WO2025149983A2 - Plateforme de distribution avec transfection non virale intégrée et traitement de cellule - Google Patents

Plateforme de distribution avec transfection non virale intégrée et traitement de cellule

Info

Publication number
WO2025149983A2
WO2025149983A2 PCT/IB2025/050324 IB2025050324W WO2025149983A2 WO 2025149983 A2 WO2025149983 A2 WO 2025149983A2 IB 2025050324 W IB2025050324 W IB 2025050324W WO 2025149983 A2 WO2025149983 A2 WO 2025149983A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
delivery
pod
population
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/050324
Other languages
English (en)
Inventor
Jorg Thommes
Michael Maguire
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Avectas Ltd
Original Assignee
Avectas Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Avectas Ltd filed Critical Avectas Ltd
Publication of WO2025149983A2 publication Critical patent/WO2025149983A2/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the aqueous solution can include 106 mM KCl.
  • the well can be configured to contain a population of non-adherent cells.
  • the non-adherent cell can include a peripheral blood mononuclear cell.
  • the non-adherent cell can include an immune cell.
  • the non-adherent cell can include a T lymphocyte.
  • the payload can include a messenger ribonucleic acid (mRNA).
  • the mRNA can encode a gene-editing composition.
  • the gene editing composition can reduce the expression of PD-1.
  • the mRNA can encode a chimeric antigen receptor.
  • the system can be used to deliver a cargo compound or composition to a mammalian cell.
  • the population of non-adherent cells can include a monolayer.
  • Some example implementations of the current subject matter can include utilizing a delivery platform that includes a chamber (e.g., a pod or bioreactor) to perform sequential unit operations (used in cell therapy processing) in a single, integrated closed system.
  • Manufacturing process steps integrated together may include viral- and non-viral cell transfection, cell expansion (static, fed batch and/or perfusion), cell washing, cell Attorney Docket No.048831-531001WO concentration and cell formulation.
  • integrating cell transfection and cell expansion in a single system can reduce the number of systems used in cell manufacturing.
  • the methods and systems described herein include a delivery platform for performing a technique of contacting cells (e.g., non-adherent cells) with a volume of aqueous solution, where the aqueous solution includes the payload/cargo to be delivered to the cells.
  • the delivery system includes a spray head, bags or reservoirs (e.g., where the bags or reservoirs may include different fluids), pumps, an interface screen, and a pod (e.g., a bioreactor or transfection chamber).
  • the pod e.g., a bioreactor or transfection chamber
  • the pod may be utilized for transfection, where cells are transferred to the transfection chamber, culture medium is removed (e.g., by centrifugation), and cargos or payloads are combined with a delivery solution (e.g., which may include sucrose, potassium chloride, HEPES and water), and subsequently delivered to the cells in the transfection chamber.
  • a delivery solution e.g., which may include sucrose, potassium chloride, HEPES and water
  • the delivery platform utilizes a method for the delivery of a payload/cargo compounds to cells (e.g., non-adherent cells).
  • an example of the delivery platform is the Single Use System (SUS) provided by Avectas Limited of Dublin, Ireland.
  • SUS Single Use System
  • the (SUS) is described in more detail below and includes a pod, which is sometimes referred to as a Single Use Assembly (SUA). It has been demonstrated that the SUA can be used to first edit T cells (using GFP mRNA), and then expand the cells (for 4 days) within the SUA. Results of the cell expansion (using the SUA) demonstrated comparable growth rates of edited cells in the SUA, as when edited cells were expanded in T Flasks (a common system used to expand T cells). As described in more detail herein, a membrane at the bottom of the closed SUA Attorney Docket No.048831-531001WO chamber can serve as a cell retention mechanism during wash and/or perfusion steps. This allows integration of wash steps into the transfection and expansion operations.
  • SUA Single Use Assembly
  • Some implementations of the systems and methods described herein can facilitate advancing from batch operation to fed batch or perfusion operation during cell expansion.
  • the rocking and vibrating function of the SUS can be utilized to enhance mass transfer during cell expansion, thus improving mass transfer, enhancing media utilization, and improving volumetric productivity.
  • Feed lines at the bottom and side of the reaction chamber can be used to add fresh media and/or remove spent media, including the applications of cell washing and cell concentration.
  • An integrated SUA solution after being developed for use in Good Manufacturing Practice (GMP) manufacturing processes, can replace existing technologies based on single step unit operations. These includes existing cell culture expansion systems and cell washing/concentration equipment and existing cell transfection technologies using Electroporation.
  • FIG. 40A and FIG. 40B show a schematic of the SUA
  • Sensors for critical parameters like pH and oxygen concentration can be added to the SUA reaction chamber, which can substantially improve process control with the concomitant benefits to process consistency and predictability. This also supports the progression from batch to fed batch and perfusion operation. In its most advanced state, this creates opportunities for adaptive control of the cell culture environment.
  • the system has the potential for automation of many process steps, which reduces manual interactions.
  • Table 1 Challenges and opportunities in cell therapy manufacturing Category Root Cause Opportunity Technical Approach Attorney Docket No.048831-531001WO lack of process improve mixing fresh media feed from the bo ⁇ om of the control and mass chamber create a stable bed of expanded transfer during cells in the upwardflow with op ⁇ mal cell expansion transport of nutrients and waste product removal through the expanded bed; use rocking mo ⁇ on to improve mixing Con ⁇ nuous or use media feed from bo ⁇ om and media Lack of semi con ⁇ nuous removal via side port predictability opera ⁇ on /variable introduce pH sensor to control CO 2 accumula ⁇ on, success rate sensors that are O 2 sensor to avoid oxygen limita ⁇ on, essen ⁇ al to measure cell concentra ⁇ on for cell process control specific media feed control “set it and forget feedback use sensors to control cri ⁇ cal parameters it” as main controlled opera ⁇ onal opera ⁇ on principle in sta ⁇ c cultures mostly manual process integrate transfec ⁇ on, expansion, washes process with integra ⁇ on and concentra ⁇ ons in a single closed mul ⁇ ple system independent High labor steps, low
  • Some implementations of the current subject matter can provide a platform for cell engineering that can provide clinical grade transfection in that treated cells have high viability and expression.
  • the delivery platform can provide smaller scale cell processing and can be used for experimental designs involving smaller quantities of cells, Attorney Docket No.048831-531001WO such as .5M- 15M cells.
  • the platform can include features that make it easy to use, for example, by having single-use pods for performing the cell engineering process that is described in more detail herein.
  • the pod can be resuable.
  • the pods can be chamber.
  • the system can include control features enabling easy to implement and repeatable cell processing. Some implementations can be particularly useful, for example, in research and development efforts.
  • the aqueous solution does not include an alcohol (e.g., the solution is in the absence of alcohol (e.g., 0% ethanol)).
  • the solution can also include an alcohol at greater than 0.2 percent (v/v) concentration.
  • the alcohol comprises ethanol (e.g., greater than 5% ethanol, greater than 10% ethanol, and the like).
  • the aqueous solution comprises between 20-30% ethanol, e.g., 27% ethanol. Other compositions are possible.
  • Attorney Docket No.048831-531001WO Cell expansion Cell expansion refers to the process of culturing isolated cells for multiple generations in order to reach the desired number of cells for a regenerative response in the patient.
  • a bioreactor may be used for cell expansion, which controls the growth of cells in a controlled environment to produce the desired products.
  • the integrated platform described herein is for both transducing and expanding cells (e.g., CAR-T cells) aimed at the manufacturer for autoimmune and immuno-oncology indications and scale out.
  • the SUA described is a self-contained bioreactor in addition to capacity to effect transfection, transduction and other manufacturing process steps.
  • the described bioreactor includes a polystyrene jacket designed with the bioreactor to include a heating element (e.g., to insulate the device). The heating element may be used to control the temperature with precision.
  • the cell culture density output may range from 3.8x10e6/mL (in 100 mL) to 3.4x10e6/mL (in 300 mL), or anywhere there between.
  • the total cells may be optimized and may range from 3.7x10e6 (6 days in the SUA) to 1x10e9 (6 days in the SUA), or anywhere there between.
  • the integrated platform described herein may also require less media than commercially available equipment. For example, the platform may use about 4-6x less media to get the same cell growth.
  • the SUA may be implemented within an incubator.
  • the SUA may be connected to an external O 2 and/or CO 2 source (2 or 3 gas mixer) via a gas regulator or mixer.
  • Some expansion may be achieved with air only while rocking, warming and pressure parameters may be optimized.
  • the current subject matter can also provide a platform that can automate the cell transfection and transduction process and allow delivery to cells to be performed at various scales.
  • the throughput of the system is limited, difficulties exist in applying to clinical process/treatment. There may be concerns for contamination and inconsistent process Attorney Docket No.048831-531001WO depending on operators and/or various environmental parameters.
  • the example pod includes an upper portion 1605, a filter plate 1610, and a lower portion 1615.
  • pods may be designed for specific cell populations and sizes.
  • pods can include different lower portions based on the culture.
  • the pod can be referred to as a chamber, a chamber assembly, a single-use assembly, or a disposable assembly, for example.
  • the pod may be manufactured as a single molding rather than having multiple parts that clip together.
  • the pod may have its filter membrane bonded into this single substrate.
  • the pod may have a filter with a smaller diameter such that a smaller population of cells may be treated.
  • the pod may have markings molded into it to indicate fill level or have molded features to ensure orientation within the platform is consistent.
  • the pod may have multiple features to enable it to be retained within a pod holder or stack outside of the apparatus.
  • the pod may have a lid feature to facilitate incubation of cells within it.
  • the pod may have a one-way check valve implemented to enable culture medium to be maintained within the cavity beneath the filter, or to support culture medium above the filter medium to keep cells in suspension post use of the pod.
  • some pods can include a hydroscopic foam located in the lower portion for pulling fluid from above the filter plate.
  • a hydroscopic foam located in the lower portion for pulling fluid from above the filter plate.
  • An example foam is 3MTM TegadermTM Foam Dressing (non- adhesive).
  • the lower portion does not include holes and can include a flat tissue cultured treated surface. Such an implementation can be suitable for adherent cell populations to enhance adherence. Such an implementation with a flat surface can be utilized for delivery to tissue explants or engineered tissues.
  • the pod can be suitable for culturing cells. Rather than immediately removing the cells from the pod, the cells can be cultured for a period of time, such as hours or days.
  • the pod can be formed of culture compatible materials and a pod lid can be provided.
  • the pod can include memory storing process parameters.
  • a pod memory can be programmed with the process parameters such that, when the pod is inserted into the cell engineering platform, a controller on the cell engineering platform reads, from the pod memory, the process parameters.
  • the cell engineering can proceed using the process critical parameters, for example, via an automated fashion (e.g., an amount of solution delivered to the cells can be determined by the controller), or via displaying instructions to the user via a display.
  • the cell engineering platform can write information such as an experiment identifier, date, time, and the like, to the pod memory for future use and/or reference.
  • pods can communicate with one another.
  • a container or housing adapted to hold multiple pods can include connections between the pods so that the container reads data from the memory of a first pod, and copies some or all of the data to the other pods contained in the container.
  • Such an approach can also improve repeatability because, once the first pod is programmed with process critical parameters, that data is replicated to the other pods without modification to some or all of the data.
  • the pod 105 can include an upper portion 1605, a filter plate 1610, and a lower portion 1615.
  • the pod 105 can provide a processing surface, via the filter plate 1610, on which cells can be provided for treatment and processing.
  • the filter plate 1610 can be configured to receive a filter for use in forming a monolayer of cells to be processed using the delivery platform 100.
  • the pod 105 can be received and positioned within the pod nest 110.
  • the atomizer nest 115 can be a fixed distance above the pod 105.
  • the atomizer nest 115 can be a fixed distance from the pod nest 110 to reduce the number of variables or degrees of freedom available to the user thereby providing a system that is easier to use.
  • the atomizer nest 115 can be fixed about 75 mm above the pod 105.
  • the pod nest 110 can include a circular opening to receive the pod 105.
  • a lower portion 1615 of the pod 105 can be mated to the filter plate 1610 by coupling the lower portion 1615 with a portion of the filter plate 1610 extending through the circular opening of the pod nest 110.
  • the pod nest 110 can provide support to the pod 105 and can maintain the position of the pod 105 during cell processing using the delivery platform 100.
  • the pod nest 110 can maintain the position of the pod 105 to ensure the treatment surface of the pod 105, e.g., the filter plate 1610, is sufficiently located to receive adequate amounts of delivery solution.
  • Attorney Docket No.048831-531001WO As further shown in FIG.
  • the delivery platform 100 includes an atomizer nest 115.
  • the atomizer nest 115 can include an atomizer coupled to a delivery solution source configured within the delivery platform 100.
  • the atomizer can atomize the delivery solution to provide the delivery solution to the pod 105 (e.g., in the form of a spray) to process or treat cells configured on the filter plate of the pod 105.
  • the atomizer nest 115 can be coupled to the delivery solution source via a valve connector 120, such as a clippard value connector.
  • the atomizer configured within the atomizer nest 115 can be configured to provide the delivery solution to the pod 105 at a predetermined pressure.
  • the delivery platform 100 also includes a sample pressure connector 125 and an air pressure connector 130.
  • the valve connector 120 serves to control delivery solution application to atomizer.
  • the sample pressure connector 125 pressurizes the gas above the fluid in the Eppendorff reservoir to drive the sample into the atomizer.
  • the gas pressure connector 130 supplies pressurized gas to the atomizer.
  • the delivery platform 100 also includes a power input 135.
  • the power input 135 can include a 2 channel direct current (DC) 24V power input 135.
  • the power input 135 can be electrically coupled to the On/Off switch 140.
  • the delivery platform 100 also includes a human machine interface (HMI) cable coupling 145.
  • the HMI cable coupling 145 can be electrically coupled to the HMI 150.
  • the HMI 150 can include a display, at least one data processor, and input devices configured to control operation of the delivery platform 100 and to perform the methods of cell treatment via delivery described herein.
  • the screws 680 can include M4x10 button stainless steel screws.
  • the HMI mounting plate 670 can include a plurality of cutouts 685 to release heat generated by the display 155 and/or the circuitry of the HMI 150.
  • FIGS.7A-7E are CAD drawings illustrating an example Eppendorf base support of the delivery platform 100 of FIG. 1.
  • the Eppendorf base support shown in FIGS. 7A-7E corresponds to the Eppendorf base support 310 shown in FIGS. 3 and 5.
  • the dimensions of the Eppendorf base 310 shown in FIGS. 7A-7E are exemplary and not intended to limit the size or configuration of the Eppendorf base support 310.
  • the Eppendorf base 310 includes a hole 715 configured to receive the screw portion of knob 525 shown and described in relation to FIG. 5.
  • FIG. 7B shows a top-down view of the Eppendorf base 310.
  • the Eppendorf base 310 includes a mounting surface 720 and a flange portion 725 extending from the mounting surface 720.
  • the mounting surface 720 can include a plurality of holes 705 configured to mount the Eppendorf base 310 to the top cover 315.
  • the mounting surface 720 includes an opening 730 configured with a notch 735 at a location of the opening 730 closest to the flange 725.
  • the opening 730 can include a recessed portion 740 extending circumferentially around a portion of the opening 730.
  • FIG. 8B shows a side view of the clippard module upper mount 510.
  • the clippard module upper mount 510 can include one or more recessed surfaces configured therein.
  • FIG.8C is an end view of a mounting surface 815 of the clippard module upper mount 510.
  • the mounting surface 815 can couple to the spray head base mounting platform 505 via one or more dowel pins 515 as shown in FIG.5.
  • the dowel pins 515 can be received within holes 820 as shown in FIG. 8B.
  • Hole 825 can be a threaded hole configured to receive the screw 520 shown in FIG. 5.
  • a liquid tubing inlet 1103 and an air tubing inlet 1104 may be formed (FIG. 10B). Accordingly, the liquid orifice 1101 is connected to a liquid reservoir through the liquid tubing inlet 1103, and the gas orifice 1102 is connected to a gas reservoir through the air tubing inlet 1104 as shown in FIG.10C.
  • the gas reservoir may be an air cylinder or an air pump, and may be provided with a valve.
  • an LB-100 nebulizer can be utilized.
  • the values at which the nebulizer is used involves the atomization of a volume between about 10-300 ⁇ l of cell delivery solution.
  • FIG. 10D-G illustrate another example atomizer.
  • an atomizer adaptor can be included, which can adjust an orientation of the atomizer. For example, some atomizers can spray in a direction 1-5 degrees off their main axis.
  • An adaptor can be included that holds the atomizer in a manner to adjust the orientation, for example, so the atomizer directs atomized solution in a direction perpendicular to the face of the pod 105 filter plate 1610.
  • FIGS. 11A-11E are CAD drawings illustrating an example spray head base mounting platform 505 of the delivery platform 100 of FIG. 1.
  • the spray head base mounting platform 505 shown in FIGS 11A-11E corresponds to the spray head base mounting platform 505 shown in FIG.5.
  • the dimensions of the spray head base mounting platform 505 shown in FIGS. 11A-11E are exemplary and not intended to limit the size or configuration of the spray head base mounting platform 505.
  • FIG. 11A is a top view of an upper surface 1105 of the spray head base mounting platform 505.
  • the spray head base mounting platform 505 includes a plurality of holes 1110 configured with respect to a circular opening 1115 and a recessed surface 1120.
  • the plurality of holes 1110 can be arranged around Attorney Docket No.048831-531001WO the circular opening 1115 and the recessed surface 1120.
  • the hole 1130 can be configured to receive screw 520, shown in FIG. 5, to aid in securing the clippard module upper mount 510 to the spray head base mounting platform 505.
  • FIG. 11C shows an end view of the spray head base mounting platform 505.
  • holes 1135 can be provided to receive dowel pins 545 shown in FIG. 5.
  • Hole 1140 can be configured to receive screw 550 to couple the spray head base mounting platform 505 to the upright mounting spine 410.
  • FIG. 11D shows a cross-sectional view of the spray head base mounting platform 505 from the perspective of lines B-B shown in FIG. 11A.
  • the holes 1105 can include a counter sink portion to receive the screws 540.
  • FIG. 11E is an isometric view of the spray head base mounting platform 505.
  • FIGS. 12A-12D are CAD drawings illustrating an example pod nest 1205 of an exemplary embodiment of the delivery platform 100 of FIG.1. The dimensions of the pod nest 1205 shown in FIGS. 12A-12D are exemplary and not intended to limit the size or configuration of the pod nest 1205.
  • FIGS. 12A-12D are CAD drawings illustrating an example pod nest 1205 of an exemplary embodiment of the delivery platform 100 of FIG.1. The dimensions of the pod nest 1205 shown in FIGS. 12A-12D are exemplary and not intended to limit the size or configuration of the pod nest 1205.
  • FIGS. 13A-13C are CAD drawings illustrating another example pod nest 1305 of an exemplary embodiment of the delivery platform 100 of FIG. 1.
  • the dimensions of the pod nest 1305 shown in FIGS. 13A-13C are exemplary and not intended to limit the size or configuration of the pod nest 1305.
  • FIG. 13A shows a top view of the pod nest 1305.
  • the pod nest 1305 includes a circular pod receiving area 1310.
  • a pod 105 can be received within the pod receiving area 1310.
  • the pod nest 1305 can include a plurality of holes 1315 configured to couple with the bushings 560 shown in FIG.5.
  • the pod nest 1305 also includes a hole 1320 configured to receive the screw 565 shown in FIG.5.
  • the pod nest 1305 can be secured to the upright mounting spine 410 via the bushings 560 and the screw 565.
  • FIG. 13A are CAD drawings illustrating another example pod nest 1305 of an exemplary embodiment of the delivery platform 100 of FIG. 1.
  • the pod nest 1305 can include a sensor 1325.
  • the sensor 1325 can include a camera, a radio frequency (RF) identification (ID) scanner, or an IR sensor.
  • the sensor 1325 can be configured to determine an event, such as sufficient drainage of delivery solution from the pod 105. In this way, event-driven Attorney Docket No.048831-531001WO workflows associated with intracellular delivery can be achieved using the delivery platform 100.
  • the sensors may be included in the pod 105, such as in upper portion 1605, filter plate 1610, and/or lower portion 1615.
  • electrical connections can be included in the pod 105 and the pod nest 110 for connecting to the sensors, for example, to provide power and/or make sensor measurements.
  • the pod nest 110, 1205, 1305 can be configured to vibrate to aid settling cells into a monolayer within the pod or to aid recovery of cells from within the pod. Vibrational functionality may be added directly to the pod nest 110 via vibrational elements added onto or into the pod nest. Examples of vibrational elements include eccentric motors or liner resonant displacement (LRD) devices.
  • vibrational elements include eccentric motors or liner resonant displacement (LRD) devices.
  • FIGS. 14A-14F are CAD drawings illustrating an example pod nest cover 1405 of the delivery platform 100 of FIG. 1. The dimensions of the pod nest cover 1405 shown in FIGS.14A-14F are exemplary and not intended to limit the size or configuration of the pod nest cover 1405.
  • the example pod nest cover 1405 is configured to engage with pod nest 1205 by slotting element 1415 into mating holes within pod nest 1205.
  • FIG. 14A shows a top view of the pod nest cover 1405.
  • the pod nest cover 1405 includes a semi-circular cutout 1410 into which a pod 105 can be received with placed within the pod nest 110.
  • FIG. 14B shows a side view of the pod nest cover 1405.
  • the pod nest cover 1405 can include a plurality of extensions 1415 protruding from the bottom of the pod nest cover 1405.
  • FIG. 14C shows a horizontal cross-sectional view of the pod cover 1405 from the perspective of lines B-B shown in FIG. 14A.
  • the extensions 1415 can include tapped M4 for the purposes of rigidly fixing pod nest cover 1405 to pod nest 1205. This can be achieved by mating 2 screws from the underside of pod nest 1205 with counter bored holes on pod nest 1205.
  • FIG. 14D shows a vertical cross-sectional view of the pod cover 1405 from the perspective of lines A-A shown in FIG. 14A.
  • FIG. 14E shows an isometric view of a top surface of the pod cover 1405.
  • FIG. 14F shows an isometric view of a bottom surface of the pod cover 1405. As shown in FIG. 14F, the bottom surface of the pod cover 1405 incudes the extensions 1415 protruding from the bottom surface.
  • FIG.15 is an image of an example embodiments of a pod assembly 1500 for use in the delivery platform 100 shown in FIG.1.
  • the pod assembly 1500 can include a plurality of mate-able components, which can be coupled and uncoupled while performing delivery Attorney Docket No.048831-531001WO to cells using the delivery platform 100.
  • portions of the pod assembly 1500 can be configured for use with the delivery platform 100.
  • portions of the pod assembly 1500 can be configured for repeated use with the delivery platform 100.
  • pods 105 maybe stacked temporarily on a frame adjacent or connected to the device.
  • the frame can organize and retain a small number of pods, for example 6 pods or 12 pods 105 ready for insertion into the machine manually or automatically.
  • Pods 105 retained in the frame may be pre-treated or preloaded with cells whilst retained within the frame.
  • the pods 105 can be in the frame for a limited time before and after the experiment or device usage.
  • the operator may manually transfer pods from the frame into the pot nest, transfected the cells within the part and move them to a second frame for retaining post transfected pods.
  • the frame may be of open construction to aid cleanability.
  • the frame may be manual, for example as shown by frame 2105 illustrated in FIG. 21A or the frame may be configured for use with a plate stacker, for example, as shown by frame 2110 illustrated in FIG. 21B.
  • Commercially available plate stackers are available from Hudson Robotics, Inc. of Springfield Township, New Jersey, USA.
  • the frame can include sensors and/or communications for communicating with a pod.
  • the frame can include position sensors and/or timers.
  • FIGS. 16A-16C are images of example embodiments of components of the pod assembly 1500 shown in FIG.15.
  • FIG.16A shows a retainer ring 1605 of the pod assembly 1500.
  • FIG. 16B shows a filter plate 1610 of the pod assembly 1500.
  • the retainer ring 1605 can be pre-formed and integrated with the remainder of the pod.
  • the retainer ring 1605 material can be similar to that of the other aspects of the pod substrate.
  • the filter plate 1610 can include a plurality of holes to allow fluid to drain therethrough.
  • the filter plate 1610 can receive a filter upon which cells can be provided for delivery of a payload.
  • the plurality of holes or apertures can be formed in a variety of non-limiting patterns suitable to provide sufficient retention of cells and draining of solutions.
  • the apertures may be aligned around an outer diameter of the filter plate and/or along multiple radial directions of the filter plate.
  • the heating and/or cooling elements associated with the cell collection tray 2340 can be configured within the base of Attorney Docket No.048831-531001WO the delivery platform 2300.
  • the base can include a scale located underneath the waste collection tray 2335 and/or the cell collection tray 2340. In this way, the delivery platform 2300 can determine a weight of collected media materials and collected cells.
  • the collection tray 2340 can include an articulating cradle in which media materials or collected cells can be held and maintained in motion to improve cell viability.
  • the delivery platform 2300 can include one or more media materials 2345. The media materials 2345 can be fluidically coupled to the chamber 2330 via one or more fluid circuits.
  • conditions to achieve a coating of a population of coated cells include delivery of a fine particle spray, e.g., the conditions exclude dropping or pipetting a bolus volume of solution on the cells such that a substantial population of the cells are soaked or submerged by the volume of fluid.
  • the mist or spray comprises a ratio of volume of fluid to cell volume.
  • the conditions comprise a ratio of volume of mist or spray to exposed cell area, e.g., area of cell membrane that is exposed when the cells exist as a confluent or substantially confluent layer on a substantially flat surface such as the bottom of a tissue culture vessel, e.g., a well of a tissue culture plate, e.g., a microtiter tissue culture plate.
  • the volume of solution to be delivered to the cells is a plurality of units, e.g., a spray, e.g., a plurality of droplets on aqueous particles.
  • the volume is described relative to an individual cell or relative to the exposed surface area of a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population.
  • the volume can be between 6.0 x 10 -7 microliter per cell and 7.4 x 10 -4 microliter per cell.
  • the adherent cells can include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) Attorney Docket No.048831-531001WO cells or immortalized cells, such as cell lines.
  • the population of cells comprises non-adherent cells, e.g., the % 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 primary cells as well as immortalized cells (e.g., cells of a cell line).
  • exemplary non-adherent/suspension cells include primary hematopoietic stem cell (HSC), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine- induced killer (CIK) cells, human cord blood CD34+ cells, B cells, or cell lines such as Jurkat T cell line.
  • the payload can include a small chemical molecule, a peptide or protein, or a nucleic acid.
  • the small chemical molecule can be less than 1,000 Da.
  • the chemical molecule can include MitoTracker® Red CMXRos, propidium iodide, methotrexate, and/or DAPI (4',6-diamidino-2-phenylindole).
  • the peptide can be about 5,000 Da.
  • the peptide can include ecallantide under trade name Kalbitor, is a 60 amino acid polypeptide for the treatment of hereditary angioedema and in prevention of blood loss in cardiothoracic surgery), Liraglutide (marketed as the brand name Victoza, is used for the treatment of type II diabetes, and Saxenda for the treatment of obesity), and Icatibant (trade name Firazyer, a peptidomimetic for the treatment of acute attacks of hereditary angioedema).
  • the protein or polypeptide be about 100-500,000 Da, e.g., 1,000- 150,000 Da.
  • the protein can include any therapeutic, diagnostic, or research protein or peptide, e.g., beta-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and/or horseradish peroxidase.
  • the protein can include a cancer-specific apoptotic protein, e.g., Tumor necrosis factor-related apoptosis inducing protein (TRAIL).
  • TRAIL Tumor necrosis factor-related apoptosis inducing protein
  • An antibody is generally about 150,000 Da in molecular mass.
  • the antibody can include an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc ab, and/or an anti-Raf antibody.
  • the payload can include a therapeutic agent.
  • a therapeutic agent e.g., a drug, or an active agent”, can mean any compound useful for therapeutic or diagnostic purposes, the term can be understood to mean any compound that is administered to a patient for the treatment of a condition. Accordingly, a therapeutic agent can include, proteins, peptides, antibodies, antibody fragments, and small molecules. Therapeutic agents described in U.S. Pat.
  • the therapeutic agent can include at least one of cisplatin, aspirin, statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chloropromazine HCl, thioridazine HCl, Polymyxin B sulfate, chloroxine, benfluorex HCl and phenazopyridine HCl), and fluoxetine.
  • the payload can include a diagnostic agent.
  • the diagnostic agent can include a detectable label or marker such as at least one of methylene blue, patent blue V, and indocyanine green.
  • the payload can include a fluorescent molecule.
  • the payload can include a detectable nanoparticle.
  • the nanoparticle can include a quantum dot.
  • the population of non-adherent cells can be substantially confluent, such as greater than 75 percent confluent. Confluency of cells refers to cells in contact with one another on a surface. For example, it can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of the surface, e.g., of a tissue culture vessel, is covered with cells, 100% means that it is entirely covered.
  • the buffering agent can include 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid.
  • the present subject matter relates to a method for delivering molecules across a plasma membrane.
  • the present subject matter finds utility in the field of intra-cellular delivery, and has application in, for example, delivery of molecular biological and pharmacological therapeutic agents to a target site, such as a cell, tissue, or organ.
  • the method of the present subject matter comprises introducing the molecule to an aqueous composition to form a matrix; atomizing the matrix into a spray; and contacting the matrix with a plasma membrane.
  • This present subject matter relates to a composition for use in delivering molecules across a plasma membrane.
  • the present subject matter finds utility in the field of intra- cellular delivery, and has application in, for example, delivery of molecular biological and pharmacological therapeutic agents to a target site, such as a cell, tissue, or organ.
  • the composition of the present subject matter comprises an alcohol; a salt; a sugar; and/or a buffering agent.
  • a delivery technique that facilitates intracellular delivery of molecules independent of the molecule and cell type. Nanoparticles, small molecules, nucleic acids, proteins and other molecules can be efficiently delivered into suspension cells or adherent cells in situ, including primary cells and stem cells, with low cell toxicity and the technique is compatible with high throughput and automated cell-based assays.
  • the example payload may include no more than 50% (v/v) of the alcohol, more preferably, the payload comprises 2-45% (v/v) of the alcohol, 5-40% of the alcohol, and 10-40% of the alcohol.
  • the payload may include 20-30% (v/v) of the alcohol.
  • the payload delivery solution includes 25% (v/v) of the alcohol.
  • the payload can include 2-8% (v/v) of the alcohol, or 2% of the alcohol.
  • the alcohol may include ethanol and the payload comprises 5, 10, 20, 25, 30, and up to 40% or 50% (v/v) of ethanol, e.g., 27%.
  • Example methods may include methanol as the alcohol, and the payload may include 5, 10, 20, 25, 30, or 40% (v/v) of the methanol.
  • the payload may include 2-45% (v/v) of methanol, 20-30% (v/v), or 25% (v/v) methanol.
  • the payload includes 20-30% (v/v) of methanol.
  • the alcohol is butanol and the payload comprises 2, 4, or 8% (v/v) of the butanol.
  • the payload is in an isotonic solution or buffer.
  • the payload may include 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 .
  • KCl concentration ranges from 2 mM to 500 mM. In some preferred embodiments, the concentration is greater than 100 mM, e.g., 106 mM.
  • the payload may include a sugar (e.g., a sucrose, or a disaccharide). According to example methods, the payload comprises less than 121 mM sugar, 6-91 mM, or 26-39 mM sugar. Still further, the payload includes 32 mM sugar (e.g., sucrose).
  • the sugar is sucrose and the payload comprises 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.
  • the payload may include a buffering agent (e.g. a weak acid or a weak base).
  • the buffering agent may include a zwitterion.
  • the buffering agent is 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid.
  • the payload may comprise less than 19 mM buffering agent (e.g., 1-15 mM, or 4-6 mM or 5 mM buffering agent).
  • the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payload comprises 1, 2, 3, 4, 5, 10, 12, 14 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid. Further preferably, the payload comprises 5 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid. According to example methods of the present subject matter, the payload includes ammonium acetate. The payload may include less than 46 mM ammonium acetate (e.g., between 2-35 mM, 10-15 mM, ore 12 mM ammonium acetate).
  • the payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.
  • the volume of aqueous solution performed by gas propelling the aqueous solution may include compressed air (e.g. ambient air), other implementations may include inert gases, for example, helium, neon, and argon.
  • Attorney Docket No.048831-531001WO the population of cells may include adherent cells (e.g., lung, kidney, immune cells such as macrophages) or non-adherent cells (e.g., suspension cells).
  • the population of cells may be substantially confluent, and substantially may include greater than 75 percent confluent.
  • the population of cells may form a single monolayer.
  • the payload to be delivered has an average molecular weight of up to 20,000,000 Da. In some examples, the payload to be delivered can have an average molecular weight of up to 2,000,000 Da. In some implementations, the payload to be delivered may have an average molecular weight of up to 150,000 Da. In further implementations, the payload to be delivered has an average molecular weight of up to 15,000 Da, 5,000 Da or 1,000 Da.
  • the payload to be delivered across the plasma membrane of a cell may include a small chemical molecule, a peptide or protein, a polysaccharide or a nucleic acid or a nanoparticle.
  • the payload comprises mRNA.
  • the payload includes 3.0 – 150.0 ⁇ M of a molecule to be delivered, more preferably, 6.6 – 150.0 ⁇ M molecule to be delivered (e.g. 3.0, 3.3, 6.6, or 150.0 ⁇ M molecule to be delivered).
  • the payload to be Attorney Docket No.048831-531001WO delivered has an average molecular weight of up to 15,000 Da, and the payload includes 3.3 ⁇ M molecules to be delivered. According to example methods, the payload to be delivered has an average molecular weight of up to 15,000 Da, and the payload includes 6.6 ⁇ M to be delivered. In some implementations, the payload to be delivered has an average molecular weight of up to 1,000 Da, and the payload includes 150.0 ⁇ M to be delivered.
  • a method for delivering molecules of more than one molecular weight across a plasma membrane including the steps of: introducing the molecules of more than one molecular weight to an aqueous solution; and contacting the aqueous solution with a plasma membrane.
  • the method includes introducing a first molecule having a first molecular weight and a second molecule having a second molecular weight to the payload, wherein the first and second molecules may have different molecular weights, or wherein, the first and second molecules may have the same molecular weights.
  • the first and second molecules may be different molecules.
  • the payload to be delivered may include a therapeutic agent, or a diagnostic agent, including, for example, cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chloropromazine HCl, thioridazine HCl, Polymyxin B sulfate, chloroxine, benfluorex HCl and phenazopyridine HCl), and fluoxetine.
  • statins e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl, chloropromazine HCl, thioridazine HCl, Polymyxin B sulfate, chloroxine, benfluorex HCl and phenazopyridine HCl
  • gentamicin e.g., amoxicillin, ampicillin
  • glycopeptides e.g., avoparcin, vancomycin
  • macrolides e.g., Attorney Docket No.048831-531001WO erythromycin, tilmicosin, tylosin
  • quinolones e.g., sarafloxacin, enrofloxin
  • streptogramins e.g., viginiamycin, quinupristin-dalfoprisitin
  • carbapenems lipopeptides, oxazolidinones, cycloserine, ethambutol, ethionamide, isoniazrid, para-aminosalicyclic acid, and pyrazinamide).
  • an anti-viral e.g., Abacavir, Aciclovir, Enfuvirtide, Entecavir, Nelfinavir, Nevirapine, Nexavir, Oseltamivir Raltegravir, Ritonavir, Stavudine, and Valaciclovir.
  • the therapeutic may include a protein-based therapy for the treatment of various diseases, e.g., cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B or C.
  • Additional exemplary payloads can also include detectable markers or labels such as methylene blue, Patent blue V, and Indocyanine green.
  • the gene editing composition comprises a gene editing protein
  • 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.
  • the gene editing protein may be a fusion proteins that combine homing endonucleases with the modular DNA binding domains of TALENs (megaTAL).
  • megaTAL may be delivered as a protein or alternatively, a mRNA encoding a megaTAL protein is delivered to the cells.
  • the gene editing composition comprises a RNA molecule, and the RNA molecule comprises a sgRNA, a crRNA, and/or a tracrRNA.
  • the gene editing composition comprises a RNP, and the RNP comprises a Cas protein and a sgRNA or a crRNA and a tracrRNA. Aspects of the present subject matter are particularly useful for controlling when and for how long a particular gene-editing compound is present in a cell.
  • the gene editing composition is detectable in a population of cells, or the progeny thereof, for (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12 or 0.5-72 hours after the population of cells is contacted with the aqueous solution, or (b) less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12 or 0.5-72 hours after the population of cells is contacted with the aqueous solution.
  • the genome of cells in the population of cells, or the progeny thereof comprises at least one site-specific recombination site for the Cre recombinase, Hin recombinase, or Flp recombinase.
  • aspects of the present invention relate to cells that comprise one gene editing compound, and inserting another gene editing compound into the cells.
  • one component of an RNP could be introduced into cells that express or otherwise already contain another component of the RNP.
  • cells in a population of cells, or the progeny thereof may comprise a sgRNA, a crRNA, and/or a tracrRNA.
  • the population of cells, or the progeny thereof expresses the sgRNA, crRNA, and/or tracrRNA.
  • cells in a population of cells, or the progeny thereof express a Cas protein.
  • Attorney Docket No.048831-531001WO Various implementations of the subject matter herein include a Cas protein.
  • the Cas protein is a Cas9 protein or a mutant thereof. Exemplary Cas proteins (including Cas9 and non-limiting examples of Cas9 mutants) are described herein.
  • the concentration of Cas9 protein may range from about 0.1 to about 25 ⁇ g.
  • the concentration of Cas9 may be about 1 ⁇ g, about 5 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, or about 20 ⁇ g.
  • the concentration of Cas9 may range from about 10 ng/ ⁇ L to about 300 ng/ ⁇ L; for example from about 10 ng/ ⁇ L to about 200 ng/ ⁇ l; or from about 10 ng/ ⁇ L to about 100 ng/ ⁇ l, or from about 10 ng/ ⁇ L to about 50 ng/ ⁇ l.
  • the gene editing composition comprises (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule; (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule, and further comprising a tracrRNA molecule; or
  • the ratio of the Cas9 protein to guide RNA may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • increasing the number of times that cells go through the delivery process may increase the percentage edit; wherein, in some embodiments the number of doses may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.
  • the first and second sgRNA or first and second crRNA molecules together comprise nucleic acid sequences complementary to target sequences flanking a gene, an exon, an intron, an extrachromosomal sequence, or a genomic nucleic acid sequence, wherein the gene, an exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100, kilobases in length or is at least about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100, kilobases in length.
  • the use of pairs of RNPs comprising the first and second sgRNA or first and second crRNA molecules may be used to create a polynucleotide molecule comprising the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.
  • the target sequence of a sgRNA or crRNA is about 12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17-23, or 18-22, nucleotides long. In some embodiments, the target sequence is 20 nucleotides long or about 20 nucleotides long.
  • the first and second sgRNA or first and second crRNA molecules are complementary to sequences flanking an extrachromosomal sequence that is within an expression vector.
  • Attorney Docket No.048831-531001WO Aspects of the present subject matter relate to the delivery of multiple components of a gene-editing complex, where the multiple components are not complexed together.
  • gene editing composition comprises at least one gene editing protein and at least one nucleic acid, wherein the gene editing protein and the nucleic acid are not bound to or complexed with each other. The present subject matter allows for high gene editing efficiency while maintaining high cell viability.
  • At least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the population of cells, or the progeny thereof become genetically modified after contact with the aqueous solution. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the population of cells, or the progeny thereof, are viable after contact with the aqueous solution.
  • the gene editing composition induces single-strand or double-strand breaks in DNA within the cells.
  • the gene editing composition further comprises a repair template polynucleotide.
  • the repair template comprises (a) a first flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on the other side of the single or double strand break; or (b) a first flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, Attorney Docket No.048831-531001WO 70, 80, or 90 base pairs on the other side of the single or double strand break.
  • the volume of aqueous solution is delivered to the population of cells in the form of a spray.
  • the volume is between 6.0 x 10 -7 microliter per cell and 7.4 x 10 -4 microliter per cell.
  • the spray comprises a colloidal or sub- particle comprising a diameter of 10 nm to 100 ⁇ m.
  • the volume is between 2.6 x 10 -9 microliter per square micrometer of exposed surface area and 1.1 x 10 -6 microliter per square micrometer of exposed surface area.
  • the RNP has a size of approximately 100 ⁇ ⁇ 100 ⁇ ⁇ 50 ⁇ or 10nm x 10nm x 5nm.
  • the size of spray particles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNPs per spray particle. For example, contacting the population of cells with the volume of aqueous solution may be performed by gas propelling the aqueous solution to form a spray.
  • the population of cells is in contact with said aqueous solution for 0.01-10 minutes (e.g., 0.110 minutes) prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells.
  • the population of cells includes at least one of primary or immortalized cells.
  • the population of cells may include mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, and Attorney Docket No.048831-531001WO human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cell (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, B cells.
  • T cells may include CD8+ or CD4+ T cells .
  • the CD8+ subpopulation of the CD3 + T cells are used.
  • CD8 + T cells may be purified from the PBMC population by positive isolation using anti-CD8 beads.
  • primary NK cells are isolated from PBMCs and GFP mRNA may be delivered by platform delivery technology (i.e., 3% expression and 96% viability at 24 hours).
  • NK cell lines e.g., NK92 may be used.
  • Cell types also include cells that have previously been modified for example T cells, NK cells and MSC to enhance their therapeutic efficacy, and use for 3-dimensional cultures, tissue explants, skin grafts, engineered tissues, and the like.
  • o SUA membrane area of 20 cm 2 -> seed cell density is 1*10 6 cells/cm 2 .
  • Optimal fluid height of a gas permeable membrane bioreactor systems is 10 cm -> add 200 mL of growth media to SUA with 20 cm 2 membrane area to mimic gas permeable membrane bioreactor conditions resulting in a starting cell density of 1*10 5 cells/mL.
  • start rocking motion at the optimal rocking rate determined in the pre-experiment. o Take daily samples to measure cell concentration and viability.
  • Example Systems and Methods described herein Modification of the SUA creates a fully closed system, introduces sensor technology as well as the ability to operate in full perfusion mode, thereby laying the foundation to get to even higher volumetric productivity and the high seed density approach.
  • Adding a gas overlay allows for control of dissolved oxygen concentration via an airflow and for removal of excess CO 2 in the gas outflow.
  • Attorney Docket No.048831-531001WO Introduce an outlet for spent media removal close to the top of the liquid surface at full operating volume of 200 mL.
  • the position of the outlet has to be adapted.
  • Some implementations can add an inlet and outlet to the gas phase, allowing for an airflow across the liquid surface.
  • Dissolved oxygen concentration can be controlled via airflow and overlay pressure. This can also facilitate the removal of excess CO 2 from the gas phase, thereby establishing a gradient that drives CO2 out of the liquid phase.
  • a fully closed system is developed that minimizes contamination risk and closed system validation is performed.
  • Some implementations can install at least pH and Oxygen sensors, and can add a sensor that allows determining the viable cell concentration in the reaction chamber. If perfusion operation is intended as the preferred mode of operation, sensors that measure liquid phase conditions can be installed in the spent media outlet channel.
  • CSTR continuous stirred tank reactor
  • FIG. 42 depicts this situation. If fed batch operation is the preferred mode of operation and/or if mixing conditions are not that of a CSTR, all sensors need to be installed in the reaction chamber. This is outlined in FIG. 43. Further renderings implement control loops for pH, O 2 , and potentially cell concentration. pH can be controlled through NaOH addition, O2 can be controlled through airflow and overlay pressure, and cell concentration measurements can be used in a Attorney Docket No.048831-531001WO feedback loop to control media feed through a cell specific media feed rate. FIG. 44 outlines the approach. The current transfection protocol operates at an optimized total cell number of 20 M transfected cells.
  • TIL Tumor Infiltrating Lymphocyte
  • pooling five transfection cycles will lead to a seed stock of 100 M cells, a five-fold increase Attorney Docket No.048831-531001WO in starting cell concentration.
  • this high seed concentration might be of concern due to mass transfer and oxygen supply limitations.
  • the advanced mass transfer and process control properties of the methods and devices described herein may be able to overcome these concerns and support healthy expansion at higher initial density.
  • Experiments should be conducted to evaluate the optimum number of transfection cycles that can be pooled while at the same time supporting healthy expansion in the methods and devices described herein.
  • the integrated system/process and viral transduction is added to the cell engineering capabilities prior to expansion.
  • the filter is coated with promotors and other materials during the transduction process.
  • Scale out One of the concerns regarding commercial viability of autologous cell manufacturing is the “one batch per patient” nature of patient specific therapy. Serving a large number of patients cannot be accomplished by traditional approaches to scale up manufacturing volume; each patient is served with their own individual manufacturing batch. Therefore, patient numbers must scale through parallel operation of individual batches, often described as a scale-out approach. Consequently, a commercial version of the methods and devices described herein need to allow operation in parallel. This will require decoupling of the transfection process from expansion.
  • Transfection could occur in the SUA using existing equipment, after which that same SUA is transferred to an Attorney Docket No.048831-531001WO expansion system, where many SUAs can be operated in parallel. These systems could be simple, requiring only basic rocking (to provide appropriate mixing), parallel pumps for media supply, and basic control functions for temperature, pH, O 2 , and media feed.
  • phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features.
  • the term “and/or” may also occur in a list of two or more elements or features.
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
  • the subject matter described herein can be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. The implementations set forth in the foregoing description do not represent all implementations consistent with the subject matter described herein.

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Abstract

La présente étude concerne une plate-forme d'ingénierie cellulaire pour la distribution sans vecteur et/ou virale de composés et de compositions de charge utile/charge dans les cellules. La plateforme permet l'administration à des cellules rapidement et d'une manière facile à utiliser. Des appareils, des systèmes, des techniques, des compositions et des articles connexes sont également décrits.
PCT/IB2025/050324 2024-01-12 2025-01-10 Plateforme de distribution avec transfection non virale intégrée et traitement de cellule Pending WO2025149983A2 (fr)

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