Electroporation Devices and Methods

Abstract

A device for filling and emptying an electroporation chamber is provided. Specifically, the device comprises a tubing set (10) having a main pipe (11) with a first end (12) for connection to a cell suspension input bag (1) and a second end for connection to an electroporation chamber (3). A first branch pipe from a first connection (16) with the main pipe has a connection at its other end to an exogenous material input bag (2). Preferably, the first connection is a T-shaped connection, and more preferably, the first branch pipe is connected to the stem of the T-shaped connection. A second connection between the first connection (16) and the connection to the electroporation chamber leads to a second branch pipe (20) for connection to an output reservoir (4). Furthermore, one or more pumps are arranged to act on the main pipe, the first branch pipe, and the second branch pipe, respectively, to transfer fluid therethrough. During operation, the one or more pumps are controlled such that mixing of the cell suspension from the cell suspension input bag with the exogenous material from the exogenous material bag occurs substantially at the first junction (16).

Description

[Technical Field]

[0001] The present invention relates to electroporation machines. In particular, the present invention relates to a modular electroporation system and method of operation thereof that is particularly adapted for clinical and industrial use. [Background technology]

[0002] Electroporation is understood as the use of electric pulses to reversibly destabilize biological membranes, such as vesicles or cell membranes, and to provide a pathway for the introduction of exogenous material into the vesicles or cells during destabilization. Typically, such introduced exogenous material can be genetic material to obtain genetically modified cells used for research or therapeutic purposes. Both the cells and the exogenous material are supplied as a liquid suspension. In recent years, great interest has been shown in the use of electroporation to introduce mRNA into cells, which is easily converted into proteins that confer additional functions to the cells. Cell therapy is a developing field of medicine that requires sterile and reliable manufacturing facilities, where primary cells derived from donors or patients can be genetically modified in large batches in accordance with strict Good Manufacturing Practices (GMP). For example, the production of clinical batches of allogeneic CAR T cells, which require several gene editing steps to remove foreign receptors and express artificial chimeric antigen receptors (CARs) (Non-Patent Document 1), is particularly challenging in this regard.

[0003] For industrial and commercial applications, it is essential that quality and consistency of results be maintained throughout and across batches of material, and that the batches be produced in a closed system, even when several electroporations are performed sequentially. In the specific context of primary cells, such as those derived from a single donor (i.e., limited numbers of donors), it is important that the cells are preserved, which means that the electroporator system must ensure uniformity of conductivity between the mixed cells and the medium.

[0004] With larger volumes of material, scale-up requires a reliable system that can be externally monitored while also being cleanable with disposable electroporation chambers.

[0005] Patent Document 1 discloses a system including a pulsed voltage waveform generator, a switching device for connecting the anode or cathode of the pulsed voltage waveform generator to an electroporation chamber, and an electrode array in the electroporation chamber for converting the pulsed voltage into a pulsed electric field in the chamber. Also disclosed is a cycle of filling the electroporation chamber with a suspension of cells and an exogenous substance, electroporating the mixture, and emptying the chamber in situations where the amount of cell suspension being processed exceeds the capacity of the chamber.

[0006] In such systems, mixing of the exogenous substance with the cell suspension is typically by injection of the exogenous substance into a container, typically a bag, of the cell suspension prior to the start of the electroporation cycle, however, this produces poor results as the exogenous substance is often unstable and is degraded by prolonged contact with the cell suspension prior to electroporation.

[0007] Patent Document 2 describes an electroporation device having an electroporation chamber with two inlet ports. The chamber also has a vent port and an outlet port. During filling of the chamber, a cell suspension is introduced through one of the inlet ports, and a liquid suspension of an exogenous substance is introduced through the other inlet port. The shape of the chamber is designed to promote efficient mixing. Contact between the two liquids occurs first in the chamber, thereby reducing contact between these liquids prior to electroporation. However, this configuration suffers from the drawback that it is difficult to ensure proper mixing and that the chamber is free of air bubbles. Such bubbles can lead to destructive arcing.

[0008] To date, all of these systems have proven unsuitable for the production of genetically modified therapeutic cells under Good Manufacturing Practice (GMP) on an industrial scale. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2006 / 0089674 [Patent Document 2] U.S. Patent Application No. 2011 / 142813 [Patent Document 3] U.S. Patent No. 6,010,613 [Non-patent literature]

[0010] [Non-Patent Document 1] Depil, S. et al. (2020) 'Off-the-shelf' allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 19:185~199 [Non-patent document 2] Schwartz J. et al., Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis:the sixth special issue (2013) J Clin Apher. 28(3):145-284 [Non-patent document 3] Bailey, SR, Maus, MV (2019) Gene editing for immune cell therapies. Nat Biotechnol 37, 1425~1434 [Non-patent document 4] Poirot et al., Cancer Research 2015 Summary of the Invention [Problem to be solved by the invention]

[0011] It is therefore an object of the present invention to propose an electroporation system that is both inexpensive to build and to operate, and that ensures good electroporation results.

[0012] In addition to the production of therapeutic-grade cells, applications can be found in various research or industrial processing areas, such as, by way of non-limiting example, the transformation of cell lines, bacteria, yeast, algae, plant cells, protoplasts for bioproduction, or the production of vaccines.

[0013] definition The system of the present invention is designed to process vesicles, which may be artificial or biological cells. The system is specifically designed for electroporation of cells, such as cell lines or primary cells.

[0014] Cells refers to living cells in culture or in suspension.

[0015] By "primary cells," we mean cells taken from living tissue (e.g., biopsy material) and designated for in vitro growth over a limited period of time, meaning they can undergo a limited number of population doublings. Primary cells are contrasted with continuously tumorigenic or artificially immortalized cell lines. Non-limiting examples of such cell lines include CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, and Molt 4 cells. Primary cells are typically used in cell therapy because they are considered more functional and less tumorigenic.

[0016] Cells that are differentiated from stem cells, such as cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSC), induced pluripotent stem cells (iPS), etc., are considered primary cells according to the present invention.

[0017] Generally, primary cells are derived directly or indirectly, preferably directly, from a donor or patient through various methods known in the art, such as, for example, by leukocyte reduction techniques such as those reviewed by Schwartz J. et al. (Non-Patent Document 2).

[0018] In this invention, in view of the production of clinical batches of therapeutic cells for cell therapy, particular attention has been paid to immune cells in accordance with the invention, such as T cells and NK cells, hematopoietic stem and progenitor cells, stem cells, such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.

[0019] By electroporation is meant the use of pulsed electric fields to create a transient permeability in cell membranes without loss of cell viability.

[0020] An exogenous substance is any substance outside of a living cell. For the purpose of this disclosure, an exogenous substance is a substance that is delivered to a cell. A non-exclusive list of substances is polypeptides, polynucleotides, pharmaceuticals, polymers, carbohydrates, and their combinations in the same molecule or different molecules. Examples of polypeptides and polynucleotides are proteins and DNA or RNA (including mRNA, RNAi), respectively. Exogenous substances for use in electroporation can include the above combinations, such as ribonucleoproteins (protein and RNA complexes).

[0021] Port: An opening through which gas or liquid can pass. The direction of flow depends on the use of the port.

[0022] PulseAgile Protocol: A sequence of at least three waveforms having one, two, or three of the following characteristics: (1) at least two of the at least three waveforms differ from each other in waveform amplitude, (2) at least two of the at least three waveforms differ from each other in waveform width, and (3) the spacing between the first waveforms for a first set of two of the at least three waveforms differs from the spacing between the second waveforms for a second set of two of the at least three waveforms. Examples of such agile pulse sequences are described in U.S. Patent No. 5,929,999.

[0023] Suspension: insoluble particles, such as biological cells, suspended in a water-based liquid. [Means for solving the problem]

[0024] The present invention relates to a device for filling and emptying an electroporation chamber. Specifically, the device includes a tubing set having a main pipe with a first end for connection to a cell suspension input bag and a second end for connection to an electroporation chamber. A first branch pipe from a first connection to the main pipe has a connection to an exogenous material input bag at its other end. Preferably, the first connection is a T-shaped connection, and more preferably, the first branch pipe is connected to the stem of the T-shaped connection. A second connection between the first connection and the connection to the electroporation chamber leads to a second branch pipe for connection to an output container. Furthermore, one or more pumps are positioned to act on the main pipe, the first branch pipe, and the second branch pipe, respectively, to transfer fluids therein. During operation, the one or more pumps are controlled so that mixing of the cell suspension from the cell suspension input bag with the exogenous material from the exogenous material bag occurs substantially at the first connection.

[0025] In a further configuration, one or more bubble detection devices are configured to detect the presence of gas or liquid in the tubing set. One or more valves may also be positioned in the main pipe, the first branch pipe, and the second branch pipe to respectively act to control fluid flow therein.

[0026] In a further aspect, the device further comprises a third branch pipe having a third connection at one end with the main pipe and having another end for connection to a wash solution input container, the third connection being positioned between the first and second connections. This aspect also provides a fourth branch pipe having a fourth connection at one end with the second branch pipe and having another end for connection to a wash solution waste bag.

[0027] The present invention also relates to an electroporation system including the electroporation device described above. Specifically, the system includes the electroporation device described above, a cell suspension bag connected to a first end of the main conduit, and an electroporation chamber having an inlet port connected to a second end of the main conduit. The electroporation chamber further includes a vent port fluidly connected to the vent conduit and electroporation electrodes. The system further includes an exogenous material input bag connected to the first branch conduit and an electroporated cell suspension output bag connected to the second branch conduit. Optionally, a bubble detection device is configured to detect the presence of fluid in the vent conduit. A control circuit is configured to provide an electroporation signal to the electroporation electrodes of the electroporation chamber and to control one or more pumps and one or more valves based at least on signals from the one or more bubble detection devices.

[0028] In yet a further aspect, the present invention relates to a method of operation of the previously described electroporation system. The method includes the steps of providing a tubing set, providing an electroporation chamber, providing a storage bag containing a cell culture in a liquid suspension, and providing a storage bag containing an exogenous substance in a liquid suspension. A predetermined amount of the cell culture and the exogenous substance are introduced into the electroporation chamber, and the cell culture and the exogenous substance are mixed in the tubing set before entering the electroporation chamber. An electroporation signal is supplied to the electroporation chamber to effect electroporation. Following this, the electroporation chamber is emptied into a storage container.

[0029] The method may further include a washing step in which a waste bag for receiving discarded wash solution and a wash solution storage bag are further provided. Following emptying the electroporation chamber, a predetermined amount of wash solution is introduced from the wash solution storage bag into the electroporation chamber. It is then expelled from the electroporation chamber into the waste bag.

[0030] In a preferred configuration, one or more pumps of the previously described system are operated based at least in part on signals provided by one or more bubble detection devices. Additionally, one or more valves are operable to prevent undesired flow of raw cell suspension from the cell suspension bag and / or exogenous material suspension from the exogenous material bag, backflow of electroporation product into the storage bag, and flow of raw cell suspension and exogenous material suspension into the second branch and thereby into the product bag.

[0031] In yet a further aspect, the invention includes the steps of introducing cells into the system, mixing the cells with an exogenous substance, and introducing the exogenous substance into the cells using the system to produce recombinant cells, particularly genetically modified therapeutic cells that are ready to be used fresh or frozen.

[0032] The configuration of the electroporation device described above allows for effective electroporation of cell suspensions and exogenous materials. Specifically, the electroporation device allows for the use of commercially available tubing and electroporation chambers or cuvettes, enabling cost reduction and economies of scale. The requirement for custom electroporation chambers is eliminated, while degradation of exogenous materials caused by prolonged contact with the cell suspension prior to electroporation is also minimized.

[0033] Other features and advantages of the present invention will become apparent from the following description.

[0034] The accompanying drawings are given by way of non-limiting example. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is a schematic diagram of an electroporation system according to the present invention. [Figure 2] FIG. 2 is a schematic diagram of the electroporation system of FIG. 1 further including provisions for cleaning the chamber after use. [Figure 2a] FIG. 1 is a schematic diagram of an electroporation chamber. [Figure 3] FIG. 2 is a schematic diagram of a tubing set forming part of the system of FIG. 1. [Figure 4] FIG. 4 is a view of a T-junction forming part of the tubing set of FIG. 3. [Figure 5] FIG. 2 is a schematic diagram of an electroporation device forming part of the system of FIG. 1. [Figure 6] 2 is a flow chart illustrating a method of performing electroporation in accordance with the system of FIG. 1. [Figure 7] 3 is a flow chart illustrating a method of performing electroporation in accordance with the system of FIG. 2. [Figure 8] FIG. 3 is a realistic illustration of an electroporation device forming part of the system of FIG. 2. [Figure 9]FIG. 1 shows the percentage of TCRab KO obtained in T cells electroporated with TRAC TALEN mRNA after each electroporation cycle performed with a GeneEngine connection to a PulseAgile electroporator. [Figure 10] Figure 1 shows the percentage of TCRab KO obtained in T cells electroporated with TRAC TALEN mRNA under different conditions detailed in Example 2, i.e., C3: condition 3, R1C1: ratio 1 and first cycle, R1C2: ratio 1 and second cycle, R2C1: ratio 2 and first cycle, R2C2: ratio 2 and second cycle. DETAILED DESCRIPTION OF THE INVENTION

[0036] 1 is a schematic diagram of an electroporation system comprising an electroporation device 100, a container 1 for holding a suspension of cells to be treated by electroporation, a container 2 for holding a suspension of exogenous material to be inserted into the cells, an electroporation chamber 3, and an output container 4 for collecting the product of the electroporation process. Typically, such containers are flexible bags made, for example, from plastic. For the purposes of this disclosure, these containers will be referred to as bags. This should not be construed as limiting.

[0037] A cell suspension bag 1 and an exogenous material bag 2 are fluidly connected to the electroporation device 100. Collectively, the cell suspension bag 1 and the exogenous material bag 2 are referred to as supply bags 1, 2. The electroporation device operates to draw a predetermined amount of a suspension of cells from the cell suspension bag 1 and a predetermined amount of a suspension of exogenous material from the exogenous material bag 2 for mixing the suspensions prior to delivery to the electroporation chamber 3.

[0038] In some configurations, the mixing device is configured to agitate the cell suspension bag 1 and its contents. The mixing device can compress the bag while vibrating it to induce a flow of liquid within the bag. Such a flow ensures that the concentration of cells within the bag remains constant and prevents the cells from settling within the bag, which would create a concentration gradient across the bag. This also prevents the cells from "sticking" to the walls of the bag. A uniform concentration within the bag ensures an unchanging concentration of cells entering the electroporation device when the bag is emptied. This is important to ensure uniform and reproducible electroporation results.

[0039] The system of Figure 1 further includes a signal generator configured to generate an electroporation signal. In a preferred configuration, the signal generator is configured to generate a signal according to the PulseAgile protocol. The signal generator is electrically connected to an electrode in the electroporation chamber configured to conduct the electroporation signal to the contents of the electroporation chamber.

[0040] An output bag 4 for collecting the products of the electroporation process is fluidly connected to the electroporation device 100 and configured to receive the products from the electroporation chamber 3 following electroporation.

[0041] In addition to the features of the electroporation system 111 depicted in Figure 1, the electroporation system 111a of Figure 2 includes a bag 5 containing a wash solution fluidly connected to the electroporation device 100a. The electroporation device 100a is configured so that following electroporation and discharge of the electroporation products, the wash solution is directed to the electroporation chamber 3. Washing is performed by filling and draining the electroporation chamber 3 with the wash solution. A wash solution waste bag 6 is provided and fluidly connected to the electroporation device for receiving waste wash solution.

[0042] 2a shows, in schematic form, the electroporation chamber 3 in more detail. The electroporation chamber comprises an active chamber 30 having a predetermined volume, an inlet port 31, and a vent port 32. Located within the active chamber 30 are electroporation electrodes 35a, 35b through which an electroporation signal can be applied to material within the active chamber 30. The electroporation electrodes are electrically connected to electrical leads 36a, 36b, respectively, for connection to a signal generating device.

[0043] The electroporation device 100 will now be described in more detail. The main component of the electroporation device 100 is a tubing set 10, shown in FIG. 3. The tubing set includes a main tube 11 having a first end 12 for connection to a cell culture input bag 1 and a second end 13 for connection to an electroporation chamber 3. Further forming components of the tubing set 10 are a first branch tube 14 and a second branch tube 20. The first branch tube 14 has a first connection 16 with the main tube 11 at one end 15 and an opposite end 17 configured for connection to an exogenous material bag 2. The second branch tube 20 has a second connection 21 with the main tube 11 at one end 18 and an opposite end 19 configured for connection to an output bag 4. The second connection 21 is positioned between the first connection 16 and the second end 13.

[0044] 4 shows a preferred configuration of the first junction 16 between the main vessel 11 and the first branch vessel 14. As shown, the first junction 16 takes the form of a "T," with the stem of the T forming the passageway of the main vessel 11 and the stem of the T forming the first branch vessel. The inventors have found that efficient mixing of the cell suspension and the exogenous material suspension occurs at such a junction. Other configurations are possible.

[0045] Pumps are used to move fluids to and from the electroporation chamber 3, as well as to provide defined volumes and appropriate amounts of cells and exogenous materials. Such pumps are typically peristaltic pumps configured to pump the contents of the tubing set 10 without directly contacting the fluids within the tubing set 10.

[0046] The pump is positioned to move material from cell culture input bag 1 through main conduit 11 to second end 13 and into electroporation chamber 3, and to move material from exogenous substance bag 2 through first branch conduit 14 to first connection 16, through a portion of the main conduit positioned between first connection 16 and second connection 21, through a portion of main conduit 11 positioned between second connection 21 and second end 13, to the second end, and into electroporation chamber 3. Additionally, the pump is positioned to move material in the electroporation chamber through a portion of the main conduit positioned between second end 13 and second connection 21, through second branch conduit 20, to second end 13, and into output bag 4. Thus, the reader will understand that the flow of material in the portion of the main tube 11 between the second end 13 and the second connection 21 is in a first direction while filling the electroporation chamber 3 and in a second, opposite direction while emptying the electroporation chamber.

[0047] The pump is electrically connected to a control circuit configured to control the operation of the pump and, in turn, the movement of fluid through the tubing set 10 .

[0048] FIG. 5 shows the arrangement of pumps disposed in the previously described tubing set 10. A first pump 22 is disposed in the main pipe 11 at a location between the first end 12 and the first connection 16. During operation of the electroporation device, operation of the first pump 22 pumps the contents of the cell suspension bag 1 into the electroporation chamber 3. A second pump 23 is disposed in the first branch pipe 14. Operation of the second pump 23 pumps the contents of the exogenous substance suspension bag 2 into the electroporation chamber 3. Operation of the first pump 22 and operation of the second pump 23 simultaneously cause mixing of the cell suspension and the exogenous substance suspension at the first connection. A third pump 24 is disposed in the second branch pipe 20. Operation of the third pump 24 pumps the contents of the electroporation chamber 3 toward the output bag 4. The first pump 22, the second pump 23, and the third pump 24 are electrically connected to a control circuit.

[0049] As previously mentioned, during filling of the electroporation chamber 3, flow of material in the portion of the main conduit 11 between the second connection 21 and the second end 13 is in a first direction, and during emptying of the electroporation chamber, flow in that portion of the main conduit 11 is in a second, opposite direction. Thus, operation of the first and second pumps simultaneously, with the third pump inactive, causes flow in the portion of the main conduit between the second connection and the second end 13 in the first direction. Operation of the third pump, with the first and second pumps inactive, causes flow in the second direction.

[0050] Several valves are used to prevent fluid movement within the tubing set. These valves are typically pinch valves that can be operated to prevent fluid flow through the tubing set 10 without directly contacting the fluid therein. FIG. 5 shows the arrangement of valves in the tubing set 10 of the electroporation device 100. A first valve 25 is disposed in the main tubing 11 between the first connection 16 and the second connection 21. When operated, the first valve 25 serves to regulate or prevent fluid flow from the electroporation chamber 3 to the supply bags 1 and 2, or from the supply bags 1 and 2 to the electroporation chamber. The first valve 25 prevents further mixing of the cells and exogenous material, allowing for a precise ratio of cells to exogenous material entering the electroporation chamber. A second valve 26 is disposed in the second branch tubing 20 between the second connection 21 and the third pump 24. When activated, the second valve 26 serves to regulate or prevent the flow of liquid, i.e., cells that have not been electroporated with exogenous material, from the main conduit 11 to the second branch conduit 20 and thus to the output bag 4.

[0051] First valve 25 and second valve 26 are electrically connected to and configured for control by a control circuit. Control of the pump and control of the valves are coordinated to achieve the desired movement of fluid through tubing set 10. Depending on the design of the pump used, in some configurations, the pump can perform the functions of both a pump and a valve. For example, a peristaltic pump will prevent liquid flow in the operating tubing when not rotating or when prevented from rotating. Using this example, the pump 24 and valve 26 combination acting on the second branch tubing can be substituted by the appropriate pump alone.

[0052] As discussed in the introduction above, the presence of bubbles in the electroporation chamber 3 is very detrimental to the electroporation process. Also, to aid in the ability to have precise control of the liquid flow and to detect the end of the electroporation process, bubble detection devices are placed at different locations in the tubing set. As shown in FIG. 5 , a first bubble detection device 27 is placed in the main pipe 11 at a location between the first pump 22 and the first junction 16. A second bubble detection device 28 is placed in the first branch pipe 14 at a location between the second pump 23 and the first junction 16.

[0053] The first bubble detector 27 and the second bubble detector 28 are electrically connected to a control circuit which causes the presence of bubbles in the piping to be registered by the control circuit.

[0054] Referring again to Figure 2a, the vent port 32 is fluidly connected to a vent conduit 33 through a third bubble detection device 34. The third bubble detection device is electrically connected to the control circuitry.

[0055] Referring again to the electroporation system 111a shown in FIG. 2, in addition to the features previously described in connection with the system 111 depicted in FIG. 1, this system includes a wash solution bag 5 and a wash solution waste bag 6.

[0056] FIG. 8 is a depiction of the electroporation device 100a of the electroporation system 111a. The illustrated embodiment is a GeneEngine™ machine. Also shown in FIG. 8 is a third bubble detection device 34 configured to be disposed in the vent tube 33 of the electroporation chamber 3 when connected to the electroporation device 100a. The tubing set includes the following features in addition to the tubing set 10 depicted in FIG. 3: A third branch tube is connected to the main tube at a third connection positioned on the main tube between the first connection 16 and the second connection 21. The third branch tube is configured to connect to the wash solution bag 5 at its other end. A fourth branch tube is connected to the connection with the second branch tube at one end and configured for connection to the wash solution waste bag 6 at the other end.

[0057] A fourth pump is disposed in the third branch. Also disposed in the third branch are a third valve and a fourth bubble detector. A fifth bubble detector may be disposed in the second branch. Additional valves may be disposed as needed to prevent unwanted liquid flow into the piping during operation of the first through fourth pumps. Each of the pumps and each of the bubble detectors are electrically connected to a control circuit.

[0058] A method 1000 of operating the system 111 described above and depicted in FIG. 1 will now be described. The flow chart of FIG. 6 depicts this method. Method 1000 includes providing 1001 a tubing set 10 and providing 1002 an electroporation chamber. Additionally, consumables storage bag 1 containing a cell culture in a liquid suspension are provided 1003, and storage bag 2 containing an exogenous substance in a liquid suspension are provided 1004. The tubing set is actuated such that 1005, by operation of pumps and valves, a predetermined amount of cell culture from cell culture bag 1 and a predetermined amount of exogenous substance from exogenous substance bag 2 are introduced into electroporation chamber 3.

[0059] Activating the first pump 22 introduces a predetermined amount of cell suspension into the electroporation chamber 3. Activating the second pump 23 introduces an exogenous substance suspension into the electroporation chamber 3. It is a feature of the method 1000 that the cell culture suspension and the exogenous substance suspension are mixed within the tubing set. More specifically, the mixing occurs at the junction 16 between the main pipe 11 and the first branch pipe 14. This can be achieved by operating the first pump 22 and the second pump simultaneously.

[0060] During operation of the first pump 22 and the second pump 23, the second valve 26 is held closed to prevent liquid from flowing into the second branch 20 and the output bag 4. In some configurations, the third pump 24 is configured to prevent flow in the second branch such that the second valve 26 is not required to provide this function.

[0061] Once the mixture of cell culture suspension and exogenous material suspension is introduced into the electroporation chamber, an electrical current comprising an electroporation signal is applied 1006 to the electroporation electrodes 35a, 35b of the electroporation chamber. Preferably, the electroporation signal follows the PulseAgile protocol as previously defined. As previously explained, the electroporation chamber includes electrodes for applying an electric field to the contents. Upon application of the electroporation signal, the exogenous material is incorporated into the cells in the manner described in the introduction above.

[0062] Following the electroporation step 1006, the electroporated contents of the electroporation chamber are emptied 1007 into the output bag 4. For the system depicted in FIG. 1, the empting step 1007 is performed by keeping the first valve closed, keeping the second valve 26 open, and operating the third pump 24.

[0063] The reader will understand that during step 1005, the flow of liquid in the section of the main pipe 11 between the second end 13 and the second connection 21 is in the opposite direction to the flow during step 1007.

[0064] The introducing step 1005, dispensing step 1006, and emptying step 1007 can be repeated as desired or until the consumable is depleted.

[0065] Figure 7 shows a method 1000a for operating the device 100a and system 111a depicted in Figures 2 and 8. System 111a differs from system 111 by the addition of a cleaning facility. The corresponding method includes the following additional steps: In step 1008, a bag 5 containing a cleaning solution is provided. A waste bag 6 is provided 1009 for receiving waste cleaning solution.

[0066] Following emptying of the electroporation chamber 3, the following additional steps may be performed to wash the electroporation chamber: A predetermined amount of wash solution is introduced into the electroporation chamber from the wash solution bag 1010. After a predetermined waiting time, the wash solution is drained from the electroporation chamber into the waste bag 6 1011. Washing is performed at the end of a batch of electroporation cycles and as needed.

[0067] The control circuit controls the supply of electroporation signals to the electroporation electrodes 35a, 35b of the electroporation chamber 3 by the signal generator, as well as the one or more pumps 22, 23, 24 and one or more valves 25, 26. The control circuit is configured to implement the methods 1000, 1000a described above. The control circuit is configured such that the control of the one or more pumps 22, 23, 24 and the control of the one or more valves 25, 26 is based in part on signals received from one or more bubble detection devices 27, 28 and / or a third bubble detection device 34. The presence of bubbles in the tubing set indicates bubbles in the electroporation chamber 3. Detection of fluid by the third bubble detection device may indicate a malfunction in the delivery sequence. As described above, bubbles in the electroporation chamber 3 are highly detrimental. For example, the control circuitry is configured such that if a bubble is detected by either the first bubble detection device 27 or the second bubble detection device 28, which indicates the presence of a bubble in the cell culture suspension and / or the exogenous material suspension, respectively, the electroporation process is terminated and a signal is sent to the user.

[0068] The electroporation system described herein can be used with any type of cell, particularly for bioproduction or research purposes, such as screening of genetically modified cells, but is particularly designed for the production of therapeutic cells. Thus, an aspect of the invention is the use of such a system to introduce exogenous material into cells and to obtain subsequent genetic modification of said cells with said exogenous material.

[0069] Exogenous substances include polypeptides or polynucleotides that can be expressed in cells, or any molecules that will affect genome expression, such as by introducing mutations. The exogenous substance can be, for example, a transgene, a protein-encoding mRNA, a vector containing an expression cassette, a single-stranded DNA or DNA template for insertion into the genome, RNAi, transcription, ribonucleoproteins (RNPs, Cas nucleases with guide RNAs, etc.), or translation inhibitors. For the purpose of engineering therapeutic immune cells, such as T cells or NK cells expressing chimeric antigen receptors (CARs) or recombinant TCRs, the exogenous substance can be a rare-cutting endonuclease or base editor that can specifically cut or introduce mutations into genomic target sequences, or a polynucleotide encoding such a substance (Non-Patent Document 3). By exogenous genetic material, we mean a polynucleotide encoding a protein that can be transiently expressed in such cells or designed to be integrated into the genome of such cells. According to some embodiments, the exogenous agent can be a mutagenic molecule such as a base analogue (eg, 5-bromouracil and 2-aminopurine) or an intercalating agent (eg, ethidium bromide, proflavine, daunorubicin).

[0070] The present invention also encompasses a method for the genetic modification of cells, preferably primary immune cells, where the cells are electroporated into and subsequently harvested from the electroporation system described herein.

[0071] Such a method may include one or more of the following steps. - Cells are provided, usually from a pre-culture, and introduced into the system as previously described. - Cells are mixed with exogenous material, such as mRNA encoding a target sequence-specific reagent, such as a rare-cutting endonuclease or base editor, and electroporated in the system previously described and shown in the examples. - Cells are harvested and cultured outside the system. - The cells can be purified and finally frozen, dehydrated, processed and used fresh or incorporated into compositions for therapeutic or non-therapeutic purposes.

[0072] More specifically, the system can be implemented to produce a batch of primary immune cells, preferably a batch of allogeneic CAR T cells, where the immune cells are provided by a donor, a patient, or extracted from stem cells. Such cells are mixed in a tubing set with a polynucleotide or ribonucleoprotein encoding a rare-cutting endonuclease or base editor that targets the T cell receptor (TCR) in the cells. The bubble-free mixture is then pushed into an electroporation chamber for electroporation. Several successive cycles may be performed. The electroporated cells are then recovered and cultured. Upon expression of the rare-cutting endonuclease or base editor that targets the TCR gene, cells that remain TCR-positive are removed from the culture of modified cells. The resulting TCR-negative cells can be transduced with a viral vector to equip the cells with a genetically engineered CAR or TCR and give them specificity for cancer markers such as, by way of non-limiting example, CD19, CD22, CD20, CS1, BCMA, CD123, ROR1, GD2, CD33, CD38, CD70, FAP, IL3RA, MUC1, mesothelin, PSCA, claudin 18.2, EGFRvIII, and to produce one or several batches of CAR-T cell therapeutic compositions for preclinical or clinical use.

[0073] The electroporation system of the present invention allows for successive cycles of electroporation without opening the system, with a view to producing a unique batch of recombinant vesicles or cells. In some embodiments, the above-described methods of the present invention can include more than 5, preferably more than 10, more preferably more than 15, 20, 25, 30, 35, or even more than 40 cycles, up to a maximum of 45 cycles. Each cycle can electroporate between 5 and 20 ml, preferably about 10 ml, particularly for T cells, NK cells, or HSCs, and can be used to electroporate 10 ml or more. 9 Furthermore, such systems can achieve up to 5.10 times the number of electroporated cells or vesicles when more than 30 cycles are performed. 10 When successive cycles are applied to produce a batch capable of reaching 10 recombinant cells, 10 More than one cell can be treated.

[0074] (Example) The following examples demonstrate the advantageous results achieved using the system of the present invention. By way of comparison, Example 1 demonstrates the detrimental effects of prolonged contact between cells and exogenous materials. Example 2 demonstrates the advantageous results obtained from the electroporation system of the present invention.

[0075] Example 1 GeneEngine uses a mixture of mRNA and T cells PBMCs were thawed on day 0 and incubated in 3.8 mL of TransACT 5X for 64 hours on day 1 at 3.17 °C. 8 CD3 + Cells were then seeded onto two GRex500 plates on day 4 for a 10-day expansion phase prior to electroporation in complete medium (OpTmizer™ CTS™ T Cell Expansion Basal Medium, Gibco + 5% human serum AB).

[0076] After 10 days of expansion, T cells were cultured at 10 per ml in Buffer T (Harvard Apparatus). 8 T cells were resuspended at 10 cells per TALEN arm. mRNA encoding the TALEN targeting the TRAC locus was prepared in Buffer T at 1 mg / mL. Electroporation was performed with T cells resuspended at 10 cells per TALEN arm. 6 Pre-mixed with mRNA at 0.5 μg per cell, electroporation was performed using a GeneEngine™ machine connected to a PulseAgile electroporator (updated to drive the GeneEngine™). Each electroporation cycle lasted for 1 minute. After each electroporation cycle, electroporated T cells were collected from the output bag and diluted to 1.9 µg per mL in complete medium. 6 T cells were seeded at 1000 x 1000 cells per well and cultured at 30°C for 18 hours. T cells were then transferred to prewarmed complete medium and cultured at 37°C for 3 days. TCRαβ knockout was then measured by flow cytometry using antibodies as described by Poirot et al. (2013) Non-Patent Document 4.

[0077] FIG. 9 shows the percentage of TCRαβ knockout obtained in T cells electroporated with TRAC TALEN mRNA after each electroporation cycle performed with a GeneEngine connection to a PulseAgile electroporator.

[0078] The results in Figure 9 show that as the number of electroporation cycles (and therefore time) increased, the efficacy of the TCRαβ knockout (TCRab KO survival) decreased, demonstrating that prolonged contact of mRNA with T cells reduced the efficacy of the TCRαβ knockout. The number of cycles shown in Figure 9 is 35. This strongly suggests that the mRNA is degraded in the presence of T cells. Therefore, if several electroporations are required, the T cells and mRNA should not be in contact for longer than necessary before electroporation.

[0079] Example 2 GeneEngine, in which mRNA and T cells are separated and mixed in a T-shaped tubing set according to the present invention just before electroporation. Jurkat cells were cultured at least 10 9 The cells were cultured in complete medium (OpTmizer™ CTS™ T Cell Expansion Basal Medium with 10% FBS (fetal bovine serum), Glutamax (1% v / v, Gibco), CTS™ OpTmizer™ T Cell Expansion Supplement (26 mL / liter of medium), and penicillin / streptomycin solution (100 U / mL, Gibco)) in flasks until reaching 10 cells. Cells were then cultured at 10 per ml in a CellBag device (Xuri). 6 Cells were resuspended in 1 L of complete medium and cultured at 5% CO2, 21% O2 with a gas inlet at 0.2 mL / min and rocking (8 rpm, 6° tilt) at 37°C. After 10 days of expansion, T cells and TRAC TALEN arm mRNA were cultured at 105.3 10 per ml in Buffer T. 6 T cells and TRAC TALEN arm mRNA were resuspended at 1000 ng / ml each (see Ratio 1, R1 below), or T cells and TRAC TALEN arm mRNA were resuspended at 125 ng / ml in Buffer T. 6 either resuspended at 1000x cells and 1mg / mL, respectively (see ratio 2:R2 below).

[0080] Next, several conditions were tested using a GeneEngine machine connected to a PulseAgile electroporator (updated to run the GeneEngine). Cells were electroporated without mRNA (negative control). Cells and mRNA were manually mixed and directly electroporated (positive control 1). Jurkat cells and mRNA were kept separate and untouched for 30 minutes, then manually mixed and directly electroporated (positive control 2). Jurkat cells and mRNA were manually mixed and incubated for 30 minutes before electroporation (condition 3: C3). Jurkat cells and mRNA were placed in separate bags in the GeneEngine, and the first cycle was performed. The cells and mRNA were mixed in a T-tube set and directly electroporated (C1). A second electroporation cycle was performed 30 minutes after the first cycle (C2).

[0081] Two different T cell:mRNA volume ratios were tested: either 9.5:0.5 (corresponding to 9.5 ml of Jurkat cells and 0.5 ml of mRNA) (R1) or 8:2 (R2); in each case, these ratios were used with the same amount of mRNA (10 per TALEN arm). 6 0.5 μg per cell) and the same amount of Jurkat cells (10 9 This resulted in electroporation of 100 cells.

[0082] After each cycle, cells were harvested from the output bag and diluted to 1.6 x 10 per mL in complete medium. 6 Cells were seeded at 1000 x 1000 cells per well and cultured at 30°C for 18 hours. Cells were then transferred to rewarmed complete medium and cultured at 37°C for 3 days. TCRαβ knockout was then measured by flow cytometry using antibodies as described by Poirot et al. (2014).

[0083] Figure 10 shows the percentage of TCRαβ knockout (TCRab KO) obtained in T cells electroporated with TRAC TALEN mRNA under the different conditions detailed above, i.e., C3: condition 3, R1C1: ratio 1 and first cycle, R1C2: ratio 1 and second cycle, R2C1: ratio 2 and first cycle, R2C2: ratio 2 and second cycle.

[0084] The results in Figure 10 show that the efficacy of TCRαβ knockout was approximately 50% when the cells were mixed manually immediately before electroporation (Positive Control 1 and Positive Control 2). However, when the cells and mRNA were mixed for 30 minutes before electroporation (C3), the efficacy of TCRαβ knockout dropped to only 10%, confirming previous observations as demonstrated in Example 1 above. When Jurkat cells and mRNA were mixed in the T-shaped tubing set of the GeneEngine device, the efficacy of TCRαβ knockout was much higher than that of the positive control, reaching between 60 and 70% efficacy, regardless of the volume ratio used (R1 or R2) and regardless of when electroporation was performed (in the first cycle C1 or the second cycle C2). These results demonstrate that mixing cells and mRNA in the T-shaped tubing set in the GeneEngine is well suited for efficient and sequential electroporation.

[0085] The reader will understand that the electroporation devices, systems, and methods of operation described above represent a welcome improvement in the cell industry. The disclosed devices, systems, and methods allow for the use of commercially available components to obtain electroporation products of superior quality than previously possible. The use of commercially available components allows for cost reduction and economies of scale. [Explanation of symbols]

[0086] 1. Container, cell culture input bag, cell culture bag, cell suspension bag, supply bag, storage bag 2. Container, exogenous material bag, exogenous material suspension bag, supply bag, storage bag 3. Electroporation Chamber 4 Output container, output bag 5 cleaning solution bags 6. Washing solution waste bag, waste bag 10 piping sets 11 Main manager 12 First End 13 Second End 14 First branch pipe 15 One end of the first branch pipe 14 16 First connection 17 the other end of the first branch pipe 14 18 One end of the second branch pipe 20 19 the other end of the second branch pipe 20 20 Second branch pipe 21 Second connection 22 First Pump 23 Second Pump 24 Third Pump 25 First Valve 26 Second Valve 27 First bubble detection device 28 Second bubble detection device 30 Activation chamber 31 Inlet port 32 ventilation ports 33 Ventilation pipe 34 Third bubble detection device 35a, 35b Electroporation electrodes 36a, 36b Electrical leads 100, 100a Electroporation device 111, 111a Electroporation system

Claims

1. A device (100) for supplying an electroporation chamber (3), comprising: A piping set (10), comprising: a main tube (11) having a first end (12) for connection to a cell suspension input bag (1) and a second end (13) for connection to an electroporation chamber (3); a first branch pipe (14) having a first connection (16) with said main pipe (11) at one end (15) and the other end (17) for connection with an exogenous material input bag (2); a second branch pipe (20) having at one end (18) a second connection (21) with the main pipe (11) located between the first connection and the second end (13), and at the other end (19) for connection to an output vessel (4); A piping set (10) comprising: one or more pumps (22, 23, 24) configured to act on the tubing set (10) and controllable to move fluids in the tubing set (10); Equipped with A device (100) characterized in that during operation, mixing of the cell suspension from the cell suspension input bag (1) with the exogenous material from the exogenous material bag (2) occurs at the first connection (16).

2. The device (100) of claim 1, further comprising one or more bubble detection devices (27, 28) configured to detect the presence of gas in the tubing set (10).

3. 2. The device (100) of claim 1, wherein the first connector (16) is a T-shaped connector, and preferably the first branch pipe (14) is connected to the stem of the T-shaped connector.

4. 4. The device (100) of any one of claims 1 to 3, further comprising one or more valves (25, 26) acting on the piping (10) configured to prevent fluid flow.

5. the one or more pumps a first pump (22) acting on the piping set at a location between the first end (12) of the main pipe (11) and the first connection (16); a second pump (23) acting on said first branch pipe (14); The device (100) of any one of claims 1 to 4, comprising:

6. 6. The device (100) of claim 5, wherein the one or more pumps further comprise a third pump (24) acting on the second branch pipe (20).

7. 7. The device (100) according to any one of claims 4 to 6, wherein the one or more valves consist of a first valve (25) positioned to act on the piping between the first connection (16) and the second connection (21), and a second valve (26) positioned to act on the second branch (20).

8. a third branch pipe having at one end a third connection with said main pipe (11) and having at its other end for connection with a cleaning solution input container (5), said third connection being positioned between said first connection (16) and said second connection (21); a fourth branch pipe having at one end a fourth connection with said second branch pipe (20) and having at its other end for connection with a cleaning solution waste bag (6); The device (100a) of any one of claims 1 to 7, further comprising:

9. A device (100, 100a) according to any one of claims 4 to 8, a cell suspension bag (1) connected to the first end (12) of the main pipe (11); An electroporation chamber (3), an inlet port (31) connected to the second end (13) of the main pipe (11); a vent port (32) fluidly connected to a vent tube (33); and Electroporation electrodes (35a, 35b) an electroporation chamber (3) having an exogenous material input bag (2) connected to the first branch pipe (14); an electroporated cell suspension output bag (4) connected to the second branch pipe (20); Optionally, a third bubble detection device (34) configured to detect the presence of fluid in said vent pipe (33); A control circuit comprising: supplying an electroporation signal to the electroporation electrodes (35a, 35b) of the electroporation chamber (3); controlling the one or more pumps (22, 23, 24) and the one or more valves (25, 26) based at least on signals from the one or more bubble detection devices (27, 28); a control circuit configured as follows: An electroporation system (111) comprising:

10. providing a tubing set (11) (1001); Providing an electroporation chamber (3) (1002); providing a storage bag (1) containing a cell culture in a liquid suspension (1003); providing a reservoir bag (2) containing an exogenous substance in a liquid suspension (1004); introducing (1005) a predetermined amount of the cell culture and the exogenous material into the electroporation chamber (3), wherein the cell culture and the exogenous material are mixed in the tubing set (11) before entering the electroporation chamber; applying an electroporation signal to the electroporation chamber (3) to effect electroporation in the contents (1006); emptying (1007) the electroporation chamber (3) into a storage container (4); 10. A method (1000) for electroporation comprising:

11. providing a waste bag (6) for receiving the discarded cleaning solution (1008); Providing (1009) a storage bag (5) for the cleaning solution; Following the step of emptying the electroporation chamber (1007), a step of introducing (1010) a predetermined amount of wash solution from the wash solution reservoir bag (5) into the electroporation chamber (3); Discharging (1011) the electroporation chamber into the waste bag (6) for receiving discarded washing solution; The method (1000a) of claim 10, further comprising:

12. providing one or more pumps (22, 23, 24) configured to pump fluid in said tubing set (11); providing one or more bubble detection devices (27, 28, 34) configured to provide a signal in response to the presence of fluid and / or air bubbles in said tubing set (11); operating the one or more pumps (22, 23, 24) to cause the introduction of and / or the emptiness of the electroporation chamber (3); further comprising 12. The method of claim 10 or 11, wherein the one or more pumps (22, 23, 24) are operated based at least in part on the signal provided by the one or more bubble detection devices (27, 28, 34).

13. providing one or more valves (25, 26) operable to prevent fluid flow through said tubing set (11); Operating the one or more valves, Undesirable flow of raw cell suspension from the cell suspension bag (1) and / or exogenous material suspension from the exogenous material bag (2); Reflux of the electroporation product into the storage bag (1, 2), and Flow of raw cell suspension and exogenous material suspension into the second branch (20) and into the product bag (4). operating the one or more valves to prevent 13. The method of any one of claims 10 to 12, further comprising:

14. 14. Application of the method according to any one of claims 10 to 13 for the production of therapeutic cells for cell therapy.

15. 15. The application according to claim 14, wherein the therapeutic cells are immune cells, in particular T cells and NK cells, or the therapeutic cells are stem cells, such as hematopoietic stem and progenitor cells, embryonic stem (ES) cells, or induced pluripotent stem (iPS) cells.

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