Methods for viral delivery to populations of cells

Abstract

The present invention provides a method for viral transfection of cells. TIFF2022535242000039.tif171157

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. Section 119(e) to U.S. Provisional Application No. 62 / 855,241, filed May 31, 2019, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present invention relates to the delivery of agents, such as viruses, into mammalian cells and the production of such viruses. [Background technology]

[0003] 2. Background of the Invention Viruses are widely used as effective gene delivery vehicles. Transduction is the process by which foreign DNA is introduced into cells by viruses or viral vectors. Transduction is a common tool used by molecular biologists to stably introduce foreign genes into host cells, e.g., mammalian genomes. Optimization of conditions for transduction is very important for various research and clinical applications. For cells that are difficult to transfect, lentiviral transduction provides a highly efficient solution to achieve good expression levels. Efficiency, time, production costs, and cell viability remain challenges in the field. Summary of the Invention

[0004] The present invention provides a solution to the problem of delivering complex cargoes, such as viruses, into cells. Viruses are infectious agents that are typically composed of a nucleic acid molecule in a protein coat. Viruses are microorganisms smaller than bacteria that cannot grow or reproduce apart from living cells, i.e., viruses can only multiply within living host cells.

[0005] The invention features a method of delivering a virus across a plasma membrane of a cell, the method comprising providing a population of cells and contacting the population of cells with a volume of an isotonic aqueous solution comprising the virus and an alcohol at a concentration greater than 2%. The step of contacting the population of cells with the volume of aqueous solution is performed by propelling the aqueous solution with a gas to form a spray. The spray comprises droplets (or a collection of droplets) comprising a diameter greater than or equal to 150 μm. For example, the spray comprises droplets (or a collection of droplets) comprising a diameter in the range of 177 μm to 590 μm.

[0006] Exemplary viruses to be delivered include lentiviruses, retroviruses, adenoviruses, adeno-associated viruses (AAV), or herpes simplex viruses (HSV). In a preferred embodiment, the virus is a lentivirus.

[0007] The population of cells includes mammalian cells, e.g., human immune cells. The population of cells may include adherent cells or suspension cells. In some examples, the population of cells includes non-adherent cells, such as T lymphocytes or natural killer (NK) cells. The population of cells may include primary cells or cell lines. For example, the population includes HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells. The method results in a transduction efficiency of cells that is at least 30%, at least 40%, at least 50%, or at least 60%.

[0008] The aqueous solution includes an alcohol, such as ethanol. For example, the aqueous solution includes more than 2% ethanol, more than 10% ethanol, for example, the aqueous solution includes 20-30% ethanol. Exemplary solutions include ethanol concentrations of 5-30%.

[0009] The aqueous solution further comprises one or more of the following components: 75-98% HO, 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). For example, the solution contains 32.5 mM sucrose, 106 mM KCl, 5 mM Hepes, and 12% v / v ethanol.

[0010] To receive the spray solution containing the cargo to be delivered, e.g., the virus, the population of cells may be present as a layer, e.g., a monolayer, of non-adherent cells on a substrate. The layer may be confluent or non-confluent. An exemplary layer, e.g., a monolayer, of cells is present on a membrane filter.

[0011] The present invention provides a solution to the problem of delivering viruses and compositions into cells. Thus, a method of delivering a virus across the plasma membrane of a cell (e.g., a suspension or adherent cell) comprises providing a population of cells and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising the virus.

[0012] Aqueous solutions for delivering viruses to cells include a salt, such as 12.5 to 500 mM potassium chloride (KCl). For example, the solution is isotonic to the cytoplasm of a mammalian cell, such as a human T cell. Such an exemplary isotonic delivery solution contains 106 mM KCl.

[0013] The method can be used to deliver any virus to adherent or non-adherent mammalian cells, and is particularly useful for delivering cargo to non-adherent cells due to the difficulties associated with doing so prior to the present invention. In some instances, the non-adherent cells include peripheral blood mononuclear cells, for example, the non-adherent cells include immune cells such as T cells (T lymphocytes), for example, activated or unactivated (also known as naive) T cells. The immune cells such as T cells are optionally activated with a ligand for CD3, CD28, or a combination thereof. For example, the ligand is an antibody or antibody fragment that binds to CD3 or CD28, or both.

[0014] The method includes delivering the virus in a delivery solution to a population of non-adherent cells, including a monolayer, e.g., a sheet of cells physically located on a support or substrate. For example, the cells form a layer, which is contacted with a spray of an aqueous delivery solution. For example, the monolayer (or layer) is contacted with a spray of an aqueous delivery solution. The method delivers the virus (compound or composition) into the cytoplasm of the cells, and the population of cells maintains a high percentage of viability after the procedure. The method also delivers the virus (compound or composition) in the form of a spray, which is a small volume. The small volume of the spray concentrates the virus at the plasma membrane of the cells.

[0015] In certain embodiments, the monolayer of non-adherent / suspended cells is present on the membrane filter.In some examples, the membrane filter is moved, for example, stirred or vibrated, after the cell monolayer is contacted with the spray of delivery solution.The membrane filter may be vibrated or stirred before, during, and / or after spraying the cell with the delivery solution.

[0016] The delivery solution includes an isotonic aqueous solution, the aqueous solution including a payload and an alcohol at a concentration greater than 2 percent (v / v). The alcohol can include ethanol. The aqueous solution can include greater than 10% ethanol. The aqueous solution can include 20-30% ethanol. The aqueous solution can include 27% ethanol. The aqueous solution can include 12.5-500 mM KCl. The aqueous solution can include 106 mM KCl. In an embodiment, the aqueous solution includes 27% ethanol. In another example, the aqueous solution includes 12% ethanol. In an example, the aqueous solution includes 32.5 mM potassium chloride (KCl) 106 mM, Hepes 5 mM, ethanol (EtOH) 12% v / v, and water for injection (WFI).

[0017] In examples, "S buffer" comprises a hypotonic physiological buffer (78 mM sucrose, 30 mM KCl, 30 mM potassium acetate, 12 mM HEPES) at 4°C for 5 minutes (Medepalli K. et al., Nanotechnology 2013; 24(20); incorporated herein by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in S buffer. S buffer is further described in International Application WO 2016 / 065341, e.g., at paragraphs

[0228] to

[0229] , incorporated herein by reference in its entirety.

[0018] The non-adherent cells can comprise peripheral blood mononuclear cells. The non-adherent cells can comprise immune cells. The non-adherent cells can comprise T lymphocytes. The population of non-adherent cells can comprise a monolayer.

[0019] In another aspect, the composition comprises an isotonic aqueous solution, the aqueous solution comprising KCl at a concentration of 10-500 mM and ethanol at a concentration of greater than 5 percent (v / v) for use in delivering a cargo compound or composition to a mammalian cell. The KCl concentration can be 106 mM and the alcohol concentration can be 27%. In an embodiment, the aqueous solution comprises 27% ethanol for Flexi (e.g., small scale). In an embodiment, the aqueous solution comprises 12% ethanol in a large scale system.

[0020] In an aspect, a method for delivering a virus across the plasma membrane of non-adherent cells is provided herein, the method comprising the steps of: providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising a virus.In an embodiment, the virus comprises a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), or a herpes simplex virus (HSV).

[0021] In some embodiments, the cell population comprises adherent cells or suspension cells. For example, the cell population comprises HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells.

[0022] Also provided herein is a method for delivering a virus across the plasma membrane of non-adherent cells, comprising the steps of: providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising a virus and an alcohol at a concentration greater than 2 percent (v / v). In embodiments, the virus comprises a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), or a herpes simplex virus (HSV).

[0023] In embodiments, the population of cells comprises adherent or suspension cells, for example, HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells.

[0024] In embodiments, the transfection (or transduction) efficiency is at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% (or higher).

[0025] In embodiments, the alcohol comprises ethanol. In other embodiments, the aqueous solution comprises greater than 10% ethanol. In embodiments, the aqueous solution comprises 20-30% ethanol. In embodiments, the aqueous solution comprises 27% ethanol. In embodiments, the aqueous solution comprises 12% ethanol, for example in larger scale systems.

[0026] In embodiments, the population of cells comprises a monolayer of non-adherent cells. In examples, the monolayer is contacted with a spray of said aqueous solution. The monolayer may further be on a membrane filter.

[0027] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps "and which do not materially affect the basic and novel characteristics" of the claimed invention.

[0028] [The present invention 1001] 1. A method of delivering a virus across the plasma membrane of a cell, comprising the steps of: Providing a population of cells; and Contacting the population of cells with a volume of an isotonic aqueous solution, the solution comprising the virus and an alcohol at a concentration greater than 2%, and contacting the population of cells with the volume of aqueous solution is carried out by propelling the aqueous solution with a gas to form a spray, the spray comprising droplets having a diameter greater than or equal to 150 μm. [The present invention 1002] The method of claim 1001, wherein the spray comprises droplets having a diameter in the range of 177 μm to 590 μm. [The present invention 1003] 1001. The method of claim 1001, wherein the virus comprises a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus (AAV), or a herpes simplex virus (HSV). [The present invention 1004] The method of claim 10, wherein the virus is a lentivirus. [The present invention 1005] 1001. The method of claim 1001, wherein said population of cells comprises mammalian cells. [The present invention 1006] 1002. The method of claim 1001, wherein said population of cells comprises adherent cells or suspension cells. [The present invention 1007] The method of claim 1006, wherein said population of cells comprises non-adherent cells. [The present invention 1008] The method of claim 1007, wherein said non-adherent cells comprise T lymphocytes. [The present invention 1009] The method of the present invention 1003, wherein the transduction efficiency is at least 30%, at least 40%, at least 50%, or at least 60%. [The present invention 1010] The method of claim 1006, wherein said population of cells comprises HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells. [The present invention 1011] 1001. The method of claim 1001, wherein said population of cells comprises natural killer cells. [The present invention 1012] 1001. The method of claim 1001, wherein the alcohol comprises ethanol. [The present invention 1013] 1001. The method of claim 1001, wherein the aqueous solution contains more than 2% ethanol. [The present invention 1014] 1001. The method of claim 10, wherein the aqueous solution contains more than 10% ethanol. [The present invention 1015] The method according to claim 10, wherein the aqueous solution contains 20 to 30% ethanol. [The present invention 1016] The method according to claim 10, wherein the aqueous solution contains an ethanol concentration of 5 to 30%. [The present invention 1017] The method of claim 10, wherein the aqueous solution further comprises one or more of the following components: 75-98% H 2 HO, 2–45% ethanol, 6–91 mM sucrose, 2–35 mM KCl, 2–35 mM ammonium acetate, and 1–14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). [The present invention 1018] 1001. The method of claim 1001, wherein said aqueous solution comprises sucrose 32.5 mM, KCl 106 mM, Hepes 5 mM, and ethanol 12% v / v. [The present invention 1019] 1002. The method of claim 1001, wherein said population of cells comprises a layer of non-adherent cells on a substrate. [The present invention 1020] The method of claim 10, wherein the layer is present on a membrane filter. Other features and advantages of the present invention will be clear from the following description of its preferred embodiment and from the claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.Methods and materials similar or equivalent to those described herein can be used in carrying out or testing the present invention, but suitable methods and materials are described below.All published foreign patents and patent applications cited herein are incorporated by reference.Registrations of GenBank and NCBI indicated by accession numbers cited herein are incorporated by reference.All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods, and examples are only illustrative and are not intended to be limiting. [Brief description of the drawings]

[0029] [Figure 1] 1 is a schematic diagram outlining a virus production protocol, where the methods described herein are incorporated during the plasmid transfection step of the protocol, as represented by the arrow. [Diagram 2] 1 is an image showing the viral entry pathway, from Biophys J. 2016 Mar 8; 110(5): 1028-1032, which is incorporated by reference in its entirety. [Diagram 3] 1 is a bar graph showing T cell viability before and after lentivirus (LV)-GFP delivery. [Figure 4] 1 is a bar graph showing the cumulative fold increase up to 96 hours post-infection. [Diagram 5] 1 is a bar graph showing GFP expression in T cells 3 and 4 days after LV-GFP delivery. [Figure 6]Figures 6A-6D illustrate representative 4x images of delivery solutions formulated with LV-eGFP (enhanced GFP) after 1 hour (Figure 6A), 2 hours (Figure 6B), 3 hours (Figure 6C), and 4 hours (Figure 6D) at room temperature. [Figure 7] 7A and 7B depict histogram profiles of CD3 (FIG. 7A) and CD3+CD25 (FIG. 7B) expression of day 3 PBMC initiated T cells. [Figure 8] 1 is a bar graph showing cell recovery following the SOLUPORE™ process with the system (n=3 technical replicates). [Figure 9] Figures 9A and 9B are line graphs showing the survival rate (Figure 9A) and cumulative fold increase (Figure 9B) up to 3 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). Statistical analysis calculated using a paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 10] Figures 10A and 10B are line graphs showing GFP expression (Figure 10A) and median fluorescence intensity (Figure 10B) up to 3 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). Statistical analysis calculated using a paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 11] 11A-11B are images of histogram profiles of CD3 (FIG. 11A) and CD3+CD25 (FIG. 11B) expression of day 3 PBMC initiated T cells. [Figure 12] 1 is a bar graph showing cell recovery following the SOLUPORE™ process involving the system described herein (n=3 technical replicates). [Figure 13]Figures 13A and 13B are line graphs showing the survival rate (Figure 13A) and cumulative fold increase (Figure 13B) up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). Statistical analysis calculated using a paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 14] Figures 14A and 14B are line graphs showing GFP expression in % (Figure 14A) and median fluorescence intensity (Figure 14B) up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). Statistical analysis calculated using a paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 15] Figures 15A and 15B are images showing histogram profiles of CD3 (Figure 15A) and CD3+CD25 (Figure 15B) expression of day 3 PBMC initiated T cells. [Figure 16] 1 is a bar graph showing cell recovery following the SOLUPORE™ process with the system (n=6). [Figure 17] Figures 17A and 17B are line graphs showing survival rates up to 3 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). The effect of virus delivery method (Figure 17A) and MOI of LV (Figure 17B) on survival rates are shown. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 18] Figures 18A and 18B are line graphs showing cumulative fold expansion up to 4 days after viral delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). The effect of viral delivery method (A) and MOI of LV (B) on expansion is shown. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 19]Figures 19A-19B are line graphs showing the effect of viral delivery method on GFP expression in % (Figure 19A) and in median fluorescence intensity (Figure 19B) up to 4 days post-infection. The effect of delivered MOI on % GFP expression (Figure 19C) and GFP MFI (Figure 19D) up to 4 days post-infection is shown. N=3 technical replicates for each MOI condition tested. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 20] Figures 20A and 20B are images of histogram profiles of CD3 (Figure 20A) and CD3+CD25 (Figure 20B) expression of day 3 PBMC initiated T cells. [Figure 21] 1 is a bar graph showing cell recovery following the SOLUPORE™ process with the system (n=6). [Figure 22] Figures 22A and 22B are line graphs showing viability up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). The effect of virus delivery method (Figure 22A) and MOI of LV (Figure 22B) on viability is shown. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Diagram 23] Figures 23A and 23B are line graphs showing cumulative fold expansion up to 4 days after virus delivery by SOLUPORE™ process or static transduction (n=3 technical replicates). The effect of virus delivery method (Figure 23A) and MOI of LV (Figure 23B) on expansion is shown. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 24]Figures 24A-24D are line graphs showing the effect of viral delivery method on GFP expression in % (Figure 24A) and in median fluorescence intensity (Figure 24B) up to 4 days post-infection. The effect of delivered MOI on % GFP expression (Figure 24C) and GFP MFI (Figure 24D) up to 4 days post-infection is shown. N=3 technical replicates for each MOI condition tested. Statistical analysis calculated using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Diagram 25] 1 is a bar graph showing CD3 and CD25 expression on day 3 PBMC initiated T cells (n=4). [Figure 26] 1 is a bar graph showing cell recovery following the SOLUPORE™ process with the system (n=18). [Figure 27] Figures 27A-27C are line graphs showing viability up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=18). The effect on viability by virus delivery method (Figure 27A), MOI of LV (Figure 27B), and the changes that occurred between run 1 / 2 and run 3 / 4 (Figure 27C) are shown. Statistical analysis calculated using three-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 28] Figures 28A-28C are line graphs showing cumulative fold expansion up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=18). Shown are the effects on expansion by virus delivery method (Figure 28A), MOI of LV (Figure 28B), and the changes that occurred between run 1 / 2 and run 3 / 4 (Figure 28C). Statistical analysis calculated using three-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 29]Figures 29A-29F are line graphs showing GFP expression (Figure 29A, Figure 29C, Figure 29E) and median fluorescence intensity (Figure 29B, Figure 29D, Figure 29F) up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). The effect on GFP expression and MFI by virus delivery method (Figure 29A, Figure 29B), MOI of LV (Figure 29C, Figure 29D), and the changes that occurred between run 1 / 2 and run 3 / 4 (Figure 29E, Figure 29F) are shown. Statistical analysis calculated using three-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Diagram 30] Figures 30A-30F are bar graphs showing fold expansion for run 1 (Figure 30A), run 2 (Figure 30B), run 3 (Figure 30C), and run 4 (Figure 30D) up to 4 days after virus delivery by the SOLUPORE™ process or static transduction (n=3 technical replicates). The effect of LV MOI on expansion is shown for run 3 (Figure 30E) and run 4 (Figure 30F). Statistical analysis calculated using paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Diagram 31] Figures 31A and 31B are bar graphs showing fold expansion up to 4 days after viral delivery by the SOLUPORE™ process or static transduction (n=18). The effect on expansion by viral delivery method (Figure 31A) and by MOI of LV (Figure 31B) is shown. Statistical analysis calculated using paired two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Diagram 32] Bar graph showing fold increase up to 4 days after virus delivery by SOLUPORE™ process or static transduction (n=6 per condition). Statistical analysis calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Diagram 33]Bar graph showing survival rate up to 4 days after virus delivery by SOLUPORE™ process or static transduction (n=6 per condition).Statistical analysis calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Diagram 34] Image of Falcon tubes during isolation of PBMCs. [Diagram 35] 13 is an image showing the assembly of the system chamber. [Diagram 36] 1 is a picture of the assembled system base on the stand. [Figure 37] 13 is an image showing wetting of the drain disk. [Figure 38] 1 is an image showing examples of bad and good filter membrane layouts. [Figure 39] 13 is an image illustrating the Elveflow module with fluidics and reservoir holder. [Diagram 40] 1 is an image showing the controller unit for the SOLUPORE™ method. [Diagram 41] 1 is an image showing the calibration cup in the system chamber. [Diagram 42] 1 is an image showing a manual pressure regulator for establishing airflow through the shower head. [Diagram 43] An image showing the connection from the control unit to the sample line. [Diagram 44] 13 is an image showing the rotation of the chamber to facilitate rinsing and cleaning of surfaces within the chamber. [Diagram 45] 1 is an image of an overview of the system chamber components. [Figure 46] 1 is an image of an overview of the system lid components. [Figure 47] 1 is an image showing the assembled system. [Figure 48] 1 is an image of a map of the LV expression plasmid carrying eGFP. [Figure 49]1 is a bar graph showing the estimated copy number of GFP per cell based on WPRE per 2 albumin (GFP%) for experiment 1. [Figure 50] 1 is a bar graph showing the estimated copy number of GFP per cell based on WPRE per 2 albumin (GFP%) for experiment 2. [Figure 51] 1 is a graph illustrating droplet size distribution (x-axis) versus frequency (y-axis). [Figure 52] 1 is a graph showing temperature (x-axis) and dynamic viscosity (y-axis) of various liquids. The graph is reproduced from Engineering ToolBoox (2008); Dynamic Viscosity of Common Liquids, which is incorporated by reference in its entirety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description Viruses are widely used as effective gene delivery vehicles. Transduction is the process by which foreign DNA is introduced into cells by viruses or viral vectors. Transduction is a common tool used by molecular biologists to stably introduce foreign genes into host cells, e.g., mammalian genomes. Optimization of conditions for transduction is very important for various research and clinical applications. For cells that are difficult to transfect, lentiviral transduction provides a highly efficient solution to achieve good expression levels. Efficiency, time, production costs, and cell viability remain challenges in the field.

[0031] Viral Delivery to Populations of Cells Using the SOLUPORE™ Process The SOLUPORE™ process allows the delivery of a wide range of cargoes to adherent and suspension cells in vitro and ex vivo. To date, these cargoes consist of molecules such as nucleic acids and proteins, as well as particles such as Qdots. The data herein show that the SOLUPORE™ process can be used to deliver non-bacterial microorganisms, such as viruses (e.g., lentiviruses), to T cells with higher efficiency than standard control transduction.

[0032] Viruses as vectors Viruses are used as vectors for the delivery of nucleic acids to cells because they can naturally infect human cells. Viruses must attach to the host cell before entry. Attachment is achieved when a specific protein on the viral capsid or viral envelope binds to a specific receptor protein on the cell membrane of the target cell. Depending on the type of virus, entry into the cell can occur in different ways. Viruses with a viral envelope can enter the cell by membrane fusion, where a hole is made in the cell membrane to further connect with the unfolded viral envelope. Viruses without a viral envelope can enter by endocytosis (Figure 2). Other viruses, such as bacteriophages, attach to the cell surface and only the viral genome is injected into the host cell.

[0033] immune cell manipulation Different types of viruses have different characteristics that need to be considered when used to transduce cells ex vivo for clinical applications. The main characteristics are: immunogenicity; target cell type; payload capacity; ability to transduce dividing versus non-dividing cells; transient versus stable genomic integration (Table 5).

[0034] Overview of viruses used in gene delivery applications TIFF0007637072000001.tif63157, the entire contents of which are incorporated herein by reference, Pharmaceutics 2020, 12, 183

[0035] For immune cell therapy applications, such as the delivery of chimeric antigen receptor (CAR) constructs for the generation of CAR-T cells and CAR-NK cells, gammaretroviruses and lentiviruses are typically used because they can transduce immune cells and result in stable integration into the genome. For example, the first two approved CAR-T cell products, Kymriah and Yescarta, were engineered using lentiviral and gammaretroviral vectors, respectively (Poorebrahim M et al. CritRev Clin Lab Sci. 2019 Sep;56(6):393-419). As with most viral vectors, these vectors have been modified to disable viral replication and improve cell targeting efficiency.

[0036] AAV vectors are widely used for other immune cell engineering applications, such as gene editing. Wild-type AAV can be stably integrated into chromosome 19, but AAV-based gene therapy vectors are modified to prevent integration and instead form episomal concatemers in the nucleus of host cells. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. In dividing cells, episomal DNA is not replicated with host cell DNA, so AAV DNA is lost through cell division. Therefore, AAV vectors are often used to deliver editing systems, such as CRISPR / Cas9 systems for gene editing or donor template DNA. In these cases, gene editing is permanent, but gene editing tools may be desired to be expressed only transiently to limit non-specific off-target gene editing that may occur if the tools are present in cells for long periods of time.

[0037] Lentivirus Gammaretrovirus and lentivirus are subtypes of retrovirus that contain RNA genome that is converted into DNA in transduced cells by the enzyme called reverse transcriptase that is encoded by the virus.For retrovirus, the entry into cells is followed by the process of uncoating, which causes some viral proteins to dissociate from the viral core.Viral RNA is reverse transcribed into double-stranded DNA.Then, viral proteins form a complex with proviral DNA, leading to nuclear transport and integration into the host genome.The integration process is assisted by vital viral proteins such as integrase, and endogenous host cell transcription factors.

[0038] Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become the primary tool for gene delivery into mammalian cells, and replication-defective recombinant lentiviruses are widely used in research and clinical applications. The modified lentiviruses can still infect cells, but the essential genes for producing new viral particles are no longer present. Lentiviral vectors are considered attractive gene delivery vehicles for several reasons: they provide long-term gene expression through stable vector integration into the host genome; they can infect both dividing and non-dividing cells; they can infect a wide range of cells, including important target cell types for gene and cell therapy; they lack immunogenic viral proteins after vector transduction; they can deliver complex genetic elements such as intron-containing sequences; and they are a relatively easy system for vector engineering and production. Lentiviral vectors have a safer integration site profile than gammaretroviral vectors and are commonly used in clinical trials of CAR T-cell therapy (McGarrity GJ et al. J. Gene Med. 2013;15:78-82). Third generation lentiviral vectors have incorporated key safety features to further enhance safety (Kim VN et al. J. Virol. 1998;72:811-816 and Dull T. et al. J. Virol. 1998;72:8463-8471).

[0039] The range of cell types that can be transduced with retroviruses has been expanded by pseudotyping retroviruses with the envelope glycoprotein G of vesicular stomatitis virus (VSV-G). VSV-G binds to phosphatidylserine, a ubiquitous membrane component, allowing VSV-G pseudotyped viruses to attach to and transduce a much broader range of cells. Currently, most lentiviral vectors are pseudotyped with VSV-G to enable robust transduction into many cell types, including neurons, lymphocytes, and macrophages.

[0040] Quiescent cells After isolation from either healthy donors or patients, cells are activated and subsequently transduced with lentiviral vectors (LVs), the majority of which are pseudotyped with the glycoprotein G of the vesicular stomatitis virus (VSV). The modified lymphocytes are then expanded and used in functional in vitro assays or in vivo applications. Stable and efficient transduction of B cells with LVs is very difficult to achieve. Resting B cells are limited to transduction with conventional VSV G pseudotyped LVs (VSV-LVs) due to the lack of expression of the low density lipoprotein receptor (LDLR), and must be activated prior to transduction. Efficient activation and culture of primary human B lymphocytes is complex, as it involves carefully titrated activation stimuli combined with cytokines, followed by co-culture with feeder cells. Even under optimal activation and culture conditions, transduction efficiency with VSV-LVs is notoriously low. The combination of all these challenges may serve as an explanation for why the number of clinical trials involving engineered B cells is much smaller compared to T cells.

[0041] In comparison, T lymphocyte engineering by lentiviral transduction is easier to achieve. Still, the cells, like B cells, must be activated before transduction with conventional VSV-LV, since they are otherwise not susceptible to transduction, again due to the lack of LDLR expression (X Geng, et al. Gene Therapy v.21, pages 444-449 (2014)). Protocols for isolation, activation, lentiviral transduction, and expansion of T cells have been greatly improved in recent years, primarily for the best success of CAR T cell therapy. Current state-of-the-art T cell activation relies on stimulation of the TCR activation pathway via CD3- and CD28-specific antibodies, combined with cytokines such as IL-7 and IL-15.

[0042] The need to activate lymphocytes prior to transduction with conventional LV has disadvantages. This adds to the complexity of the entire procedure, increasing the duration and cost of the manufacturing process. In addition, the stimuli applied for activation, combined with the long-term ex vivo culture, are likely to alter the cells, which may have a negative impact on the quality of the final product. As a result, naive cells may differentiate into a less preferred phenotype that exhibits a higher degree of attrition, a lower proliferation capacity, a shorter in vivo persistence, and a lower functionality. This may have very important implications for therapeutic success. As an example, it has been shown that the central memory (CD45RO+ / CD45RA+ / CD62L+) or stem cell memory (CD45RO+ / CD45RA / CD62L+) phenotype is beneficial for the persistence and function of T cells in vivo. In this regard, a positive correlation between CAR T cell central memory phenotype and positive clinical responses has been observed in several clinical studies, and as a result, the infusion of purified central memory CAR T cells is currently considered. Similarly, the central memory phenotype results in functionally superior TCR-modified T cells. Thus, minimal manipulation of lymphocytes during genetic modification has significant clinical relevance.

[0043] Spinoculation Centrifugation inoculation, or spinoculation, is widely used in virology research to enhance virus infection. The procedure involves centrifuging a mixture of virus and target cells at high speed for an extended period of time, e.g., 800×g for 30 min at 32°C. The method was thought to enhance transduction rates by concentrating the virus at the cell membrane. However, spinoculation has been shown to induce dynamic actin and cofilin activity, likely due to the cellular response to centrifugation stress (Jia Guo, et a. J. Virology Oct. 2011, p. 9824-9833). This actin activity also leads to upregulation of cell membrane receptors that may enhance virus binding and entry. It has been suggested that spinoculation cannot be simply explained by a virus concentration effect, but rather is coupled to spin-induced cytoskeletal dynamics that promote receptor recruitment, virus entry, and post-entry processes. Thus, spinoculation may affect the biology of target cells in unknown or undesirable ways.

[0044] Limitations of viral vectors Although viruses are useful for genetically manipulating cells as described above, there are limitations to their utility.

[0045] The high cost manufacturing process of chimeric antigen receptor (CAR) T-cell therapy is prohibitively expensive. Due to the cost of the virus, transduction is the main cost driver in CAR T-cell manufacturing. Several bioprocessing parameters have been identified as potentially playing a role in transduction efficiency, such as the physical proximity of lentiviral particles to T cells. This proximity can be manipulated through the number of cells and viral particles in suspension; the duration of agitation to promote uniformity; and the surface-to-volume ratio in the transduction vessel. However, limited research has been conducted on identifying and optimizing the critical process parameters of transduction. During the SOLUPORE™ process, a small amount of delivery solution is applied directly onto the exposed target cells. In this way, the cargo is brought into direct contact with the cells in a gentle manner. Delivering the virus to the cells in this way results in the concentration of the material at the cell membrane. This process enhances the virus attachment to the cell membrane and enhances the rate of entry into the cell, making the process more efficient. In turn, smaller doses of virus are used, lowering costs.

[0046] The SOLUPORE™ process is a gentle method of concentrating viruses at the cell membrane, which has significant advantages over existing concentration methods such as spinoculation, which can affect cell structure. Moreover, unlike spinoculation, the SOLUPORE™ process is designed to be compatible with cell therapy manufacturing processes.

[0047] Concentration of virus at the cell membrane also compensates for the low level of viral receptor expression on certain cell types, such as non-activated T cells, thus enhancing transduction efficiency in these cells.

[0048] The efficiency of lentiviral vector transduction of non-activated T and B cells is typically very low. It is highly desirable to improve these efficiencies, and the SOLUPORE™ process offers a solution to this problem, with high efficiency rates coupled with conditions compatible with preserving cell viability and function.

[0049] Viruses can only deliver nucleic acids, which means that they are limited in the type of cargo they can deliver. If viruses can be delivered simultaneously with other types of cargo, this can enhance the utility of viruses in engineering for next-generation cell therapy products. However, currently, there is no method that has been demonstrated to simultaneously deliver viruses with other types of cargo. Again, the SOLUPORE™ process described herein provides a solution to this problem by allowing the efficient delivery of many different cargo types, either sequentially or simultaneously. The following materials and methods were used to produce the date described herein.

[0050] LV-GFP vector The LV-GFP vector used herein carries the vesicular stomatitis virus-G (VSV-G) envelope protein, which is known to target a wide variety of cell types.

[0051] Stability of LV-GFP in delivery solution The stability of LV-GFP in the delivery solution was assessed over a 1 hour period by assessing precipitation under a microscope.

[0052] LV-GFP delivery Primary human PBMCs were thawed and activated with Miltenyi's CD3 and CD28 antibodies for 3 days. After 3 days of activation culture, cells underwent SOLUPORE™ process or static transduction with LV-GFP (MOI=2.5). Cells were allowed to recover for 72 hours before GFP expression was assessed by flow cytometry.

[0053] Stability of LV-GFP in delivery solution Prior to the present invention, the SOLUPORE™ process delivery had not previously been combined with a viral preparation, and therefore it was not known whether there would be solubility issues.

[0054] When the solutions were viewed under a microscope during stability analysis, no aggregation or precipitation was observed. Additionally, certain other cargoes showed low levels of aggregation when combined with delivery solutions, causing the Solupore® nebulizer to block during spraying. When the delivery solutions containing the virus were loaded into the Solupore® nebulizer and sprayed, no blocking occurred at any time over the course of the study, further indicating that no aggregation or precipitation occurred.

[0055] Cell viability, expansion, and GFP expression following LV-GFP delivery LV-GFP was delivered to T cell cultures by the SOLUPORE™ process and compared to standard static methods of LV transduction.

[0056] Cell viability was measured at various time points before and after viral delivery. At all time points, the viability of soluporated cells was comparable to that of control transduced cells (Figure 3).

[0057] The cumulative fold expansion of T cells was measured up to 96 hours after viral delivery. The expansion of solupore-treated cells was comparable to that of control-transduced cells (Figure 4).

[0058] GFP expression was measured on days 3 and 4 after delivery. GFP expression efficiency was higher in solupore-treated T cells compared to control-transduced cells (Figure 5). On day 3, the efficiency was 39.73±2.83% and the comparator 25.2±1.48% for solupore-treated and control-transduced cells, respectively. On day 4, the efficiency was 40.27±2.67% and the comparator 26.83±1.38% for solupore-treated and control-transduced cells, respectively.

[0059] Efficient viral delivery of virus to a population of cells The data herein demonstrate that the SOLUPORE™ process is compatible with the delivery of viruses to activated T cells. No precipitation or aggregation was observed when mixed with the SOLUPORE™ process delivery solution. The virus solution could be sprayed and the target cells were successfully transduced. The viability and expansion rate of solupore-treated T cells were not affected.

[0060] GFP expression was higher in solupore-treated T cells compared to control-transduced cells, indicating that the SOLUPORE™ process enhances viral transduction of T cells.

[0061] Taken together, these findings demonstrate that the SOLUPORE™ process is suitable for delivery of viruses to cells and is superior to standard methods.

[0062] Because the SOLUPORE™ process is suitable for viral delivery, it is possible to use the solupore treatment in cell therapy manufacturing processes that include viral transduction. Due to the cost of the virus, transduction is the main cost driver in CAR T cell manufacturing. Because the SOLUPORE™ process enhances viral transduction, it is now possible to achieve similar levels of transduction efficiency using less virus, thus lowering costs.

[0063] Various cargoes can be delivered simultaneously by the SOLUPORE™ process. The demonstration herein that the SOLUPORE™ process is compatible with virus delivery means that the SOLUPORE™ process can be used to co-deliver viruses with other cargoes. These other cargoes can be other viruses, or can be proteins, nucleic acids, small molecules, or complexes thereof. The ability to co-deliver cargoes means that the manipulation steps that would otherwise occur in different process steps can be combined into a single process step. This process has great benefits for the manufacturing process, including cost, time, and labor. In addition, fewer process steps means less handling and contamination risk, as well as simplification of the process. Alternatively, the virus is delivered in sequence before or after the other cargoes.

[0064] SOLUPORE ™ process allows cargo to be delivered to non-activated T cells. Lentiviral vectors have very low transduction efficiency in non-activated T cells. Therefore, SOLUPORE ™ process increases the transduction efficiency of lentivirus in non-activated T cells.

[0065] A core feature of the SOLUPORE™ process device is the ease of medium exchange. Once the cell-containing solution is transferred into the device, the liquid can be drained and replaced with a different liquid. In this way, liquid handling steps are possible. Such liquid handling steps can include, for example, washing steps. In viral transduction and other cell manufacturing processes, washing steps are often required. The Solupore® device allows for the incorporation of such steps into the manufacturing process.

[0066] The Solupore® technology is also scalable, which means that viral transduction using this method can be carried out on small scale for early and preclinical studies, and on larger scales for process development and clinical applications.

[0067] Advantages of the method and surprising results How viruses differ from other cargoes Viruses are microorganisms, i.e. submicroscopic infectious agents that replicate only inside the living cells of an organism. Viruses can infect all types of living organisms, from animals and plants to microorganisms, including bacteria and archaea. Viruses consist of a core of genetic material, either DNA or RNA, surrounded by a protective coat called a capsid, made up of proteins. Sometimes the capsid is surrounded by an additional spikey coat called an envelope. Viruses are able to latch onto a host cell and enter its interior.

[0068] Previously, the cargo used in Solupore™ was either a single class of molecule (such as a protein or nucleic acid), or a mixture of molecules, or a complex of molecules (such as the Cas9 ribonucleoprotein (RNP)). Viruses are more complex agents in terms of structure and function. In terms of structure, the genetic material is a single-stranded RNA that is contained within a protein capsid and is therefore encapsulated. Lentiviral virions contain 2% nucleic acid, 60% protein, and 35% lipids and 3% carbohydrates. It is expected that the structure must remain intact for the virus to successfully infect a cell. In terms of function, it is expected that once inside the cell, the virus must remain capable of releasing its genetic material in order to express the transgene. It is also expected that the host cell must remain viable and functional in order for the viral transgene to be subsequently expressed.

[0069] In terms of size, lentiviruses range from 80-100 nm in diameter. In contrast, with respect to the components of a typical RNP, the SpCas9 protein is approximately 7.5 nm in hydrodynamic diameter (160 kDa) with a net positive surface charge, and the sgRNA is 5.5 nm in hydrodynamic diameter (approximately 31 kDa) and negatively charged (Bioconjug Chem. 2017 April 19; 28(4): 880-884). Thus, RNPs are significantly smaller than lentiviral viroids.

[0070] Surprising Results and Unexpected Benefits of Viral Transduction Using the Solupore™ Process The Solupore™ process has been used to deliver relatively simple cargo molecules (as opposed to complex cargo such as microorganisms) into mammalian cells. The process involves multiple steps, and prior to the present invention, it was not known whether one, some, and / or all of these steps would be compatible with successful viral infection as outlined above.

[0071] 1. Mixing the cargo with the delivery solution It was not known whether the virus preparation described herein was compatible with the delivery solution. Occasionally, other cargoes have been observed to aggregate and thus impede the process. Such aggregation causes several problems, such as loss of cargo functionality and blockage of the Solupore™ fluidic pathway. Sometimes, the extent of this aggregation is substantial, so that it is immediately evident and the experiment is aborted. However, sometimes, aggregation is undetectable during the Solupore™ experiment and is only identified when analytical assays are performed to test for cargo activity in the target cells. Subsequent troubleshooting led to the detection of a partial blockage of the fluidic pathway, which was not enough to interfere with the completion of the experiment, but likely altered the results.

[0072] For this study, compatibility testing of the delivery solution was performed and, surprisingly, no aggregation was observed under the microscope. Additionally, no blockage of the device was observed. Additionally, a successful outcome was achieved, i.e., expression of the GFP transgene. These findings indicate that the viral preparation was compatible with the delivery solution. This was surprising given the complexity of the viral material. Furthermore, the lack of observable blockage of the fluid pathway indicates that no aggregation occurred within the tubing or while the solution was being transferred through the system. Again, this was surprising given the complexity and size of the viral material.

[0073] 2. Form droplets of a certain size and velocity It was not known whether the virus preparation described herein was compatible with the aerosolization process.During the process, a custom-made nebulizer that has never been used with viruses before is used to break the delivery solution into droplets.However, the results described herein show that the virus preparation was compatible.

[0074] 3. Move those droplets a specific distance These droplets (e.g., delivery solution that has been divided into droplets using a custom-made nebulizer) are then advanced toward target cells over a distance of 75 mm. During the transition, the droplets will be subject to evaporation and condensation, similar to a typical aerosolization process. Prior to this study, it was not known whether this would adversely affect the virus's ability to (1) remain active, (2) enter cells, and (3) continue to express the transgene. The results show that the virus preparation was compatible with all three steps of the process.

[0075] 4. Droplets land on target cells The droplets landed on the target cells with a force of approximately 17 g, and it was not known whether this was compatible with preserving the integrity and functionality of the viral particles. The results showed that the viral preparation was compatible with this process.

[0076] 5. Incubate the cells with the applied solution The delivery solution is applied onto the cells and allowed to incubate with the cells for 30 seconds. 50-100 microliters is 2827.43 mm 2 Because only a very small volume is applied to a relatively large area, evaporation and drying was expected, and it was not known whether this would adversely affect the ability of the virus to infect cells. The results show that the virus preparation was compatible with this process.

[0077] 6. Applying the second solution After the incubation step, a second solution is applied onto the cells. It was not known whether this would adversely affect the virus's ability to infect cells. It could result in dilution of the extracellular virus or adversely affect the virus in some other way. The results indicate that the virus preparation was compatible with this process.

[0078] 7. Cells are removed from the SOLUPORE™ process chamber To remove the cells from the chamber and place them in culture, it is necessary to flush the chamber several times and agitate the cell suspension. It was not known whether this was likely to interfere in any way with the viral infection process, for example by damaging the cells so that they could not successfully express the viral transgene. The results show that the viral preparation was compatible with this process.

[0079] 8. Culture the cells for several days During viral infection, cytoplasmic nucleic acids from the virus are recognized by intracellular specific sensors of the host. The efficacy of this recognition system is crucial for triggering innate host defenses, which in turn stimulate a more specific adaptive immune response against the virus (Lee, H., Chathuranga, K. & Lee, J. Intracellular sensing of viral genomes and viral evasion. Exp Mol Med 51, 1-13 (2019)). Prior to this study, it was not known whether the Solupore™ process described above affected cells in some manner that would cause them to be more sensitive than normal to the presence of the virus. If this occurred, cell viability could be compromised, and the cells could not survive the four-day culture period post-infection. Alternatively, the cells could survive, but their health could be compromised such that they could not express the transgene. Surprisingly, the cells into which the virus was delivered experienced approximately 90% survival.

[0080] Characterization of the viral infection process and droplets Atomization of the lentivirus in the transfection chamber is a separate process from the SOLUPORE™ process. As described herein, the cargo delivered to the population of cells is a biologically active and viable virus (e.g., a lentivirus).

[0081] Typical titers of lentivirus are 10 per milliliter. 6 ~10 7Transducing units (TU / ml) of lentivirus at these concentrations are in the range of 1000 to 15000, and the viscosity of lentivirus at these concentrations is highly dynamically viscous compared to water / ethanol mixtures. To illustrate, (Table 1) the dynamic viscosity of water at room temperature is close to 1 mPa s, the dynamic viscosity of ethanol is close to 0.1 mPa s, the dynamic viscosity of olive oil is close to 60 0.1 mPa s, and the dynamic viscosity of castor oil is close to 600 0.1 mPa s. The dynamic viscosity of lentivirus is reported by Tran, Reginald, PhD Thesis, Georgia Tech (2016) to be 6913 mPa s, which is close to being 1 log more viscous than castor oil. Sterile filtered 1% bovine serum albumin (BSA) has been found by some to reduce molecular interactions that can cause viral particles to "stick" to injection devices (Jasnow A. et al. Methods Mol Biol. "Construction of Cell-Type Specific Promoter Lentiviruses for Optically Guiding Electrophysiological Recordings and for Targeted Gene Delivery" 2009; 515: 199-213, incorporated herein by reference in its entirety).

[0082] Dynamic viscosity is an important factor in atomization. Experimental studies of atomization in an internally mixed twin-fluid atomizer, such as that used in the SOLUPORE™ process, over a wide range of liquid viscosities, gas supply pressures, and gas-to-liquid mass ratios (GLR) have been conducted. See, for example, Li, Z. et al. "Effect of liquid viscosity on atomization in an internal-mixing twin-fluid atomizer" Fuel vol. 103; Jan 2013 pages 486-494, which is incorporated herein by reference in its entirety. Among all test conditions, the finest spray was obtained at an axial distance of 150 mm. However, when the viscosity was increased to 120 mPa s, the droplet size distribution changed significantly. Higher viscosity droplets produced larger droplets (e.g., 1-2 logs larger than the present droplets produced by the SOLUPORE™ process, measured droplet size distribution, FIG. 51). Larger droplets represented a larger proportion of the droplet population (distribution) and the dropoff in droplet velocity along the spray axis was stronger at higher viscosities.

[0083] A table showing the dynamic viscosities of common liquids is provided below (and a graph is provided in Figure 52).

[0084] The absolute or dynamic viscosities of some common liquids at a temperature of 300 K are given below: TIFF0007637072000002.tif177150

[0085] It can be concluded that nebulization of lentivirus in the SOLUPORE™ process produces larger, slower moving droplets that experience a different layer of cells below than the previously described SOLUPORE™ process. Consequently, this nebulization process resulted in transfection levels approaching 30%, a surprising and unexpected observation.

[0086] Virus infection process The dynamic viscosity of water is close to 1 mPa s (millipascal second). The dynamic viscosity of ethanol / water mixtures is also close to 1 mPa s. The dynamic viscosity of aqueous solutions that can contain ethanol concentrations of 5-30%. Aqueous solutions contain 75-98% H 2 HO, 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), with a viscosity in the region of 2 mPa s.

[0087] Titer 10 7 ~10 8 The dynamic viscosity of lentivirus in TU / mL is close to 6913 mPa s. As the viscosity of a fluid increases, it tends to form larger droplets when sprayed at a given spray pressure, e.g., 1.7 bar. A spray consisting of smaller droplets has a much larger surface area per volume than a spray consisting of larger droplets. Furthermore, the droplets have a lower surface tension than water, and therefore the droplets are even larger. The cells then undergo an entirely different process. As such, finer sprays can spread better over their target surface. This effect is relatively small for fluids with viscosities below 10 mPa s, but becomes more pronounced at higher dynamic viscosities. A fluid with a higher dynamic viscosity than water or a water / ethanol mixture will have a higher average droplet size for any given flow rate and pressure. The relationship between the mechanical properties of fluids can be calculated by the following commonly accepted formula: TIFF0007637072000003.tif26128In formula, D f = corrected droplet size for the fluid, D w = calculated droplet size for water, V f = viscosity of the fluid (viscosity in mPa s; water = 1.0 mPa s, lentivirus = 6913 mPa s).

[0088] From equation [1], lentivirus droplets sprayed under the same pressure and flow conditions as water / ethanol mixtures (e.g., virus and ethanol concentrations between 5 and 30% and 75 and 98% H2O). 2 A droplet containing a volume of an aqueous solution containing one or more of: 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) can be calculated to have a droplet size close to 5.9 times larger than a water / ethanol droplet. As described in International Application WO 2016 / 065341 (incorporated herein by reference in its entirety), droplets in the size range of 30 μm to 100 μm and 50 μm to 80 μm in diameter have been described. Generally, in the methods described herein, when the aqueous solution being sprayed contains a virus (e.g., a lentivirus), the droplet size range is about 150 μm to 600 μm in diameter, or about 177 μm to 590 μm in diameter. In other examples, the droplet diameter size is 200 μm to 600 μm, or about 300 μm to 600 μm, or about 400 μm to 600 μm, or about 500 μm to 600 μm. In other examples, the droplet size of the invention herein can be greater than 600 μm, for example, about 600 μm to 1000 μm in diameter, or about 600 μm to 900 μm, or about 600 μm to 800 μm, or about 600 μm to 700 μm in diameter. In some examples, the droplet size can be characterized by a diameter of up to 1000 μm, for example, 150 μm to 1000 μm.

[0089] These droplets are much larger than anticipated or described in WO 2016 / 065341, where it is noted that "some of the colloidal droplets created may be too large for a given intracellular delivery application. Some of the colloidal droplets created may be so large that cell death may occur despite the creation of colloidal droplets of appropriate size." See paragraph

[0172] of WO 2016 / 065341. It was therefore unexpected and surprising that cells (e.g., cells contacted with an aqueous solution containing the virus) tolerated such a different process compared to the SOLUPRE™ process described in WO 2016 / 065341, and that the cells were infected with lentivirus and viable.

[0090] Droplet size characteristics The larger diameter droplets of the present invention described herein have larger volume and weight, move slower, and impact cell layers with greater force.For example, when diameter increases by 5.9 times, droplet volume increases by nearly 206.8 times.Therefore, the fluid dynamics of this system is separate from that described with reference to WO 2016 / 065341, and constitutes a new virus infection process.

[0091] Delivery of cargo to cells The difficulty of transfecting molecules into cells has plagued research and treatment, such as cell therapy, gene therapy, and genetic modification, for decades.The present invention provides a solution to such problems for complex cargo entities such as viruses.Generally, a method for delivering a payload across the plasma membrane of a cell includes the steps of providing a population of cells, and contacting the population of cells with a volume of aqueous solution, the aqueous solution comprising the payload and a concentration of alcohol greater than 2 percent, the volume being a function of (i) the exposed surface area of ​​the population of cells; or (ii) the number of cells in the population of cells, and contacting the population of cells with the volume of aqueous solution is performed by propelling the aqueous solution with gas to form a spray.

[0092] The reason for the difficulty in transfecting certain types of cells may be that non-adherent cells lack cell surface heparan sulfate proteoglycans, which are molecules responsible for the adhesion of cells to the extracellular matrix.Transfection methods such as electroporation and / or nucleofection have the drawback of impairing cell viability, the ability of cells to resume proliferation after treatment, and cell function, such as the immune activity of lymphocytes.The transfection / transduction compositions and methods described herein are characterized by not having such drawbacks and therefore having significant advantages over previous methods of introducing cargo molecules into mammalian cells, such as non-adherent / suspension cells that are difficult to transfect.

[0093] The present invention is based on the surprising discovery that a compound or a mixture of compounds (compositions) is delivered into the cytoplasm of eukaryotic cells by contacting the cells with a solution containing a virus and an agent that reversibly permeates or dissolves cell membrane.Preferably, the solution is delivered to the cells in the form of a spray, for example, aqueous particles (see, for example, PCT / US2015 / 057247 and PCT / IB2016 / 001895, which are incorporated herein by reference in their entirety).For example, the cells are coated with a spray, but are not immersed or submerged in the solution that contains the delivery compound.Exemplary agents that permeate or dissolve the membrane of eukaryotic cells include alcohol and detergents, for example ethanol and Triton X-100, respectively. Other exemplary surfactants, e.g., surface active agents, include polysorbate 20 (e.g., Tween 20), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), sodium dodecyl sulfate (SDS), and octylglucoside.

[0094] Examples of conditions for achieving coating of the population of cells to be coated include delivery of a fine particle spray, e.g., conditions exclude dripping or pipetting a bolus volume of solution onto the cells such that a substantial population of cells is immersed or submerged by the volume of fluid.Thus, the mist or spray includes a ratio of the volume of fluid to the volume of cells.Alternatively, the conditions include a ratio of the volume of mist or spray to the exposed cell area, e.g., to the area of ​​the cell membrane exposed when cells are present as a confluent or substantially confluent layer on a substantially flat surface such as a tissue culture vessel, e.g., a well of a tissue culture plate, e.g., the bottom of a microtiter tissue culture plate, or on a filter membrane, e.g., a filter plate, and the cells are exposed by removing the medium.

[0095] Advantages of the claimed method of the present invention The methods described herein offer numerous advantages over known techniques and applications. Exemplary advantages include: 1) the same number of cells can be transfected with less virus (or alternatively, reducing the amount of virus required to infect a population of cells); 2) the ability to transfect more cells using the same amount of virus (or increase the number of cells transfected using the same amount of virus used in current protocols); 3) increased viral uptake, 4) Increased cell viability, 5) increased transfection efficiency, and 6) Production efficiency (the ability to carry out multiple processes in a single process or area).

[0096] "Cargo" or "payload" is a term used to describe a compound, or composition, that is delivered across the cell plasma membrane via aqueous solution into the interior of a cell.

[0097] In one aspect, delivering a virus across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the virus and an alcohol content greater than 2 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.

[0098] In another aspect, a composition for delivering a virus across the plasma membrane of a cell includes an aqueous solution containing the virus, an alcohol concentration greater than 2 percent (e.g., greater than 5 percent), more than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffer. For example, the concentration of alcohol, e.g., ethanol, does not exceed 50%.

[0099] In another aspect, a composition for delivering a virus across the plasma membrane of a cell includes an aqueous solution comprising the virus, more than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffer. For example, the aqueous solution does not include alcohol.

[0100] One or more of the following characteristics may be included in any workable combination: The volume of solution delivered to the cells is in units, e.g., multiple droplets on a spray, e.g., aqueous particles. The volume is stated relative to individual cells, or relative to the exposed surface area of ​​a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, the volume is 6.0×10 per cell. -7 ~ 7.4 x 10 cells per microliter -4 The volume can be 4.9 x 10 cells per microliter. -6 ~2.2 x 10 cells per microliter -3 The volume is 9.3 x 10 cells per microliter. -6 ~2.8 x 10 cells per microliter -5The volume can be about 1.9×10 cells per microliter. -5 The volume can be in microliters, with about being within 10 percent. The volume is 6.0 x 10 cells per -7 ~2.2 x 10 cells per microliter -3 The volume is 2.6 x 10 per square micrometer of exposed surface area. -9 microliters ~ 1.1 x 10 per square micrometer of exposed surface area -6 The volume can be from 5.3×10 microliters per square micrometer of exposed surface area to 1.6×10 microliters per square micrometer of exposed surface area. -7 The volume can be about 1.1×10 per square micrometer of exposed surface area. -7 It can be microliters. About can be within 10 percent.

[0101] Cell confluency refers to cells that are in contact with each other on a surface. For example, it can be expressed as an estimated (or counted) percentage, for example, 10% confluency means that 10% of the surface, for example of a tissue culture vessel, is covered with cells, and 100% means that it 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 spun down, pulled down by vacuum or tissue culture medium suction from the top of the cell mass, or removed by suction or vacuum removal from the bottom of the vessel with a filter setting.

[0102] The step of contacting the population of cells with the volume of aqueous solution can be carried out by propelling the aqueous solution with a gas to form a spray. The gas can include nitrogen, ambient air, or an inert gas. The spray can include a volume of discrete units ranging in size from greater than 150 μm in diameter.

[0103] A total volume of 20 μl of the aqueous solution was sprayed onto approximately 1.9 cm 2 The total volume of 10 μl of aqueous solution can be delivered to a cell-occupied area of ​​approximately 0.95 cm, e.g., one well of a 24-well culture plate. 2 The spray is delivered to an area occupied by cells of, for example, one well of a 48-well culture plate. The spray is optionally delivered to a larger area, for example, the size of a Petri dish or even larger, of any size appropriate for the diameter of the area covered by the spray emitted from the sprayer.

[0104] Typically, the aqueous solution contains the virus to be delivered across the cell membrane and into the cells, and the second volume is a buffer or culture medium that does not contain a payload. Alternatively, the second volume (buffer or medium) can also contain the virus. In some examples, the second volume contains a different type of cargo, such as a nucleic acid, protein, or chemical compound (i.e., a non-viral cargo). Alternatively, the first solution contains a non-viral cargo and the second solution contains a viral cargo. The viral and non-viral cargo may be delivered sequentially or simultaneously, i.e., in the same delivery solution, as described above. In some embodiments, the aqueous solution contains a payload and an alcohol, and the second volume does not contain alcohol (and optionally does not contain a payload). The population of cells can be contacted with the aqueous solution for 0.1 to 10 minutes before adding a second volume of buffer or culture medium to sink or suspend the population of cells. The buffer or culture medium can be phosphate buffered saline (PBS). The population of cells can be contacted with the aqueous solution for 2 seconds to 5 minutes before adding a second volume of buffer or culture medium to sink or suspend the population of cells. The population of cells can be contacted with an aqueous solution containing, for example, a virus for 30 seconds to 2 minutes before adding a second volume of buffer or culture medium without, for example, a virus to sink or suspend the population of cells. The population of cells can be contacted with the spray for about 1 to 2 minutes before adding a second volume of buffer or culture medium to sink or suspend the population of cells. During the time between the spray of the cells and the addition of the buffer or culture medium, the cells remain hydrated by a layer of moisture from the spray volume.

[0105] The aqueous solution may contain ethanol concentrations of 2-30%, 2-40%, or 2-50%. 2The delivery solution may include one or more of the following: 106 mM KCl, 2-45% ethanol, 6-91 mM sucrose, 2-500 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). For example, the delivery solution contains 106 mM KCl and 27% ethanol. In embodiments, the aqueous solution includes 27% ethanol for Flexi (e.g., small scale). In embodiments, the aqueous solution includes 12% ethanol in large scale systems.

[0106] The population of cells 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, neural cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, induced pluripotent stem cells (iPSCs), and human embryonic kidney (HEK) cells, or immortalized cells such as cell lines. In a preferred embodiment, the population of cells includes non-adherent cells, for example, the percentage of non-adherent cells 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 and immortalized cells (e.g., cells of cell lines). Exemplary non-adherent / suspension cells include primary hematopoietic stem cells (HSCs), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, dendritic cells, tumor infiltrating lymphocytes (TILs), or cell lines such as Jurkat T cell lines. In other examples, NK cell lines are used, including NK92 and KHYG1.

[0107] The population of non-adherent cells can be substantially confluent, e.g., greater than 75 percent confluent. Cell confluency refers to cells in contact with each other on a surface. For example, this can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of the surface, e.g., of a tissue culture vessel, is covered with cells, and 100% means complete coverage. 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 spinning down, pulling down by vacuum or tissue culture medium aspiration from the top of the cell population, or by aspiration or vacuum removal from the bottom of the vessel. Additional removal methods may include gravity, or the use of magnetic beads and magnets. The population of cells can form a monolayer of cells.

[0108] The alcohol may be selected from methanol, ethanol, isopropyl alcohol, butanol, and benzyl alcohol. The salt may be selected from NaCl, KCl, Na 2 HPO 4 , K.H. 2 PO 4 , and C 2 H 3 O 2 NH. In a preferred embodiment, the salt is KCl. The sugar can include sucrose. The buffer can include 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid.

[0109] The subject matter relates to a method for delivering viruses across plasma membranes. The subject matter finds utility in the field of intracellular delivery, and has applicability in, for example, the delivery of molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues, or organs. The subject matter's method includes: introducing molecules into an aqueous composition to form a matrix; atomizing the matrix into a spray; and contacting the matrix with a plasma membrane.

[0110] The subject matter relates to compositions for use in the delivery of viruses across plasma membranes.The subject matter finds utility in the field of intracellular delivery, and has applicability in the delivery of molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues, or organs.The subject matter compositions include alcohol; salt; sugar; and / or buffering agents.

[0111] Exemplary methods described herein include aqueous solutions containing alcohol. The term "alcohol" refers to a polyatomic organic compound that contains a hydroxyl (-OH) functional group attached to at least one carbon atom. The alcohol may be a monohydric alcohol and may contain at least one carbon atom, such as methanol. The alcohol may contain at least two carbon atoms (e.g., ethanol). In other aspects, the alcohol may contain at least three carbons (e.g., isopropyl alcohol). The alcohol may contain at least four carbon atoms (e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol). Exemplary payloads may contain up to 50% (v / v) alcohol, more preferably, the payloads include 2-45% (v / v) alcohol, 5-40% alcohol, and 10-40% alcohol. The aqueous solutions may contain 20-30% (v / v) alcohol.

[0112] In some aspects of the subject matter, the virus is present in an isotonic solution or buffer.

[0113] In some examples, "S buffer" comprises a hypotonic physiological buffer (78 mM sucrose, 30 mM KCl, 30 mM potassium acetate, 12 mM HEPES) at 4°C for 5 minutes (Medepalli K. et al., Nanotechnology 2013; 24(20); incorporated herein by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in S buffer. S buffer is further described in International Application WO 2016 / 065341, e.g., at paragraphs

[0228] to

[0229] , incorporated herein by reference in its entirety.

[0114] According to the present subject matter, the aqueous solution may include at least one salt. The salt may be NaCl, KCl, Na 2 HPO 4 , C 2 H 3 O 2 NH 4 , and K.H. 2 PO 4 For example, the KCl concentration is in the range of 2 mM to 500 mM. In some preferred embodiments, the concentration is higher than 100 mM, for example, 106 mM.

[0115] In an example, the aqueous solution contains 32.5 mM potassium chloride (KCl) 106 mM, Hepes 5 mM, ethanol (EtOH) 12% v / v, and water for injection (WFI).

[0116] According to exemplary methods of the present subject matter, the aqueous solution may include a sugar (e.g., sucrose, or a disaccharide). According to exemplary methods, the payload includes less than 121 mM sugar, 6-91 mM, or 26-39 mM sugar. Still further, the aqueous solution (e.g., including the virus) includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload includes 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

[0117] According to an exemplary method of the present subject matter, the aqueous payload (e.g., including the virus) may include a buffer (e.g., a weak acid or a weak base). The buffer may include a zwitterion. According to an exemplary method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The aqueous solution (e.g., including the virus) may include less than 19 mM of the buffer (e.g., 1-15 mM, or 4-6 mM, or 5 mM of the buffer). According to an exemplary method, 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 aqueous solution (e.g., including the virus) includes 5 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0118] According to an exemplary method of the present subject matter, the aqueous solution (e.g., containing a virus) comprises ammonium acetate. The aqueous solution (e.g., containing a virus) may comprise less than 46 mM ammonium acetate (e.g., 2-35 mM, 10-15 mM, or 12 mM ammonium acetate). The aqueous solution (e.g., containing a virus) may comprise 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.

[0119] The volume of aqueous solution that is effected by injecting the aqueous solution with a gas may include compressed air (eg, ambient air), while other implementations may include inert gases such as helium, neon, and argon.

[0120] In certain aspects of the present subject matter, the population of cells may include adherent cells (eg, lung, kidney, immune cells, such as macrophages) or non-adherent cells (eg, suspension cells).

[0121] In certain aspects of the present subject matter, the population of cells may be substantially confluent, including substantially greater than 75 percent confluent. In preferred implementations, the population of cells may form a single monolayer.

[0122] In an aspect, the step of contacting the population of cells with the volume of aqueous solution can be performed by propelling the aqueous solution with a gas 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, 10 minutes) before adding a second volume of buffer or culture medium to submerge or suspend the population of cells.

[0123] In various embodiments, the population of cells includes at least one of primary cells or immortalized cells. For example, the population of cells may include mesenchymal stem cells, lung cells, neural cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSCs), 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 may include CD8+ T cells or CD4+ T cells. In some aspects, CD3 + The CD8+ subpopulation of T cells is used. CD8 + T cells can be purified from PBMC population by positive isolation using anti-CD8 beads or negative selection using anti-CD4 beads.In some aspects, primary NK cells can be isolated from PBMC, umbilical cord-derived NK, iPSC-derived NK, and GFP mRNA can be delivered by platform delivery technology.In additional aspects, NK cell line, for example, NK92, can be used.

[0124] Cell types also include cells that have been previously modified to enhance their therapeutic efficacy, e.g., T cells, NK cells, and MSCs. For example: T cells or NK cells expressing chimeric antigen receptors (CAR T cells, CAR NK cells, respectively); endosomes; transduced cells and endosomes derived from them, e.g., MSCs; T cells expressing modified T cell receptors (TCRs); MSCs that have been virally or non-virally modified to overexpress therapeutic proteins that complement their innate properties (e.g., delivery of Epo with lentiviral vectors or BMP-2 with AAV-6) (reviewed in Park et al, Methods, 2015 Aug;84-16.); MSCs that have been primed with non-peptide drugs or magnetic nanoparticles for enhanced efficacy and external control of targeting, respectively (Park et al., 2015); MSCs that have been functionalized with targeting moieties to enhance their homing to the treatment site using enzymatic modification (e.g., fucosyltransferase), chemical conjugation (e.g., modification of SLeX on MSCs by using N-hydroxy-succinimide (NHS) chemistry), or non-covalent interactions (e.g., engineering of the cell surface with palmitated proteins that act as hydrophobic anchors for subsequent conjugation of antibodies) (Park et al., 2015). For example, T cells, e.g., primary T cells or T cell lines that have been modified to express a chimeric antigen receptor (CAR T cells), may 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, with the aim of editing the gene encoding the CAR and thereby reducing or stopping the expression of the CAR in the modified T cells. In other examples, the methods and systems herein are used to edit various genes in engineered cells to enhance the activity of CAR-T cells, such as editing PD-1 to enable CAR-T cells to circumvent immune system checkpoint blockade. In other examples, the methods and systems herein are used to engineer a mixture of cell types in a single step.For example, a mixture of different T cell populations, or a mixture of different modified T cells, or a mixture of T cells and NK cells is used.

[0125] Aspects of the present invention relate to the viral delivery of gene editing compounds and complexes to cells and tissues, such as the delivery of Cas-gRNA ribonucleoprotein for genome editing in primary human T cells, hematopoietic stem cells (HSCs), and mesenchymal stromal cells (MSCs).In some instances, the mRNA encoding such proteins is delivered to cells.

[0126] In some embodiments, the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or an Flp recombinase.In additional embodiments, the gene editing protein can be a fusion protein (megaTAL) that combines a homing endonuclease and the modular DNA binding domain of TALEN.For example, the megaTAL can be delivered as a protein, or alternatively, the mRNA that codes for the megaTAL protein is delivered to the cell.

[0127] Various aspects of the CRISPR-Cas system are known in the art. Non-limiting aspects of this system are described, for example, in U.S. Patent No. 9,023,649, issued May 5, 2015; U.S. Patent No. 9,074,199, issued July 7, 2015; U.S. Patent No. 8,697,359, issued April 15, 2014; U.S. Patent No. 8,932,814, issued January 13, 2015; PCT International Patent Application Publication No. WO 2015 / 071474, published August 27, 2015; Cho et al., (2013) Nature Biotechnology Vol 31 No 3 pp 230-232 (including supplementary information); and Jinek et al., (2012) Science Vol 337 No 6096 pp 816-821, the entire contents of each of which are incorporated herein by reference.

[0128] 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, Cs The Cas9 enzymes include m4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, or their homologs or modified versions. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein can be found in SwissProt database under accession number Q99ZW2 and in NCBI database under accession number Q99ZW2.1. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of Cas9 protein amino acid sequence. Another non-limiting example is the Streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme, such as Cas9, has DNA cleavage activity. In certain embodiments, the CRISPR enzyme is Cas9, which may be from S. pyogenes or S. pneumoniae. In various embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, such as 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 base pairs, or more, from the first or last nucleotide of the target sequence.In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cut one or both strands of the target polynucleotide containing the target sequence.For example, the aspartic acid to alanine substitution in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cuts both strands to a nickase (cuts a single strand).Other examples of the mutation that makes Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A.In an aspect of the present invention, nickase can be used for genome editing via homologous recombination.

[0129] In certain embodiments, Cas9 nickases can be used in combination with guide sequences, such as two guide sequences that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ.

[0130] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to create a mutated Cas9 that substantially lacks all DNA cleavage activity. D10A mutation can be combined with one or more of H840A, N854A, or N863A mutations to create a Cas9 enzyme that substantially lacks all DNA cleavage activity. In certain embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less than its non-mutated counterpart. Other mutations may be useful; if Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations can be made at the corresponding amino acids to achieve similar effects.

[0131] In certain embodiments, the delivered protein (such as a Cas protein or variant thereof) may include an intracellular localization signal. For example, a Cas protein in an RNP may include an intracellular localization signal. Depending on the context, for example, a fusion protein comprising Cas9 and a nuclear localization signal may be referred to herein as "Cas9" without specifying that it comprises a nuclear localization signal. In some embodiments, the payload (such as an RNP) comprises a fusion protein comprising a localization signal. For example, the fusion protein may contain a nuclear localization signal, a nucleolar localization signal, or a mitochondrial targeting signal. Such signals are known in the art, and non-limiting examples are described in Kalderon et al., (1984) Cell 39 (3 Pt 2): 499-509;Makkerh et al., (1996) Curr Biol. 6 (8): 1025-7;Dingwall et al., (1991) Trends in Biochemical Sciences 16 (12): 478-81;Scott et al., (2011) BMC Bioinformatics 12:317 (7 pages);Omura T (1998) J Biochem. 123(6): 1010-6;Rapaport D (2003) EMBO Rep. 4(10):948-52;and Brocard & Hartig (2006) Biochimica et Biophysica Acta (BBA) -Molecular Cell Research 1763(12): 1565-1573, the contents of each of which are incorporated herein by reference. In various embodiments, the Cas protein may contain more than one localization signal, such as two, three, four, five, or more nuclear localization signals. In some embodiments, the localization signal is present at the N-terminus of the Cas protein, and in other embodiments, the localization signal is present at the C-terminus of the Cas protein.

[0132] In some embodiments, the enzyme coding sequence encoding CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells.Eukaryotic cells can be or be derived from specific organisms, such as mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates.Generally, codon optimization refers to the process of modifying nucleic acid sequences to enhance expression in target host cells by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of native sequence with the codon that is more frequently or most frequently used in the gene of the host cell, while maintaining native amino acid sequence.Different species show a certain bias for certain codons of certain amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codons being translated and on the availability of specific transfer RNA (tRNA) molecules. The predominance of tRNAs selected in a cell is generally a reflection of the codons most frequently used in peptide synthesis.

[0133] Thus, genes can be adjusted for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in "codon usage databases," and these tables can be adapted in a number of 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 are also available for codon-optimizing a particular sequence for expression in a particular host cell, for example, Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0134] Generally, guide sequence is any polynucleotide sequence that has sufficient complementarity with target polynucleotide sequence to hybridize with target sequence and direct sequence-specific binding of CRISPR complex to target sequence.In some embodiments, the degree of complementarity between guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or is greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, when optimally aligned using suitable alignment algorithm.In some embodiments, the degree of complementarity is 100%. Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which 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, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length, or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In certain embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length, or fewer.The ability of guide sequence to direct sequence-specific binding of CRISPR complex to target sequence can be evaluated by any suitable assay.For example, the components of CRISPR system sufficient to form CRISPR complex, including the guide sequence to be tested, can be provided to host cell with corresponding target sequence, for example, by transfection of vectors encoding the components of CRISPR sequence, and then evaluating preferential cleavage in target sequence, such as by Surveyor assay as described herein.Similarly, the cleavage of target polynucleotide sequence can be evaluated in test tube by providing the components of CRISPR complex, including target sequence, guide sequence to be tested, and control guide sequence different from test guide sequence, and comparing the binding or cleavage rate at target sequence between test guide sequence reaction and control guide sequence reaction.

[0135] CRISPR-Cas technology, which facilitates genome engineering in a wide range of cell types, is rapidly evolving. It has been recently shown that delivery of Cas9-gRNA editing tools in the form of ribonucleoproteins (RNPs) produces several benefits compared to delivery of plasmids encoding Cas9 and gRNA. The benefits include faster and more efficient editing, fewer off-target effects, and less toxicity. RNPs have been delivered by lipofection and electroporation, but the remaining limitations of these delivery methods, especially for certain clinically relevant cell types, include toxicity and low efficiency. Therefore, there is a need to provide a delivery approach for delivering biologically relevant payloads, such as RNPs, across the plasma membrane and into cells. "Cargo" or "payload" is a term used to describe a microorganism, such as a non-bacterial microorganism, such as a virus, a compound, or a composition that is delivered across the cell plasma membrane and into the interior of the cell via an aqueous solution.

[0136] The subject matter relates to a delivery technique that facilitates the delivery of a wide range of payloads to cells with low toxicity. Genome editing can be achieved by delivering RNP to cells using some aspects of the subject matter. Levels are then reduced until Cas9 is no longer detectable. The delivery technique itself does not adversely affect the viability or functionality of Jurkat T cells and primary T cells. The subject matter allows Cas9 RNP-mediated gene editing in clinically relevant cell types with minimal toxicity.

[0137] Transient and direct delivery of CRISPR / Cas components such as Cas and / or gRNA has advantages compared to expression vector-mediated delivery.For example, a certain amount of Cas, gRNA, or RNP can be added at a more precise timing and for a limited time compared to using expression vector.The components expressed from vector may be produced in different amounts and for different times, making it difficult to achieve consistent gene editing without off-target editing.In addition, the preformed complex of Cas and gRNA (RNP) cannot be delivered with expression vector.

[0138] In one aspect, the subject matter describes cells attached to a solid support (e.g., a strip, a polymer, a bead, or a nanoparticle). The support or scaffold may be a porous or non-porous solid support. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the carrier can be either soluble or insoluble to some degree for the purposes of the subject matter. The support material can have virtually any possible structural shape. Thus, the support shape may be spherical, as in a bead, or cylindrical, as in the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet or test strip. A preferred support includes polystyrene beads.

[0139] In other aspects, the solid support comprises a polymer with which the cells are chemically bound, immobilized, dispersed, or associated. The polymer support may be a polymer network and may be prepared in bead form (e.g., by suspension polymerization). The cells on such scaffolds can be sprayed with an aqueous solution containing a payload according to the present invention to deliver the desired compound to the cytoplasm of the scaffold. Exemplary scaffolds include stents and other implantable medical devices or structures. EXAMPLES

[0140] The following examples illustrate certain specific aspects of the present invention and are not meant to limit the scope of the invention.

[0141] The embodiments herein are further illustrated by the following examples and detailed protocols.However, the examples are merely intended to illustrate the embodiments and should not be interpreted as limiting the scope of the present specification.The contents of all references and published patents and patent applications cited throughout this application are incorporated herein by reference.

[0142] Example 1: Delivery of a virus as a payload Non-bacterial microorganisms, such as viruses, are delivered to eukaryotic cells.In some examples, commercially available viruses, such as adeno-associated viruses (AAV), lentiviruses, or retroviruses, are used.For example, lentiviruses contain nucleic acids that code model (test) cargo, such as GFP.Viruses (encoding GFP) are delivered using the methods and systems herein, the systems described herein.

[0143] Multiplicities of infection (MOI) (or the number of virions (virus particles) added per cell in an infection) of 0.1, 1, 10, and 100 were performed. The volume of the methods and systems herein relative to the number of cells was evaluated.

[0144] A range of MOIs were tested, and volumes ranging from about 1 μL to about 1000 mL were tested.

[0145] Additionally, 1×10 5 ~1×10 7 Various membrane holder sizes were tested to allow for removal of medium from a range of cell numbers. Cells can be cultured after transduction to reach volumes required for cell therapy, e.g., up to 10 9 grow into cells.

[0146] For example, the commercially available lentiviral vector encoding GFP, which is transduced by spinoculation, is used as a control.The control is used according to the standard protocol for spinoculation of lentiviral vector to suspension cells.In the examples, the commercially available spinoculation method is used to transduce suspension cells (Jurkat T cells, PBMC, PBL, B cells, etc.).

[0147] Transduction and characterization of suspension cells For normal transduction of suspension cells, the virus was added to activated T cells in culture flasks and bags as a control. After viral transduction, the cells are cultured and the virus is washed out. After a few days, the cells are harvested and evaluated for viability and % transduction (virus encoding GFP) with a flow cytometer.

[0148] The life span of transduction efficiency was monitored over 14 days.

[0149] Vector copy number (VCN) was assessed to determine low copies / cell, e.g., 0.5-1 copy / cell. Exemplary release criteria include (0.5-5 per Levine 2006). VCN is established using quantitative PCR.

[0150] Phenotypic changes were assessed by flow cytometry, including the maintenance of naïve, central, and effector memory.

[0151] Transducing T cells and other cell types We also evaluate viral delivery to T cells in multiple activation states. For example, it is not uncommon to transduce activated T cells. We carried out a study to determine whether naive T cells can be used, which would reduce COG (cost of goods sold). There is no need for activation, and therefore the advantages of the methods described herein include reduced processing time. In other examples, we also evaluate delivery to previously modified T cells, such as CAR-T cells or gene-edited T cells.

[0152] Transduction Additionally, standard transduction of virus was performed. For example, HEK293 adherent cells were used. This method eliminates the need to remove medium through a filter. For example, cells can be seeded in 6-well plates, the medium is manually removed to expose the cells, and then the cells are permeabilized directly on the plate. Additionally, some of the problems of putting the system into a virus containment laboratory and isolating all the equipment are eliminated.

[0153] AAV, retroviruses, etc. are also transduced using such methods. Lentiviruses are advantageous as they are the simplest ex vivo transduction method, and AAV is a common virus used in commercial settings.

[0154] Example 2: Delivery of virus via spray Virus can be delivered via the SOLUPORE™ device, e.g., small, medium, or large systems, including replicates of the described procedures, including various membranes, device sizes, etc.

[0155] In this method, the medium is temporarily removed from the cells forming a cell monolayer to allow the dropletized virus to come into intimate contact with the cells for a specified incubation period, after which the medium is returned to the cells and the cells are cultured for downstream applications.

[0156] The methods and systems herein may include a filter membrane (with or without a drain disk) through which the medium is temporarily removed from the cells (by centrifugation, gravity flow, or vacuum). After monolayer formation, various cargoes are aerosolized in a buffer solution and the droplets are applied onto the cells in a controlled manner (variable volume, height, etc.). Various cargoes include mRNA, DNA, CRISPR RNP, and viral particles (lentivirus, retrovirus, AAV, etc.) that allow gene transfer to cells or editing of genetic material in cells.

[0157] In some implementations, the process includes T cells, NK cells, and the like.

[0158] In some implementations, the cells are treated with an SMA nebulizer, a Conikal nebulizer, or the like.

[0159] The system is used to transduce T cells. Additionally, the system is used to deliver cargo and virus for simultaneous or sequential editing. The system is also used to engineer T cells with RNA, DNA, and siRNA. The system is also used to knock in or knock out genes, which can be done separately or in parallel with the introduction of genetic information with viruses. The system is used to edit cells with multiple cell editing platforms, such as CRISPR / Cas9, Cas12, MegaTAL, TALEN, ZFN, etc., all of which can be delivered alone, in parallel with viruses, or sequentially.

[0160] In some implementations, cells are treated with multiple nebulizers at once or in sequence. In other iterations, one nebulizer is used in isolation. Using a nebulizer to aerosolize virus particles improves the contact between cells and virus, improving transduction. The virus is dropletized and lands on the surface of the membrane where the cells are located. Dropletized virus allows a much smaller volume to be administered to the cells. After a short incubation, the medium is replaced. This medium may also contain a low dose of virus.

[0161] Test multiple cell types.Additionally, add various media and additives, such as cytokines, including, for example, polybrene or PGE2 to enhance transduction.Add other commercially available fibronectin peptides (Retronectin) or Lentiboost to enhance the interaction between virus and cells, and add in various iterations.

[0162] Spray speed / duration / pressure are also evaluated. Cell recovery by various methods (eg, bottom-up / pipette) is tested.

[0163] The cargo for genetic engineering may be suspended in a variety of buffers, none of which limits the scope of this patent, which contains many variations of what cargo can be delivered to what cell types.

[0164] Furthermore, T cell transduction can be highly variable and this method of controlled delivery can make viral transduction more consistent and therefore more advantageous.

[0165] Example 3: Gene Therapy Viral Vectors The table below shows the main differences between the various types of viruses used in gene therapy of eukaryotic cells.

[0166] Overview of common viruses used to generate gene therapy viral vectors TIFF0007637072000004.tif67163

[0167] Overview of adherent and suspension cells in the use of viral vectors TIFF0007637072000005.tif57161

[0168] Retroviruses have a number of disadvantages, therefore AAV and lentiviruses are more widely used.

[0169] Example 4: Viral delivery to a population of cells using the SOLUPORE™ process The SOLUPORE™ process allows the delivery of a wide range of cargoes to adherent and suspension cells in vitro and ex vivo. To date, these cargoes consist of molecules such as nucleic acids and proteins, as well as particles such as Qdots. The data herein show that the SOLUPORE™ process can be used to deliver non-bacterial microorganisms, such as viruses (e.g., lentiviruses), to T cells with higher efficiency than standard control transduction.

[0170] Viruses as vectors Viruses are used as vectors for the delivery of nucleic acids to cells because they can naturally infect human cells. Viruses must attach to the host cell before entry. Attachment is achieved when a specific protein on the viral capsid or viral envelope binds to a specific receptor protein on the cell membrane of the target cell. Depending on the type of virus, entry into the cell can occur in different ways. Viruses with a viral envelope can enter the cell by membrane fusion, where a hole is made in the cell membrane to further connect with the unfolded viral envelope. Viruses without a viral envelope can enter by endocytosis (Figure 2). Other viruses, such as bacteriophages, attach to the cell surface and only the viral genome is injected into the host cell.

[0171] immune cell manipulation Different types of viruses have different characteristics that need to be considered when used to transduce cells ex vivo for clinical applications. The main characteristics are: immunogenicity; target cell type; payload capacity; ability to transduce dividing versus non-dividing cells; transient versus stable genomic integration (Table 5).

[0172] Overview of viruses used in gene delivery applications TIFF0007637072000006.tif63158, the entire contents of which are incorporated herein by reference, Pharmaceutics 2020, 12, 183

[0173] For immune cell therapy applications, such as the delivery of chimeric antigen receptor (CAR) constructs for the generation of CAR-T cells and CAR-NK cells, gammaretroviruses and lentiviruses are typically used because they can transduce immune cells and result in stable integration into the genome. For example, the first two approved CAR-T cell products, Kymriah and Yescarta, were engineered using lentiviral and gammaretroviral vectors, respectively (Poorebrahim M et al. CritRev Clin Lab Sci. 2019 Sep;56(6):393-419). As with most viral vectors, these vectors have been modified to disable viral replication and improve cell targeting efficiency.

[0174] AAV vectors are widely used for other immune cell engineering applications, such as gene editing. Wild-type AAV can be stably integrated into chromosome 19, but AAV-based gene therapy vectors are modified to prevent integration and instead form episomal concatemers in the nucleus of host cells. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. In dividing cells, episomal DNA is not replicated with host cell DNA, so AAV DNA is lost through cell division. Therefore, AAV vectors are often used to deliver editing systems, such as CRISPR / Cas9 systems for gene editing or donor template DNA. In these cases, gene editing is permanent, but gene editing tools may be desired to be expressed only transiently to limit non-specific off-target gene editing that may occur if the tools are present in cells for long periods of time.

[0175] Lentivirus Gammaretrovirus and lentivirus are subtypes of retrovirus that contain RNA genome that is converted into DNA in transduced cells by the enzyme called reverse transcriptase that is encoded by the virus.For retrovirus, the entry into cells is followed by the process of uncoating, which causes some viral proteins to dissociate from the viral core.Viral RNA is reverse transcribed into double-stranded DNA.Then, viral proteins form a complex with proviral DNA, leading to nuclear transport and integration into the host genome.The integration process is assisted by vital viral proteins such as integrase, and endogenous host cell transcription factors.

[0176] Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become the primary tool for gene delivery into mammalian cells, and replication-defective recombinant lentiviruses are widely used in research and clinical applications. The modified lentiviruses can still infect cells, but the essential genes for producing new viral particles are no longer present. Lentiviral vectors are considered attractive gene delivery vehicles for several reasons: they provide long-term gene expression through stable vector integration into the host genome; they can infect both dividing and non-dividing cells; they can infect a wide range of cells, including important target cell types for gene therapy and cell therapy; they lack immunogenic viral proteins after vector transduction; they can deliver complex genetic elements such as intron-containing sequences; and they are a relatively easy system for vector engineering and production. Lentiviral vectors have a safer integration site profile than gammaretroviral vectors and are commonly used in clinical trials of CAR T cell therapy (McGarrity GJ et al. J. Gene Med. 2013;15:78-82). Third generation lentiviral vectors have incorporated key safety features to further enhance safety (Kim VN et al. J. Virol. 1998;72:811-816 and Dull T. et al. J. Virol. 1998;72:8463-8471).

[0177] The range of cell types that can be transduced with retroviruses has been expanded by pseudotyping retroviruses with the envelope glycoprotein G of vesicular stomatitis virus (VSV-G). VSV-G binds to phosphatidylserine, a ubiquitous membrane component, allowing VSV-G pseudotyped viruses to attach to and transduce a much broader range of cells. Currently, most lentiviral vectors are pseudotyped with VSV-G to enable robust transduction into many cell types, including neurons, lymphocytes, and macrophages.

[0178] Quiescent cells After isolation from either healthy donors or patients, cells are activated and subsequently transduced with lentiviral vectors (LVs), the majority of which are pseudotyped with the glycoprotein G of the vesicular stomatitis virus (VSV). The modified lymphocytes are then expanded and used in functional in vitro assays or in vivo applications. Stable and efficient transduction of B cells with LVs is very difficult to achieve. Resting B cells are limited to transduction with conventional VSV G pseudotyped LVs (VSV-LVs) due to the lack of expression of the low density lipoprotein receptor (LDLR), and must be activated prior to transduction. Efficient activation and culture of primary human B lymphocytes is complex, as it involves carefully titrated activation stimuli combined with cytokines, followed by co-culture with feeder cells. Even under optimal activation and culture conditions, transduction efficiency with VSV-LVs is notoriously low. The combination of all these challenges may serve as an explanation for why the number of clinical trials involving engineered B cells is much smaller compared to T cells.

[0179] In comparison, T lymphocyte engineering by lentiviral transduction is easier to achieve. Still, the cells, like B cells, must be activated before transduction with conventional VSV-LV, since they are otherwise not susceptible to transduction, again due to the lack of LDLR expression (X Geng, et al. Gene Therapy v.21, pages 444-449 (2014)). Protocols for isolation, activation, lentiviral transduction, and expansion of T cells have been greatly improved in recent years, primarily for the best success of CAR T cell therapy. Current state-of-the-art T cell activation relies on stimulation of the TCR activation pathway via CD3- and CD28-specific antibodies, combined with cytokines such as IL-7 and IL-15.

[0180] The need to activate lymphocytes prior to transduction with conventional LV has disadvantages. This adds to the complexity of the entire procedure, increasing the duration and cost of the manufacturing process. In addition, the stimuli applied for activation, combined with the long-term ex vivo culture, are likely to alter the cells, which may have a negative impact on the quality of the final product. As a result, naive cells may differentiate into a less preferred phenotype that exhibits a higher degree of attrition, a lower proliferation capacity, a shorter in vivo persistence, and a lower functionality. This may have very important implications for therapeutic success. As an example, it has been shown that the central memory (CD45RO+ / CD45RA+ / CD62L+) or stem cell memory (CD45RO+ / CD45RA / CD62L+) phenotype is beneficial for the persistence and function of T cells in vivo. In this regard, a positive correlation between CAR T cell central memory phenotype and positive clinical responses has been observed in several clinical studies, and as a result, the infusion of purified central memory CAR T cells is currently considered. Similarly, the central memory phenotype results in functionally superior TCR-modified T cells. Thus, minimal manipulation of lymphocytes during genetic modification has significant clinical relevance.

[0181] Spinoculation Centrifugation inoculation, or spinoculation, is widely used in virology research to enhance virus infection. The procedure involves centrifuging a mixture of virus and target cells at high speed for an extended period of time, e.g., 800×g for 30 min at 32°C. The method was thought to enhance transduction rates by concentrating the virus at the cell membrane. However, spinoculation has been shown to induce dynamic actin and cofilin activity, likely due to the cellular response to centrifugation stress (Jia Guo, et a. J. Virology Oct. 2011, p. 9824-9833). This actin activity also leads to upregulation of cell membrane receptors that may enhance virus binding and entry. It has been suggested that spinoculation cannot be simply explained by a virus concentration effect, but rather is coupled to spin-induced cytoskeletal dynamics that promote receptor recruitment, virus entry, and post-entry processes. Thus, spinoculation may affect the biology of target cells in unknown or undesirable ways.

[0182] Limitations of viral vectors Although viruses are useful for genetically manipulating cells as described above, there are limitations to their utility.

[0183] The high cost manufacturing process of chimeric antigen receptor (CAR) T-cell therapy is prohibitively expensive. Due to the cost of the virus, transduction is the main cost driver in CAR T-cell manufacturing. Several bioprocessing parameters have been identified as potentially playing a role in transduction efficiency, such as the physical proximity of lentiviral particles to T cells. This proximity can be manipulated through the number of cells and viral particles in suspension; the duration of agitation to promote uniformity; and the surface-to-volume ratio in the transduction vessel. However, limited research has been conducted on identifying and optimizing the critical process parameters of transduction. During the SOLUPORE™ process, a small amount of delivery solution is applied directly onto the exposed target cells. In this way, the cargo is brought into direct contact with the cells in a gentle manner. Delivering the virus to the cells in this way results in the concentration of the material at the cell membrane. This process enhances the virus attachment to the cell membrane and enhances the rate of entry into the cell, making the process more efficient. In turn, smaller doses of virus are used, lowering costs.

[0184] The SOLUPORE™ process is a gentle method of concentrating viruses at the cell membrane, which has significant advantages over existing concentration methods such as spinoculation, which can affect cell structure. Moreover, unlike spinoculation, the SOLUPORE™ process is designed to be compatible with cell therapy manufacturing processes.

[0185] Concentration of virus at the cell membrane also compensates for the low level of viral receptor expression on certain cell types, such as non-activated T cells, thus enhancing transduction efficiency in these cells.

[0186] The efficiency of lentiviral vector transduction of non-activated T and B cells is typically very low. It is highly desirable to improve these efficiencies, and the SOLUPORE™ process offers a solution to this problem, with high efficiency rates coupled with conditions compatible with preserving cell viability and function.

[0187] Viruses can only deliver nucleic acids, which means that they are limited in the type of cargo they can deliver. If viruses can be delivered simultaneously with other types of cargo, this can enhance the utility of viruses in engineering for next-generation cell therapy products. However, currently, there is no method that has been demonstrated to simultaneously deliver viruses with other types of cargo. Again, the SOLUPORE™ process described herein provides a solution to this problem by allowing the efficient delivery of many different cargo types, either sequentially or simultaneously. The following materials and methods were used to produce the date described herein.

[0188] LV-GFP vector The LV-GFP vector used herein carries the vesicular stomatitis virus-G (VSV-G) envelope protein, which is known to target a wide variety of cell types.

[0189] Stability of LV-GFP in delivery solution The stability of LV-GFP in the delivery solution was assessed over a 1 hour period by assessing precipitation under a microscope.

[0190] LV-GFP delivery Primary human PBMCs were thawed and activated with Miltenyi's CD3 and CD28 antibodies for 3 days. After 3 days of activation culture, cells underwent SOLUPORE™ process or static transduction with LV-GFP (MOI=2.5). Cells were allowed to recover for 72 hours before GFP expression was assessed by flow cytometry.

[0191] Stability of LV-GFP in delivery solution Prior to the present invention, the SOLUPORE™ process delivery had not previously been combined with a viral preparation, and therefore it was not known whether there would be solubility issues.

[0192] When the solutions were viewed under a microscope during stability analysis, no aggregation or precipitation was observed. Additionally, certain other cargoes showed low levels of aggregation when combined with delivery solutions, causing the Solupore® nebulizer to block during spraying. When the delivery solutions containing the virus were loaded into the Solupore® nebulizer and sprayed, no blocking occurred at any time over the course of the study, further indicating that no aggregation or precipitation occurred.

[0193] Cell viability, expansion, and GFP expression following LV-GFP delivery LV-GFP was delivered to T cell cultures by the SOLUPORE™ process and compared to standard static methods of LV transduction.

[0194] Cell viability was measured at various time points before and after viral delivery. At all time points, the viability of solupore-treated cells was comparable to that of control-transduced cells (Figure 3).

[0195] The cumulative fold expansion of T cells was measured up to 96 hours after viral delivery. The expansion of solupore-treated cells was comparable to that of control-transduced cells (Figure 4).

[0196] GFP expression was measured on days 3 and 4 after delivery. GFP expression efficiency was higher in solupore-treated T cells compared to control-transduced cells (Figure 5). On day 3, the efficiency was 39.73±2.83% and the comparator 25.2±1.48% for solupore-treated and control-transduced cells, respectively. On day 4, the efficiency was 40.27±2.67% and the comparator 26.83±1.38% for solupore-treated and control-transduced cells, respectively.

[0197] Efficient viral delivery of virus to a population of cells The data herein demonstrate that the SOLUPORE™ process is compatible with the delivery of viruses to activated T cells. No precipitation or aggregation was observed when mixed with the SOLUPORE™ process delivery solution. The virus solution could be sprayed and the target cells were successfully transduced. The viability and expansion rate of solupore-treated T cells were not affected.

[0198] GFP expression was higher in solupore-treated T cells compared to control-transduced cells, indicating that the SOLUPORE™ process enhances viral transduction of T cells.

[0199] Taken together, these findings demonstrate that the SOLUPORE™ process is suitable for delivery of viruses to cells and is superior to standard methods.

[0200] Because the SOLUPORE™ process is suitable for viral delivery, it is possible to use the solupore treatment in cell therapy manufacturing processes that include viral transduction. Due to the cost of the virus, transduction is the main cost driver in CAR T cell manufacturing. Because the SOLUPORE™ process enhances viral transduction, it is now possible to achieve similar levels of transduction efficiency using less virus, thus lowering costs.

[0201] Various cargoes can be delivered simultaneously by the SOLUPORE™ process. The demonstration herein that the SOLUPORE™ process is compatible with virus delivery means that the SOLUPORE™ process can be used to co-deliver viruses with other cargoes. These other cargoes can be other viruses, or can be proteins, nucleic acids, small molecules, or complexes thereof. The ability to co-deliver cargoes means that the manipulation steps that would otherwise occur in different process steps can be combined into a single process step. This process has great benefits for the manufacturing process, including cost, time, and labor. In addition, fewer process steps means less handling and contamination risk, as well as simplification of the process. Alternatively, the virus is delivered in sequence before or after the other cargoes.

[0202] SOLUPORE™ process allows cargo to be delivered to non-activated T cells. Lentiviral vectors have very low transduction efficiency in non-activated T cells. Therefore, SOLUPORE™ process increases the transduction efficiency of lentivirus in non-activated T cells.

[0203] A core feature of the SOLUPORE™ process device is the ease of medium exchange. Once the cell-containing solution is transferred into the device, the liquid can be drained and replaced with a different liquid. In this way, liquid handling steps are possible. Such liquid handling steps can include, for example, washing steps. In viral transduction and other cell manufacturing processes, washing steps are often required. The Solupore® device allows for the incorporation of such steps into the manufacturing process.

[0204] The Solupore® technology is also scalable, which means that viral transduction using this method can be carried out on small scale for early and preclinical studies, and on larger scales for process development and clinical applications.

[0205] Example 5: Delivery of Lentiviral Vectors (LV) to T Cells by SOLUPORE™ - Process with Solupore™ A data set was generated for LV delivery via the SOLUPORE™ process compared to a static transduction control.

[0206] We have developed a viral delivery technology for cell and gene therapy. The platform relies on reversible cell permeabilization for payload delivery with a functionally closed device. Using this approach, effective nucleic acid and gene editing was demonstrated in therapeutically relevant cell types with minimal impact on cell viability, proliferation, gene expression, or phenotype. Utility for delivering viral vectors to target cells was also demonstrated. Data describes lentiviral vector delivery by the SOLUPORE™ process to T cells.

[0207] PBMC isolation Half of a fresh leukapheresis pack (donor ID: RG1083) was obtained from StemCell Technologies. PBMCs were isolated and cryopreserved as described in "PBMC-derived T cell isolation, initiation, and cell culture" (provided in Example 5). To generate a peripheral blood mononuclear cell (PBMC) cell bank, cells were counted and viability assessed by Nucleocounter NC-200. 61.5×10 6 Viable cells / mL were frozen in 1 mL aliquots to -100 °C with VIA Freeze at a controlled rate of -1 °C / min. A total of 43 vials were banked and all frozen vials were transferred from the VIA Freeze to liquid N for permanent storage. 2The PBMC bank was qualified by thawing three random vials and assessing cell viability and cell recovery upon thawing. The thawing procedure was performed as described in "PBMC-derived T cell isolation, initiation, and cell culture" (provided). For each vial, 1 × 10 6 Viable cell density of 4 x 10 cells / mL 6 Cells were also seeded in 6-well plates and activated with CD3 and CD28 antibodies. Three days after activation, cells were harvested and CD3 and CD25 expression was assessed as described in "Cell thawing, culture, and preparation of cells for experimental use" (provided in Example 7).

[0208] Thawing and initiation of PBMCs Complete media was prepared and cryopreserved PBMCs were thawed and activated for 3 days using the protocol described in "PBMC-derived T cell isolation, initiation, and cell culture" (provided in Example 5). Representative 10x images were taken using an inverted microscope to capture cell clumps and overall morphology. To determine whether CD3 and CD25 expression following activation met the release criteria for the SOLUPORE™ process, activated PBMC-initiated T cells were collected and stained with CD3- and CD25-conjugated antibodies for flow acquisition and analysis, as outlined in Example 8. CD3 and CD25 expression was verified to be >90% before cells were released for experimental use.

[0209] Lentivirus (LV-eGFP; "high sensitivity GFP") Three lots of LV-eGFP, supplied in 50 μL aliquots, were obtained from Tailored Genes and stored at −80° C. LV batches were titrated and adapted on K562 cells.

[0210] To investigate the stability of LV in the delivery solution formulation, 75 μL of delivery solution with LV-eGFP as the payload was prepared in a 96-well plate. To evaluate possible precipitation, 4x, 10x, and 20x images were taken every hour for 4 hours using an inverted microscope. The delivery solution contained sucrose 32.5 mM, KCl 106 mM, Hepes 5 mM, EtOH 12% v / v, and water for injection.

[0211] The required number of aliquots were thawed on ice and briefly centrifuged to collect any liquid that had spilled over the sides of the vial. The LV-eGFP aliquots were pooled before use to ensure sufficient volume for the experiment. Once the payload delivery solution was prepared at 0, the remaining LV-eGFP was kept at RT until static transduction was performed.

[0212] Specifications of the three LV-eGFP lots used in all experimental runs TIFF0007637072000007.tif40160

[0213] Preparation of PBMC initiating T cells As outlined in "Cell Thawing, Culture, and Cell Preparation" (provided in Example 7), the required number of PBMC-initiated T cells were pelleted by centrifugation and resuspended in basal medium (CTS OpTmizer T Cell Expansion SFM containing supplements) to produce the desired cell density listed for each experiment. A post-dilution cell count was performed and 30 mL aliquots of cell suspension were prepared in 50 mL Falcon tubes and kept in a 37° C. incubator until use. Immediately prior to the SOLUPORE™ process, each sample was weighed and assessed for both cell number and viability to ensure that the cells remained at the desired cell density. Each sample was then transferred to a 50 mL syringe in preparation for the SOLUPORE™ process, as described in "SOLUPORE™ Process of PBMC-Initiated T Cell Cultures" (described herein).

[0214] Static transduction For each multiplicity of infection (MOI) condition, the required number of T cells were diluted in complete medium to produce the desired volume, and a post-dilution cell count was performed to confirm cell density. The prepared cell suspension was then aliquoted into Nunc™ EasYFlasks™ TC-treated T25 flasks, with three technical replicates and one untreated control per MOI condition. Cells were incubated at 37° C., 5% CO until static transduction. 2 Kept in an incubator.

[0215] Static transduction parameters tested in each experiment TIFF0007637072000008.tif51160

[0216] LV was added to each stationary transformation sample in parallel with the completion of the SOLUPORE™ process to capture any potential degradation of LV over time. Immediately after virus addition, flasks were incubated at 37° C., 5% CO 2 Returned to the incubator.

[0217] For each run, the complete medium was doubled one day after virus delivery. For run 1 / 2, a larger volume was given and samples were transferred to T75 flasks and cultured flat. For run 3 / 4, 3 mL of complete medium was added to each T25 flask. For run 1, the final analysis was performed 3 days after the SOLUPORE™ process. For runs 2 and onwards, 0.5×10 cultures 3 days after infection were cultured after time point analysis was performed. 6 Dilution to VCD in cells / mL was performed to allow further analysis the next day. Post-dilution cell counts were performed to track cumulative fold expansion up to 4 days after the SOLUPORE™ process. All post-recovery and post-SOLUPORE™ process time point analyses were completed.

[0218] Assembly, Calibration, and the SOLUPORE™ Process with the Solupore™ System Following the "SOLUPORE™ Process of PBMC-Initiated T-Cell Culture" (described herein), the sterilized components were aseptically assembled in a biosafety cabinet (BSC). Pressure leak testing was completed to ensure the device was properly sealed and the pressure profile was preserved. Calibration and stop solutions were prepared according to the "SOLUPORE™ Process of PBMC-Initiated T-Cell Culture" (described in Example 6) and kept on ice until use.

[0219] Due to the increased payload volume to achieve an MOI of 2.5, the system was calibrated to deliver 75-80 µL in run 1 / 2. Because the LV-eGFP payload solution had a higher viscosity than the calibration solution, a significant amount of payload delivery solution remained after both runs. To deliver the desired amount of LV, the system was then calibrated to deliver 95-100 µL to adjust for insufficient spray volume.

[0220] Parameters tested for the SOLUPORE™ process in each experiment SOLUPORE™ process parameters tested in all experiments TIFF0007637072000009.tif61160

[0221] 1×10 9 Assuming a viral titer in TU / mL, the volume (μL) of LV-eGFP payload required for each spray was calculated for all experiments as follows: TIFF0007637072000010.tif10128

[0222] Based on the total delivery volume, the final concentration (%) of LV-eGFP payload was determined as: The total volume of delivery solution needed for each experiment was determined by the following calculation: Derived using TIFF0007637072000012.tif11142.

[0223] For all experiments, delivery solutions were formulated based on the final concentrations of each component listed below.

[0224] Payload and delivery solution composition for syringes and Elveflow reservoirs. The % of WFI (water for injection) was adjusted to reflect the increased LV-eGFP composition. TIFF0007637072000013.tif26160

[0225] For all SOLUPORE™ processes, cells were loaded, solupore-processed, and harvested in 20 mL of complete medium. Cleaning and priming between samples was performed as described in "SOLUPORE™ Process of PBMC-Initiated T Cell Cultures" (described herein). Post-recovery and post-SOLUPORE™ process time point analyses were completed.

[0226] In some examples, "S buffer" comprises a hypotonic physiological buffer (78 mM sucrose, 30 mM KCl, 30 mM potassium acetate, 12 mM HEPES) at 4°C for 5 minutes (Medepalli K. et al., Nanotechnology 2013; 24(20); incorporated herein by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in S buffer. S buffer is further described in International Application WO 2016 / 065341, e.g., at paragraphs

[0228] to

[0229] , incorporated herein by reference in its entirety.

[0227] Post-SOLUPORE™ process and post-transduction analyses completed for each experiment. ◆ represents the time point analysis tested. TIFF0007637072000014.tif70160

[0228] For all post-SOLUPORE™ process time points, samples were weighed, counted and assessed for viability by Nucleocounter NC-200 before collection for downstream analysis.

[0229] For each run, the complete medium was doubled one day after virus delivery. For run 1 / 2, the larger volume was given and samples were transferred to T75 flasks and cultured flat, while for run 3 / 4, 3 mL of complete medium was simply added to each T25 flask. For run 1, the final analysis was performed 3 days after the SOLUPORE™ process. For runs 2 and onwards, after time point analysis was performed, 5×10 cultures were cultured 3 days after the SOLUPORE™ process. 5 Dilution to VCD in cells / mL allowed further analysis the next day. Post-dilution cell counts were performed to track cumulative fold expansion up to 4 days after the SOLUPORE™ process. All post-recovery and post-SOLUPORE™ process time point analyses were completed as listed above.

[0230] Decontamination of the Solupore(TM) System Payload solution was removed from the Elveflow valve and properly purged before disposal. The sprayer was then rinsed by purging twice with 1 mL of PREempt RTU, twice with 1 mL of WFI, and twice with 1 mL of 70% IPA (isopropanol). Finally, the sprayer was purged again in the same order with 1 mL of each reagent. The system was disassembled and the PREempt RTU was sprayed. 20 L of Citranox 1% v / v in tap water was prepared and the system components were submerged and rinsed with DI (deionized) H2O as described. 2 The gaskets and O-rings were placed in autoclavable bags and autoclaved on a robust cycle. The UV light in the BSC was activated to ensure all non-autoclavable components were sterilized. The ethylene oxide (EtO) sterilization step was omitted. Each component was then sprayed with 70% IPA and dried using an air gun before being placed back into the BSC. The gaskets and O-rings were placed in autoclavable bags and autoclaved on a robust cycle. The UV light in the BSC was activated to ensure all non-autoclavable components were sterilized. The ethylene oxide (EtO) sterilization step was omitted.

[0231] Sample preparation and 7-aminoactinomycin D (7-AAD) viability staining and %GFP data acquisition For each time point, 200 μL of cells from each sample were collected into a 96-well V-bottom polypropylene plate. The cells were then pelleted by centrifugation at 300×g for 7 min, and the supernatant was removed using a multichannel pipette. During centrifugation, a master mix of 7-AAD viability dye in FACS solution (5 μL of 7-AAD in 200 μL FACS solution per sample) was prepared in a 15 mL Falcon tube. Each sample was resuspended in 200 μL of staining solution and incubated at RT for 5 min, protected from light. 7-AAD viability staining and GFP expression were acquired using CytoFlex and quantified using FlowJo, following the acquisition and gating strategy.

[0232] Droplet digital polymerase chain reaction (ddPCR): Sample collection, DNA extraction, and PCR sample preparation For each time point analysis, 200 μL of cells from each sample were collected into a 96-well V-bottom polystyrene plate. The cells were then pelleted by centrifugation at 300×g for 7 min, and the supernatant was removed using a multichannel pipette. During centrifugation, 1× lysis buffer was prepared as follows:

[0233] Preparation of 1x in-house cell lysis buffer TIFF0007637072000015.tif26160

[0234] 50 μL of 1× lysis buffer was added to each sample. All reactions were incubated at room temperature (RT) for 20 minutes before being gently transferred (to avoid air bubbles) to a standard 96-well PCR plate. The reaction plate was sealed with clear plastic film and then incubated at 56° C. for 15 minutes and 95° C. for 10 minutes in a thermocycler and cooled to 4° C. Samples were then stored at −20° C. until additional samples were collected from all experiments.

[0235] To prepare samples for PCR, all DNA templates were diluted 20-fold in a standard 96-well plate using PCR-grade DNase- / RNase-free water as diluent. 1x ddPCR Super Master Mix was prepared in 15 mL Falcon tubes with the following composition for each reagent / reaction:

[0236] Preparation of 1x ddPCR Super Master Mix. The volume of each reagent was scaled up according to the total number of samples. A 10% volume variance (for pipetting error) and two extra reactions were also included as NTC controls. TIFF0007637072000016.tif31160

[0237] 18 μL of master mix was added to each reaction well of a 96-well Bio-Rad ddPCR plate in addition to 4 μL of 20-fold diluted DNA. For the NTC control, 4 μL of PCR-grade DNase / RNase-free water was added. A pierceable foil heat seal was placed directly on top of the prepared plate and the plate was sealed using a plate sealer. The reactions were then vortexed and spun down briefly to collect the scattered liquid at the bottom before being transferred to an automated droplet generator (Bio-Rad AutoDG). Once droplets were generated, the plate was sealed again using a pierceable foil heat seal and transferred to a Bio-Rad C1000 thermocycler to start the PCR using the following cycle: 95°C for 10 min (ramp 2°C / s), [94°C for 30 s (ramp 2°C / s), 60°C for 1 min (ramp 2°C / s)] × 39, 98°C for 10 min, and finally hold at 4°C. Upon completion of the PCR cycles, the reactions were transferred to a Bio-Rad QX200 droplet plate reader to quantify VCN by the ratio of Alb / WPRE (woodchuck hepatitis virus post-transcriptional regulatory element). The position of the threshold line was determined using the analysis software QuantaSoft to produce a clear separation between the positive and negative populations in both the x and y directions. Based on the Poisson distribution, the concentrations (copy numbers / μL) of both Alb and WPRE were obtained and %GFP was calculated using the following equation: This was derived by applying the .TIFF2007637072000017.tif15128.

[0238] Software / Statistical Analysis TIFF0007637072000018.tif50160

[0239] LV stability Based on qualitative analysis using 4x, 10x, and 20x magnification, there was no evidence of any precipitation in the delivery solutions up to 4 hours after formulation.

[0240] LV Delivery Run 1 CD3 and CD25 expression on activated PBMC-derived T cells, measured by flow analysis, was 92.3% and 91.3%, respectively.

[0241] Following the SOLUPORE™ process, the average total recovery from the SOLUPORE™ process was 63%±12% and the viable recovery was 60%±13% for three replicates (FIG. 8).

[0242] For both viral delivery methods, cell number and viability were assessed immediately after infection, and at days 1 and 3 post-infection. As shown in FIG. 9A, the viability of solupore-treated cells 1 day after viral delivery was significantly lower (p<0.05) than that of statically transduced cells (98.5% and 93.8%, respectively). Nevertheless, the viability of solupore-treated and statically transduced cells was comparable 3 days after infection (99.3% for both delivery methods).

[0243] Cells harvested from the system after the SOLUPORE™ process had a cumulative fold expansion of 4.9 after 3 days, which was significantly lower than the statically transduced cells, which had a cumulative fold expansion of 9.5 (p<0.01) (FIG. 9B). As seen in FIG. 30A, a significant difference in fold expansion was observed on day 1.

[0244] To assess viral delivery efficiency, GFP expression and median fluorescence intensity were quantified by flow cytometry. After 3 days, %GFP expression was 18.1% for solupore-treated T cells and 16.8% for stationary transduced T cells (Figure 10A). Solupore-treated T cells had significantly higher %GFP expression up to 1 day after viral delivery (p<0.01).

[0245] The MFI of expressed GFP was significantly higher for solupore-treated T cells immediately after viral delivery (p<0.01) (Figure 10B). However, this decreased over time, and after 3 days of culture, solupore-treated T cells showed a significantly lower MFI than stationary transduced T cells (p<0.001).

[0246] LV Delivery Run 2 A second run of virus delivery by both the SOLUPORE™ process with the system and static transduction was performed similarly to the first run. Three days after activation from PBMC, the pre-delivery cell population was 96.7% CD3 + and 99.5% CD3 + CD25 + (Figures 11A and 11B).

[0247] Following virus delivery via the SOLUPORE™ process, the average total cell recovery was 67%±6% and the viable recovery was 65%±6% for three technical replicates (FIG. 12).

[0248] As indicated, analysis was performed on this run 4 days after infection to verify the GFP expression observed at earlier time points. Cell viability was greater than 90% for both viral delivery methods 1 day after infection (Figure 13A), but the viability of solupore-treated cells was significantly lower (p<0.01) than statically transduced cells (92.0% and 98.6%, respectively). At later time points, solupore-treated cells recovered and no significant difference in viability was observed between the two delivery methods.

[0249] Cells harvested from the system after the SOLUPORE™ process had a cumulative fold expansion of 7.7 after 4 days, which was significantly lower than the statically transduced cells, which had a cumulative fold expansion of 12.5 (p<0.01) (FIG. 13B). In addition, the fold expansion at each post-delivery time point was significantly lower in the solupore-treated cells when compared to the statically transduced cells (p<0.01) (FIG. 30B).

[0250] As shown in Figure 14A, %GFP expression quantified by flow cytometry 3 days after infection was 15.2% for solupore-treated T cells and 19.1% for statically transduced T cells. After an additional day, %GFP expression was maintained for statically transduced cells (19.4%), while GFP expression in solupore-treated cells was significantly reduced to 11.5% (p<0.05). Solupore-treated T cells had significantly higher %GFP expression up to 1 day after virus delivery (p<0.01).

[0251] The MFI of expressed GFP was significantly higher for solupore-treated T cells immediately after viral delivery (p<0.01) (FIG. 14B). However, the MFI in solupore-treated T cells decreased over time. After 3 and 4 days of culture, T cells from static transduction had a significantly higher MFI (p<0.001).

[0252] LV Delivery Run 3 Three days after activation from PBMC, the pre-delivery cell population was 90.6% CD3+ and 94.2% CD3+CD25+ (FIGS. 15A and 15B).

[0253] Immediately after the SOLUPORE™ process, the average total recovery was 62%±9% and the viable recovery was 58%±8% (FIG. 16). Despite the difference in the number of cells loaded, there was no significant difference in the recovery (p>0.05; paired two-tailed t-test).

[0254] As shown in FIG. 17A, the viability of solupore-treated cells after 1 day was significantly lower (p<0.01) than that of cells receiving virus delivery by static transduction (90.7% and 99.5%, respectively). At later time points, the viability of solupore-treated cells recovered to greater than 99% and was significantly higher (p<0.05) than that of statically transduced cells at day 4 (98.7% and 97.9%, respectively). These findings were independent of the MOI used, which did not appear to have any effect on viability (FIG. 17B).

[0255] Cells harvested from the system after the SOLUPORE™ process had a cumulative fold expansion of 9.6 after 4 days, significantly lower (p<0.05) than cells from static transduction, which had a cumulative fold expansion of 13.2 (FIG. 13A). Regardless of the method of viral delivery, cells transduced at the lower MOI of 2.5 had significantly better expansion over 4 days (p<0.05) (FIG. 13B).

[0256] At each time point after infection, representative samples were taken to evaluate the efficiency of virus delivery in both delivery methods. As quantified by flow cytometry, %GFP after 3 days was 26.2% for solupore-treated T cells and 25.8% for statically transduced T cells (Figure 19A). After another day, %GFP decreased (p<0.01) for both the SOLUPORE™ process and static transduction to 22.8% and 22.9%, respectively. Solupore-treated T cells had significantly higher %GFP expression until 1 day after virus delivery (p<0.01). There was no statistical difference in %GFP between MOIs of 2.5 or 5, regardless of LV delivery method (Figure 19C).

[0257] The median fluorescence intensity (MFI) of expressed GFP was significantly higher for solupore-treated T cells immediately after virus delivery (p<0.001) (Figures 19A-19D and Figure 10B). However, the MFI of solupore-treated T cells decreased over time, and after 3 and 4 days of culture, T cells from static transduction had a significantly higher MFI (p<0.001). There was no statistical difference in GFP MFI between multiplicities of infection (MOI) of 2.5 or 5, regardless of LV delivery method (Figure 19D).

[0258] LV Delivery Run 4 The fourth run of virus delivery by both the SOLUPORE™ process with the system and static transduction was performed similarly to run 3. Three days after activation from PBMC, the pre-delivery cell population was 90.8% CD3+ and 95.1% CD3+CD25+ (Figures 20A and 20B).

[0259] After virus delivery by the SOLUPORE™ process, the average total cell recovery was 63%±10% and the viable recovery was 59%±10% for six replicates ( FIG. 21 ). There was no significant difference in recovery despite the difference in the number of cells loaded (p>0.05; paired two-tailed t-test).

[0260] The viability of statically transduced cells at all post-infection time points was higher than 98%. In comparison, at day 1 post-infection, the viability of solupore-treated cells was significantly lower (p<0.001) than that of statically transduced cells (89.6% and 99%, respectively, FIG. 22A). At later time points, the viability of solupore-treated cells recovered to higher than 99% and at day 4 was significantly higher (p<0.05) than that of statically transduced cells (99.7% and 99.0%, respectively). Regardless of the method of virus delivery, cells transduced with the lower MOI of 2.5 had significantly better viability at day 1 post-infection (p<0.05) (FIG. 22B). At later time points, cell viability at both MOIs was comparable (>98%), with the higher MOI of 5 having a significantly higher (p<0.05) viability at day 4 compared to the lower MOI of 2.5 (99.6% and 99.1%, respectively).

[0261] Cells harvested from the system after the SOLUPORE™ process had a cumulative fold expansion of 9.0 after 4 days (FIG. 23A), which was significantly lower (p<0.001) than cells from static transduction, which had a cumulative fold expansion of 22.5. There was no statistical difference in expansion between MOIs of 2.5 or 5, regardless of the method of viral delivery (FIG. 23B).

[0262] Three days after delivery, % GFP expression was significantly higher (p<0.01) in solupore-treated T cells (34.3%) than in statically transduced cells (25.4%) (Figure 24A). After one more day, GFP expression was maintained at 36.1% and 26.7% for both the SOLUPORE™ process and static transduction, respectively. As shown in Figure 24C, the method of viral delivery and MOI conditions did not have any significant effect on % GFP expression.

[0263] The median fluorescence intensity (MFI) of expressed GFP was significantly higher for solupore-treated T cells immediately after viral delivery (p<0.001) (Figure 24B). However, the MFI of solupore-treated T cells decreased over time, and after 3 days of culture, the MFI was significantly lower than in stationary transduced T cells (p<0.001). Similarly, there was no statistical difference in GFP MFI regardless of the method and MOI of viral delivery used (Figure 24D).

[0264] T cell purity and activation To meet the release criteria for experimental use, PBMC initiated T cells were required to have greater than 90% expression for both CD3 and CD3 / CD25. In all runs, cells passed QC with an average of 93.1%±4% CD3+ and 95.0%±3% CD3+CD25+ and were released for the SOLUPORE™ process (FIG. 25).

[0265] Post-recovery analysis During technology transfer of the system to the facility, minimum thresholds of 50% ± 10% total cell recovery and 30% ± 10% viable cell recovery were used to define a successful SOLUPORE™ process run. Cumulatively, total cell recovery was 63% ± 9% and viable cell recovery was 60% ± 9% (n = 18 sprays, Figure 26). 6 cells and 12 × 10 6 Different cell loads of cells were tested for the third and fourth runs, but there was no significant difference in the recovery percentage between the two cell load conditions (p>0.05; paired two-tailed t-test).

[0266] Survival rate after viral delivery An additional criterion for the success of the SOLUPORE™ process was a viability of 70%±10% or more after one day of the SOLUPORE™ process. The average viability of all four runs after one day of the SOLUPORE™ process was 90.0%±5.3% (n=18 sprays). However, this was lower (p<0.001) than the day 1 viability of statically transduced T cells (99.0%, FIG. 27A). The lower viability observed after one day of infection may be due to the reduced viability typically seen immediately after the SOLUPORE™ process, e.g., during the recovery period (88.3%; n=18 sprays). Despite this observation, there was no significant difference in day 3 viability (>98% for both virus delivery methods) when compared to statically transduced cells. This demonstrates that solupore-treated T cells can recover over time. Furthermore, 4 days after the SOLUPORE™ process, solupore-treated cells showed significantly (p<0.01) higher cell viability (99.2%) compared to statically transduced cells (98.4%).

[0267] Regardless of the method used for virus delivery, a lower MOI of 2.5 resulted in significantly higher (p<0.01) viability one day after infection when compared to an MOI of 5 (96.0% and 91.7%, respectively, FIG. 27B). This was observed in cells that underwent the SOLUPORE™ process and not for static transduction (FIG. 33). There was no statistical difference in viability between runs 1 / 2 and 3 / 4, regardless of the changes made to the calibration and post-infection culture conditions (FIG. 27C).

[0268] Growth kinetics after viral delivery Following viral delivery by either the SOLUPORE™ process or static transduction, cell counts were performed up to 4 days after infection to assess the impact of viral delivery on T cell proliferation. After 4 days, a mean fold expansion of 16.8 was observed in statically transduced T cells. This was significantly higher (p<0.001) than solupore-treated T cells, which experienced a fold expansion of 9.0 (Figure 28A). The difference in cumulative fold expansion observed in all four runs was mainly due to the recovery period required by the cells after the SOLUPORE™ process, which is further complicated at later days by the multiplicative nature of the cumulative fold expansion. Daily fold expansion is shown in Figures 31A and 31B.

[0269] In addition, one day after delivery, T cells receiving a lower MOI of 2.5 had a higher (p<0.001) expansion than those receiving an MOI of 5, regardless of delivery method (Figure 28B). However, this difference was not observed 3 or 4 days after delivery. Again, this was observed primarily in solupore-treated cells, but not in statically transduced cells (Figure 32), which may also be related to the lower viability observed after the SOLUPORE™ process (recovery period).

[0270] As shown in FIG. 28C, runs 3 and 4 had significantly higher expansion compared to runs 1 and 2 at 1 day (p<0.001) and 3 days (p<0.01) after delivery. However, this effect was not observed 4 days after delivery. The observed difference in expansion kinetics may be due to the changes made to the cell culture technique in runs 3 and 4, specifically the volume to surface area ratio. The reduction in the volume to surface ratio in runs 3 and 4 likely allowed for reasonable oxygen diffusion through the cell culture, thereby facilitating cell growth. This reduced volume to surface area ratio was achieved throughout the entire period of culture starting immediately after the SOLUPORE™ process in runs 3 and 4. For runs 1 and 2, the volume to surface area ratio was reduced 1 day after the SOLUPORE™ process when the culture was transferred from an upright T25 flask to a flat-laid T75 flask. The day 4 data suggest that these changes may not have a sustained long-term effect on T cell expansion.

[0271] GFP expression by flow cytometry In all runs, GFP expression immediately after the SOLUPORE™ process was significantly higher (p<0.001) than in statically transduced T cells (44.7% and 1.1%, respectively, FIG. 29A). This significant difference was also observed one day after delivery (p<0.001). Considering that the packaging of LV-eGFP used in this study was a crude virus preparation, this difference in GFP expression at early time points can be explained by the fact that cells likely received GFP as protein cargo from reversible permeabilization of the cell membrane during the SOLUPORE™ process, whereas statically transduced cells were not subjected to this procedure. As the data suggests (FIG. 29A), this difference diminishes over time because the protein cargo is only transiently present and is diluted as cells expand. To address the possibility of protein cargo delivery, an additional time point after 4 days of SOLUPORE™ processing was introduced for runs 2 onwards to assess whether the % GFP observed at the 3 day time point was an accurate depiction of LV delivery success as opposed to residual GFP protein uptake during the SOLUPORE™ process. In runs 2, 3 and 4, the average GFP% after 3 and 4 days of SOLUPORE™ processing was 25.7% and 25.9%, with no difference between the two time points (p>0.05; paired two-tailed t-test), suggesting that the results collected after 3 days of SOLUPORE™ processing are an accurate assessment of LV delivery efficiency.

[0272] Regardless of the viral delivery method, no significant differences in GFP% or MFI based on MOI were observed (Figures 29C and 29D), which is in line with what was observed in the historical data generated and further validates the suggestion of using an MOI of 2.5.

[0273] overview - Viable cell recovery and total cell recovery following the SOLUPORE™ process across 18 samples was 60% and 63%, respectively. - The viability after 24 hours of the SOLUPORE™ process was 90% (n=18 samples). - The system is capable of delivering LV-eGFP to T cells with an average GFP expression of 25.7% across 18 SOLUPORE™ process experiments.

[0274] By modifying the calibration to dispense the appropriate payload solution volume and optimizing the cell culture parameters (i.e., volume to surface area ratio), GFP expression was significantly improved.

[0275] Example 6: Isolation, initiation, and cell culture of PBMC-derived T cells Isolation, initiation, and cell culture of PBMC-derived T cells Methods herein describe the isolation, initiation, and cell culture of T cells derived from PBMCs. The exemplary methods outlined below cover T cells initiated from PBMCs, including the isolation, culture, and initiation of these cells.

[0276] Related acronyms: AB serum: Human serum from an AB donor that lacks antibodies to A and B blood group antigens. APC: Allophycocyanin BSC: Biological Safety Cabinet DMSO: Dimethyl sulfoxide DPBS: Dulbecco's phosphate buffered saline IL-2: Interleukin 2 is a cytokine required for T cell proliferation and survival IU: International Units FBS-HI: Fetal bovine serum-heat inactivated mRNA: messenger RNA PBMC: peripheral blood mononuclear cells RT: room temperature TCGM: T cell growth medium 7-AAD: 7-aminoactinomycin D

[0277] material TIFF0007637072000019.tif58160

[0278] device TIFF0007637072000020.tif32128

[0279] procedure PBMC isolation Preparation of dilution buffer: TIFF0007637072000021.tif12160

[0280] Preparation of CTS T cell culture medium (TCGM) TIFF0007637072000022.tif24160 * IL-2 is added on day 0. Once IL-2 is added it is used within one week and is always made fresh on the day of initiation. 1. Upon opening a bottle of CTS, remember to immediately add CTS supplement. 2. Add serum or equivalent and filter using a 50 ml syringe and a 0.2 μm filter or using a 0.2 μm SteriCup. 3. Once filtered, add L-glutamine and IL-2. 4. Use the medium within one week.

[0281] Isolation of PBMCs 1. Dilute blood with DPBS (called dilution buffer) containing 1% heat inactivated FBS in sterile Duran. 1.1. Dilute the buffy coat in a 1:1 ratio (blood:dilution buffer). 1.2. Dilute the contents of the Leukopak in a 1:2 ratio (blood:dilution buffer). 2. Add 10 ml of Lymphoprep solution to a 50 ml Falcon (the number of Falcon is the number of ml of total diluted blood divided by 40). 3. Carefully layer the diluted blood onto the prepared Lymphoprep by holding the 50 ml tube perpendicular to the 25 ml stripette. 4. Centrifuge at 400 g for 22 minutes with acceleration set to 6 and brake off (deceleration set to 0 - minimum). See Figure 34. 5. Remove the top (serum) layer using a sterile Pasteur pipette and discard all but 3 ml of serum layer in the tube. 6. Carefully transfer the PBMC layer (the cloudy white layer below the serum and on top of the Lymphoprep) to a clean tube, making sure not to remove any of the red blood cell pellet. 7. Top up the buffy coat layer to 50 ml with DPBS+1% HI-FBS, mix and centrifuge at 450 x g for 7 minutes with the brake on (acceleration 9, deceleration 9 - use these settings for the remainder of the protocol). 8. Carefully pour off the supernatant without disturbing the PBMC pellet. Resuspend the pellet in a small volume (<1 ml or the amount of remaining liquid). Vortex. 9. Top up the PBMCs to 50 ml with DPBS+1% HI-FBS, mix and centrifuge at 450 g for 7 min.8.9. 10. Discard the supernatant by pouring. Resuspend the pellet in 30 ml DPBS + 1% HI-FBS. This is the isolated PBMC. 11. Count the cells: In the Eppendorf, add 50 μl of cell suspension to 950 μl of DPBS + 1% HI-FBS to make a 1:20 dilution. Using a Via1 cassette, take the diluted cell suspension and add it to the Nucleocounter. Count the cells under the "Cell Count and Viability" program, making sure to add the cell dilution to the program. See WI-6 NC-3000 NucleoCounter Operation & Maintenance. 12. The PBMCs can be cryopreserved at this point by following the freezing steps in the "Freezing" section below. PBMCs should be frozen at 50 million cells per ml.

[0282] Freezing of PBMCs 1. To cryopreserve PBMCs, centrifuge the cell suspension at 400 g for 5 minutes and remove the supernatant. 2. Prepare freezing medium as 90% HI-FBS + 10% dimethyl sulfoxide (DMSO) and store on ice. Make 1 ml of freezing medium for every 50 million cells. 3. Cells can be frozen using a Mr. Frosty or a controlled rate freezer.

[0283] Mr. Frosty 1. Mr. Frosty should refill the container with room temperature isopropanol up to the mark (refill monthly) to ensure the freezing process is as quick as possible. 2. The cryovial should be pre-labeled and pre-opened in the BSC. 3. Using a 25 ml stripette, begin to slowly drip the Freezing Medium onto the cell pellet, making sure to swirl at the same time. 4. Using the same 25 ml stripette, remove most of the cell and freezing media solution and quickly add 1 ml to the prepared cryovial. 5. Quickly cap the tubes and add them to the Mr. Frosty container, then move the Mr. Frosty to a -80°C freezer. 6. Cells are cultured the next day. On dry ice only Then it should be transferred to liquid nitrogen.

[0284] Controlled Rate Freezer 1. 1-2 days prior to isolation, ensure the pressurized liquid nitrogen tank is filled and check the liquid nitrogen level before use. 2. If cryovials are used, they should be pre-labeled and pre-opened in the BSC. 3. Centrifuge the cell suspension. 4. Follow the instructions for a WI-10 control rate freezer equipped with a low pressure liquid nitrogen supply tank and activate the "T cell" setting on the control rate freezer. 5. Using a 25 ml stripette, slowly add the Freezing Medium dropwise onto the cell pellet while swirling. 6. Using the same 25 ml stripette, remove most of the cell and freezing media solution and quickly add 1 ml into the vial. 7. While still in the BSC, quickly cap the tube. 8. When a temperature of 4°C is reached, add the samples to the controlled rate freezer. 9. Immediately after the freezer has completed its run (ie, when it reaches -180°C), the cells should be transferred to liquid nitrogen.

[0285] T cell initiation Day 0 - Thawing This protocol follows procedures for the use of a single donor and different donors should not be mixed. 1. Prepare medium and warm to 37°C. 2. Remove the required number of PBMC vials from liquid nitrogen. 2.1. Due to donor-to-donor variability in PBMC proliferation, it may be necessary to initiate more PBMC than the number of T cells required on day 3 due to different degrees of PBMC expansion. 3. In the BSC, prepare one 50 ml tube containing 10 ml of pre-warmed media (10 ml for each vial of cells to be thawed). 4. Thaw the PBMC vials in a 37° C. water bath (thaw no more than two vials at a time) with occasional gentle swirling until only small pieces of ice remain. 5. Return the vial to the BSC and slowly add 1 ml of warm media to the vial, pipetting up and down once to mix. 6. Slowly transfer the contents of the vial into an appropriately labeled 50 ml falcon pre-warmed medium. 7. Rinse the vial with medium and transfer it to a 50 ml tube. 8. Bring the volume to 50 ml with T Cell Growth Medium (TCGM). 9. Centrifuge at 300 x g for 7 minutes at RT. 10. In the BSC, aspirate and discard the supernatant. 11. Resuspend in 10 ml of TCGM. 12. Count the cells using a Nucleocounter. 12.1. To count the cells, take an aliquot (50 μl) of the cell suspension and dilute with 450 μl of medium in an Eppendorf to a final volume of 500 μl. 12.2. Count cells using the Via-1 cassette and Nucleocounter - include dilution on the software (i.e. enter "50" in the sample box and "450" ​​in the dilution box - Nucleocounter will calculate the dilution factor). 12.3. Visually inspect the gates on the software and, if necessary, modify them to include the body of the cells and record cell viability and concentration. 13. Increase the cell volume with TCGM to 1 x 10 6 / ml. 14. Add 0.5 μl per million cells of both CD3 and CD28 antibodies. 15. To initiate, seed the cells into appropriately labeled flasks. 15.1. Maximum 30 ml / T25. 15.2. Maximum 80 ml / T75. 16. Place the flask in 5% CO 2 Place on your side (horizontally) in a buffered water-jacketed incubator at 37°C. 17. Cells should be left untouched for approximately 72 / 96 hours (seed on Monday for Thursday use or seed on Friday for Monday / Tuesday use). 18. The cells will look "flaky" or "snow-like" after 3 days in the flask and are starting well.

[0286] Example 7: Solupore™ System for PBMC-Initiated T Cell Cultures The SOLUPORE™ process of PBMC initiated T cells is carried out in a functionally closed instrument.

[0287] Related Acronyms BSC: Biosafety Cabinet d: Diameter EtOH: Ethanol GFP: Green fluorescent protein PBS: Phosphate-buffered saline Pen / Strep: Penicillin / Streptomycin PETE: Polyester track etching PC: Polycarbonate PS: Polysulfone SS: Stainless steel Al: Aluminum WFI: Water for injection mRNA: messenger ribonucleic acid

[0288] Materials and Equipment System Equipment TIFF0007637072000023.tif64154

[0289] System Components TIFF0007637072000024.tif126144

[0290] System Consumables TIFF0007637072000025.tif110143

[0291] System Consumables TIFF0007637072000026.tif92165

[0292] Preparation of solutions All solution preparations must be performed in a BSC. 1. Stop solution : Prepare 50 mL of stop solution by mixing 25 mL of PBS and 25 mL of WFI in a labeled 50 mL falcon tube and keep on ice until needed. Do not use PBS that has been opened for more than one month. Record the date opened in the run log. 2. Cell culture media : Calculate the amount of medium needed based on the total number of samples and the resuspension volume. Prepare complete cell culture medium (+0.5% Pen / Strep) or obtain an aliquot of previously prepared medium (120 mL aliquot). Keep at room temperature. 3. Calibration Solutions : Prepare a minimum 10 mL volume of payload-free delivery solution for calibration of the nebulizer by adding the following volumes in the order detailed in (Table 5): first WFI, then EtOH (final 12%), and finally S Buffer (prepared at 20x) to a 50 mL Falcon tube labeled "Calibration". Note: Do not add S Buffer and EtOH solutions together as this will result in precipitation of excipients in S Buffer. Make sure to add WFI first. 3.1. Elveflow Reservoir : In the BSC, transfer 1 mL of "Calibration" solution to a labeled 1.5 mL sterile Eppendorf tube. If the number of samples (number of sprays) for the SOLUPORE™ Process is known, proceed to 4. SOLUPORE™ Process Solution Preparation. If the number of samples (number of sprays) for the SOLUPORE™ Process is not known, proceed to 5. Assembly and Calibration, and once the number of samples is known, prepare the SOLUPORE™ Process Solutions.

[0293] Payload-free delivery solution for nebulizer calibration TIFF0007637072000027.tif20128

[0294] 4. SOLUPORE™ Process Solutions : Using the calculations in Table 6, an example is given for 10 sprays, 2.4 x 10 7 Calculate the volume of delivery solution required for the SOLUPORE™ process of T cells.

[0295] Calculating the Total Volume of Delivery Solution Required for the SOLUPORE™ Process TIFF0007637072000028.tif29128 * Note that varying the delivery volume of the nebulizer may alter the priming volume and dead volume, and the above calculations will be modified accordingly. ** The excess volume corresponds to the 10% excess allowed during calibration. 4.1. Calculate the required volumes of payload solution and delivery solution components for the required number of experimental sprays and record in the run record, with reference to Table 7. An example is given for 10 sprays at GFP concentrations of 0.1 μg / mL and 0.4 μg / mL. 4.2. In the BSC, add the components of the delivery solution to a 1.5 ml Eppendorf tube labeled "SOLUPORE™ Process" and store on ice until needed. Note: Do not add the S Buffer and EtOH solutions together as this will result in precipitation of the excipients in the S Buffer. Make sure to add the WFI first. Do not add the payload (GFP mRNA) at this stage.

[0296] Payload and delivery solution composition for syringes and Elveflow reservoirs TIFF0007637072000029.tif42160

[0297] 5. Assembling the system, calibrating the nebulizer, and priming the filter membrane 5.1. Using the checklist provided, transfer all necessary bags containing components and consumables from the EtO cabinet into the BSC to assemble the system unit (Figure 35 and Figure 36). 5.2. Using the checklist provided, aseptically transfer consumables and labware needed for one experimental run into the BSC. 5.3. Unpack the sterile components and dispose of the bag. 5.4. To assemble the lid (Figure 36): 5.4.1. Place the X-ring to seal the atomizer (a). 5.4.2. Insert the atomizer equipped with an airline (b). 5.4.3. Add the cap to secure the sprayer in place (c). 5.4.4. Screw on the sprayer cap using the Allen key provided. (d). 5.4.5. Install the bracket containing the Clippard valve as shown in (e). 5.4.6. Fix the bracket by fastening with screws (f). 5.4.7. Install the reservoir holder and Elve flow module (g). 5.4.8 Connect the silicone tubing to the reservoir needle (h). Select Settings on the Unit Control and press Purge, which opens the clippard valve, then insert a segment of silicone tubing into the Clippard valve. 5.4.9. Connect the other end of the silicone tubing to the needle in the nebulizer sample channel. Ensure that the silicone tubing below the clippard valve is straight. If not, press purge to open the clippard valve and straighten it. 5.4.10. Connect the Clippard valve wires as shown in the diagram (j). 5.4.11. Connect the atomizer air line to the solenoid valve. 5.4.12. Connect the vent tube and filter (k). 8.5.5. Place the System Base (Figure 35) on the tilt stand as in Figure 36. 5.6. Screw the three 1 / 4-28 Luer locks provided into the drain port, bleed port, and cell inlet / outlet ports. 5.7. Connect the luer locks to each of the tubing, ensuring all clamps are closed. 5.8. Connect a 0.2 μm filter to the end of the bleed port tubing and connect a waste bag to the drain port tubing. 5.9. Insert o-ring 1 (the thicker o-ring) into the groove around the drain area (Figures 35 and 36). 5.10. Place the membrane holder on the base (Figures 35-37). 5.11. Place gasket 1 onto the membrane holder (Figures 35-38). 5.12. If assembling for filter priming with SOLUPORE™ process, add the drain disk (Figures 35-39) and proceed to assembly for calibration or loading of SOLUPORE™ process solution, adding the blue filter divider in place of the drain disk and filter membrane (Figures 35-39). Continue with 5.15. 5.13. Wet the disk by pipetting 1 ml of 1x PBS. Ensure that the drain port is open. Gently press down on the edge of the drain disk to facilitate even wetting of the drain disk and rotate the disk to distribute the liquid, as shown in Figure 37. Do not touch the drain disk within the area bounded by gasket 1. 5.14. Place the PETE filter membrane on top of the drain disc. 5.15. If preparing only a calibration, place Gasket 2 on top of the filter membrane (Figures 35-40) or blue filter divider. Note for the SOLUPORE™ Process: When placing Gasket 2, be careful to avoid wrinkling the filter. If the filter is visible to be wrinkled (Figure 38), dispose of it and start again with a new filter. Note for the SOLUPORE™ Process: To aid in positioning the filter membrane between sample runs, an empty syringe can be connected to the drain tubing. Use this syringe to pull a gentle vacuum to help maintain a flat filter membrane before placing Gasket 2 on top. 5.16. Insert O-ring 2 (Figures 35-41). 5.17. Place the PS or SS mask (Figures 35-42). 5.18. Insert O-ring 3 (Figure 35-42). 5.19. Open the clamp on the drain tube. 5.20. Place the lid over the unit without clamping. 5.21. Go to 5.22 for calibration and 8.7 for filter priming for the SOLUPORE™ process.

[0298] calibration 5.22. Prime the nebulizer with calibration solution. 5.23. Unscrew the base of the Elveflow unit counterclockwise (Figure 39) and remove the 1.5 mL Eppendorf tube (to protect the needle during storage). Place the "calibration" tube in the base and screw the Elveflow unit back on. 5.24. On the Controller (Figure 40), select "Air" and set the pressure to 1730 mbar, then select "Sample" and set the pressure to 50 mbar. 5.25. Remove the lid and place the calibration cup, which has been cleaned with 70% IPA, under the sprayer. 5.26. Activate the "Spray" button until a spray appears at the tip of the sprayer (usually 2-3 times). 5.27. Set the “Sample” pressure to 100 mbar, or start with the calibrated setting from an experimental run the previous day. 5.28. Activate the “Spray” button twice, verify by visual inspection that spray has occurred, and remove the waste container. 5.29. To calibrate the atomizer: 5.30. Using scissors, cut the top edge of the calibration cup that has been cleaned with 70% IPA. 5.31. Tare the cup on the analytical balance. 5.32. Place the cup into the MID 1T chamber as shown in Figure 41. 5.33. Place the lid over the unit and simultaneously close the two opposing clamps with even pressure. 5.34. Ensure that the clamp on the vent port is open. 5.35. Close the clamps on the drain tubing, cell inlet / outlet tubing, and bleed port tubing. 5.36. Nebulizer calibration: 5.36.1. Ensure that the air pressure is set to 1730 mbar. 5.36.2. Set the sample pressure to the last calibration value recorded on the run record. 5.36.3. Using the manual pressure regulator, set the airflow through the showerhead to the required pressure, e.g. 90 mbar (Figure 42). 5.36.4. Open the clamp on the airflow line to the showerhead. 5.36.5. Activate the “Spray” button. 5.36.6. Close the clamp on the airflow line to the showerhead. 5.36.7. Immediately unclamp the lid, remove the calibration cup and replace the lid onto the unit. 5.36.8. Immediately weigh the calibration collection cup. 5.36.9. The weight will be recorded in the run record. 5.36.10. Spray the cup liberally with 70% IPA and wipe dry. 5.36.11. Weight 55 mg * If above (55 μL), reduce sample pressure by 10 mbar. Dispense one spray into waste cup and repeat spray and measurement. If weight is below 50 mg (50 μL), increase fluid pressure by 10 mbar. Dispense one spray into waste cup and repeat spray and measurement. 5.36.13. * Assumptions: The density of the delivery solution is approximately 1 g / ml. 5.37. Repeat the calibration measurement until three consecutive 50-55 mg readings are obtained, ensuring that the calibration solution does not run out and that the entire length of the sample line is always filled with liquid. 5.38. Record the optimum pressure and calibration weight in the run record. Once calibration is complete, place the waste cup under the nebulizer. Select System Settings, increase the pressure setting to 500 mbar, select Purge, Sample and press to expel the calibration solution from the nebulizer. Repeat Purge one more time and discard the waste cup.

[0299] SOLUPORE™ Process 6. Loading of payload solution "SOLUPORE™ Process Solution" 6.1. Disconnect the sample line from the control unit line. Figure 43. 6.2. Load the required volume of GFP into the SOLUPORE™ process solution. 6.3. Unscrew the base of the Elveflow unit counterclockwise (Figure 39) and take out a 1.5 mL Eppendorf tube (calibration). Place the "SOLUPORE™ Process" tube in the base and screw the Elveflow unit back on. 6.4. Reconnect the sample line to the control unit line. 6.5. On the Controller (Figure 40), select "Air" and ensure the pressure is set to 1730 mbar, then select "Sample" and set the pressure to 50 mbar. 6.6. Remove the lid and place the calibration cup, which has been cleaned with 70% IPA, on top of the blue filter membrane divider under the sprayer. 6.7. Activate the "Spray" button until a spray appears at the tip of the sprayer (usually 2-3 times). 6.8. Set the sample pressure to the calibrated value and actuate the “Spray” button once. Verify by visual inspection that spray occurs. 6.9. Remove the blue disk divider and assemble the filter drain disk and filter membrane as described in 5.13-5.20 and proceed to 7 for filter priming for the SOLUPORE™ process.

[0300] 7. Filter Priming for the SOLUPORE™ Process 7.1. Place the lid onto the unit and lock the opposing clamps together to seal the system chamber. 7.2. Ensure that you have: 4 x 60 ml syringes 4 x Syringe Caps 1 x 150 ml sterile bag 1 x Luer spike interconnector 1 x 0.2 μm filter, on the bleed port tube before moving on to priming the filter membrane 7.3. Take a 60 mL syringe, label it "Prime", remove the plunger, insert the syringe cap and fill the syringe with 60 mL of 1x PBS. Insert the plunger, remove the cap and connect the syringe to the cell inlet port. 7.4. Ensure that the clamps on the vent and drain tubes are closed. 7.5. Open the clamp on the cell inlet tube and load 60 mL of PBS into the chamber. 7.6. Open the pinch clamp on the airflow tube. 7.7. Open the drain port clamp and allow approximately 30 mL of PBS to drain, which is approximately 50% of the PBS. 7.8. Close the pinch clamp on the airflow tube and immediately open the clamp on the vent tube. 7.9. Verify that gravity drainage occurs. If gravity drainage is impeded, repeat from step 8.7.3. 7.10. If required, reserve 1 mL of priming solution at room temperature for sterility testing.

[0301] 8. Cell loading 8.1. Record on the run record. The time at which the cell sample was aliquoted by cell culture Sample volume Cell concentration, e.g., 24 x 10 6 cells / 30 mL Time to load cells into chamber 8.2. Ensure that the cell suspension provided in the basal medium is at the required cell concentration. 8.3. Mix homogenously using a 5 mL pipette set to 4 mL and gently disrupt any cell clumps by pipetting up and down 10 times. 8.4. Using a 200 μL pipette, take 2×200 μL of cell suspension and transfer each aliquot into a 1.5 mL Eppendorf tube labeled “BS (before SOLUPORE™ process) + sample number” for counting and flow cytometry analysis. Repeat for all samples. 8.5. For flow cytometry, place all samples in 1.5 mL tubes on ice and perform analysis at the end of the SOLUPORE™ process experiment. 8.6. Count according to WI-06. Briefly: Mix the sample gently by pipetting up and down 5 times with a P200 pipette. Do not foam the sample. Count using Via 1 cassettes on an NC3000 / NC200 (following WI-06). Record the time of counting in the run record. Make sure to use the same nucleocounter to count all samples before and after the SOLUPORE™ process. 8.7. Obtain a temperature reading of the Stop Solution prior to first use and record in the run record. 8.8. Disconnect and recap the syringe used for priming. Save it for the next run. 8.9. Take a sterile 60 mL syringe, remove the plunger, connect the syringe to the cap and gently pour the cell suspension into the syringe. 8.10. Insert the plunger, invert the syringe, remove the cap and gently expel the air. 8.11. Connect the syringes to the cell inlet / outlet tubing. 8.12. Ensure that the clamp on the drain tube is closed. 8.13. Open the clamps on the cell inlet / outlet tubing and gently push the plunger to load the cell suspension into the chamber. Hold the syringe plunger down to fill the inlet tubing and once filled, rotate the syringe 180° and add the remaining cell suspension. Once the cells are loaded, remove the syringe and purge the inlet line with air from the air-filled syringe to push any remaining liquid in the line into the chamber. Avoid introducing air bubbles by gently tilting the chamber to the left. 8.14. Close the clamps on the cell inlet / outlet tubing and discard the syringe. 8.15. Prepare a labeled syringe containing 5 mL of Stop Solution and another labeled syringe containing 20 mL of cell culture medium; cap the syringes and set aside. 8.16. Open the drain clamp and start the timer to measure the filtration time. 8.17. The filter appears dry to the eye and liquid flows through the tube. When there are no more, the filtration is considered complete. 8.18. Record the time of filtration in the run record. 8.19. Once no more liquid is flowing through the drain tube, open the bleed port clamp and hold the bleed port tube in an upright position above the unit base to observe the remaining liquid flowing through the drain tube. Ensure that all liquid is removed from the drain tube. 8.20. Close all clamps on the drain line, waste bag, and bleed port tubing. 8.21. Disconnect and set aside the waste bag. Connect the syringe containing the harvest solution, cell culture medium. 8.22. Connect the syringe containing the stop solution to the inlet tube. 8.23. Turn on the airflow through the shower head (e.g., set to 90 mabr), open the clamp on the airflow line, and immediately activate the "Spray" button and start a 30 second timer. 8.24. Close the clamps on the airflow lines. 8.25. After the 30 second incubation, open the clamp on the inlet line and add Stop Solution and incubate for an additional 30 seconds. 8.26. Open the drain clamp and rapidly flush 20 mL of medium into the chamber. 8.27. Tilt the chamber and gently aspirate the cell suspension into the syringe in and out of the chamber 14 times. Avoid the formation of air bubbles by gently pulling the plunger up and down to slowly aspirate the solution in and out of the syringe. Ensure that the entire filter is washed with the recovery solution. 8.28. Tilt the chamber back to the neutral position. 8.29. Collect the cell suspension by slowly tilting the chamber to its maximum angle while slowly aspirating the cell suspension into the syringe. If air bubbles are observed on the mask, return to neutral position and tilt the chamber again to aspirate the air bubbles into the syringe. 8.30. Close the inlet ramp and disconnect the syringe. 8.31. Transfer the cell suspension into a T-25 flask. 8.32. Reconnect the syringe to the inlet port and aspirate any remaining cell suspension and transfer into the T-25 flask.

[0302] 9. Post-recovery analysis 9.1. Using a 200 μL pipette, take 2×200 μL of cell suspension and transfer each aliquot to a 1.5 mL Eppendorf tube labeled “AS (after SOLUPORE™ process) + sample number” for counting and flow cytometry analysis and set aside at room temperature. 9.2. Weigh the cell culture dish containing the Solupore processed cell suspension and record the weight / volume in the run record. 9.3. Ensure cell culture dishes are labeled: sample name; volume recorded; operator name; date. 9.4. Keep the cell culture dishes at 37 ± 2°C and 5% CO 2 , and place in an upright position in an incubator at 95% relative humidity. Count the "Post-Sol" sample set aside for counting, count according to WI-06 and record the time of counting in the run record. Be sure to use the same nucleocounter to count the "Pre-Sol" and "Post-Sol" samples.

[0303] 10. Cleaning between samples 10.1. Dispose of waste bags between experimental replicates. 10.2. Unclamp and remove the lid, disassemble the filtration unit, remove and discard the drain disc and filter membrane. 10.3. Reassemble the instrument without inserting the filter. 10.4. Clamp the chamber lid. 10.5. Ensure that the drain port clamp and bleed port clamp are closed. 10.6. Fill a sterile, labeled syringe with 60 mL of WFI and connect to the inlet tubing to load the water into the chamber. 10.7. Hold the instrument at both ends and rotate the chamber to facilitate rinsing of all surfaces (Figure 44). Tilt the unit to its maximum angle to ensure that the mask and lid walls are cleaned. 10.8. Remove the liquid by aspirating with a syringe connected to the drain tube. 10.9. Repeat 10.6-10.8 using 60 mL of 1x PBS. 10.10. The chamber is now ready to be reassembled with a new drain disc and filter membrane for a new sample. 11. After assembly of the unit with new drain discs and filter membranes, repeat from section 5.13 for all other samples. 12. Record the sample names and conditions tested in the run record. 13. Once the experiment is complete, move on to the cleaning system. 14. Export all required NC3000 / NC200 files and save in the appropriate experiment folder for analysis. 15. Cells are placed in culture for 24 hours and analyzed for: Number of cells recovered (NC3000 / NC200) Cell viability (NC3000 / NC200) Transfection efficiency (%GFP - flow cytometry)

[0304] Example 8: Cell thawing and cell preparation The methods described herein provide standard protocols for the preparation of cell culture medium, thawing cell stocks, culturing cells, and preparing cells for experimental use. The methods cover the preparation of cell culture medium and procedures for thawing cell stocks stored in liquid nitrogen, as well as culturing cells in preparation for experimental use.

[0305] Related Acronyms DMSO - Dimethyl Sulfoxide FBS-HI - Fetal Bovine Serum-Heat Inactivated SDS - Safety Data Sheet PBS - Phosphate Buffered Saline SFM: Serum-free medium P / S: Penicillin-Streptomycin TC: tissue culture BSC: Biosafety Cabinet

[0306] material TIFF0007637072000030.tif156160

[0307] device TIFF0007637072000031.tif61160

[0308] procedure 1. Preparation of CD3+ T cell culture medium TIFF0007637072000032.tif37160

[0309] 1. To prepare complete medium for CD3+ T cells, add a full bottle of CTS Optimizer supplement to a full bottle of CTS Optimizer medium and record the lot number for each on the cell culture template. 2. Add the required volume of L-glutamine as in the table above. 3. Before adding 0.1% IL-2 (200 U / ml), filter-sterilize the medium using a vacuum filtration system (hereinafter referred to as Stericup Image). 4. P / S is optional for post-transfection medium and is user specified. 5. To use the filtration system, spray the vacuum and stericup with 70% ethanol before placing in the BSC. The vacuum pump has two outlets, ensure that the tubing is connected to air at the outlet. 6. Plug the vacuum device into power and ensure that the power is on at the BSC. 7. Remove the stericup from its sterile packaging and attach the nozzle of the vacuum pump to the stericup. 8. Remove the transparent lid from the stericup and add the pre-prepared medium to the top of the stericup and replace the lid on the stericup. 9. Turn on the vacuum by using the switch on the front of the pump. 10. The medium will then pass through the filter and once it has passed the stericup, turn off the pump and remove the nozzle from the stericup. 11. Remove the top of the stericup with a twisting motion and twist the sterile lid (blue) onto the bottom of the stericup containing the filter-sterilized medium. It will click when fully closed. 12. For large batch preparation of media, store the media at 4°C in clearly labeled stericups before aliquoting the media in 50 / 120 ml falcons if required. Note that media cannot be stored at 4°C for longer than 7 days. 13. Add 0.1% IL-2 (200 U / ml) for T cell culture. 14. To prepare IL-2, add 2 ml of sterile water to one lyophilized vial of 50 μg recombinant human IL-2, mix and divide into 500 μl aliquots. Store at -20°C. 15. Keep all cytokines and antibiotics (optional after transfection) on ice during thawing or prior to use. 16. After use, return reagents to storage, e.g., -20°C or 4°C. 17. Media should be warmed in a water bath set to 37°C for at least 15 minutes prior to use. 18. Record the water bath temperature on the cell culture template.

[0310] Preparation of PBMC-initiated CD3+ T cell culture medium TIFF0007637072000033.tif42163

[0311] 1. To prepare TCGM complete medium for PBMC initiated CD3+ T cells, add a full bottle of CTS Optimizer supplement to a full bottle of CTS Optimizer medium and record the lot number for each on the cell culture template. 2. Add the required volumes of Human Ab Serum and Physiologix Serum as in the table above. 3. Add the required volume of L-glutamine as per 8.3. in the table above. 4. Add the required volume of HEPES buffer as in the table above. 5. Prior to addition of P / S and IL-2, filter sterilize the medium using a vacuum filtration system. 6. To use the filtration system, spray the filtration system and stericup with 70% ethanol before placing in the BSC. The vacuum pump has two outlets, ensure that the tubing is connected to air at the outlet. 7. Plug the filtration system into a power source and ensure that the power is on in the BSC. 8. Remove the stericup from its sterile packaging and attach the nozzle of the filtration system to the stericup. 9. Remove the clear lid from the stericup and add the pre-prepared medium to the top of the stericup and replace the lid on the stericup. 10. Turn on the filtration system by using the switch on the front of the filtration system. 11. The medium will then pass through the filter and once it has passed the stericup, the vacuum is turned off and the nozzle is removed from the stericup. 12. Remove the top of the stericup with a twisting motion and twist the sterile lid (blue) onto the bottom of the stericup containing the filter-sterilized medium. It will click when fully closed. 13. For large batch preparation of media, store media at 4°C in clearly labeled stericups before preparing aliquots in 50 / 120 ml falcon and before addition of P / S and IL-2, if necessary. Note that media cannot be stored at 4°C for longer than 7 days. 14. Add IL-2 (200 U / ml) for T cell culture. To prepare IL-2, add 2 ml of sterile water to one lyophilized vial of 50 μg recombinant human IL-2, mix and divide into 500 μl aliquots. Store at -20°C. 15. Optionally, add P / S (10,000 U / ml) (250 μl per 50 ml of medium) after transfection. 16. Before use, warm the medium for at least 15 minutes using a water bath set to 37°C.

[0312] Thawing and activation of cells 1. Prepare CD3+ T cell medium or complete TCGM medium and warm to 37°C. 2. Prepare CTS™ OpTmizer™ T Cell Expansion SFM containing supplements only (this is the base medium) and warm to 37°C. 3. Record the water bath temperature on the cell culture template. 4. In the BSC, prepare one 50 ml tube containing 10 ml of pre-warmed media (10 ml for each vial of cells to be thawed). 5. Remove the required vials / bags from the Liquid Nitrogen / -150°C freezer. (Please note that all appropriate PPE must be worn while working with Liquid Nitrogen or the -150°C freezer.) 5.1. Due to donor-to-donor variability in PBMC proliferation, it may be necessary to initiate more PBMCs than the number of T cells required on day 3 due to differences in the extent of PBMC expansion. This number should be assessed the first few times a new donor is thawed. 5.2. Due to loss of CD3+ T cells after activation, it is necessary to thaw and activate at least twice the number of cells required. TIFF0007637072000034.tif161606. Thaw the cell vials in a 37°C water bath (thaw no more than two vials at a time) with occasional gentle swirling until only small pieces of ice remain. Be sure not to let the rim of the vial come into contact with water from the water bath as this is a risk of contamination. 7. When "ice pebbles" are visible, move to the BSC, spray and wipe the vial with 70% EtOH and rapidly dilute the cells in culture media by adding 1 ml of warmed media to the cryovial using a P1000 pipette or 3 ml to the cryobag using a Luer-lock syringe. 8. Add the contents of the vial / bag to the previously prepared media in falcon. Note: DMSO is toxic to cells, so thawed cells should not be left in the freezing mixture for any extended period of time. 9. Rinse the vial with another 1 ml of media to ensure all cells are collected and transfer to the same falcon containing the cells. 10. Centrifuge the falcons at 300 × 8.10 g for 7 min at RT. 11. Check for cell pellet and remove the supernatant without disturbing the pellet and discard into a waste beaker. This can be done by pouring or by removing with a strip or Pasteur pipette. 12. Resuspend the pellet in culture medium to a cell density of greater than 1×10 6 / ml (usually 10 ml for CD3+ T cell vials, 20 ml for PBMC vials, and 30 ml for bags). 13. Count cells using the Via-1 cassette (see WI-6 NC-3000 NucleoCounter Operation & Maintenance or outline below). 13.1. To count the cells, take 50 μl of cell suspension and dilute with 150 μl of medium in an Eppendorf to a final volume of 200 μl. 13.2. Count cells using the Via-1 cassette and Nucleocounter - include dilution on the software (i.e. enter "50" in the sample box and "150" in the dilution box - Nucleocounter will calculate the dilution factor). 13.3. Visually inspect the gates on the software and, if necessary, modify them to include the body of the cells and record cell viability and concentration (cells / ml) and size on the cell culture template. 14. Calculate the total cell number and record all calculations and cell numbers on the cell culture template. 15. Seed the cells into appropriately labeled cell culture flasks or bags at a density of 1 x 10 6 / ml. TIFF0007637072000035.tif2116616. For CD3+ T cells, place TC flask upright in the incubator. 16.1. For CD3+ T cells, rest the cells in a 37° C. incubator after the thawing process for at least 4 hours before proceeding with the activation step. 16.2. To activate CD3+ T cells, add a 2:1 ratio of CTS Grade Dynabeads:cells (5 μl per million cells). 16.2.1. For example, for 100 x 106 cells, add 500 μl of CTS Dynabeads to the cells and gently swirl the flask to ensure that the Dynabeads are evenly distributed amongst the entire cell population in the TC flask. 16.3. Place the cells back in the incubator upright until they are required for experimental use. 16.4. Unless otherwise stated, prepare cells for experimental use 19 hours after activation. 17. PBMC are activated immediately after thawing with soluble CD3 pure functional grade, human (clone: ​​OKT3) and CD28 pure functional grade, human (clone: ​​15E8) antibodies. 18. Ensure that the CD3 and CD28 antibodies remain at 4°C and store on ice until time of activation. 19. Ensure that CD3 and CD28 antibodies are dated within one month of first opening, color labeled, and contain the user's initials. Upon opening, the date and user's initials must be written on the vial. Every user must have their own set of CD3 and CD28 antibodies. Sharing between users is not permitted. 20. Record the CD3 and CD28 lot numbers and expiration dates on the cell culture template. 20.1. For 1× PBMC activation, add 0.5 μl each of CD3 and CD28 antibodies per million cells. 20.1.1. For example, for 1x activation of 100 x 106 PBMCs, add 50 μl of CD3 antibody and 50 μl of CD28 antibody to the falcon cells and gently invert the 50 ml tube to ensure that the antibodies are evenly distributed amongst the entire cell population. 20.2. For 2x PBMC activation, add 1 μl each of CD3 and CD28 antibodies per million cells. 20.3. To initiate, cells are seeded into appropriately labeled T75 flasks. 20.4. Gently place the flask on its side (horizontally) in the incubator. 20.5. Cells should be left undisturbed for approximately 72-96 hours. 20.6. Cells will appear "flaky" or "snow-like" 72 hours after initiation. 20.7. Always examine cells under a microscope for the appearance of healthy clumps of PBMC.

[0313] Preparation of CD3+ T cells for experimental use 1. 19 hours after activation, remove the flask containing the CD3+ T cells from the incubator to the BSC. 2. Gently mix the cell-Dynabead suspension with a stripette and transfer to a 50 ml falcon tube. 3. Wash the flask with 5 ml of medium and transfer to the same falcon tube. 4. Vortex the falcon tube thoroughly and place in the 50 ml magnet. 5. Wait 2 minutes. 6. Using a 25 ml stripette, carefully pipette down to the 5 ml mark without touching the end of the falcon to collect most of the media. This media contains the de-beaded cells and will be collected into freshly labeled sterilin. 7. Leave approximately 5 ml of medium in the bottom of the falcon, while avoiding disturbing the beads. 8. Remove the falcon tube from the magnet and add an additional 5 ml of medium and mix again. 8.9. 9. Repeat incubation of the falcon tube in the 50 ml magnet and collection of medium (steps 5-9). 10. Remove the 50 ml falcon from the magnet for the second time. 11. Using a P1000 pipette, collect the dynabeads with the medium remaining at the bottom of the falcon tube. 12. Place this medium containing the beads into a 1.5 ml Eppendorf and place in the Eppendorf (small) magnet for 2 minutes. 13. Remove the medium and pipette it into sterilin. 14. Add 1 ml of medium to the Eppendorf, mix and wash. Place on the Eppendorf magnet and remove the supernatant to sterilin. 15. Discard the Eppendorf containing the beads. 16. Centrifuge the sterilin containing the cells at 300 x g for 7 minutes. 17. The expected number of cells / ml is 50 x 10 6 If there are fewer than cells, resuspend the cells in 10 ml of fresh medium. When resuspending, always use a P1000 first to disrupt the cell pellet. 18. Expected number of cells / 8.18 ml is 100 x 10 6 If there are more than cells, resuspend the cells in 20 ml of fresh medium. 19. In an Eppendorf, prepare dilutions of cells by adding 50 μl of cell suspension to 150 μl of medium and count using a Via-1 cassette. All counts should be done in duplicate for accuracy. 20. Record the number of cells / ml after de-bead, cell viability, and cell size on the cell culture template. 21. Calculate the total cells / ml and volume of cell suspension needed for the experiment. 21.1. For example, when counting cells, 4 x 10 6 cells / ml, and the total cell count was 80 × 10 6 20 x 10 in a 10 ml aliquot 6 The two parameters in are preferred. 21.2. Need / have = 20 x 10 6 / 4×10 6= 5 x 2.5 parameters = 12.5 ml of cell suspension to 12.5 ml of CTS OpTmizer medium + supplements. This is the master mix, always make up an extra 0.5 of the condition. For example, if there are 2 parameters, always make up 2.5x, so a final volume of 25 ml if calling for 20 ml. This allows for some pipetting error. 22. Prepare a cell master mix containing cell suspension (added using P5000) and CTS OpTmizer medium + supplements in either a 120 ml falcon or 500 ml falcon (depending on desired volume), mix gently by inversion, and aliquot 10 ml of cell suspension into white cap 30 ml tubes using a P5000 or P10000 pipette for precision. After aliquoting every 3 tubes, close and invert the master mix several times to ensure a homogenous cell suspension is maintained. 23. Ensure that the 30 ml white cap sterlins are clearly labeled with the experiment number, donor number, cell number, and the initials of the person who prepared the sample. 24. For untreated cell samples, ensure that cells are prepared in T25 TC flasks containing CD3+ T cell complete medium, not CTS OpTmizer medium + supplements. UT cells are cultured at 0.5 x 10 6 / ml, i.e. 10 × 10 in 20 ml 6 Cells are growing exponentially to ensure good viability on day 1 after seeding for experiments.

[0314] Preparation of PBMC-initiated CD3+ T cells for experimental use 1. 72 hours (3 days) after initiation, remove a T75 flask of PBMC-initiated CD3+ T cells from the incubator to the BSC. Visually observe for clumps, which are a sign of properly initiated cells. 2. Transfer the cell suspension to a 50 ml centrifuge falcon. 3. Centrifuge the 50 ml falcon containing the cells at 300 x g for 7 minutes. 4. Resuspend the cells in 20-50 ml of fresh pre-warmed medium, using a P1000 to first break up the cell pellet. 6 20 ml if fewer than 100 x 10 cells are expected 6 If more than one piece is required, use 50 ml. 5. Prepare a dilution of the cells by adding 50 μl of cell suspension to 150 μl of medium in an eppendorf and count the cells using a Via-1 cassette (see WI-6 NC-3000 NucleoCounter Operation & Maintenance). 6. To ensure that the PBMCs are activated and primed, the cells are stained with CD3 (T cell purity) and CD25 (T cell activation) antibodies for flow cytometry analysis. The cells cannot be released for experimental use before this staining is fully analyzed on the flow cytometer. 6.1. To stain for CD3 and CD25 expression, 1 x 10 6 Calculate the volume of cell suspension needed for the cells. 6.2. 1×10 6 The volume of cells that will give the cells is removed, transferred to a 1.5 ml eppendorf and centrifuged at 300 xg for 5 minutes. 6.3. After centrifugation, remove the cell supernatant and resuspend the cell pellet in 100 μl of FACs buffer. 6.4. To optimize fluorescence compensation settings for flow cytometry analysis, use compensation beads when staining for CD25 and CD3 antibodies. 6.4.1. Prepare compensation beads for both CD25 and CD3 antibodies as follows: 6.4.1.1. Label two 1.5 ml eppendorf tubes CD25 comp and CD3 comp. Mix the compensation beads by vortexing. Add 1 drop of compensation beads to each tube, then add 0.5 μl of CD25 antibody or CD3 antibody to the appropriate tube. 6.5. Once compensation beads are prepared, add 5 μl of both CD25 and CD3 antibodies to 1×10 6 Add to eppendorf containing cells. 6.6. Incubate cells with antibody for 10 minutes at 4 degrees. 6.7. After 10 minutes, wash the cells by adding 1 ml of FACs buffer and centrifuge at 300×g for 5 minutes. 6.8. After centrifugation, discard the cell supernatant and resuspend the cell pellet in 100 μl of FACs buffer. 6.9. Analyze CD25 and CD3 expression using flow cytometry by acquiring 10,000 events for both bead and cell samples. 6.9.1. Obtain compensation bead samples before cell samples. 7. If the cell sample yields >90% for both CD25+ and CD3+ staining on the flow cytometer, the cells are released for experimental use. 8. If a cell sample yields <90% in both CD25+ and CD3+ staining on the flow cytometer, notify the end user and do not release the cells for experimental use unless otherwise indicated. 9. Once confirmed for release, calculate the total cells / ml and volume of cell suspension needed for the experiment based on the nucleocounter count. All counts should be done in duplicate for accuracy. (These calculations should be done during flow incubation, but do not prepare cells until they have passed QC for experimental release). 9.1. For example, when counting cells, 4 x 10 6 cells / ml, and the total cell count was 80 × 10 6 20 x 10 in a 10 ml aliquot 6 The two parameters in are preferred. 9.2. Need / have = 20 x 10 6 / 4×10 6 = 5 x 2.5 parameters = 12.5 ml of cell suspension to 12.5 ml of CTS OpTmizer medium + supplements. This is the master mix, always make up an extra 0.5 of the condition. For example, if there are 2 parameters, always make up 2.5x, so a final volume of 25 ml if calling for 20 ml. This allows for some pipetting error. 10. Prepare a cell master mix containing cell suspension (added using P5000) and CTS OpTmizer medium + supplements in either a 120 ml falcon or 500 ml falcon (depending on desired volume), mix gently by inversion, and aliquot 10 ml of cell suspension into white cap 30 ml tubes using a P5000 or P10000 pipette for precision. After aliquoting every 3 tubes, close and invert the master mix several times to ensure a homogenous cell suspension is maintained. 11. Ensure that the 30 ml white cap sterlins are clearly labeled with the experiment number, donor number, cell number, and the initials of the person who prepared the sample. 12. For untreated cell samples, ensure that cells are prepared in T25 TC flasks containing TCGM complete medium, not CTS OpTmizer medium + supplements. UT cells are cultured at 0.5 x 10 6 / ml, i.e. 10 × 10 in 20 ml 6 Cells are growing exponentially to ensure good viability on day 1 after seeding for experiments.

[0315] Example 9: LV-eGFP (high sensitivity GFP) vector A map of the LV expression plasmid vector with eGFP is provided herein in FIG.

[0316] Example 10: Incorporation of GFP after viral infection The number of integrated copies of GFP per cell was examined using ddPCR.

[0317] At 3 and 4 days after infection, 200 uL of cells were collected in a 96-well V-bottom plate. After centrifugation and removal of the supernatant, the cells were lysed with 50 uL of 1× in-house cell lysis buffer for 20 min at RT and then transferred to the PCR plate. The cells were incubated at 56C for 15 and 10 min in a thermocycler. The lysate was then diluted 20-fold with water and run with Alb / WPRE primers to determine GFP%.

[0318] Estimated copy number of GFP per cell based on WPRE per 2 albumin (GFP%) for experiment 1 (see Figures 49 and 50).

[0319] The results show that a significant increase in GFP incorporation was achieved using the SOLUPORE™ process compared to static transduction (both days 3 and 4) (see Figures 49 and 50).

[0320] Example 11: Viral infection process and droplet characteristics Atomization of the lentivirus in the transfection chamber is a separate process from the SOLUPORE™ process. As described herein, the cargo delivered to the population of cells is a biologically active and viable virus (e.g., a lentivirus).

[0321] Typical titers of lentivirus are 10 per milliliter. 6 ~10 7Transducing units (TU / ml) of lentivirus at these concentrations are in the range of 1000 to 15000, and the viscosity of lentivirus at these concentrations is highly dynamically viscous compared to water / ethanol mixtures. To illustrate, (Table 1) the dynamic viscosity of water at room temperature is close to 1 mPa s, the dynamic viscosity of ethanol is close to 0.1 mPa s, the dynamic viscosity of olive oil is close to 60 0.1 mPa s, and the dynamic viscosity of castor oil is close to 600 0.1 mPa s. The dynamic viscosity of lentivirus is reported by Tran, Reginald, PhD Thesis, Georgia Tech (2016) to be 6913 mPa s, which is close to being 1 log more viscous than castor oil. Sterile filtered 1% bovine serum albumin (BSA) has been found by some to reduce molecular interactions that can cause viral particles to "stick" to injection devices (Jasnow A. et al. Methods Mol Biol. "Construction of Cell-Type Specific Promoter Lentiviruses for Optically Guiding Electrophysiological Recordings and for Targeted Gene Delivery" 2009; 515: 199-213, incorporated herein by reference in its entirety).

[0322] Dynamic viscosity is an important factor in atomization. Experimental studies of atomization in an internally mixed twin-fluid atomizer, such as that used in the SOLUPORE™ process, over a wide range of liquid viscosities, gas supply pressures, and gas-to-liquid mass ratios (GLR) have been conducted. See, for example, Li, Z. et al. "Effect of liquid viscosity on atomization in an internal-mixing twin-fluid atomizer" Fuel vol. 103; Jan 2013 pages 486-494, which is incorporated herein by reference in its entirety. Among all test conditions, the finest spray was obtained at an axial distance of 150 mm. However, when the viscosity was increased to 120 mPa s, the droplet size distribution changed significantly. Higher viscosity droplets produced larger droplets (e.g., 1-2 logs larger than the present droplets produced by the SOLUPORE™ process, measured droplet size distribution, FIG. 51). Larger droplets represented a larger proportion of the droplet population (distribution) and the dropoff in droplet velocity along the spray axis was stronger at higher viscosities.

[0323] A table showing the dynamic viscosities of common liquids is provided below (and a graph is provided in Figure 52).

[0324] The absolute or dynamic viscosities of some common liquids at a temperature of 300 K are given below: TIFF0007637072000036.tif177150

[0325] It can be concluded that nebulization of lentivirus in the SOLUPORE™ process produces larger, slower moving droplets that experience a different layer of cells below than the previously described SOLUPORE™ process. Consequently, this nebulization process resulted in transfection levels approaching 30%, a surprising and unexpected observation.

[0326] Virus infection process The dynamic viscosity of water is close to 1 mPa s (millipascal second). The dynamic viscosity of ethanol / water mixtures is also close to 1 mPa s. The dynamic viscosity of aqueous solutions that can contain ethanol concentrations of 5-30%. Aqueous solutions contain 75-98% H 2 HO, 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), with a viscosity in the region of 2 mPa s.

[0327] Titer 10 7 ~10 8 The dynamic viscosity of lentivirus in TU / mL is close to 6913 mPa s. As the viscosity of a fluid increases, it tends to form larger droplets when sprayed at a given spray pressure, e.g., 1.7 bar. A spray consisting of smaller droplets has a much larger surface area per volume than a spray composed of larger droplets. Furthermore, the droplets have a lower surface tension than water, and therefore the droplets are even larger. The cells then undergo an entirely different process. As such, finer sprays can spread better over their target surface. This effect is relatively small for fluids with viscosities below 10 mPa s, but becomes more pronounced at higher dynamic viscosities. A fluid with a higher dynamic viscosity than water or a water / ethanol mixture will have a higher average droplet size for any given flow rate and pressure. The relationship between the mechanical properties of fluids can be calculated by the following commonly accepted formula: TIFF0007637072000037.tif26128In formula, D f = corrected droplet size for the fluid, D w = calculated droplet size for water, V f = viscosity of the fluid (viscosity in mPa s; water = 1.0 mPa s, lentivirus = 6913 mPa s).

[0328] From equation [1], lentivirus droplets sprayed under the same pressure and flow conditions as water / ethanol mixtures (e.g., virus and ethanol concentrations between 5 and 30% and 75 and 98% H2O). 2 A droplet containing a volume of an aqueous solution containing one or more of: 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) can be calculated to have a droplet size close to 5.9 times larger than a water / ethanol droplet. As described in International Application WO 2016 / 065341 (incorporated herein by reference in its entirety), droplets in the size range of 30 μm to 100 μm and 50 μm to 80 μm in diameter have been described. Generally, in the methods described herein, when the aqueous solution being sprayed contains a virus (e.g., a lentivirus), the droplet size range is about 150 μm to 600 μm in diameter, or about 177 μm to 590 μm in diameter. In other examples, the droplet diameter size is 200 μm to 600 μm, or about 300 μm to 600 μm, or about 400 μm to 600 μm, or about 500 μm to 600 μm. In other examples, the droplet size of the invention herein can be greater than 600 μm, for example, about 600 μm to 1000 μm in diameter, or about 600 μm to 900 μm, or about 600 μm to 800 μm, or about 600 μm to 700 μm in diameter. In some examples, the droplet size can be characterized by a diameter of up to 1000 μm, for example, 150 μm to 1000 μm.

[0329] These droplets are much larger than anticipated or described in WO 2016 / 065341, where it is noted that "some of the colloidal droplets created may be too large for a given intracellular delivery application. Some of the colloidal droplets created may be so large that cell death may occur despite the creation of colloidal droplets of appropriate size." See paragraph

[0172] of WO 2016 / 065341. It was therefore unexpected and surprising that cells (e.g., cells contacted with an aqueous solution containing the virus) tolerated such a different process compared to the SOLUPRE™ process described in WO 2016 / 065341, and that the cells were infected with lentivirus and viable.

[0330] Droplet size characteristics The larger diameter droplets of the present invention described herein have larger volume and weight, move slower, and impact cell layers with greater force.For example, when diameter increases by 5.9 times, droplet volume increases by nearly 206.8 times.Therefore, the fluid dynamics of this system is separate from that described with reference to WO 2016 / 065341, and constitutes a new virus infection process.

[0331] Other Aspects Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, and not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0332] The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0333] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. 1. An ex vivo method of delivering a viable virus across the plasma membrane of a cell, comprising the steps of: Contacting the population of cells with a volume of an isotonic aqueous solution, the solution comprising the virus and an alcohol at a concentration greater than 2%, and contacting the population of cells with the volume of the aqueous solution is carried out by propelling the aqueous solution with a gas to form a spray, the spray comprising droplets having a diameter greater than or equal to 150 μm.

2. 10. The method of claim 1, wherein the spray comprises droplets comprising a diameter in the range of 177 μm to 590 μm.

3. 2. The method of claim 1, wherein the virus comprises a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus (AAV), or a herpes simplex virus (HSV).

4. The method of claim 1, wherein the virus is a lentivirus.

5. The method of claim 1 , wherein the population of cells comprises mammalian cells.

6. The method of claim 1 , wherein the population of cells comprises adherent cells or suspension cells.

7. The method of claim 6, wherein said population of cells comprises non-adherent cells.

8. The method of claim 7, wherein the non-adherent cells comprise T lymphocytes.

9. 4. The method of claim 3, wherein the transduction efficiency is at least 30%, at least 40%, at least 50%, or at least 60%.

10. 7. The method of claim 6, wherein said population of cells comprises HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells.

11. The method of claim 1 , wherein said population of cells comprises natural killer cells.

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

13. 10. The method of claim 1, wherein the aqueous solution contains greater than 2% ethanol.

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

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

16. 2. The method of claim 1, wherein the aqueous solution comprises an ethanol concentration of 5 to 30%.

17. 2. The method of claim 1, wherein the aqueous solution further comprises one or more of the following components: 75-98% H 2 O, 6–91 mM sucrose, 2–35 mM KCl, 2–35 mM ammonium acetate, and 1–14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES).

18. 2. The method of claim 1, wherein the aqueous solution comprises sucrose 32.5 mM, KCl 106 mM, Hepes 5 mM, and ethanol 12% v / v.

19. The method of claim 1 , wherein the population of cells comprises a layer of non-adherent cells on a substrate.

20. The method of claim 1 , wherein the layer is on a membrane filter.

21. 10. The method of claim 1, wherein the droplets comprise a viscosity of 120 mPas.

22. 10. The method of claim 1, wherein the droplets comprise a viscosity of 7000 mPas.

23. The method of claim 1, wherein the aqueous solution further comprises one or more of the following components: 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES).

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