure through the tubing connector on the lid of the stirred cell. The pressure was applied for a set time (10 - 60 seconds) or until the filter membrane appeared dry visually. In some cases, a 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. A pressure of 100 - 200 mbar was applied to form the monolayer and completely remove 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, with the original unmodified membrane holder, monolayers were formed in a 63.5 mm stirred cell using 50 - 100×10 6 T cells. For a PES (polyethylene sulfone) membrane, it was possible to remove the medium by applying 150 mbar for 20 - 30 seconds for a cell concentration of 50×10 6 cells, but for 60×10 6 cells, 30 - 60 seconds and 200 mbar were required to remove the medium. For a PCTE (polycarbonate track etched) membrane, for the lower cell concentration, 250 mbar for 60 seconds was required. For 100×10 6In the case of individual cells, even after 2 minutes at 250 mbar, the medium is still thought to be present on the filter membrane. To remove this, the pressure was increased to 1 bar in 10 seconds and then decreased to the normal level (100 mbar). This was done several times to assist in medium removal.

[0394] Positive pressure was tested with an unmodified filter holder (Figure 109) in 63.5 mm and 44.5 mm stirred cells, using approximately 50×10^6 cells and a hydrophilic PCTE membrane with 2.0 μm pores. At no stage in this mechanism was all the medium removed (0 - 600 bar, 0 - 6 minutes).

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

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

[0397] (Table) TIFF2025084859000015.tif35128

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

[0399] PES filters were examined and it was found that these filters enabled the formation of uniform monolayers and efficient removal of the culture medium. However, the cell recovery rate from the filter membrane was low (50%). The PCTE track edge filter resulted in an improvement in cell recovery rate from the filter membrane (50 - 90%) (Figure 121). However, the efficiency of removing the culture medium was slower compared to the PES filter (the time required to remove the medium at 150 mbar was 10 seconds for the PES filter, while it was 45 seconds for the PCTE filter). The best recovery rate achieved was approximately 87% at 100 mbar for 60 seconds with PCTE 0.4 hydrophobic 20×10 6 It was about 87% at 100 mbar for 60 seconds (Figure 122).

[0400] Atomization to enable intracellular delivery of mRNA to T cells When the LB - 100 spray head was examined, a target area with a diameter of 60 mm was revealed. A force analysis of LB - 100 was performed (Figure 120). To optimize the delivery of 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 the LB - 100 spray delivery, the height of the spray head to the target area was adjusted. Distances in the range of 30 mm to 160 mm from the tip of the spray head to the target area were examined. The optimal distance range was found to be within 60 - 100 mm from the tip of the spray head to the target area. The uptake increased as the distance decreased and decreased as the distance increased.

[0402] To optimize LB-100 spray delivery, the air pressure was varied in the range of 1-6 bar, and it was found that 2.5-3.0 bar is the optimal range for intracellular delivery. GFP mRNA delivery was reduced at pressures lower than 2.5 bar, and cell viability was reduced at pressures higher than 3.0 bar. Increasing the air pressure increased the effective target area.

[0403] To optimize LB-100 spray delivery, the delivered volume was varied in the range of 10-300 μl, and it was found that a volume of 80-100 μl is optimal for intracellular delivery. 5-10 ml of a cell suspension of human primary T cells in the range of 0.4-10×10 6 cells / ml was added to a stirred cell unit (Merck Millipore; PCTE filter, pore size 0.4 μm or 1 μm). A positive pressure in the range of 100-150 mbar was applied for 20-50 seconds to form a cell monolayer. 10-300 μl of a delivery solution containing 0.1 μg / μl of GFP mRNA was sprayed onto the cell monolayer and incubated for 2 minutes. A stop solution (1 ml) was added, then incubated for 30 seconds, and culture medium (4 ml) was added to the filter membrane. The cells were incubated overnight in a humidified incubator at 37 °C and 5% CO 2 and evaluated for GFP fluorescence by flow cytometry 17-24 hours later.

[0404] To increase the delivery efficiency, an evaluation of the optimal number of hits was performed by comparing the one-hit strategy and the two-hit strategy. The data showed an increase in delivery efficiency when the cells received a second hit. 0.4-10×10 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 at a density of 13×10 3 cells / mm 2 were seeded into a stirred cell unit (Merck Millipore; PCTE filter, pore size 0.4 μm or 1 μm). A positive pressure in the range of 100 - 200 mbar was applied for 10 - 50 seconds to form a cell monolayer. 80 - 100 μl of a delivery solution containing 0.1 μg / μl of GFP mRNA was sprayed onto the cell monolayer and incubated for 2 minutes. A 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 overnight in a humidified incubator at 37 °C and 5% CO 2 , and after 17 - 24 hours, GFP fluorescence was evaluated by flow cytometry.

[0408] The spray duration was examined. This was achieved by adjusting the opening time of a valve that controls the flow of air and payload to the atomizer. Spray durations of 280 - 700 ms were tested.

[0409] Figure 134 shows an exemplary embodiment of a delivery system configured to facilit...

Claims

1. 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:

2. The method of claim 1 , wherein the alcohol comprises ethanol.

3. The method of claim 2, wherein the aqueous solution comprises more than 10% ethanol.

4. The method of claim 2, wherein the aqueous solution comprises 20-30% ethanol.

5. 2. The method of claim 1, wherein the aqueous solution comprises 27% ethanol.

6. 2. The method of claim 1, wherein the aqueous solution comprises 12.5 to 500 mM KCl.

7. 2. The method of claim 1, wherein the aqueous solution comprises 106 mM KCl.

8. The method of claim 1 , wherein the non-adherent cells comprise peripheral blood mononuclear cells.

9. The method of claim 1 , wherein the non-adherent cells comprise immune cells.

10. The method of claim 1 , wherein the non-adherent cells comprise T lymphocytes.

11. The method of claim 9, wherein the immune cells are activated by a ligand for CD3, a ligand for CD28, or a combination thereof.

12. The method of claim 1, wherein the population of non-adherent cells constitutes a monolayer.

13. The method of claim 1 , wherein the monolayer is contacted with a spray of the aqueous solution.

14. 2. The method of claim 1, wherein the method delivers the payload into the cytoplasm of the cells and the population of cells comprises a greater survival percentage compared to delivery of the payload by electroporation.

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

16. The method of claim 5, wherein the mRNA encodes a gene editing composition.

17. 17. The method of claim 16, wherein the gene editing composition reduces expression of PD-1.

18. The method of claim 13, wherein the monolayer is present on a membrane filter.

19. 14. The method of claim 13, wherein the membrane filter is vibrated after contact with the spray.

20. The method of claim 15, wherein the mRNA encodes a chimeric antigen receptor.

21. 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:

22. 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 21 , configured to:

23. 23. The system of claim 22, 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.

24. 23. The system of claim 22, 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.

25. 22. The system of claim 21, wherein the delivery solution application device comprises a nebulizer.

26. 22. The system of claim 21, wherein the delivery solution application device is configured to deliver between 10 and 300 microliters of the delivery solution per actuation.

27. 22. The system of claim 21, 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.

28. 22. The system of claim 21, further comprising an enclosure configured to control the environment of the plate containing the wells.

29. 22. The system of claim 21, 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 forcing a liquid portion of the culture medium out of the well such that a layer of cells remains within the well.

30. 22. The system of claim 21, wherein the stop solution application device comprises a needle emitter configured to couple to a stop solution reservoir.

31. 22. The system of claim 21, wherein the culture medium application device comprises a needle emitter configured to couple to a culture medium reservoir.

32. 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.

22. The system of claim 21, further comprising a controller configured to:

33. 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.

23. The system of claim 22, further configured to:

34. Activating a positive pressure system to remove the supernatant from the well and generate a cell monolayer within the well.

22. The system of claim 21, further comprising a controller configured to:

35. 22. The system of claim 21, 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.

36. 36. The system of claim 35, wherein the delivery solution application device comprises a spray head and a film surrounding a distal end of the spray head.

37. 22. The system of claim 21, further comprising a vibration system coupled to the membrane holder and configured to vibrate the membrane.

38. a plate, the wells of which are configured to contain a population of non-adherent cells.

22. The system of claim 21, further comprising:

39. 22. The system of claim 21, wherein the delivery solution comprises an isotonic aqueous solution comprising a payload and an alcohol at a concentration greater than 5 percent (v / v).

40. 39. The system of claim 38, wherein the alcohol comprises ethanol.

41. 40. The system of claim 39, wherein the aqueous solution comprises greater than 10% ethanol.

42. 40. The system of claim 39, wherein the aqueous solution comprises 20-30% ethanol.

43. 39. The system of claim 38, wherein the aqueous solution comprises 27% ethanol.

44. 39. The system of claim 38, wherein the aqueous solution comprises 12.5 to 500 mM KCl.

45. 39. The system of claim 38, wherein the aqueous solution comprises 106 mM KCl.

46. 38. The system of claim 37, wherein the non-adherent cells comprise peripheral blood mononuclear cells.

47. The system of claim 37 , wherein the non-adherent cells comprise immune cells.

48. The system of claim 37, wherein the non-adherent cells comprise T lymphocytes.

49. 40. The system of claim 38, wherein the payload comprises messenger ribonucleic acid (mRNA).

50. The system of Claim 49, wherein the mRNA encodes a gene editing composition.

51. The system of Claim 50, wherein the gene editing composition reduces expression of PD-1.

52. The system of claim 49, wherein the mRNA encodes a chimeric antigen receptor.

53. 22. The system of claim 21 for use in delivering a cargo compound or cargo composition to a mammalian cell.

54. The system of claim 37, wherein the population of non-adherent cells constitutes a monolayer.

55. A composition comprising an isotonic aqueous solution comprising KCl at a concentration of 10-500 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.

56. 56. The composition of claim 55, wherein the KCl concentration is 106 mM and the alcohol concentration is 27%.

57. Apparatuses, systems, techniques, compositions, and articles described or illustrated herein.

Images