Methods of viral delivery to population of cells

Abstract

To provide a method of viral transfection of cells.SOLUTION: A method includes the steps of: providing a population of cells; and bringing the population of cells into contact with a certain volume of an isotonic aqueous solution containing a virus and alcohol at a concentration greater than 2%. The step of contacting with the isotonic aqueous solution is performed by injecting the aqueous solution with a gas to form a spray. The spray includes droplets (or a collection of droplets) with a diameter greater than or equal to 150 μm. For example, the spray includes droplets (or a collection of droplets) with a diameter in a range of 177 μm to 590 μm.SELECTED DRAWING: Figure 1

Description

Technical Field

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

[0002] Field of the Invention The present invention relates to the delivery of agents (e.g., viruses) into mammalian cells and the production of such viruses.

Background Art

[0003] 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 the genome of host cells, e.g., mammalian cells. Optimization of the conditions for transduction is very important for a variety of research and clinical applications. For cells that are difficult to transfect, lentiviral transduction provides a highly efficient solution for achieving good expression levels. Efficiency, time, production cost, and cell viability remain challenges in the art.

Summary of the Invention

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

[0005] The present invention features a method for delivering a virus across a cell's plasma membrane, 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 higher than 2%. The step of contacting the population of cells with the volume of the aqueous solution is performed by injecting the aqueous solution with a gas so as to form a spray. The spray comprises droplets (or aggregates of droplets) having a diameter greater than 150 μm or including 150 μm. For example, the spray comprises droplets (or aggregates of droplets) having a diameter in the range of 177 μm to 590 μm.

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

[0007] The population of cells includes mammalian cells, such as human immune cells. The population of cells may include adherent cells or floating 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 the 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 an ethanol concentration of 5 - 30%.

[0009] The aqueous solution further comprises one or more of the following components: 75 - 98% H2O, 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 comprises 32.5 mM sucrose, 106 mM KCl, 5 mM Hepes, and 12% v / v ethanol.

[0010] To receive a spray solution containing a cargo, e.g., a virus, to be delivered, a population of cells may be present as a layer of non-adherent cells on a substrate, e.g., as a monolayer. The layer may be confluent or non-confluent. An exemplary layer of cells, e.g., a monolayer, 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 floating or adherent cell) comprises providing a population of cells and contacting the population of cells with a volume of an isotonic aqueous solution, wherein the aqueous solution comprises the virus.

[0012] An aqueous solution for delivering a virus to a cell comprises a salt, e.g., 12.5 - 500 mM potassium chloride (KCl). For example, the solution is isotonic with respect to the cytoplasm of mammalian cells such as human T cells. Such an exemplary isotonic delivery solution contains 106 mM KCl.

[0013] The method is 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 examples, the non - adherent cells include peripheral blood mononuclear cells, for example, the non - adherent cells include immune cells such as T cells (T lymphocytes), for example, activated or non - activated (also known as naive) T cells. Immune cells such as T cells are optionally activated with ligands of 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 that includes a monolayer, for example, a sheet of cells physically located on a support or substrate. For example, the cells form a layer and this is contacted with a spray of the aqueous delivery solution. For example, the monolayer (or layer) is contacted with a spray of the 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 and the spray is in a small volume. The small - volume 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 a membrane filter. In some examples, after contacting the cell monolayer with a spray of the delivery solution, the membrane filter is moved, for example, agitated or vibrated. The membrane filter may be vibrated or agitated before, during, and / or after spraying the delivery solution onto the cells.

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

[0017] In an example, "S buffer" contains 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); which is hereby incorporated by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in the S buffer. The S buffer is further described in international application WO 2016 / 065341, for example, paragraphs

[0228] -

[0229] , which is hereby incorporated by reference in its entirety.

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

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

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

[0021] In an embodiment, the population of cells includes adherent or floating cells. For example, the population of cells includes 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, the method comprising providing a population of non-adherent cells; and contacting the population of cells with a volume of isotonic aqueous solution, wherein the aqueous solution contains the virus and an alcohol at a concentration higher than 2% (v / v). In an embodiment, the virus includes a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), or a herpes simplex virus (HSV).

[0023] In an embodiment, the population of cells includes adherent or floating cells, such as HEK293 cells, HEK293T cells, Lenti-x 293T cells, or HEK293F cells.

[0024] In an embodiment, 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 an embodiment, the alcohol comprises ethanol. In other embodiments, the aqueous solution comprises more than 10% ethanol. In an embodiment, the aqueous solution comprises 20 - 30% ethanol. In an embodiment, the aqueous solution comprises 27% ethanol. In an embodiment, the aqueous solution comprises 12% ethanol, for example, in a larger scale system.

[0026] In an embodiment, the cell population comprises a monolayer of non - adherent cells. In an example, the monolayer is contacted with a spray of the aqueous solution. The monolayer may further be present 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 of the invention recited in the claim "and those that do not materially affect the basic and novel characteristics".

[0028] Other features and advantages of the invention will be apparent from the following description of its preferred embodiments and from the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. GenBank and NCBI entries indicated by accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Brief Description of the Drawings

[0029]

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Mode for Carrying Out the Invention

[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 the genome of host cells, such as mammalian cells. Optimization of the conditions for transduction is very important for a variety of research and clinical applications. For cells that are difficult to transfect, lentiviral transduction provides a highly efficient solution for achieving good expression levels. Efficiency, time, production cost, and cell viability remain challenges in the art.

[0031] Virus delivery to a population of cells using the SOLUPORE (trademark) process The SOLUPORE™ process enables delivery of a wide range of cargos to adherent and suspension cells in vitro and ex vivo. To date, these cargos have consisted 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] Virus as a vector Since viruses can infect human cells naturally, they are used as vectors for the delivery of nucleic acids to cells. Before entry, viruses must attach to the host cell. Attachment is achieved when specific proteins on the viral capsid or viral envelope bind to specific receptor proteins 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 opens in the cell membrane and the unfolded viral envelope is further connected. 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 as follows: immunogenicity; target cell type; payload capacity; ability to transduce non-dividing cells compared to dividing cells; transience versus stable genomic integration (Table 5).

[0034] Overview of viruses used in gene delivery applications TIFF2025081453000002.tif63157, which is incorporated herein by reference in its entirety, Pharmaceutics 2020, 12, 183

[0035] For the application of immunotherapy, such as the delivery of chimeric antigen receptor (CAR) constructs for the generation of CAR-T cells and CAR-NK cells, gamma-retroviruses 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 a lentiviral vector and a gamma-retroviral vector, respectively (Poorebrahim M et al. Crit Rev Clin Lab Sci. 2019 Sep;56(6):393-419). Similar to most viral vectors, these vectors were modified to disable viral replication and improve cell targeting efficiency.

[0036] For the application of other immune cell manipulations, such as gene editing, AAV vectors are widely used. 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 lifetime 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 the CRISPR / Cas9 system or donor template DNA for gene editing. In these cases, gene editing is permanent, but it may be desirable to express the gene editing tool transiently only to limit off-target gene editing that can occur when the tool is present in cells over a long period of time.

[0037] Lentivirus Gamma retroviruses and lentiviruses are subtypes of retroviruses that contain an RNA genome which is converted to DNA in transduced cells by an enzyme called reverse transcriptase encoded by the virus. For retroviruses, after entry into the cell, the uncoating process continues, whereby several viral proteins dissociate from the viral core. The viral RNA is reverse transcribed into double-stranded DNA. The viral proteins then complex with the proviral DNA, leading to nuclear translocation and integration into the host genome. The integration process is assisted by very important viral proteins such as integrase, and endogenous host cell transcription factors.

[0038] Lentiviral vectors derived from human immunodeficiency virus (HIV-1) have become a major tool for gene delivery into mammalian cells, and replication-defective recombinant lentiviruses are widely used in research and clinical applications. Modified lentiviruses can still infect cells but no longer contain the essential genes for producing new virus particles. 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 gene elements such as intron-containing sequences; and they are a relatively easy system for vector manipulation 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 G.J. et al. J. Gene Med. 2013;15:78-82). Third-generation lentiviral vectors incorporate key safety features and further enhance safety (Kim V.N. 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 by retroviruses has been expanded by pseudotyped retroviruses with the vesicular stomatitis virus envelope glycoprotein G (VSV-G). VSV-G binds to phosphatidylserine, a ubiquitous membrane component, which allows VSV-G pseudotyped viruses to attach to and transduce a much wider range of cells. Currently, most lentiviral vectors are pseudotyped with VSV-G to allow robust transduction into many cell types, including neurons, lymphocytes, and macrophages.

[0040] Quiescent cells After isolation from either healthy donors or patients, the cells are activated and subsequently transduced with a lentiviral vector (LV) that is pseudotyped with the glycoprotein G of vesicular stomatitis virus (VSV) for the majority of them. The modified lymphocytes are then expanded and used in functional in vitro assays or for in vivo applications. Stable and efficient transduction of B cells by LV is very difficult to achieve. Resting B cells are restricted from transduction by conventional VSV G pseudotyped LV (VSV-LV) due to the lack of expression of 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 activation stimuli combined with cytokines that are carefully titered and subsequent co-culture with feeder cells. Even under optimal activation and culture conditions, the transduction efficiency with VSV-LV is notoriously low. The combination of all these challenges could account for the fact that the number of clinical trials involving engineered B cells is much less compared to T cells.

[0041] In comparison, engineering of T lymphocytes by lentiviral transduction is easier to achieve. Still, the cells, like B cells, must be activated prior to transduction with conventional VSV-LV due to the lack of LDLR expression, rendering them otherwise not readily transducible (X Geng, et al. Gene Therapy v.21, pages444-449(2014)). Primarily due to the great success of CAR T cell therapy, the protocols for T cell isolation, activation, lentiviral transduction, and expansion have been significantly improved in recent years. Current state-of-the-art T cell activation relies on stimulation of the TCR activation pathway via CD3-specific antibodies and CD28-specific antibodies in combination with cytokines such as IL-7 and IL-15.

[0042] The need to activate lymphocytes prior to transduction in conventional LV has disadvantages. This adds complexity to the overall procedure, increasing the duration and cost of the manufacturing process. In addition, the stimuli applied for activation, combined with long-term ex vivo culture, are likely to change the cells, which can have a negative impact on the quality of the final product. As a result, naive cells can differentiate into less preferred phenotypes that exhibit a higher degree of exhaustion, lower proliferative capacity, shorter in vivo persistence, and lower functionality. This can have very important implications for therapeutic success. As an example, the phenotypes of central memory (CD45RO+ / CD45RA+ / CD62L+) or stem cell memory (CD45RO+ / CD45RA / CD62L+) have been shown to be beneficial for T cell persistence and function in vivo. In this regard, a positive correlation between the 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 being considered. Similarly, the central memory phenotype results in functionally superior TCR-engineered T cells. Therefore, minimal manipulation of lymphocytes during gene modification has significant clinical validity.

[0043] Spinoculation Centrifugal inoculation, i.e., spinoculation, is widely used in virological research to enhance virus infection. The procedure involves centrifuging a mixture of virus and target cells at high speed for an extended period, e.g., 800×g for 30 minutes at 32°C. The method was thought to enhance transduction efficiency by concentrating the virus at the cell membrane. However, spinoculation has been shown to induce the activities of dynamic actin and cofilin, probably due to the cellular response to centrifugal stress (Jia Guo, et al. J. Virology Oct. 2011, p. 9824-9833). This actin activity also leads to the upregulation of cell membrane receptors that can enhance virus binding and entry. Spin-mediated enhancement cannot be simply explained by the virus concentration effect; rather, it is suggested to be coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, virus entry, and post-entry processes. Therefore, spinoculation may affect the biology of target cells in unknown or undesirable ways.

[0044] Limitations of viral vectors As described above, viruses are useful for genetic manipulation of cells, but their utility has limitations.

[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, such as the physical proximity of lentiviral particles to T cells, have been identified as potentially playing a role in transduction efficiency. This proximity can be manipulated through the number of cells and viral particles in suspension; the duration of agitation to promote homogeneity; and the surface-to-volume ratio in the transduction vessel. However, limited research has been conducted on the identification and optimization of 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 substance at the cell membrane. This process enhances virus attachment to the cell membrane and increases the rate of entry into the cell, making the process more efficient. Consequently, a smaller dose of virus is used and the cost is reduced.

[0046] Since the SOLUPORE™ process is a gentle way to concentrate the virus at the cell membrane, it has significant advantages over existing concentration methods such as spinoculation that can affect cell structure. Furthermore, the SOLUPORE™ process is designed to be compatible with the cell therapy manufacturing process, unlike spinoculation.

[0047] Concentration of the virus at the cell membrane also compensates for the low level of virus receptor expression for 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 cells and B cells is typically very low. It is highly desirable to improve these efficiencies, and the SOLUPORE™ process provides a solution to this problem at a high rate of efficiency in conditions compatible with the preservation of cell viability and function.

[0049] Viruses are only capable of delivering nucleic acids, which means that the types of cargo they can deliver are limited. If viruses could be co-delivered with other types of cargo, this could enhance the utility of viruses in the manipulation of next-generation cell therapy products. However, currently, there is no method that has been demonstrated to co-deliver viruses with other types of cargo. Again, the SOLUPORE™ process described herein provides a solution to this problem by enabling the efficient delivery of numerous different cargo types, either sequentially or simultaneously. The following materials and methods were used to generate the dates 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 evaluated over a period of 1 hour by assessing precipitation under a microscope.

[0052] LV-GFP delivery Primary human PBMCs were thawed and activated with Miltenyi's CD3 antibody and CD28 antibody for 3 days. Three days after the activation culture, the cells were subjected to the SOLUPORE™ process with LV-GFP (MOI = 2.5) or static transduction. The cells were allowed to recover for 72 hours before GFP expression was evaluated by flow cytometry.

[0053] Stability of LV-GFP in delivery solution Prior to the present invention, since SOLUPORE™ process delivery had not been previously combined with viral preparations, it was not known whether there were solubility issues.

[0054] When the solution was viewed under a microscope during the stability analysis, no aggregation or precipitation was observed. Additionally, certain other cargos showed low levels of aggregation when combined with the delivery solution, causing the Solupore® nebulizer to clog during spraying. Loading the virus-containing delivery solution into the Solupore® nebulizer and spraying it further demonstrated that no clogging occurred and no aggregation or precipitation took place at any point during the course of the study.

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

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

[0057] The cumulative expansion rate of T cells was measured up to 96 hours after virus 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. The 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 25.2 ± 1.48% for solupore-treated cells and control transduced cells, respectively. On day 4, the efficiency was 40.27 ± 2.67% and 26.83 ± 1.38% for solupore-treated cells and control transduced cells, respectively.

[0059] Efficient viral delivery of virus to a population of cells The data in this specification demonstrated that the SOLUPORE™ process is compatible with the delivery of virus to activated T cells. No precipitation or aggregation was observed when mixed with the delivery solution of the SOLUPORE™ process. The virus solution could be sprayed and successfully transduced the target cells. The survival rate and growth 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 enhanced viral transduction of T cells.

[0061] Collectively, these findings demonstrate that the SOLUPORE™ process is suitable for virus delivery to cells and is superior to standard methods.

[0062] Since the SOLUPORE™ process is suitable for virus delivery, it is possible to use solupore treatment in the cell therapy manufacturing process involving viral transduction. Due to the cost of the virus, transduction is a major cost driver in CAR T cell manufacturing. The SOLUPORE™ process can now achieve a similar level of transduction efficiency using less virus to enhance viral transduction, thus reducing costs.

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

[0064] The SOLUPORE™ process enables delivery of cargos to non-activated T cells. Lentiviral vectors have very low transduction efficiency in non-activated T cells. Thus, the SOLUPORE™ process increases the transduction efficiency of lentiviruses in non-activated T cells.

[0065] A core feature of the SOLUPORE™ process device is the ability to facilitate media exchange. When a solution containing cells is transferred into the device, the liquid is drained and can be replaced with a different liquid. Thus, the process of handling liquids is possible. Such a process of handling liquids can include, for example, a washing step. In virus transduction and other cell manufacturing processes, a washing step is often required. The Solupore® device enables incorporation of such steps into the manufacturing process.

[0066] The Solupore® technology is also scalable, meaning that viral transduction using this method can be carried out on a small scale for initial and preclinical studies, as well as on a larger scale for process development and clinical applications.

[0067] Advantages and surprising results of the method How viruses differ compared to other cargo Viruses are microorganisms, for example, submicroscopic infectious agents that replicate only inside the living cells of organisms. Viruses can infect all types of living things, from animals and plants to microorganisms including bacteria and archaea. A virus consists of a core of genetic material, either DNA or RNA, surrounded by a protective coat called a capsid made of protein. Sometimes the capsid is surrounded by an additional outer spikey coat called an envelope. Viruses can latch onto and enter host cells.

[0068] Previously, the cargo used with Solupore™ was either a single class of molecule (such as a protein or nucleic acid), or a mixture of molecules, or a molecular complex (such as a Cas9 ribonucleoprotein (RNP)). Viruses are more complex agents in terms of structure and function. Structurally, the genetic material is single-stranded RNA contained within a protein capsid and thus encapsulated. A lentiviral virion contains 2% nucleic acid, 60% protein, and 35% lipid, and 3% carbohydrate. It is expected that the structure must remain intact for the virus to successfully infect a cell. Functionally, once inside the cell, the virus is expected to be able to release 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 expressed subsequently.

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

[0070] Surprising Results and Unexpected Advantages of Viral Transduction Using the Solupore™ Process The Solupore™ process has been used to deliver relatively simple cargo molecules (not 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 were compatible with successful viral infection as outlined above.

[0071] 1. Mix the cargo and the delivery solution It was not known whether the viral preparations described herein were compatible with the delivery solution. Occasionally, it has been observed that other cargoes aggregate and thus interfere with the process. Such aggregation causes several problems such as loss of cargo functionality and blockage of the Solupore™ fluid pathway. Sometimes, the degree of this aggregation is substantial and thus immediately apparent, interrupting the experiment. However, sometimes the aggregation is undetectable during Solupore™ experiments and is only identified when an analytical assay is performed to test for cargo activity in the target cells. Subsequent troubleshooting has not been sufficient to interfere with the completion of the experiment but has resulted in the detection of a partial blockage of the fluid pathway that likely altered the results.

[0072] For this study, compatibility tests of the delivery solution were performed, and surprisingly, no aggregation was observed under the microscope. Additionally, no blockage of the device was observed. Additionally, successful results, namely the expression of the GFP transgene, were achieved. These findings indicate that the virus preparation was compatible with the delivery solution. Considering the complexity of the viral material, this was surprising. Furthermore, the absence of observable blockage of the fluid path indicates that no aggregation occurred either within the tube or while the solution was being transferred through the system. Again, considering the complexity and size of the viral material, this was surprising.

[0073] 2. Form droplets of a specific size and velocity It was not known whether the virus preparation described herein was compatible with the aerosolization process. During the process, the delivery solution was divided into droplets using a custom sprayer that had not been previously used with viruses. However, the results described herein indicate that the virus preparation was compatible.

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

[0075] 4. Land the droplets on the 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 maintaining the integrity and functionality of the virus particles. The results showed that the virus preparation was compatible with this process.

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

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

[0078] 7. Remove the cells from the SOLUPORE (trademark) process chamber To remove the cells from the chamber and place them in culture, it is necessary to wash out the chamber several times and agitate the cell suspension. It was not known whether this would likely interfere with the virus infection process by damaging the cells in some way, for example, preventing successful expression of the virus transgene. The results indicate that the virus preparation was compatible with this process.

[0079] 8. Culture the cells for several days During viral infection, viral-derived cytoplasmic nucleic acids are recognized by host intracellular specific sensors. The efficacy of this recognition system is crucial for inducing innate host defense, which in turn stimulates 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 above Solupore™ process affected cells in any way to make them more sensitive to the presence of the virus. If this occurred, cell viability could be impaired and the cells might not survive during the 4-day culture period after infection. Alternatively, the cells could survive but their health could be impaired such that they were unable to express the transgene. Surprisingly, the cells into which the virus was delivered experienced an approximate 90% survival rate.

[0080] Virus infection process and droplet characteristics The nebulization of lentivirus within the transfection chamber is a process separate from the SOLUPORE™ process. As described herein, the cargo delivered to a population of cells is a biologically active and viable virus (e.g., lentivirus).

[0081] A typical titer of lentivirus is 10 6 ~10 7is in the range of transduction units (TU / ml), and the viscosity of lentivirus at these concentrations is highly and dynamically viscous compared to the water / ethanol mixture. For illustration, (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 600.1 mPa s, and the dynamic viscosity of castor oil is close to 6000.1 mPa s. The dynamic viscosity of lentivirus has been reported to be 6913 mPas by Tran, Reginald, PhD Thesis, Georgia Tech (2016), which is nearly 1 log more viscous than castor oil. Sterile filtered 1% bovine serum albumin (BSA) has been found by some to reduce the molecular interactions that can result in virus particles "sticking" to the injection device (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, which is hereby incorporated by reference in its entirety).

[0082] Dynamic viscosity is an important factor in atomization. An experimental study on atomization in an internal mixing two-fluid atomizer, such as used in the SOLUPORE™ process, has been conducted over a wide range of liquid viscosities, gas supply pressures, and gas-to-liquid mass ratios (GLR). 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 hereby incorporated by reference in its entirety. Among all test conditions, the finest sprays were obtained at an axial distance of 150 mm. However, as the viscosity 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 current droplets produced by the measured droplet size distribution for the SOLUPORE™ process, Figure 51). Larger droplets corresponded to a large proportion of the droplet population (distribution), and the attenuation of droplet velocity along the spray axis was stronger at higher viscosities.

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

[0084] The absolute or dynamic viscosity of some common liquids at a temperature of 300 K is shown below. TIFF2025081453000003.tif177150

[0085] Atomization of lentivirus within the SOLUPORE™ process produced larger, more slowly moving droplets, and it can be concluded that the experience of the underlying cell layer is different from the previously described SOLUPORE™ process. Consequently, this atomization process resulted in a transfection level of nearly 30%, which is 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 an ethanol / water mixture is also close to 1 mPa s. The dynamic viscosity of an aqueous solution that can contain an ethanol concentration of 5 - 30%. The aqueous solution contains 75 - 98% H 2 O, 2 - 45% ethanol, 6 - 91 mM sucrose, 2 - 35 mM KCl, 2 - 35 mM ammonium acetate, and one or more of 1 - 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and the viscosity is in the region of 2 mPa s.

[0087] Titer 10 7 ~10 8 TU / mL of lentivirus has a dynamic viscosity close to 6913 mPa s. As the viscosity of the fluid increases, at a given spray pressure, for example 1.7 bar, when sprayed, there is a tendency to form larger droplets. 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 thus the droplets become even larger. Next, the cells experience a completely different process. By itself, a finer spray 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. Fluids with higher dynamic viscosities than water or water / ethanol mixtures will have higher average droplet sizes for any given flow rate and pressure. The relationship between the mechanical properties of the fluid can be calculated by the following generally accepted formula. TIFF2025081453000004.tif26128where, D f = the corrected droplet size for the fluid, D w = the calculated droplet size for water, V f = the viscosity of the fluid (viscosity in mPa s; water = 1.0 mPA s, lentivirus is 6913 mPa s).

[0088] From Equation [1], droplets of lentivirus sprayed under the same pressure and flow conditions as the water / ethanol mixture (e.g., droplets containing the virus, an ethanol concentration of 5-30%, and 75-98% H 2 O, 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) in a certain volume of aqueous solution) can be calculated to have a droplet size nearly 5.9 times larger than that of water / ethanol droplets. As described in International Application WO 2016 / 065341, which is hereby incorporated by reference in its entirety, droplets in the size ranges of 30 μm to 100 μm and 50 μm to 80 μm in diameter were described. Generally, in the methods described herein, when the aqueous solution to be sprayed contains a virus (e.g., lentivirus), the droplet size range is from about 150 μm to 600 μm in diameter, or from about 177 μm to 590 μm in diameter. In other examples, the diameter size of the droplets 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 is greater than 600 μm, for example, it can be from about 600 μm to 1000 μm in diameter, or from about 600 μm to 900 μm, or from about 600 μm to 800 μm, or from 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 would be expected or described in "Some of the produced colloidal droplets may be too large for a given intracellular delivery application. Because some of the produced colloidal droplets are too large, cell death may occur despite producing colloidal droplets of the appropriate size." as predicted in WO 2016 / 065341. See paragraph

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

[0090] Droplet size characteristics The larger-diameter droplets of the invention described herein have a larger volume and weight, move more slowly, and impact the cell layer with a greater force. For example, when the diameter increases by 5.9-fold, the volume of the droplet increases nearly 206.8-fold. Thus, the hydrodynamics of this system are distinct from those described with reference to WO 2016 / 065341 and constitute a new virus infection process.

[0091] Delivery of cargo to cells The difficulty of transfecting molecules into cells has plagued research and therapy, e.g., cell therapy, gene therapy, 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 a cell's plasma membrane comprises providing a population of cells and contacting the population of cells with a volume of an aqueous solution, wherein the aqueous solution comprises the payload and an alcohol at a concentration higher than 2 percent, and the volume is 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 the aqueous solution is effected by injecting the aqueous solution with a gas so as to form a spray.

[0092] The reason why it is difficult to transfect a certain type of cell may be that non - adherent cells lack heparan sulfate proteoglycan on the cell surface, which is a molecule responsible for cell adhesion to the extracellular matrix. Transfection methods such as electroporation and / or nucleofection have the drawbacks of impairing cell viability, the ability of cells to resume proliferation after treatment, and cell functions, such as the immunological activity of lymphocytes. The transfection / transduction compositions and methods described herein do not have such drawbacks and thus are characterized by 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 mixture of compounds (composition) 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 lyses the 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 the spray but not immersed or submerged in the solution containing the delivery compound. Exemplary agents that permeate or lyse the membrane of eukaryotic cells include alcohols and surfactants, such as ethanol and Triton X - 100, respectively. Other exemplary surfactants, such as 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 octyl glucoside.

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

[0095] Advantages of the method recited in the claims of the present invention The methods described herein provide a number of advantages over well-known techniques and applications. Exemplary advantages include the following: 1) The ability to transfect the same number of cells with fewer viruses (or reduce the amount of virus required to infect a population of cells), 2) The ability to transfect more cells with the same amount of virus (or increase the number of cells transfected with the same amount of virus used in current protocols), 3) Increased virus uptake, 4) Increased cell viability, 5) Increased transfection efficiency, and 6) Production efficiency (the ability to perform multiple steps in a single process or area).

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

[0097] In one aspect, delivering a virus across the plasma membrane of a cell involves providing a population of cells and contacting the population of cells with an aqueous solution. The aqueous solution includes the virus and an alcohol content at a concentration higher than 2%. 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 at a concentration higher than 2% (e.g., higher than 5%), a salt more than 46 mM, a sugar less than 121 mM, and a buffer less than 19 mM. For example, the concentration of the alcohol, such as 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 containing the virus, a salt more than 46 mM, a sugar less than 121 mM, and a buffer less than 19 mM. For example, the aqueous solution does not contain alcohol.

[0100] One or more of the following characteristics can be included in any feasible combination. The volume of the solution delivered to the cells is a plurality of units, such as a spray, such as a plurality of droplets on aqueous particles. The volume is described with respect to individual cells or 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%) population of cells. For example, the volume can be 6.0×10 -7 microliters per cell to 7.4×10 -4 microliters per cell. The volume can be 4.9×10 -6 microliters per cell to 2.2×10 -3 microliters per cell. The volume can be 9.3×10 -6 microliters per cell to 2.8×10 -5It can be in microliters. The volume is about 1.9×10 per cell -5 It can be in microliters, and "about" means within 10 percent. The volume is 6.0×10 per cell -7 microliters ~ 2.2×10 per cell -3 microliters. The volume is 2.6×10 per square micrometer of the exposed surface area -9 microliters ~ 1.1×10 per square micrometer of the exposed surface area -6 It can be in microliters. The volume is 5.3×10 - 8 microliters per square micrometer of the exposed surface area ~ 1.6×10 per square micrometer of the exposed surface area -7 It can be in microliters. The volume is about 1.1×10 per square micrometer of the exposed surface area -7 It can be in microliters. "About" can mean within 10 percent.

[0101] Cell confluence refers to cells that are in contact with each other on the surface. For example, this can be expressed as an estimated (or counted) percentage. For example, 10% confluence means that, for example, 10% of the surface of a tissue culture vessel is covered with cells, and 100% means 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 removed by spin - down, or by suction from above the cell population with a vacuum or aspiration of the tissue culture medium, or by aspiration or vacuum removal from the bottom of the container in a filter setup.

[0102] The step of contacting a population of cells with an aqueous solution of the above volume can be carried out by injecting the aqueous solution with a gas so as to form a spray. The gas can include nitrogen, ambient air, or an inert gas. The spray can include discrete unit volumes over a range of sizes larger than 150 μm in diameter.

[0103] The total volume of 20 μl of the aqueous solution can be delivered by spraying to an area occupied by cells of about 1.9 cm 2 for example, to one well of a 24-well culture plate. The total volume of 10 μl of the aqueous solution is delivered to an area occupied by cells of about 0.95 cm 2 for example, to 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 an even larger area, of any size suitable for the diameter of the area covered by the spray emitted from an atomizer.

[0104] Typically, the aqueous solution contains the virus to be delivered into the cells across the cell membrane, and the second volume is a buffer or culture medium that does not contain the payload. Alternatively, the second volume (buffer or medium) can also contain the virus. In some examples, the second volume contains different types of cargo, such as nucleic acids, proteins, or chemical compounds (i.e., non-viral cargo). Alternatively, the first solution contains non-viral cargo and the second solution contains viral cargo. The viral cargo and non-viral cargo may be delivered sequentially or simultaneously as described above, i.e., in the same delivery solution. In some embodiments, the aqueous solution contains the payload and alcohol, and the second volume does not contain alcohol (and optionally does not contain the payload). The cell population can be contacted with the aqueous solution for 0.1 to 10 minutes before adding the second volume of buffer or culture medium to sink or suspend the cell population. The buffer or culture medium can be phosphate buffered saline (PBS). The cell population can be contacted with the aqueous solution for 2 seconds to 5 minutes before adding the second volume of buffer or culture medium to sink or suspend the cell population. The cell population 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 that does not contain the virus to sink or suspend the cell population. The cell population can be contacted with the spray for about 1 to 2 minutes before adding the second volume of buffer or culture medium to sink or suspend the cell population. During the time between the cell spray and the addition of the buffer or culture medium, the cells remain hydrated by a layer of water from the spray volume.

[0105] The aqueous solution can contain an ethanol concentration of 2-30%, 2-40%, or 2-50%. The aqueous solution is 75-98% H 2It can contain one or more of O, 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 an embodiment, the aqueous solution contains 27% ethanol for Flexi (e.g., small scale). In an embodiment, the aqueous solution contains 12% ethanol in a large-scale system.

[0106] The cell population can include adherent cells or non-adherent cells. Adherent cells can include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, nerve cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, induced pluripotent stem cells (iPSCs), and human embryonic kidney (HEK) cells, or immortalized cells such as cell lines. In a preferred embodiment, the cell population includes non-adherent cells. For example, the percentage of non-adherent cells in the population is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% non-adherent cells. 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 line. In other examples, NK cell lines including NK92 and KHYG1 are used.

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

[0108] The alcohol can be selected from methanol, ethanol, isopropyl alcohol, butanol, and benzyl alcohol. The salt can be selected from NaCl, KCl, Na 2 HPO 4 、KH 2 PO 4 、and C 2 H 3 O 2 NH. 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 relates to a method for delivering a virus across a plasma membrane. The subject finds utility in the field of intracellular delivery and has applicability, e.g., in the delivery of molecular biological and pharmacological therapeutic agents to target sites such as cells, tissues, or organs. The method of the subject includes introducing a molecule into an aqueous composition to form a matrix; spraying the matrix to make a spray; and contacting the matrix with the plasma membrane.

[0110] The subject relates to a composition for use in the delivery of viruses across the plasma membrane. The subject finds utility in the field of intracellular delivery and has applicability, for example, in the delivery of molecular biological and pharmacological therapeutic agents to target sites such as cells, tissues, or organs. The compositions of the subject include alcohol; salts; sugars; and / or buffers.

[0111] The exemplary methods described herein include an aqueous solution containing alcohol. The term "alcohol" means a polyatomic organic compound containing 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 contains 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). The exemplary payload may contain up to 50% (v / v) alcohol, more preferably, the payload contains 2 - 45% (v / v) alcohol, 5 - 40% alcohol, and 10 - 40% alcohol. The aqueous solution may contain 20 - 30% (v / v) alcohol.

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

[0113] In some examples, "S buffer" contains 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); which is incorporated herein by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in the S buffer. The S buffer is further described in International Application WO 2016 / 065341, for example, paragraphs

[0228] -

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

[0114] According to the present subject matter, the aqueous solution may contain at least one salt. The salt may be selected from NaCl, KCl, Na 2 HPO 4 、C 2 H 3 O 2 NH 4 、and KH 2 PO 4 and may be selected from. For example, the KCl concentration ranges from 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, 106 mM potassium chloride (KCl), 5 mM Hepes, 12% v / v ethanol (EtOH), and water for injection (WFI).

[0116] According to an exemplary method of the present subject matter, the aqueous solution may contain a sugar (e.g., sucrose, or a disaccharide). According to an exemplary method, the payload contains less than 121 mM sugar, 6 - 91 mM, or 26 - 39 mM sugar. Additionally, (e.g., containing a virus) the aqueous solution contains 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload contains 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, an aqueous solution payload (e.g., containing a virus) may contain a buffer (e.g., a weak acid or a weak base). The buffer may contain zwitterions. According to the exemplary method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The aqueous solution (e.g., containing a virus) may contain less than 19 mM of the buffer (e.g., 1 - 15 mM, or 4 - 6 mM, or 5 mM of the buffer). According to the exemplary method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payload contains 1, 2, 3, 4, 5, 10, 12, 14 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. More preferably, the aqueous solution (e.g., containing a virus) contains 5 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

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

[0119] The volume of the aqueous solution achieved by injecting the aqueous solution with a gas may contain compressed air (e.g., ambient air), and other embodiments may contain an inert gas, e.g., helium, neon, and argon.

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

[0121] In certain aspects of the subject matter, the cell population may be substantially confluent or may comprise confluence greater than substantially 75 percent. In preferred embodiments, the cell population may form a single monolayer.

[0122] In an aspect, the step of contacting the cell population with the aqueous solution of the volume may be performed by injecting the aqueous solution with a gas so as to form a spray. In certain embodiments, the cell population 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 sink or suspend the cell population.

[0123] In various embodiments, the cell population comprises at least one of primary cells or immortalized cells. For example, the cell population may comprise mesenchymal stem cells, lung cells, nerve cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells. Non-limiting examples of T cells 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 the PBMC population by positive isolation using anti-CD8 beads or by negative selection using anti-CD4 beads. In some aspects, primary NK cells are isolated from PBMC, cord blood-derived NK, iPSC-derived NK, and GFP mRNA can be delivered by platform delivery technology. In additional aspects, an NK cell line, e.g., NK92, may be used.

[0124] Cell types also include cells that have been previously modified to enhance their therapeutic efficacy, such as T cells, NK cells, and MSCs. For example: T cells or NK cells expressing chimeric antigen receptors (CAR T cells and CAR NK cells, respectively); endosomes; transduced cells, and endosomes derived from them, such as MSCs; T cells expressing modified T cell receptors (TCRs); MSCs modified virally or non-virally to overexpress therapeutic proteins that complement their innate properties (e.g., delivery of Epo using a lentiviral vector or BMP-2 using AAV-6) (reviewed in Park et al, Methods, 2015 Aug;84-16.); MSCs primed with non-peptidic drugs or magnetic nanoparticles for enhanced efficacy and external control of targeting, respectively (Park et al., 2015); MSCs functionalized with targeting moieties to enhance their homing to the site of treatment 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., manipulation of the cell surface with palmitated proteins acting as hydrophobic anchors for subsequent conjugation of antibodies) (Park et al., 2015). For example, T cells modified to express a chimeric antigen receptor, such as primary T cells or T cell lines (CAR T cells), may be further processed 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 for the purpose 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 for editing various genes in modified cells to enhance the activity of CAR-T cells, such as editing of PD-1 to enable CAR-T cells to avoid immune checkpoint blockade. In other examples, the methods and systems herein are used for manipulating 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 invention relate to the expression viral delivery of gene editing compounds and complexes to cells and tissues, for example, the delivery of Cas-gRNA ribonucleoproteins for genome editing in primary human T cells, hematopoietic stem cells (HSCs), and mesenchymal stromal cells (MSCs). In some examples, mRNA encoding such proteins is delivered to the 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 a Flp recombinase. In additional embodiments, the gene editing protein may be a fusion protein (megaTAL) that combines a homing endonuclease and the modular DNA binding domain of a TALEN. For example, the megaTAL may be delivered as a protein, or alternatively, mRNA encoding the megaTAL protein is delivered to the cells.

[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, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, or homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2 and in the NCBI database under accession number Q99ZW2.1. Accession numbers A0A0G4DEU5 and CDJ55032 in the UniProt database provide another example of the Cas9 protein amino acid sequence. Another non-limiting example is the Streptococcus thermophilus Cas9 protein, whose amino acid sequence can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, unmodified CRISPR enzymes such as Cas9 have DNA cleavage activity. In certain embodiments, the CRISPR enzyme is Cas9 and may be Cas9 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 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 base pairs, 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 such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). Other examples of mutations that convert Cas9 to a nickase include, without limitation, H840A, N854A, and N863A. In aspects of the invention, the nickase can be used for genome editing via homologous recombination.

[0129] In certain embodiments, a Cas9 nickase can be used in combination with two guide sequences, for example, two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows for introducing nicks in both strands and can be used to induce NHEJ.

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

[0131] In certain embodiments, the protein to be delivered (such as a Cas protein or a variant thereof) may include an intracellular localization signal. For example, the Cas protein within the 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 includes the nuclear localization signal. In some embodiments, the payload (such as an RNP) includes a fusion protein that includes a localization signal. For example, the fusion protein may contain a nuclear localization signal, a nucleolar localization signal, or a mitochondrial targeting signal. Such signals are known in the art, and non-limiting examples are described in Kalderon et al., (1984) Cell 39 (3 Pt 2): 499-509; Makkerh et al., (1996) Curr Biol. 6 (8): 1025-7; Dingwall et al., (1991) Trends in Biochemical Sciences 16 (12): 478-81; Scott et al., (2011) BMC 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 include 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 a CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon of a native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons, or more codons than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more) with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage frequency between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, inter alia, on the properties of the codons being translated and 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. Tables of codon usage frequencies are readily available, for example, in the "Codon Usage Database", 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, and for example, Gene Forge (Aptagen; Jacobus, Pa.) is also available. 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 the CRISPR enzyme correspond to the codons most frequently used for a particular amino acid.

[0134] Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence sufficient to hybridize with the target sequence and direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm. In some embodiments, the degree of complementarity is 100%. Optimal alignment may be determined by use of 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 nucleotides, or more, or 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 nucleotides, or more nucleotides. In certain embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides, or fewer nucleotides.The ability of a guide sequence to direct the sequence-specific binding of a CRISPR complex to a target array may be evaluated by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence by, for example, transfection of a vector encoding the components of the CRISPR sequence, and subsequent evaluation of preferential cleavage within the target sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in vitro by providing the components of a CRISPR complex comprising the target sequence, the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction.

[0135] CRISPR-Cas technology, which facilitates genome engineering in a wide range of cell types, is rapidly evolving. Delivery of the Cas9-gRNA editing tool in the form of ribonucleoprotein (RNP) has recently been shown to confer several benefits compared to delivery of plasmids encoding Cas9 and gRNA. Benefits include faster and more efficient editing, fewer off-target effects, and less toxicity. RNPs have been delivered by lipofection and electroporation, but these delivery methods remain limited, particularly for certain clinically relevant cell types, including 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 like 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 relates to delivery techniques that facilitate the delivery of a wide range of payloads to cells with low toxicity. Genome editing can be achieved by delivering RNPs to cells using some aspects of the subject. The level then drops until Cas9 can no longer be detected. The delivery technique itself does not have a detrimental effect on the viability or functionality of Jurkat T cells and primary T cells. The subject enables gene editing via Cas9 RNPs in clinically relevant cell types with minimal toxicity.

[0137] The 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 the use of expression vectors. Components expressed from vectors can be produced in various amounts and over various times, making it difficult to achieve consistent gene editing without off-target editing. Additionally, pre-formed complexes of Cas and gRNA (RNPs) cannot be delivered with expression vectors.

[0138] In one aspect, the subject describes cells attached to a solid support (e.g., a strip, polymer, bead, or 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, amylases, natural and modified celluloses, polyacrylamide, pumice, and magnetite. The nature of the carrier can be either soluble or insoluble to some extent for the purposes of the subject. The support material can have virtually any possible structural shape. Thus, the support shape may be spherical as in beads, or cylindrical as on the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat such as in a sheet or test strip. Preferred supports include polystyrene beads.

[0139] In other aspects, the solid support comprises a polymer to which the cells chemically bind, immobilize, disperse, or associate. The polymeric support may be a polymer network and may be prepared in bead form (e.g., by suspension polymerization). To deliver a desired compound to the cells on such a scaffold, an aqueous solution containing the payload according to the invention can be sprayed. Exemplary scaffolds include stents and other implantable medical devices or structures.

Examples

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

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

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

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

[0144] A range of MOI was tested, and volumes in the range of about 1 μL to about 1000 mL were tested.

[0145] Additionally, 1×10 5 ~1×10 7 Various membrane holder sizes were tested to enable removal of the medium from a range of cell numbers of cells. When cells are cultured after transduction, they grow to the volume required for cell therapy, for example up to 10 9 cells.

[0146] For example, a commercially available lentiviral vector encoding GFP, transduced using spinoculation, was used as a control. The control is used according to the standard protocol for spinoculation of lentiviral vectors against floating cells. In the examples, a commercially available spinoculation method was used for transduction of floating cells (such as Jurkat T cells, PBMCs, PBLs, B cells, etc.).

[0147] Transduction and evaluation / analysis of floating cells For normal transduction of floating cells, the virus was added to activated T cells in culture flasks and bags as a control. The cells are cultured after virus introduction and the virus is washed. After several days, the cells are collected and the viability and % transduction (virus encoding GFP) are evaluated by flow cytometry.

[0148] The lifespan of the transduction efficiency was monitored over 14 days.

[0149] The vector copy number (VCN) was evaluated to determine low copy / cell numbers, for example, 0.5 - 1 copy / cell. Exemplary release criteria include (0.5 - 5 for Levine 2006). VCN is established using quantitative PCR.

[0150] Changes in phenotype were evaluated by flow cytometry. For example, changes including maintenance of naive, central memory, effector memory were evaluated.

[0151] Perform transduction on T cells and other cell types Viral delivery to T cells in multiple activation states was also evaluated. For example, it is not uncommon to transduce activated T cells. Studies were conducted to determine whether naive T cells, which would result in a reduction in COG (cost of goods), could be used. Activation is not required, and thus, advantages of the methods described herein include a reduction in processing time. In other examples, delivery to previously modified T cells, such as CAR-T cells or gene-edited T cells, is also evaluated.

[0152] Transduction Additionally, standard viral transduction was performed. For example, HEK293 adherent cells were used. This method eliminates the need to remove media through a filter. For example, cells can be seeded in a 6-well plate, the media manually removed to expose the cells, and then the cells directly transfected on the plate. Additionally, some issues regarding containment of the system within a viral containment laboratory and isolation of all equipment are eliminated.

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

[0154] Example 2: Delivery of virus via spray Viruses can be delivered via the SOLUPORE™ device. For example, small, intermediate, or large systems, including iterations of the described procedures, including various membranes, device sizes, etc.

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

[0156] The methods and systems described 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 cargos are aerosolized in a buffer and the droplets are applied to the cells in a controlled manner (variable such as volume, height, etc.). The various cargos include mRNA, DNA, CRISPR RNP, and viral particles (such as lentivirus, retrovirus, AAV, etc.) that enable gene transfer to cells or editing of genetic material in cells.

[0157] In some embodiments, the process includes T cells, NK cells, etc.

[0158] In some embodiments, the cells are handled using an SMA nebulizer, a Conikal nebulizer, etc.

[0159] The system is used for transducing T cells. Additionally, the system is used for delivery to simultaneously or sequentially edit cargos and viruses. This system is also used for T cell manipulation using RNA, DNA, and siRNA. This system is further used for knocking in or knocking out genes, which can be done separately or in parallel with the introduction of genetic information using a virus. This system is used for editing cells using a number of cell editing platforms such as CRISPR / Cas9, Cas12, MegaTAL, TALEN, ZFN, etc., all of which can be delivered alone, in parallel with a virus, or sequentially.

[0160] In some implementations, cells are handled by multiple nebulizers either simultaneously or sequentially. In other iterations, one nebulizer is used in isolation. Using a nebulizer to aerosolize virus particles improves the contact between the cells and the virus and improves transduction. The virus is dropletized and lands on the surface of the membrane where the cells are located. The dropletized virus allows for a much smaller volume to be administered to the cells. After a short incubation, the medium is exchanged. 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 polybrene or PGE2, for example, to enhance transduction. Other commercially available fibronectin peptides (Retronectin) or Lentiboost are also added to enhance the interaction between the virus and the cells and are added in various iterations.

[0162] Also evaluate the spray rate / duration / pressure. Test the recovery of cells by various methods (e.g., bottom-up / pipetting).

[0163] The cargo for gene manipulation may be suspended in various buffers, none of which limit the scope of this patent, which contains many variations of what cargo can be delivered to what cell types.

[0164] Furthermore, transduction of T cells can be very variable, and with this method of controlled delivery, viral transduction can be made more consistent and thus more advantageous.

[0165] Example 3: Gene therapy viral vectors The following table shows the main differences between various types of viruses used in eukaryotic gene therapy.

[0166] Overview of common viruses used to generate gene therapy viral vectors TIFF2025081453000005.tif67163

[0167] Overview of adherent and floating cells in the use of viral vectors TIFF2025081453000006.tif57161

[0168] Retroviruses have many disadvantages and, thus, AAV and lentiviruses are more widely used.

[0169] Example 4: Virus Delivery to a Population of Cells Using the SOLUPORE™ Process The SOLUPORE™ process enables delivery of a wide range of cargos to adherent and suspension cells in vitro and ex vivo. To date, these cargos have consisted of molecules such as nucleic acids and proteins as well as particles such as Qdots. The data herein demonstrate 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] Virus as a Vector Viruses are used as vectors for delivery of nucleic acids to cells because they can infect human cells naturally. Viruses must attach to host cells prior to entry. Attachment is achieved when specific proteins on the viral capsid or viral envelope bind to specific receptor proteins 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 holes are opened in the cell membrane and the unfolded viral envelope is further connected. 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 as follows: immunogenicity; target cell type; payload capacity; ability to transduce non-dividing cells relative to dividing cells; transience versus stable genomic integration (Table 5).

[0172] Overview of Viruses Used in Gene Delivery Applications TIFF2025081453000007.tif63158 which is incorporated herein by reference in its entirety, Pharmaceutics 2020, 12, 183

[0173] For applications of immunotherapy such as the delivery of chimeric antigen receptor (CAR) constructs for the generation of CAR-T cells and CAR-NK cells, gamma-retroviruses and lentiviruses are typically used, as 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 a lentiviral vector and a gamma-retroviral vector, respectively (Poorebrahim M et al. Crit Rev Clin Lab Sci. 2019 Sep;56(6):393-419). Similar to most viral vectors, these vectors were modified to disable viral replication and improve cell targeting efficiency.

[0174] For the application of other immune cell manipulations such as gene editing, AAV vectors are widely used. 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 the CRISPR / Cas9 system or donor template DNA for gene editing. In these cases, gene editing is permanent, but it may be desirable to express the gene editing tool transiently only to limit off-target gene editing that can occur when the tool is present in cells over a long period of time.

[0175] Lentivirus Gamma-retroviruses and lentiviruses are subtypes of retroviruses that contain an RNA genome that is converted to DNA in transduced cells by an enzyme called viral-encoded reverse transcriptase. For retroviruses, after entry into the cell, the uncoating process continues, whereby some viral proteins dissociate from the viral core. The viral RNA is reverse transcribed into double-stranded DNA. The viral proteins then complex with the proviral DNA and result in nuclear translocation and integration into the host genome. The integration process is assisted by very important viral proteins such as integrase and endogenous host cell transcription factors.

[0176] Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become a major tool for gene delivery into mammalian cells, and replication-defective recombinant lentiviruses are widely used in research and clinical applications. Modified lentiviruses can still infect cells, but no longer contain the essential genes for producing new viral particles. 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 gene elements such as intron-containing sequences; and they are a relatively easy system for vector manipulation 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 G.J. et al. J. Gene Med. 2013;15:78-82). Third-generation lentiviral vectors incorporate key safety features and further enhance safety (Kim V.N. 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 by retroviruses has been expanded by pseudotyped retroviruses with the vesicular stomatitis virus envelope glycoprotein G (VSV-G). VSV-G binds to phosphatidylserine, a ubiquitous membrane component, which allows VSV-G pseudotyped viruses to attach to and transduce a much wider 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, the cells are activated and subsequently transduced with a lentiviral vector (LV) whose majority is pseudotyped with the glycoprotein G of vesicular stomatitis virus (VSV). The modified lymphocytes are then expanded and used in functional in vitro assays or for in vivo applications. Stable and efficient transduction of B cells by LV is very difficult to achieve. Resting B cells are restricted from transduction by conventional VSV G pseudotyped LV (VSV-LV) due to the lack of expression of low density lipoprotein receptor (LDLR) and must be activated prior to transduction. Efficient activation and culture of primary human B lymphocytes is complex because it involves activation stimuli combined with cytokines that are carefully titered and subsequent co-culture with feeder cells. Even under optimal activation and culture conditions, the transduction efficiency with VSV-LV is notoriously low. The combination of all these difficulties may account for the fact that the number of clinical trials involving engineered B cells is much less compared to T cells.

[0179] In contrast, manipulation of T lymphocytes by lentiviral transduction is easier to achieve. Still, the cells, like B cells, must be activated prior to transduction with conventional VSV-LV because of the lack of LDLR expression, rendering them otherwise not readily transducible (X Geng, et al. Gene Therapy v.21, pages 444 - 449(2014)). Primarily due to the greatest success of CAR T cell therapy, the protocols for T cell isolation, activation, lentiviral transduction, and expansion have been significantly improved in recent years. Current state-of-the-art T cell activation relies on the stimulation of the TCR activation pathway via CD3-specific antibodies and CD28-specific antibodies combined with cytokines such as IL-7 and IL-15.

[0180] The need to activate lymphocytes prior to transduction in conventional LV has disadvantages. This adds to the complexity of the overall procedure, increasing the duration and cost of the manufacturing process. Additionally, the stimuli applied for activation, combined with long-term ex vivo culture, are likely to change the cells, which can have a negative impact on the quality of the final product. As a result, naive cells can differentiate into less preferred phenotypes that exhibit a higher degree of exhaustion, lower proliferative capacity, shorter in vivo persistence, and lower functionality. This can have very important implications for therapeutic success. As an example, the phenotypes of central memory (CD45RO+ / CD45RA+ / CD62L+) or stem cell memory (CD45RO+ / CD45RA / CD62L+) have been shown to be beneficial for the persistence and function of T cells in vivo. In this regard, a positive correlation between the 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 being considered. Similarly, the central memory phenotype leads to functionally superior TCR-modified T cells. Therefore, minimal manipulation of lymphocytes during gene modification has significant clinical validity.

[0181] Spinoculation Centrifugal inoculation, i.e., spinoculation, is widely used in virological research to enhance virus infection. The procedure involves centrifuging a mixture of virus and target cells at high speed for an extended period, for example, at 800×g for 30 minutes at 32°C. The method was thought to enhance transduction efficiency by concentrating the virus at the cell membrane. However, spinoculation has been shown to induce the activities of dynamic actin and cofilin, presumably due to the cellular response to centrifugal stress (Jia Guo, et al. J. Virology Oct. 2011, p. 9824-9833). This actin activity also leads to upregulation of cell membrane receptors that can enhance virus binding and entry. Spin-mediated enhancement cannot be simply explained by the virus concentration effect; rather, it is suggested to be coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, virus entry, and post-entry processes. Therefore, spinoculation may affect the biology of target cells in an unknown or undesirable manner.

[0182] Limitations of Virus Vectors As described above, viruses are useful for genetic manipulation of cells, but their utility has limitations.

[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, such as the physical proximity of lentiviral particles to T cells, have been identified as potentially playing a role in transduction efficiency. This proximity can be manipulated through the number of cells and virus particles in suspension; the duration of agitation to promote homogeneity; and the surface-to-volume ratio in the transduction vessel. However, limited research has been conducted on the identification and optimization of 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 substance at the cell membrane. This process enhances virus attachment to the cell membrane and the rate of entry into the cell, making the process more efficient. Consequently, a smaller dose of virus is used and the cost is reduced.

[0184] Since the SOLUPORE™ process is a gentle way to concentrate the virus at the cell membrane, it has significant advantages over existing concentration methods such as spinoculation that can affect cell structure. Furthermore, the SOLUPORE™ process is designed to be compatible with the cell therapy manufacturing process, unlike spinoculation.

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

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

[0187] Viruses are only capable of delivering nucleic acids, which means that the types of cargo they can deliver are limited. If viruses could be co-delivered with other types of cargo, this could enhance the utility of viruses in the manipulation of next-generation cell therapy products. However, currently, there is no method that has been demonstrated to co-deliver viruses with other types of cargo. Again, the SOLUPORE™ process described herein provides a solution to this problem by enabling the efficient delivery of numerous different cargo types, either sequentially or simultaneously. The following materials and methods were used to generate the dates 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 evaluated over 1 hour by assessing precipitation under the microscope.

[0190] LV-GFP Delivery Primary human PBMCs were thawed and activated with Miltenyi's CD3 antibody and CD28 antibody for 3 days. Three days after the activation culture, the cells were subjected to the SOLUPORE™ process with LV-GFP (MOI = 2.5) or static transduction. The cells were allowed to recover for 72 hours before GFP expression was evaluated by flow cytometry.

[0191] Stability of LV-GFP in Delivery Solution Prior to the present invention, it was not known whether there were solubility issues because SOLUPORE™ process delivery had not been previously combined with viral preparations.

[0192] When the solution was viewed under a microscope during the stability analysis, no aggregation or precipitation was observed. Additionally, certain other cargos showed low levels of aggregation when combined with the delivery solution, causing the Solupore® nebulizer to clog during spraying. Loading and spraying the virus-containing delivery solution into the Solupore® nebulizer further demonstrated that no clogging occurred at any point during the course of the study, and no aggregation or precipitation took place.

[0193] Cell Viability, Expansion, and GFP Expression after LV-GFP Delivery LV-GFP was delivered to T cell cultures by the SOLUPORE™ process and compared to the standard static method of LV transduction.

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

[0195] The cumulative expansion rate of T cells was measured up to 96 hours after virus 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. The 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 25.2 ± 1.48% for solupore-treated cells and control-transduced cells, respectively. On day 4, the efficiency was 40.27 ± 2.67% and 26.83 ± 1.38% for solupore-treated cells and control-transduced cells, respectively.

[0197] Efficient Virus Delivery of Virus to a Population of Cells The data in this specification demonstrated that the SOLUPORE™ process is compatible with the delivery of virus to activated T cells. No precipitation or aggregation was observed when mixed with the delivery solution of the SOLUPORE™ process. The virus solution could be sprayed and was successful in transducing target cells. The survival rate and growth 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] Collectively, these findings demonstrate that the SOLUPORE™ process is suitable for virus delivery to cells and is superior to standard methods.

[0200] Since the SOLUPORE™ process is suitable for virus delivery, it is possible to use solupore treatment in cell therapy manufacturing processes involving viral transduction. Due to the cost of the virus, transduction is a major cost driver in CAR T cell manufacturing. The SOLUPORE™ process can now achieve similar levels of transduction efficiency using less virus to enhance viral transduction and thus reduce costs.

[0201] A variety of cargos can be delivered simultaneously by the SOLUPORE™ process. The demonstration in this specification that the SOLUPORE™ process is compatible with virus delivery means that the SOLUPORE™ process can be used to co-deliver viruses with other cargos. These other cargos can be other viruses, or can be proteins, nucleic acids, small molecules, or complexes thereof. The ability to co-deliver cargos 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 manufacturing processes including cost, time, and labor. In addition, fewer process steps mean less handling and contamination risk, as well as process simplification. Alternatively, the virus can be delivered sequentially, either before or after the other cargos.

[0202] The SOLUPORE™ process enables delivery of cargos to non-activated T cells. Lentiviral vectors have very low transduction efficiency in non-activated T cells. Thus, the SOLUPORE™ process increases the transduction efficiency of lentiviruses in non-activated T cells.

[0203] A core feature of the SOLUPORE™ process device is the ability to facilitate media exchange. When a solution containing cells is transferred into the device, the liquid is drained and can be replaced with a different liquid. Thus, the process of handling liquids is possible. Such a process of handling liquids can include, for example, a washing step. In virus transduction and other cell manufacturing processes, a washing step is often required. The Solupore® device enables the incorporation of such steps into the manufacturing process.

[0204] The Solupore® technology is also scalable, meaning that virus transduction using this method can be performed on a small scale for initial and preclinical studies, as well as on a larger scale for process development and clinical applications.

[0205] Example 5: Delivery of Lentiviral Vector (LV) to T Cells by SOLUPORE™ - The Process Using Solupore™ A dataset on LV delivery by the SOLUPORE™ process was generated, comparing it to a static transduction control.

[0206] Viral delivery technologies for cell therapy and gene therapy have been developed. The platform relies on a reversible cell permeation process for payload delivery using a functionally closed device. Using this approach, effective nucleic acids and gene editing have been demonstrated in treatment-relevant cell types with minimal impact on cell viability, proliferation, gene expression, or phenotype. The utility for delivering viral vectors to target cells has also been demonstrated. The data illustrate 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 "Isolation, Initiation, and Cell Culture of PBMC-Derived T Cells" (provided in Example 5). To generate a peripheral blood mononuclear cell (PBMC) cell bank, the cells were counted and viability was assessed using a Nucleocounter NC-200. 61.5×10 6 viable cells / mL were frozen at a controlled rate of -1°C / min to -100°C using VIA Freeze in 1 mL aliquots. A total of 43 vials were banked, and all frozen vials were transferred from VIA Freeze to liquid N for permanent storage. 2Transferred to the tank. The eligibility of the PBMC bank was confirmed by thawing three random vials and evaluating the cell viability and cell recovery during thawing. The thawing procedure was carried out as described in "Isolation, Initiation, and Cell Culture of PBMC-Derived T Cells" (provided). For each vial, 4×10 6 cells at a live cell density of 1×10 6 cells / mL were also seeded into 6-well plates and activated using anti-CD3 and anti-CD28 antibodies. Three days after activation, the cells were collected and the expression of CD3 and CD25 was evaluated as described in "Preparation of Cells for Cell Thawing, Culture, and Experimental Use" (provided in Example 7).

[0208] Thawing and Initiation of PBMCs Complete medium was prepared using the protocol described in "Isolation, Initiation, and Cell Culture of PBMC-Derived T Cells" (provided in Example 5), and cryopreserved PBMCs were thawed and activated for 3 days. Representative 10× images were taken using an inverted microscope to capture cell clumping and overall morphology. To determine whether the expression of CD3 and CD25 after activation meets the release criteria for the SOLUPORE™ process, activated PBMC starting T cells were collected and stained with anti-CD3 conjugated antibody and anti-CD25 conjugated antibody for flow acquisition and analysis as outlined in Example 8. Before the cells were released for experimental use, the expression of CD3 and CD25 was verified to be >90%.

[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. The LV batches were titered and adapted for K562 cells.

[0210] To investigate the stability of LV in the delivery solution formulation, 75 μL of the 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 contains 32.5 mM sucrose, 106 mM KCl, 5 mM Hepes, 12% v / v EtOH, and water for injection.

[0211] The required number of aliquots were thawed on ice and briefly centrifuged to collect the liquid scattered on the side of the vial. The LV-eGFP aliquots were pooled before use to ensure there was 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 three LV-eGFP lots used in all experimental runs TIFF2025081453000008.tif40160

[0213] Preparation of PBMC-Initiated T Cells As outlined in "Cell Thawing, Culturing, and Cell Preparation" (provided in Example 7), the required number of PBMC-derived T cells were pelleted by centrifugation and resuspended in the basal medium (CTS OpTmizer T Cell Expansion SFM containing supplements) to yield the desired cell density listed for each experiment. A post-dilution cell count was performed and 30 mL aliquots of the 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 evaluated for both cell number and viability to ensure 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 for PBMC-derived T Cell Cultures" (described herein).

[0214] Static Transduction For each multiplicity of infection (MOI) condition, the required number of T cells was diluted in complete medium to yield the desired volume, and cell counting was performed after dilution to confirm cell density. Subsequently, the prepared cell suspension was aliquoted into Nunc™ EasYFlasks™ TC-treated T25 flasks, with three technical replicate experiments and one untreated control per MOI condition. The cells were maintained in a 37 °C, 5% CO 2 incubator until static transduction was performed.

[0215] Static transduction parameters tested in each experiment TIFF2025081453000009.tif51160

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

[0217] For each run, the complete medium was doubled 1 day after virus delivery. A larger volume was given for runs 1 / 2, and the samples were transferred to T75 flasks and cultured flat. For runs 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 later, after performing time point analysis, the cultures 3 days after infection were diluted to a viable cell density (VCD) of 0.5 × 10 6 cells / mL to enable further analysis the next day. Cell counting was performed after dilution to track the cumulative magnification factor 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 Using the Solupore™ System After the "SOLUPORE™ process for PBMC - initiated T - cell cultures" described in this specification, the sterilized components were aseptically assembled in a biosafety cabinet (BSC). A pressure leak test was completed to ensure that the device was properly sealed and the pressure profile was preserved. Calibration and stop solutions were prepared according to the "SOLUPORE™ process for PBMC - initiated T - cell cultures" (described in Example 6) and placed on ice until use.

[0219] For the increase in payload volume to achieve an MOI of 2.5, the system was calibrated to deliver 75 - 80 μL in Runs 1 / 2. Since 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 the insufficient spray volume.

[0220] Parameters tested for the SOLUPORE™ process in each experiment SOLUPORE™ process parameters tested in all experiments TIFF2025081453000010.tif61160

[0221] 1×10 9 Assuming a viral titer of 1×10 TU / mL, the volume (μL) of LV - eGFP payload required for each spray was calculated for all experiments as follows. TIFF2025081453000011.tif10128

[0222] Based on the total delivery volume, the final concentration (%) of the LV - eGFP payload was determined by TIFF2025081453000012.tif11128. The total volume of delivery solution required for each experiment was derived using the following calculation: TIFF2025081453000013.tif11142.

[0223] For all experiments, the delivery solution was formulated based on the final concentration of each component listed below.

[0224] Composition of payload and delivery solution for syringe and Elveflow reservoir. The % of WFI (Water for Injection) was adjusted to reflect the increase in the LV-eGFP composition. TIFF2025081453000014.tif26160

[0225] For all SOLUPORE™ processes, cells were loaded, solupore-treated, and collected in 20 mL of complete medium. Cleaning and priming between samples were performed as described in the “SOLUPORE™ process for PBMC-initiated T cell cultures” (described herein). Endpoint analysis was completed after recovery and after the SOLUPORE™ process.

[0226] In some examples, “S buffer” contains 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); which is hereby incorporated by reference in its entirety). In some examples, potassium acetate is replaced with ammonium acetate in the S buffer. The S buffer is further described in International Application WO 2016 / 065341, for example, paragraphs

[0228] -

[0229] , which is hereby incorporated by reference in its entirety.

[0227] Analysis after the SOLUPORE™ process and after transduction completed for each experiment. ◆ represents the endpoint analysis tested. TIFF2025081453000015.tif70160

[0228] For all time points after the SOLUPORE™ process, samples were weighed, counted, and viability was assessed using a Nucleocounter NC-200 prior to collection for downstream analysis.

[0229] For each run, complete media was doubled one day after virus delivery. A larger volume was given for Runs 1 / 2, and samples were transferred to T75 flasks, laid flat, and cultured, while for Runs 3 / 4, 3 mL of complete media was simply added to each T25 flask. For Run 1, final analysis was performed 3 days after the SOLUPORE™ process. For Runs 2 and later, after time point analysis, the cultures 3 days after the SOLUPORE™ process were diluted to a VCD of 5×10 5 cells / mL to enable further analysis the next day. Post-dilution cell counts were performed to track the cumulative magnification 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™ System The payload solution was removed from the Elveflow valve and properly removed prior to disposal. The nebulizer was then rinsed by purging it 2 times with 1 mL of PREempt RTU, 2 times with 1 mL of WFI, and 2 times with 1 mL of 70% IPA. Finally, the nebulizer was purged again in the same order using 1 mL of each reagent. The system was disassembled and the PREempt RTU was sprayed. 1% v / v Citranox in 20 L of tap water was prepared and the system components were immersed and rinsed with DI H 2 O. Each component was then sprayed with 70% IPA and dried using an air gun prior to being placed back in the BSC. Gaskets and O-rings were placed in an autoclaveable bag and autoclaved in a robust cycle. The UV light in the BSC was activated to ensure that all non-autoclaveable components were sterilized. The ethylene oxide (EtO) sterilization step was omitted.

[0231] Sample Preparation and Acquisition of 7-Aminoactinomycin D (7-AAD) Viability Discrimination Staining and % GFP Data 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 minutes, and the supernatant was removed using a multi-channel pipette. During centrifugation, a master mix of the 7-AAD viability dye in the FACS solution (5 μL of 7-AAD in 200 μL of FACS solution per sample) was prepared in a 15 mL Falcon tube. Each sample was resuspended in 200 μL of the staining solution and incubated for 5 minutes at RT while protected from light. 7-AAD viability staining and GFP expression were acquired using a CytoFlex according to the acquisition and gating strategy and quantified using FlowJo.

[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 minutes, and the supernatant was removed using a multi-channel pipette. During centrifugation, 1× lysis buffer was prepared as follows.

[0233] Preparation of 1× In-house Cell Lysis Buffer TIFF2025081453000016.tif26160

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

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

[0236] Preparation of 1× ddPCR Supermix. The volume of each reagent was scaled up according to the total number of samples. 10% volume contingency (for pipetting errors) and two extra reactions were also included as NTC controls. TIFF2025081453000017.tif31160

[0237] 18 μL of master mix was added to 4 μL of 20-fold diluted DNA and added to each reaction well of a 96-well Bio-Rad ddPCR plate. 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 reaction was then vortexed and briefly spun down to collect any dispersed liquid at the bottom before transferring it to an automated droplet generator (Bio-Rad AutoDG). Once the droplets were generated, the plate was sealed again using a pierceable foil heat seal and transferred to a Bio-Rad C1000 thermal cycler to initiate PCR using the following cycle: 10 minutes at 95°C (ramp 2°C / second), [30 seconds at 94°C (ramp 2°C / second), 1 minute at 60°C (ramp 2°C / second)] × 39, 10 minutes at 98°C, and finally held at 4°C. Once the PCR cycle was complete, the reaction was 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 number / μL) of both Alb and WPRE were obtained and %GFP was derived by applying the following equation: TIFF2025081453000018.tif15128 was derived by applying

[0238] Software / Statistical Analysis TIFF2025081453000019.tif50160

[0239] Stability of LV Based on qualitative analysis using magnifications of 4-fold, 10-fold, and 20-fold, there was no evidence of any precipitation in the delivery solution up to 4 hours post-formulation.

[0240] LV Delivery Run 1 The expression levels of CD3 and CD25 in activated PBMC-derived T cells measured by flow analysis were 92.3% and 91.3%, respectively.

[0241] After the SOLUPORE™ process, for three repeated experiments, the average total recovery rate from the SOLUPORE™ process was 63% ± 12%, and the viable recovery rate was 60% ± 13% (Figure 8).

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

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

[0244] To evaluate virus delivery efficiency, GFP expression and median fluorescence intensity were quantified by flow cytometry. The % GFP expression three days later was 18.1% for solupore-treated T cells and 16.8% for statically transduced T cells (Figure 10A). Solupore-treated T cells had a significantly higher % GFP expression until one day after virus delivery (p < 0.01).

[0245] The MFI of the 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 statically transduced T cells (p<0.001).

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

[0247] After viral delivery by the SOLUPORE™ process, for three technical replicate experiments, the average total cell recovery rate was 67%±6% and the viable recovery rate was 65%±6% (Figure 12).

[0248] As shown, an analysis was performed for this run 4 days after infection to verify the GFP expression observed at earlier time points. The cell viability was higher than 90% for both viral delivery methods 1 day after infection (Figure 13A), but the viability of solupore-treated cells was significantly lower than that of statically transduced cells (p<0.01) (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 collected from the system after the SOLUPORE (trademark) process had a cumulative magnification of 7.7 four days later. This was significantly lower than that of the statically transfected cells, which had a cumulative magnification of 12.5 (p<0.01) (Figure 13B). Additionally, the magnification at each time point after delivery was significantly lower in the solupore-treated cells compared to the statically transfected cells (p<0.01) (Figure 30B).

[0250] As shown in Figure 14A, the % GFP expression quantified by flow cytometry three days after infection was 15.2% for the solupore-treated T cells and 19.1% for the statically transfected T cells. One day later, the % GFP expression was maintained for the statically transfected cells (19.4%), but the GFP expression in the solupore-treated cells decreased significantly to 11.5% (p<0.05). The solupore-treated T cells had significantly higher % GFP expression until one day after virus delivery (p<0.01).

[0251] The MFI of the expressed GFP was significantly higher for the solupore-treated T cells immediately after virus delivery (p<0.01) (Figure 14B). However, the MFI in the solupore-treated T cells decreased over time. Three and four days after culture, the T cells derived from static transfection had 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+ (Figures 15A and 15B).

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

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

[0255] Cells collected from the system after the SOLUPORE™ process had a significantly lower (p < 0.05) cumulative expansion ratio of 9.6 compared to cells from static transfection with a cumulative expansion ratio of 13.2 at 4 days (Figure 13A). Regardless of the method of virus delivery, cells transfected at a lower MOI of 2.5 had a significantly better expansion over 4 days (p < 0.05) (Figure 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 transfected T cells (Figure 19A). One day later, % GFP decreased (p < 0.01) for both the SOLUPORE™ process and static transfection, 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). Regardless of the LV delivery method, there was no statistical difference in % GFP between MOIs of 2.5 or 5 (Figure 19C).

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

[0258] LV Delivery Run 4 A fourth run of virus delivery by both the SOLUPORE™ process with the system and resting transduction was performed as in the third run. 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, for six replicate experiments, the mean total cell recovery rate was 63%±10% and the viable recovery rate was 59%±10% (Figure 21). Despite differences in the number of cells loaded, there was no significant difference in the recovery rates (p>0.05; paired two-sided t-test).

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

[0261] The cells collected from the system after the SOLUPORE™ process had a significantly lower (p<0.001) cumulative expansion ratio of 9.0 compared to the cells derived from stationary transfection that had a cumulative expansion ratio of 22.5 4 days later (Figure 23A). Regardless of the method of virus delivery, there was no statistical difference in expansion between MOIs of 2.5 or 5 (Figure 23B).

[0262] Three days after delivery, the GFP expression in % was significantly higher (p<0.01) in the solupore-treated T cells (34.3%) compared to the cells transduced by stationary transfection (25.4%) (Figure 24A). One day later, the GFP expression was maintained at 36.1% and 26.7% respectively for both the SOLUPORE™ process and stationary transfection. As shown in Figure 24C, the method of virus delivery and the MOI conditions had no significant effect on the % GFP expression.

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

[0264] Purity and Activation of T Cells For experimental use, PBMC starting T cells were required to have expression higher than 90% for both CD3 and CD3 / CD25. In all runs, cells passed QC on average at 93.1%±4% CD3+ and 95.0%±3% CD3+CD25+ and were released for the SOLUPORE™ process (Figure 25).

[0265] Analysis after Recovery During technology transfer of the system to the facility, minimum thresholds of a total cell recovery rate of 50%±10% or more and a viable cell recovery rate of 30%±10% or more were used to define the success of SOLUPORE™ process runs. Cumulatively, the total cell recovery rate was 63%±9% and the viable cell recovery rate was 60%±9% (n = 18 sprays, Figure 26). 24×10 6 cells and 12×10 6 cells with different cell loads were tested for the third and fourth runs, but there were no significant differences in the recovery percentages between the two cell load conditions (p>0.05; paired two-sided t-test).

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

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

[0268] Growth Kinetics after Virus Delivery After viral delivery by either the SOLUPORE (trademark) process or static transfection, cell counts were performed up to 4 days post-infection to evaluate the effect of viral delivery on T cell proliferation. At 4 days, an average fold increase of 16.8 was observed in statically transfected T cells. This was significantly higher than the solupore-treated T cells, which experienced a fold increase of 9.0 (p<0.001) (Figure 28A). The difference in cumulative fold increase observed in all 4 runs was mainly due to the recovery period required by the cells after the SOLUPORE (trademark) process, which becomes even more complex at a later date due to the multiplicative nature of the cumulative fold increase. The daily fold increases are shown in Figures 31A and 31B.

[0269] In addition, T cells that received a lower MOI of 2.5 one day post-delivery had a higher (p<0.001) increase than those that received an MOI of 5, regardless of the delivery method (Figure 28B). However, this difference was not observed 3 or 4 days post-delivery. Again, this was mainly observed in solupore-treated cells but not in statically transfected cells (Figure 32) and may similarly be related to the lower viability observed after the SOLUPORE (trademark) process (recovery period).

[0270] As shown in Figure 28C, Runs 3 and 4 had significantly higher increases compared to Runs 1 and 2 one day (p<0.001) and three days (p<0.01) after delivery. However, this effect was not observed four days after delivery. The differences observed in the growth rate theory 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 area ratio in Runs 3 and 4 likely enabled proper oxygen diffusion through the cell culture, thereby promoting cell growth. This reduced volume to surface area ratio was achieved throughout the entire culture period starting immediately after the SOLUPORE™ process in Runs 3 and 4. For Runs 1 and 2, the volume to surface area ratio decreased one day after the SOLUPORE™ process when the culture was transferred from an upright T25 flask to a flat T75 flask. The data on day 4 suggest that these changes may not have a long-lasting effect on T cell growth.

[0271] GFP Expression by Flow Cytometry In all runs, GFP expression immediately after the SOLUPORE™ process was significantly higher than in statically transduced T cells (p<0.001) (44.7% and 1.1% respectively, Figure 29A). This significant difference was also observed 1 day after delivery (p<0.001). Considering that the package of LV-eGFP used in this study was a crude virus preparation, this difference in GFP expression at the early time point is likely due to the fact that cells received GFP as a protein cargo from the reversible permeability of the cell membrane during the SOLUPORE™ process, whereas statically transduced cells were not subjected to this procedure. As the data suggest (Figure 29A), this difference decreases over time because the protein cargo is only transiently present and diluted as the cells grow. To address the possibility of protein cargo delivery, an additional time point was introduced 4 days after the SOLUPORE™ process for runs 2 and later to evaluate whether the %GFP observed at the 3-day time point was an accurate depiction of the success of LV delivery as opposed to the uptake of residual GFP protein during the SOLUPORE™ process. In runs 2, 3, and 4, the average GFP% at 3 and 4 days after the SOLUPORE™ process was 25.7% and 25.9% with no difference between the two time points (p>0.05; paired two-sided t-test), suggesting that the results collected 3 days after the SOLUPORE™ process are an accurate assessment of LV delivery efficiency.

[0272] Irrespective of the virus delivery method, no significant differences in GFP% or MFI based on MOI were observed (Figure 29C and Figure 29D). This is in line with what was observed in the generated historical data and further validates the proposal to use an MOI of 2.5.

[0273] Overview The viable cell recovery rate and total cell recovery rate after the SOLUPORE™ process across 18 samples were 60% and 63% respectively. The viability after 24 hours of the ‐SOLUPORE™ process was 90% (n = 18 samples). The ‐system can deliver LV-eGFP to T cells with an average GFP expression of 25.7% over 18 ‐SOLUPORE™ process experiments.

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

[0275] Example 6: Isolation, Initiation, and Cell Culture of PBMC-Derived T Cells Isolation, Initiation, and Cell Culture of PBMC-Derived T Cells The methods herein describe the isolation, activation, and cell culture of PBMC-derived T cells. The exemplary methods outlined below cover T cells starting from PBMC and include the isolation, culture, and activation of these cells.

[0276] Related Acronyms: AB serum: Human serum from AB donors lacking antibodies to the 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 unit 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] Materials TIFF2025081453000020.tif58160

[0278] Equipment TIFF2025081453000021.tif32128

[0279] Procedures PBMC Isolation Preparation of Dilution Buffer: TIFF2025081453000022.tif12160

[0280] Preparation of CTS T Cell Culture Medium (TCGM) TIFF2025081453000023.tif24160 * Add IL-2 on day 0. Use it within 1 week after adding IL-2 and always freshly prepare it on the starting day. 1. When opening the CTS vial, be sure to immediately add the CTS supplement. 2. Add serum or equivalent and filter using a 50 ml syringe and a 0.2 μm filter or a 0.2 μm SteriCup. 3. After filtering, add L-glutamine and IL-2. 4. Use the medium within 1 week.

[0281] Isolation of PBMCs 1. Dilute the blood with DPBS (referred to as dilution buffer) containing 1% heat-inactivated FBS in sterile Duran. 1.1. Dilute the buffy coat at a ratio of 1:1 (blood:dilution buffer). 1.2. Dilute the contents of the Leukopak at a ratio of 1:2 (blood:dilution buffer). 2. Add 10 ml of Lymphoprep solution to a 50 ml Falcon (the number of Falcons is the number obtained by dividing the total ml of diluted blood by 40). 3. Hold the 50 ml tube vertically against the 25 ml stripette and carefully layer the diluted blood onto the prepared Lymphoprep. 4. Centrifuge at 400 g for 22 minutes with the acceleration set to 6 and brake-off (set to 0 - minimum deceleration). See Figure 34. 5. Using a sterile Pasteur pipette, remove the upper (serum) layer and discard, leaving 3 ml of the serum layer in the tube. 6. Carefully transfer the PBMC layer (the white cloudy layer beneath the serum and above the Lymphoprep) to a clean tube, making sure not to take any of the red blood cell pellet. 7. Supplement the buffy coat layer with DPBS + 1% HI-FBS to 50 ml, mix, turn on the brake, and centrifuge at 450×g for 7 minutes (acceleration 9, deceleration 9 - use this setting for the rest 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. Supplement the PBMC with DPBS + 1% HI-FBS to 50 ml, mix, and centrifuge at 450 g for 7 minutes as in 8.9. 10. Discard the supernatant by pouring it off. Resuspend the pellet in 30 ml of DPBS + 1% HI-FBS. This is the isolated PBMC. 11. Count the cells: In an eppendorf, add 50 μl of the cell suspension to 950 μl of DPBS + 1% HI-FBS to make a 1:20 dilution. Take the diluted cell suspension using a Via1 cassette and add it to the Nucleocounter. Make sure to add the cell dilution to the program and count the cells under the "Cell Count and Viability" program. See WI-6 NC-3000 NucleoCounter Operation & Maintenance. 12. PBMC can be cryopreserved at this point by following the cryopreservation process in the "Cryopreservation" section below. PBMC should be cryopreserved at 50 million cells per ml.

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

[0283] Mr. Frosty 1. Mr. Frosty should be filled with room temperature isopropanol to the mark on the container so that the freezing process is as rapid as possible (refill monthly). 2. The cryovial should be pre-labeled and pre-opened in the BSC. 3. Using a 25 ml stripette, slowly start to drip the cryopreservation medium onto the cell pellet and always swirl simultaneously. 4. Using the same 25 ml stripette, take a large amount of the cell and cryopreservation medium solution and quickly add 1 ml to the prepared cryovial. 5. Quickly cap the tubes, add them to the Mr. Frosty container, and then move Mr. Frosty to an -80°C freezer. 6. The cells should be Only on Dry Ice transferred to liquid nitrogen the next day.

[0284] Controlled Rate Freezer 1. Ensure that the pressurized liquid nitrogen tank is filled 1 - 2 days before isolation 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 WI-10 controlled-rate freezer with an attached low-pressure liquid nitrogen supply tank and move the "T cell" setting of the controlled-rate freezer. 5. Using a 25 ml stripette, slowly drip the freezing medium onto the cell pellet while swirling. 6. Using the same 25 ml stripette, take a large amount of the cell and freezing medium solution and quickly add 1 ml into the vial. 7. Still in the BSC, quickly cap the tube. 8. When the temperature reaches 4°C, add the sample to the controlled-rate freezer. 9. Immediately after the freezer completes its run (i.e., when it reaches -180°C), the cells should be transferred to liquid nitrogen.

[0285] Initiation of T Cells Day 0 - Thawing This protocol follows the procedure for single donor use and different donors should not be mixed. 1. Prepare the medium and warm it to 37°C. 2. Remove the required number of PBMC vials from liquid nitrogen. 2.1. Due to donor-to-donor variability in PBMC expansion, it may be necessary to start with more PBMC than the number of T cells required on day 3, due to differences in the degree of PBMC expansion. 3. In the BSC, prepare one 50 ml tube containing 10 ml of pre-warmed medium (10 ml per vial of cells to be thawed). 4. Thaw the PBMC vials in a 37°C water bath, swirling gently occasionally until small pieces of ice remain (thaw no more than two vials at a time). 5. Return the vial to the BSC, slowly add 1 ml of warmed medium to the vial, and pipette up and down once to mix. 6. Slowly transfer the contents of the vial to the pre-warmed medium in a properly labeled 50 ml falcon. 7. Rinse the vial with the medium and transfer it to a 50 ml tube. 8. Adjust the volume to 50 ml with T cell growth medium (TCGM). 9. Centrifuge at 300×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 it with 450 μl of medium in an Eppendorf to a final volume of 500 μl. 12.2. Count the cells using a Via-1 cassette and a Nucleocounter - including dilution on the software (i.e., enter "50" in the sample box and "450" in the dilution box - the Nucleocounter calculates the dilution factor). 12.3. Visually inspect the gates on the software and, if necessary, change to include the cell body and record the cell viability and concentration. 13. Increase the volume of the cells with TCGM to 1×10 6 / ml. 14. Add 0.5 μl of both CD3 antibody and CD28 antibody per million cells. 15. Seed the cells into a properly labeled flask for initiation. 15.1. Maximum 30 ml / T25. 15.2. Maximum 80 ml / T75. 16. Place the flask horizontally at 37 °C in an incubator with a water jacket with buffered water. 2 (Horizontal) 17. Cells should be left untouched for approximately 72 / 96 hours (seed on Monday for use on Thursday, or seed on Friday for use on Monday / Tuesday). 18. Cells will appear "flaky" or "snowy" in flasks after 3 days and will then be starting well.

[0286] Example 7: Solupore™ System for PBMC-Initiated T Cell Cultures The SOLUPORE™ process for PBMC-started T cells is performed in a functionally closed device.

[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 TIFF2025081453000024.tif64154

[0289] System Components TIFF2025081453000025.tif126144

[0290] System Consumables TIFF2025081453000026.tif110143

[0291] System Consumables TIFF2025081453000027.tif92165

[0292] Preparation of Solutions All solution preparations must be carried out 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 place on ice until needed. Do not use PBS that has been opened for more than 1 month. Record the opening date in the run log. 2. Cell Culture Medium : Calculate the required medium based on the total number of samples and 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 Solution : Prepare a payload-free delivery solution for nebulizer calibration with a minimum volume of 10 mL by adding the volumes of WFI first, then EtOH (final 12%), and finally S buffer (prepared at 20×) in that order to a 50 mL Falcon tube labeled "Calibration" as detailed in (Table 5). Note: Do not add the S buffer and EtOH solutions together. This is to avoid precipitation of excipients in the S buffer. Make sure to add WFI first. 3.1. Elveflow Reservoir : In the BSC, transfer 1 mL of the "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 prepare the solutions for 4 SOLUPORE™ processes. If the number of samples (number of sprays) for the SOLUPORE™ process is not known, proceed to step 5 for assembly and calibration and then prepare the solutions for the SOLUPORE™ process once the number of samples is known.

[0293] Payload-free delivery solution for nebulizer calibration TIFF2025081453000028.tif20128

[0294] 4. Solution for the SOLUPORE™ Process : Using the calculations in Table 6, which are examples given for 10 sprays, calculate the volume of the delivery solution required for the SOLUPORE™ process for 2.4 × 10 7 cells of T cells at a spray volume of 50 μL.

[0295] Calculation of the total volume of the delivery solution required for the SOLUPORE™ process TIFF2025081453000029.tif29128 * Note that changing the delivery volume of the nebulizer may change the priming volume and dead volume, and accordingly the above calculations will be modified. ** The excess volume corresponds to a 10% excess allowed during calibration. 4.1. Referring to Table 7, calculate the required volumes of the components of the payload solution and the delivery solution for the required number of experimental sprays and record them in the run log. This is an example 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 required. Note: Do not add the S buffer and EtOH solution together. This is because it will result in precipitation of the excipient in the S buffer. Make sure to add WFI first. Do not add the payload (GFP mRNA) at this stage.

[0296] Composition of the payload and delivery solutions for the syringe and Elveflow reservoir TIFF2025081453000030.tif42160

[0297] 5. Assembly of the System, Calibration of the Atomizer, and Priming of the Filter Membrane 5.1. Using the provided checklist, transfer all necessary bags containing components and consumables from the EtO cabinet into the BSC to assemble the system unit (Figures 35 and 36). 5.2. Using the provided checklist, aseptically transfer the consumables and experimental instruments required for a single experimental run into the BSC. 5.3. Unpack the sterilized components and dispose of the bags. 5.4. To assemble the lid (Figure 36): 5.4.1. Place the X-ring and seal the nebulizer (a). 5.4.2. Insert the nebulizer equipped with the air line (b). 5.4.3. Add the cap and fix the nebulizer in place (c). 5.4.4. Using the provided Allen key, tighten the screw of the nebulizer cap (d). 5.4.5. Attach the bracket containing the Clippard valve as shown in (e). 5.4.6. Fix the bracket by screwing it in (f). 5.4.7. Attach the reservoir holder and the Elve flow module (g). 5.4.8. Connect the silicon tube to the reservoir needle (h). When selecting the setting on the unit control and pressing Purge, the Clippard valve opens, and then insert the segment of the silicon tube into the Clippard valve. 5.4.9. Connect the other end of the silicon tube to the needle in the nebulizer sample channel. Make sure the silicon tube under the Clippard valve is straight. If not, press Purge to open the Clippard valve and straighten it. 5.4.10. Connect the Clippard valve wire as shown in the figure (j). 5.4.11. Connect the air line of the nebulizer to the solenoid valve. 5.4.12. Connect the vent tube and filter (k). 8.5.5. As shown in Figure 36, place the system base (Figure 35) on the tilt stand. 5.6. Screw in 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 their respective tubes, ensuring that all clamps are closed. 5.8. Connect a 0.2 μm filter to the end of the bleed port tube and connect the waste bag to the drain port tube. 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 on the membrane holder (Figures 35 - 38). 5.12. When assembling for filter priming of the SOLUPORE™ process, add the drain disk (Figures 35 - 39), proceed to assembly for calibration or loading of the SOLUPORE™ process solution, and add a blue filter divider instead of the drain disk and filter membrane (Figures 35 - 39). Proceed to 5.15. 5.13. Wet the disk by pipetting 1 ml of 1×PBS. Ensure that the drain port is open. As shown in Figure 37, gently press down on the edge of the drain disk to facilitate uniform wetting of the drain disk and rotate the disk to disperse the liquid. Do not touch the drain disk within the area delimited by gasket 1. 5.14. Place the PETE filter membrane on top of the drain disk. 5.15. When preparing for calibration only, place gasket 2 on the filter membrane (Figs. 35 - 40) or the blue filter divider. Note regarding the SOLUPORE™ process: When placing gasket 2, take care to avoid wrinkles in the filter. If wrinkles are visible in the filter (Fig. 38), dispose of it and restart with a new filter. Note regarding the SOLUPORE™ process: To assist in the placement of the filter membrane during the sample run, an empty syringe can be connected to the drain tube. Use this syringe to gently draw a vacuum and help maintain a flat filter membrane before placing gasket 2 on top. 5.16. Insert O-ring 2 (Figs. 35 - 41). 5.17. Place the PS or SS mask (Figs. 35 - 42). 5.18. Insert O-ring 3 (Figs. 35 - 42). 5.19. Open the clamp on the drain tube. 5.20. Place the lid on the unit without clamping. 5.21. For calibration, proceed to 5.22; for filter priming of the SOLUPORE™ process, move to 8.7.

[0298] Calibration 5.22. Prime the nebulizer with the calibration solution. 5.23. Loosen the base screw of the Elveflow unit counterclockwise (Fig. 39) and remove a 1.5 mL Eppendorf tube (to protect the needle during storage). Place the "calibration" tube in the base and return the Elveflow unit and tighten with the screw. 5.24. On the controller (Fig. 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 washed with 70% IPA under the nebulizer. 5.26. Activate the "Spray" button until the spray appears at the tip of the nebulizer (usually 2 - 3 times). 5.27. Set the "Sample" pressure to 100 mbar or start with the calibrated settings from the previous day's experimental run. 5.28. Activate the "Spray" button twice, confirm the occurrence of the spray by visual inspection, and remove the waste container. 5.29. To calibrate the nebulizer: 5.30. Use scissors to cut the upper edge of the calibration cup washed with 70% IPA. 5.31. Weigh the tare of the cup on a chemical balance. 5.32. Place the cup in the MID 1T chamber as shown in Figure 41. 5.33. Place the lid on the unit and simultaneously close the two opposite clamps with uniform pressure. 5.34. Confirm that the clamp on the vent port is open. 5.35. Close the clamps on the drain tube, cell inlet / outlet tube, and bleed port tube. 5.36. Nebulizer calibration: 5.36.1. Confirm that the air pressure is set to 1730 mbar. 5.36.2. Set the sample pressure to the last calibrated value recorded in the run record. 5.36.3. Using the manual pressure regulator, set the air flow through the shower head to the required pressure, e.g., 90 mbar (Figure 42). 5.36.4. Open the clamp on the air flow line to the shower head. 5.36.5. Activate the "Spray" button. 5.36.6. Close the clamp on the air flow line to the shower head. 5.36.7. Immediately remove the lid clamp, take out the calibration cup, and return the lid to the unit. 5.36.8. Immediately weigh the calibration collection cup. 5.36.9. Record the weight in the run record. 5.36.10. Spray the cup liberally with 70% IPA and wipe dry. 5.36.11. If the weight is above 55 mg * (55 μL), reduce the sample pressure by 10 mbar. Dispense one spray into the waste cup and repeat spraying and measurement. 5.36.12. If the weight is below 50 mg (50 μL), increase the hydraulic pressure by 10 mbar. Dispense one spray into the waste cup and repeat spraying and measurement. 5.36.13. * Assumption: The density of the delivery solution is approximately 1 g / ml. 5.37. Repeat the calibration measurement until three consecutive measurements in the range of 50 - 55 mg are obtained. Ensure that the calibration solution is not depleted and that the entire length of the sample line is always filled with liquid. 5.38. Record the optimal pressure and calibration weight in the run log. 5.39. Once calibration is complete, place the waste cup under the nebulizer. Select the system settings, increase the pressure setting to 500 mbar, select and press purge, sample to expel the calibration solution from the nebulizer. Repeat purge one more time and dispose of 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. Loosen the base screw of the Elveflow unit counterclockwise (Figure 39), remove the 1.5 mL Eppendorf tube (calibration). Place the "SOLUPORE™ process" tube into the base, and return and tighten the Elveflow unit with the screw. 6.4. Reconnect the sample line to the control unit line. 6.5. On the controller (Figure 40), select "Air", ensure that 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 washed with 70% IPA on top of the blue filter membrane divider under the nebulizer. 6.7. Activate the "Spray" button (usually 2 - 3 times) until spray appears at the tip of the nebulizer. 6.8. Set the sample pressure to the calibration value and activate the "Spray" button once. Confirm that spray has occurred by visual inspection. 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 of the SOLUPORE™ process.

[0300] 7. Filter Priming of the SOLUPORE™ Process 7.1. Place the lid on the unit and lock the opposite clamps together to seal the system chamber. 7.2. Ensure having the following: 4 × 60 ml syringes 4 × syringe caps 1 × 150 ml sterile bag 1 × Luer spike interconnector 1 × 0.2μm filter on the bleed port tube before moving on to prime 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 1×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 tube and drain tube 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 drain approximately 30 mL, which is about 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. For the sterility test, take 1 mL of the priming solution at room temperature if required.

[0301] 8. Loading of Cells 8.1. Record on the run record. Time when the cell sample was aliquoted by cell culture Volume of the sample Cell concentration, e.g., 24×10 6 Cells / 30 mL Time to load the cells into the chamber 8.2. Ensure that the cell suspension provided in the basal medium is at the required cell concentration. 8.3. Using a 5 mL pipette set to 4 mL, mix uniformly 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 the 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. Flow cytometry: Place all samples in 1.5 mL tubes on ice and perform the analysis at the end of the SOLUPORE™ process experiment. 8.6. Count according to WI-06. Briefly, Gently mix the sample by pipetting 5 times using a P200 pipette. Do not foam the sample. Count using 1 cassette of Via 1 on NC3000 / NC200 (in accordance with WI-06). Record the counting time in the run log. Ensure that the same nucleocounter is used to count all samples before and after the SOLUPORE™ process. 8.7. Obtain the temperature reading of the stop solution before first use and record it in the run log. 8.8. Disconnect the syringe used for priming, recap it, and set it aside 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 syringe to the cell inlet / outlet tube. 8.12. Ensure that the clamp on the drain tube is closed. 8.13. Open the clamp on the cell inlet / outlet tube and gently push the plunger to load the cell suspension into the chamber. Keep the syringe plunger down to fill the inlet tube. 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 an 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 clamp on the cell inlet / outlet tube and dispose of 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 them 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 the liquid flowing through the tube When there is no more left, filtration is considered complete. 8.18. Record the filtration time in the run log. 8.19. When the liquid has stopped 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 any 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 tube. 8.21. Disconnect and set aside the waste bag. Connect a syringe containing the cell culture medium as the recovered solution. 8.22. Connect a 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, immediately activate the "spray" button, and start a 30 - second timer. 8.24. Close the clamp on the airflow line. 8.25. After 30 seconds of incubation, open the clamp on the inlet line, add the stop solution, and incubate for an additional 30 seconds. 8.26. Open the drain clamp and rapidly pour 20 mL of medium into the chamber. 8.27. Tilt the chamber and gently aspirate the cell suspension 14 times from the inside and outside of the chamber up to the syringe. Avoid forming air bubbles by gently pulling the plunger up and down to slowly aspirate the solution inside and outside the syringe. Ensure that the entire filter is rinsed away by the recovered solution. 8.28. Tilt the chamber back to the neutral position. 8.29. Collect the cell suspension by slowly tilting the chamber to the maximum angle while slowly aspirating the cell suspension into the syringe. If air bubbles are observed on the mask, return to the neutral position and tilt the chamber again to aspirate the air bubbles into the syringe. 8.30. Close the inlet lamp and disconnect the syringe. 8.31. Transfer the cell suspension into a T-25 flask. 8.32. Reconnect the syringe to the inlet port, aspirate any residual cell suspension, and transfer it into the T-25 flask.

[0302] 9. Analysis after Recovery 9.1. Using a 200 μL pipette, take 2 × 200 μL of the cell suspension and transfer each aliquot into 1.5 mL Eppendorf tubes labeled "AS (after SOLUPORE™ process) + sample number" for counting and flow cytometry analysis, and keep them at room temperature. 9.2. Weigh the cell culture dish containing the Solupore-treated cell suspension and record the weight / volume in the run record. 9.3. Ensure that the cell culture dish is labeled: sample name; recorded volume; operator name; date. 9.4. Place the cell culture dish upright in an incubator at 37 ± 2 °C, 5% CO 2 , 95% relative humidity. 9.5. Count the "Post-Sol" samples taken for counting, count them according to WI-06, and record the counting time in the run record. Ensure that the same nucleocounter is used to count the "Pre-Sol" and "Post-Sol" samples.

[0303] 10. Cleaning between Samples 10.1. Dispose of the waste bag during the repetition of the experiment. 10.2. Remove the lid clamp and remove the lid, disassemble the filtration unit, take out the drain disk and the filter membrane, and dispose of them. 10.3. Reassemble the instrument without inserting the filter. 10.4. Clamp the chamber lid. 10.5. Ensure that the drain port clamp and the bleed port clamp are closed. 10.6. Fill a sterilized and labeled syringe with 60 mL of WFI, connect it to the inlet tube, and load 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 the maximum extent to ensure that the mask and the lid wall are washed. 10.8. Suction with a syringe connected to the drain tube to remove the liquid. 10.9. Repeat steps 10.6 - 10.8 using 60 mL of 1×PBS. 10.10. The chamber is now ready to be reassembled again with a new drain disk and filter membrane for a new sample. 11. After assembling the unit with a new drain disk and filter membrane, repeat from item 5.13 for all other samples. 12. Record the tested sample names and conditions in the run log. 13. When the experiment is completed, move on to the cleaning system. 14. Export all required NC3000 / NC200 files and save them in the appropriate experiment folder for analysis. 15. Incubate the cells for 24 hours and analyze for the following. Number of cells recovered (NC3000 / NC200) Cell viability (NC3000 / NC200) Transfection efficiency (% GFP - flow cytometry)

[0304] Example 8: Cell Thawing Culture and Preparation of Cells The methods described herein provide standard protocols for the preparation of cell culture media, thawing of cell stocks, culturing of cells, and preparation of cells for experimental use. The methods cover the preparation of cell culture media and procedures for thawing cell stocks during liquid nitrogen storage, as well as culturing of 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] Materials TIFF2025081453000031.tif156160

[0307] Equipment TIFF2025081453000032.tif61160

[0308] Procedures 1. Preparation of CD3+ T Cell Culture Medium TIFF2025081453000033.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 shown 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 the Stericup image). 4. P / S is optional for the medium after transfection and is specified by the user. 5. To use the filtration system, spray 70% ethanol on the vacuum device and the Stericup before placing them in the BSC. The vacuum pump has two outlets, make sure the tube is connected to the air at the outlet. 6. Plug the vacuum device into the power supply and make sure the power is on in the BSC. 7. Remove the Stericup from the sterile packaging and attach the nozzle of the vacuum pump to the Stericup. 8. Remove the transparent lid from the Stericup, add the pre-prepared medium to the top of the Stericup, and return the lid to the Stericup. 9. Turn on the vacuum device by using the switch on the front of the pump. 10. The medium will then pass through the filter. Once it has passed through the Stericup, turn off the pump and remove the nozzle from the Stericup. 11. Remove the top of the Stericup by twisting, and screw on the sterile lid (blue) on top of the lower part of the Stericup containing the filter-sterilized medium. There will be a clicking sound when it is fully closed. 12. For large batch preparation of the medium, if necessary, store the medium at 4°C in a clearly labeled Stericup before aliquoting the medium in 50 / 120 ml falcons. Note that the medium cannot be stored at 4°C for more 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 vial of lyophilized recombinant human IL-2 at 50 μg, 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 before use. 16. After use, return the reagents to the storage location, e.g., -20°C or 4°C. 17. Warm the medium using a water bath set at 37°C for at least 15 minutes before use. 18. Record the temperature of the water bath on the cell culture template.

[0310] Preparation of PBMC-Initiated CD3+ T Cell Culture Medium TIFF2025081453000034.tif42163

[0311] 1. To prepare TCGM complete medium for PBMC starting CD3+ T cells, add the full bottle of CTS Optimizer supplement to the 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 shown in the table above. 3. Add the required volume of L-glutamine as shown in 8.3. in the table above. 4. Add the required volume of HEPES buffer as shown in the table above. 5. Filter-sterilize the medium using a vacuum filtration system before adding P / S and IL-2. 6. To use the filtration system, spray 70% ethanol on the filtration system and stericup before placing it 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 the power supply and ensure that the power is on in the BSC. 8. Remove the stericup from the sterile packaging and attach the nozzle of the filtration system to the stericup. 9. Remove the clear lid from the stericup, add the pre-prepared medium to the top of the stericup, and return the lid to 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. Once it has passed through the stericup, turn off the vacuum device and remove the nozzle from the stericup. 12. Remove the top of the stericup by twisting motion and screw on the sterile lid (blue) on top of the bottom part of the stericup containing the filter-sterilized medium. It will make a clicking sound when fully closed. 13. For large batch preparation of the medium, store the medium at 4°C in a clearly labeled stericup if necessary, before aliquoting in 50 / 120 ml falcon and before addition of P / S and IL-2. Note that the medium 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 vial of lyophilized recombinant human IL-2 (50 μg), mix, and aliquot into 500 μl. Store at -20°C. 15. Optionally, add P / S (10,000 U / ml) (250 μl per 50 ml of medium) after transfection. 16. Warm the medium using a water bath set at 37°C for at least 15 minutes before use.

[0312] Thawing and Activation of Cells 1. Prepare CD3+ T cell medium or complete TCGM medium and warm it to 37°C. 2. Prepare CTS™ OpTmizer™ T Cell Expansion SFM which contains only supplements (this is the basal medium) and warm it to 37°C. 3. Record the temperature of the water bath on the cell culture template. 4. In the BSC, prepare one 50 ml tube containing 10 ml of pre-warmed medium (10 ml per vial of cells to be thawed). 5. Remove the required vials / bags from the liquid nitrogen / -150 °C freezer. (Note that all appropriate PPE must be worn while handling liquid nitrogen or the -150 °C freezer.) 5.1. Due to variability between donors in the expansion of PBMCs, it may be necessary to start with more PBMCs than the number of T cells required on day 3, due to differences in the degree of PBMC expansion. This number should be evaluated for 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. 6. Thaw the cell vials in a 37 °C water bath, swirling gently occasionally until small pieces of ice remain (thaw no more than two vials at a time). Ensure that the edges of the vials do not come into contact with water from the water bath, as this is a risk of contamination. 7. When "ice chips" are visible, transfer to the BSC, spray 70% EtOH onto the vial and wipe, and rapidly dilute the cells in culture medium by adding 1 ml of warmed medium 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 pre-prepared medium in the falcon. Caution: Since DMSO is toxic to cells, thawed cells should not be placed in the freezing mixture for any extended period. 9. Rinse the vial with another 1 ml of medium and transfer to the same falcon containing the cells, to ensure that all cells are recovered. 10. Centrifuge the falcon at 300 × g for 7 minutes at RT. 11. Check for the cell pellet and remove the supernatant without disturbing the pellet, discarding it into a waste beaker. This can be done by pouring or by removing using a strip or Pasteur pipette. 12. Resuspend the pellets in the culture medium to a cell density greater than 1×10⁶ / ml (usually 10 ml for CD3+ T cell vials, 20 ml for PBMC vials, and 30 ml for bags). 13. Count the cells using a Via-1 cassette. (See WI-6 NC-3000 NucleoCounter Operation & Maintenance or the following overview.) 13.1. To count the cells, take 50 μl of the cell suspension and dilute it with 150 μl of medium in an Eppendorf to a final volume of 200 μl. 13.2. Count the cells using a Via-1 cassette and a Nucleocounter - including dilution on the software (i.e., enter "50" in the sample box and "150" in the dilution box - the Nucleocounter calculates the dilution factor). 13.3. Visually inspect the gates on the software and, if necessary, modify to include the cell bodies, and record the cell viability and concentration (cells / ml) and size in the cell culture template. 14. Calculate the total cell number and record all calculations and cell numbers in the cell culture template. 15. Seed the cells at a density of 1×10⁶ / ml into appropriately labeled cell culture flasks or bags. TIFF2025081453000036.tif2116616. For CD3+ T cells, place the TC flask upright in the incubator. 16.1. For CD3+ T cells, allow the cells to rest in a 37°C incubator for at least 4 hours after the thawing process before proceeding to the activation step. 16.2. To activate CD3+ T cells, add CTS Grade Dynabeads at a ratio of 2:1: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 among the entire cell population in the TC flask. 16.3. Return the cells to the incubator and place them upright until they are required for experimental use. 16.4. Unless otherwise specified, prepare the cells for experimental use 19 hours after activation. 17. Activate PBMC 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 them on ice until the time of activation. 19. Ensure that the CD3 and CD28 antibodies are dated within 1 month from the time they were first opened, are labeled with color, and contain the user's initials. At the time of opening, the date and the user's initials must be written on the vial. All users must have their own set of CD3 and CD28 antibodies. Sharing between users is not permitted. 20. Record the lot numbers and expiration dates of CD3 and CD28 on the cell culture template. 20.1. For 1x 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 PBMC, 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 among the entire cell population. 20.2. For 2x PBMC activation, add 1 μl each of CD3 and CD28 antibodies per million cells. 20.3. At the start, seed the cells into appropriately labeled T75 flasks. 20.4. Place the flask horizontally (lying on its side) gently in the incubator. 20.5. The cells should be left undisturbed for approximately 72 - 96 hours. 20.6. The cells will appear "flaky" or "snow-like" after 72 hours from the start. 20.7. Always examine the cells under the microscope for the appearance of healthy clumps of PBMCs.

[0313] Preparation of CD3+ T cells for experimental use 1. After 19 hours of activation, remove the flask containing CD3+ T cells from the incubator to the BSC. 2. Gently agitate the cell-Dynabead suspension with a stripette and transfer it to a 50 ml falcon tube. 3. Wash the flask with 5 ml of medium and transfer it to the same falcon tube. 4. Vortex the falcon tube well and place it on a 50 ml magnet. 5. Wait for 2 minutes. 6. Using a 25 ml stripette, carefully pipette down to the 5 ml mark without touching the side of the falcon to collect most of the medium. This medium contains the de-beaded cells and collect this into a new labeled sterilin. 7. Leave approximately 5 ml of medium at the bottom of the falcon, avoiding disturbing the beads. 8. Remove the falcon tube from the magnet, add another 5 ml of medium and mix again. 8.9. 9. Repeat the incubation of the falcon tube in the 50 ml magnet and collection of the medium (Steps 5 - 9). 10. Remove the 50 ml falcon from the magnet for the second time. 11. Use a P1000 pipette to collect the dynabeads using the medium remaining at the bottom of the falcon tube. 12. Place this medium containing beads into a 1.5 ml Eppendorf and place it on an Eppendorf (small) magnet for 2 minutes. 13. Remove the medium and transfer it to a sterilin using a pipette. 14. Add 1 ml of the medium to the Eppendorf, mix and wash. Add it to the Eppendorf magnet and remove the supernatant to the sterilin. 15. Discard the Eppendorf containing the beads. 16. Centrifuge the sterilin containing the cells at 300×g for 7 minutes. 17. If the expected number of cells / ml is less than 50×10 6 cells, resuspend the cells in 10 ml of fresh medium. When resuspending, always use a P1000 first to break up the cell pellet. 18. If the expected number of cells / 8.18. ml is more than 100×10 6 cells, resuspend the cells in 20 ml of fresh medium. 19. In an eppendorf, prepare a cell dilution by adding 50 μl of the cell suspension to 150 μl of the medium, and count this using a Via-1 cassette. To increase accuracy, all counts should be done in duplicate. 20. Record the number of cells / ml, cell viability, and cell size after bead removal (de-bead) in a cell culture template. 21. Calculate the total number of cells / ml and volume of the cell suspension required for the experiment. 21.1. For example, at the time of cell counting, it is 4×10 6 cells / ml in 20 ml, and the total number of cells is 80×10 6 . 20×10 6 in a 10 ml aliquot with 2 parameters is desirable. 21.2. Required / have = 20×10 6 / 4×10 6=5×2.5 parameter = 12.5 ml of cell suspension to 12.5 ml of CTS OpTmizer medium + supplement. This is the master mix and always constitutes 0.5 of the condition extra. For example, if there are 2 parameters, it always constitutes 2.5 times, so if 20 ml is required, the final volume is 25 ml. This allows for pipetting errors. 22. Prepare the cell master mix containing cell suspension (added using P5000) and CTS OpTmizer medium + supplement in either a 120 ml falcon or 500 ml falcon (depending on the desired volume), gently mix by inverting, and aliquot 10 ml of the cell suspension into 30 ml white cap tubes using a P5000 or P10000 pipette for accuracy. After aliquoting every 3 tubes, close the master mix and invert several times to ensure a homogeneous cell suspension is maintained. 23. Ensure that the 30 ml white cap sterlin is 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 the cells are prepared in a T25 TC flask containing CD3+ T cell complete medium instead of CTS OpTmizer medium + supplement. UT cells are exponentially growing to ensure good viability on day 1 after seeding at 0.5×10 6 / ml, i.e., 10×10 6 cells in 20 ml.

[0314] Preparation of PBMC-started CD3+ T cells for experimental use 1. After 72 hours (3 days) of start, remove the T75 flask of PBMC-started CD3+ T cells from the incubator to the BSC. Visually observe for clumping, which is a sign of properly started cells. 2. Transfer the cell suspension to a 50 ml falcon for centrifugation. 3. Centrifuge the 50 ml falcon containing the cells at 300×g for 7 minutes. 4. First, resuspend the cells in 20 - 50 ml of fresh pre-warmed medium using a P1000 to disrupt the cell pellet. If less than 50×10 6 cells are expected, use 20 ml; if more than 100×10 6 cells, use 50 ml. 5. In an eppendorf, prepare a cell dilution by adding 50 μl of the cell suspension to 150 μl of medium and count the cells using a Via-1 cassette (refer to WI-6 NC-3000 NucleoCounter Operation & Maintenance). 6. To ensure that the PBMCs are activated and starting, stain the cells with CD3 (T cell purity) antibody and CD25 (T cell activation) antibody for flow cytometry analysis. The cells cannot be released for experimental use until this staining has been fully analyzed on a flow cytometer. 6.1. Calculate the volume of the cell suspension required for 1×10 6 cells for staining for CD3 and CD25 expression. 6.2. Take out the volume of cells that gives 1×10 6 cells, transfer them to a 1.5 ml eppendorf, and centrifuge at 300×g for 5 minutes. 6.3. After centrifugation, remove the cell supernatant and resuspend the cell pellet in 100 μl of FACs buffer. 6.4. Use compensation beads during staining for CD25 antibody and CD3 antibody to optimize the fluorescence compensation settings for flow cytometry analysis. 6.4.1. Prepare compensation beads for both CD25 antibody and CD3 antibody as follows. 6.4.1.1. Label two 1.5 ml eppendorf tubes as CD25 comp and CD3 comp. 6.1.1.2. Mix the correction beads by vortexing. Add 1 drop of correction beads to each tube and then add 0.5 μl of CD25 antibody or CD3 antibody to the appropriate tube. 6.5. Once the correction beads are prepared, add both 5 μl of CD25 antibody and CD3 antibody to the eppendorf containing 1×10 6 cells. 6.6. Incubate the cells with the antibody at 4°C for 10 minutes. 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 the expression of CD25 and CD3 by acquiring 10,000 events for both the bead sample and the cell sample using flow cytometry. 6.9.1. Acquire the correction bead sample before the cell sample. 7. If the cell sample results in >90% for both CD25+ staining and CD3+ staining on the flow cytometer, release the cells for experimental use. 8. If the cell sample results in <90% for both CD25+ staining and CD3+ staining on the flow cytometer, notify the end user and, unless otherwise instructed, do not release the cells for experimental use. 9. Once release is confirmed, calculate the total number of cells / ml and the volume of the cell suspension required for the experiment based on nucleocounter counting. To increase accuracy, all counts should be done in duplicate. (These calculations should be done during the flow incubation, but do not prepare the cells until they pass QC for experimental release.) 9.1. For example, during cell counting, if there are 4×10 6 cells / ml in 20 ml, the total number of cells is 80×10 6 . 20×10 6 in a 10 ml aliquot with 2 parameters is desirable. 9.2. Necessary / Have = 20×10 6 / 4×10 6 = 5×2.5 Parameter = 12.5 ml of cell suspension to 12.5 ml of CTS OpTmizer medium + supplement. This is the master mix and always constitutes an extra 0.5 of the condition. For example, if there are 2 parameters, it always constitutes 2.5 times, so if 20 ml is required, the final volume is 25 ml. This allows for pipetting errors. 10. Prepare a cell master mix containing cell suspension (added using P5000) and CTS OpTmizer medium + supplement in either a 120 ml falcon or 500 ml falcon (depending on the desired volume), gently mix by inverting, and aliquot 10 ml of the cell suspension into white cap 30 ml tubes using a P5000 or P10000 pipette for accuracy. After aliquoting every 3 tubes, close the master mix and invert several times to ensure a homogeneous cell suspension is maintained. 11. Ensure that the 30 ml white cap sterlin is 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 the cells are prepared in a T25 TC flask containing TCGM complete medium instead of CTS OpTmizer medium + supplement. UT cells are exponentially growing to ensure good viability on day 1 after experimental seeding at 0.5×10 6 / ml, i.e., 10×10 in 20 ml 6 cells.

[0315] Example 9: LV-eGFP (high-sensitivity GFP) vector The map of the LV expression plasmid vector having eGFP is provided in FIG. 48 herein.

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

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

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

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

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

[0321] A typical titer of lentivirus is 10 6 ~10 7is in the range of transduction units (TU / ml), and the viscosity of lentivirus at these concentrations is highly and dynamically viscous compared to the water / ethanol mixture. For illustration, (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 600.1 mPa s, and the dynamic viscosity of castor oil is close to 6000.1 mPa s. The dynamic viscosity of lentivirus has been reported to be 6913 mPas by Tran, Reginald, PhD Thesis, Georgia Tech (2016), which is nearly 1 log more viscous than castor oil. Sterile-filtered 1% bovine serum albumin (BSA) has been found by some to reduce the molecular interactions that can result in virus particles "sticking" to the injection device (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, which is hereby incorporated by reference in its entirety).

[0322] Dynamic viscosity is an important factor in atomization. An experimental study on atomization in an internal mixing two-fluid atomizer, such as used in the SOLUPORE™ process, has been conducted over a wide range of liquid viscosities, gas supply pressures, and gas-to-liquid mass ratios (GLR). 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 hereby incorporated by reference in its entirety. Among all the test conditions, the finest spray was obtained at an axial distance of 150 mm. However, as the viscosity increased to 120 mPa s, the droplet size distribution changed significantly. The higher viscosity droplets produced larger droplets (e.g., 1-2 logs larger than the current droplets produced by the measured droplet size distribution for the SOLUPORE™ process, Figure 51). The larger droplets corresponded to a large proportion of the droplet population (distribution), and the attenuation of droplet velocity along the spray axis was stronger at higher viscosities.

[0323] A table showing the dynamic viscosities of common liquids is presented 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 shown below. TIFF2025081453000037.tif177150

[0325] Atomization of lentivirus within the SOLUPORE™ process produced larger, more slowly moving droplets, and it can be concluded that the experience of the underlying cell layer was different from the previously described SOLUPORE™ process. Consequently, this atomization process resulted in a transfection level of nearly 30%, which is 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 the ethanol / water mixture is also close to 1 mPa s. The dynamic viscosity of an aqueous solution that can contain an ethanol concentration of 5 - 30%. The aqueous solution can contain one or more of 75 - 98% H 2 O, 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), and the viscosity is in the region of 2 mPa s.

[0327] Titer 10 7 ~10 8 TU / mL of lentivirus has a dynamic viscosity close to 6913 mPa s. As the viscosity of the fluid increases, at a given spray pressure, for example 1.7 bar, when sprayed, there is a tendency to form larger droplets. 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 thus the droplets become even larger. Next, the cells experience a completely different process. By itself, a finer spray 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. Fluids with a higher dynamic viscosity than water or water / ethanol mixtures will have a higher average droplet size for any given flow rate and pressure. The relationship between the mechanical properties of the fluid can be calculated by the following generally accepted equation. TIFF2025081453000038.tif26128Where D f = the corrected droplet size for the fluid, D w = the calculated droplet size for water, V f = the viscosity of the fluid (viscosity in mPa s; water = 1.0 mPA s, lentivirus is 6913 mPa s).

[0328] From Equation [1], droplets of lentivirus sprayed under the same pressure and flow conditions as the water / ethanol mixture (e.g., droplets containing the virus, an ethanol concentration of 5-30%, and 75-98% H 2 O, 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) in a certain volume of aqueous solution) can be calculated to have a droplet size nearly 5.9 times larger than that of water / ethanol droplets. As described in International Application WO 2016 / 065341, which is incorporated herein by reference in its entirety, droplets in the size ranges of 30 μm to 100 μm and 50 μm to 80 μm in diameter were described. Generally, in the methods described herein, when the aqueous solution being sprayed contains a virus (e.g., lentivirus), the droplet size range is from about 150 μm to 600 μm in diameter, or from about 177 μm to 590 μm in diameter. In other examples, the diameter size of the droplets 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 is greater than 600 μm, for example, it can be from about 600 μm to 1000 μm in diameter, or from about 600 μm to 900 μm, or from about 600 μm to 800 μm, or from 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 in diameter.

[0329] These droplets are much larger than what was predicted or described in WO 2016 / 065341, which states that "some of the produced colloidal droplets may be too large for a given intracellular delivery application. Because some of the produced colloidal droplets are too large, cell death may occur despite producing colloidal droplets of the appropriate size." See paragraph

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

[0330] Characteristics of droplet size The larger diameter droplets of the invention described herein have a larger volume and weight, move more slowly, and impact the cell layer with a greater force. For example, when the diameter is increased by 5.9-fold, the volume of the droplet increases nearly 206.8-fold. Thus, the hydrodynamics of this system are distinct from those described with reference to WO 2016 / 065341 and constitute a new virus infection process.

[0331] Other aspects While the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not 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 referred to in this specification establish knowledge available to those skilled in the art. All U.S. patents and U.S. patent applications, whether published or unpublished, cited in this specification are hereby incorporated by reference. All published foreign patents and patent applications cited in this specification are hereby incorporated by reference into this specification. All other published references, documents, manuscripts, and scientific literature cited in this specification are hereby incorporated by reference into this specification.

[0333] Although the invention has been shown and described with respect to particular 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 defined by the appended claims.

Claims

1. 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 performed 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 , 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).

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.

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