阅读说明：本技术 细胞工程平台 (Cell engineering platform ) 是由 M·麦圭尔 于 2019-05-31 设计创作，主要内容包括：本发明主题内容提供了一种诊断和临床应用规模的细胞工程平台,用于将有效载荷/货物化合物和组合物无载体传递或者病毒传递到非粘附细胞中。该平台可以实现快速的向一个封闭系统中传递大量细胞。这里还描述了相关的仪器、系统、技术、物品和组合物。(The present subject matter provides a diagnostic and clinical application scale cellular engineering platform for vector-free delivery or viral delivery of payload/cargo compounds and compositions into non-adherent cells. The platform can realize rapid transfer of a large number of cells into a closed system. Related apparatus, systems, techniques, articles, and compositions are also described herein.)

1. A method, comprising:

filling a mixture of cells and a first culture medium into a chamber of a cell engineering platform; and is

Discharging the first culture medium from the chamber through a filter, allowing the cells to deposit on the filter,

wherein the cell engineering platform comprises:

a chamber;

a cover disposed at a first end of the chamber;

a base disposed at a second end of the chamber; and

a filter holder disposed within the chamber.

2. The method of claim 1, further comprising spraying a delivery solution comprising an osmotic agent and a payload onto the cells deposited on the filter.

3. The method of claim 2, further comprising applying a stop solution in the chamber.

4. The method of claim 3, further comprising filling the chamber with a second medium to resuspend the cells from the filter.

5. The method of claim 4, wherein the drained first culture medium is reused as the second culture medium.

6. The method of claim 4, further comprising agitating the chamber.

7. The method of claim 4, further comprising extracting the resuspended cells from the chamber.

8. The method of claim 4, wherein filling the chamber is accomplished automatically by a pump and controller.

9. The method of claim 1, further comprising culturing cells within the chamber.

10. The method of claim 1, wherein the first medium is drained from the chamber by providing a positive pressure into the chamber.

11. The method of claim 1, wherein the first medium is drained from the chamber by gravity.

12. The method of claim 3, wherein a stop solution is applied to wash the cells.

13. The method of claim 4, wherein filling the chamber with the second medium is performed as at least one of a cell washing process, a cell concentration changing process, and/or a cell culture medium changing process.

14. A system, comprising:

a housing configured to receive a filter plate, the filter plate including an aperture therein;

a differential pressure applicator configured to apply different pressures into the bore;

a delivery solution applicator configured to deliver an atomized delivery solution into the well;

a stop solution applicator configured to deliver a stop solution to the well; and

a media applicator configured to deliver media into the well.

15. The system of claim 14, wherein the housing comprises:

a chamber;

a cover disposed at a first end of the chamber; and

a base disposed at a second end of the chamber;

wherein the filter plate is disposed within the chamber.

16. The system of claim 15, wherein the differential pressure applicator is coupled to a cover of the housing and includes a showerhead having a plurality of holes at an end thereof.

17. The system of claim 15, wherein a stop solution applicator delivers the stop solution into the well through a septum disposed on a lid of the housing.

18. The system of claim 15, wherein the medium applicator can deliver the medium into the well through a septum disposed in a lid of the housing or through a port of the chamber.

19. The system of claim 14, wherein the housing is configured to tilt the filter plate.

20. The system of claim 14, wherein the delivery solution applicator comprises a robotic arm and a spray head, the robotic arm being magnetically coupled to the spray head.

21. The system of claim 20, further comprising a soft elastic barrier surrounding the filter plate and the showerhead, the soft elastic barrier separating the robotic arm and the showerhead.

22. The system of claim 20, wherein the robotic arm is configured to transport the spray head to a plurality of locations on the filter plate.

23. The system of claim 14, wherein the stop solution applicator and the media applicator are integrally formed as a port within a housing, and the housing forms a container.

24. The system of claim 23, wherein the container includes a spent media outlet for collecting spent media.

25. The system of claim 14, further comprising a soft elastomeric barrier enclosing the filter pad and the transfer solution applicator within the housing, the soft elastomeric barrier and the housing forming a bioreactor.

26. The system of claim 14, wherein the housing comprises a filter plate base configured to tilt, rotate, and/or vibrate the filter plate.

27. The system of claim 14, wherein the delivery solution applicator comprises a robotic arm and a spray head, the spray head being a disposable device.

28. The system of claim 14, wherein the filter plate is sized to accommodate 10⁷More than one T cell.

29. The system of claim 14, wherein the system is configured to automatically:

providing cells to a filter plate in a culture medium;

removing the culture medium to form a cell monolayer on top of the filter plate;

applying a nebulized delivery solution on the cell monolayer;

culturing the cell;

applying a stop solution to the cultured monolayer of cells; and

providing new media to the cell monolayer.

30. The system of claim 29, wherein the system is configured to automatically tilt, vibrate, and/or rotate the filter plate to resuspend the cells in new media.

31. The system of claim 29, wherein the system is configured to repeatedly apply the nebulized delivery solution, the culturing, and the administration stop solution.

32. The system of claim 14, wherein the delivery solution applicator comprises an atomizer.

33. The system of claim 32, wherein the delivery solution applicator further comprises a mass flow controller or a volumetric flow controller to regulate the flow of gas to operate the nebulizer.

34. The system of claim 14, wherein the delivery solution applicator is configured to deliver 10-300 microliters of delivery solution per actuation.

35. The system of claim 14, further comprising a temperature control system configured to control a temperature of the transfer solution and/or the filter sheet comprising pores.

36. The system of claim 14, wherein the delivery solution comprises an aqueous solution comprising a payload and an alcohol at a concentration greater than 2% (v/v).

37. The system of claim 36, wherein the alcohol comprises ethanol.

38. The system of claim 36, wherein the aqueous solution comprises greater than 5% ethanol.

39. The system of claim 36, wherein the aqueous solution comprises 5-30% ethanol.

40. The system of claim 36, wherein the aqueous solution comprises 12% or 25% ethanol.

41. The system of claim 36, wherein the aqueous solution comprises 12.5-500mM KCl.

42. The system of claim 36, wherein the aqueous solution comprises 106mm kcl.

43. The system of claim 14, further comprising the filter plate and the well can be configured to contain a plurality of non-adherent cells.

44. The system of claim 43, wherein the non-adherent cells comprise peripheral blood mononuclear cells.

45. The system of claim 43, wherein the non-adherent cells comprise immune cells.

46. The system of claim 43, wherein the non-adherent cells comprise T lymphocytes.

47. The system of claim 36, wherein the payload may comprise a messenger ribonucleic acid (mRNA).

48. The system of claim 47, wherein the mRNA encodes a gene editing composition.

49. The system of claim 48, wherein the gene-editing composition reduces the expression of PD-1.

50. The system of claim 47, wherein the mRNA encodes a chimeric antigen receptor.

51. The system of claim 14, for delivering a cargo compound or composition to a mammalian cell.

52. The system of claim 43, wherein the population of non-adherent cells is a monolayer.

53. A system, comprising:

a chamber;

a cover disposed at the first end of the chamber;

a base disposed at a second end of the chamber; and

a filter holder disposed within the chamber.

54. The system of claim 53, wherein the filter holder comprises a plurality of holes arranged in a predetermined pattern.

55. The system of claim 54, wherein the filter holder comprises a plurality of targets, wherein the plurality of wells are arranged to allow cells to be deposited on the filter at areas corresponding to the plurality of wells.

56. The system of claim 53, further comprising a gasket disposed between the filter holder and the base.

57. The system of claim 55, further comprising a filter holder insert positioned within the filter holder to receive the filter between the filter holder and the filter holder insert.

58. The system of claim 57, wherein the filter holder insert comprises a concave top surface.

59. The system of claim 57, wherein the filter holder insert comprises a plurality of openings corresponding to the plurality of targets.

60. The system of claim 59, wherein the filter holder comprises three targets.

61. The system of claim 59, wherein the filter holder comprises seven targets.

62. The system of claim 59, wherein the filter holder comprises 19 targets.

63. The system of claim 57, wherein the plurality of targets are arranged in a square pattern, a rectangular pattern, a triangular pattern, or a linear pattern.

64. The system of claim 53, further comprising a controller configured to operate at least one of a pump, a valve, a heating element, a cooling element, and a stirring device.

65. The system of claim 64, wherein the pump is a peristaltic pump or a volumetric pump.

66. The system of claim 53, wherein the cap further comprises a pressure port and an aperture.

67. The system of claim 53, wherein the base comprises a port for receiving or discharging the medium.

68. The system of claim 66, wherein the cap further comprises a septum.

69. The system of claim 68, wherein the pressure port comprises a showerhead including a plurality of holes at an end thereof.

70. The system of claim 66, wherein a 0.2 micron filter is connected to the pressure port.

71. The system of claim 70, further comprising a pinch valve configured to open and close a pressure port to atmosphere through the 0.2 micron filter.

72. The system of claim 53, wherein the chamber of the system comprises a port for extracting the processed cells.

73. The system of claim 53, wherein the system is sized to handle sizes greater than 1x10⁹The T cell of (1).

74. Apparatus, systems, techniques, compositions, and articles of manufacture as described or illustrated herein.

Technical Field

The subject matter described herein refers to cellular engineering platforms that do not utilize vector delivery.

Background

There are differences in cell transfection efficiency between different cell types. Transfection of suspension cells, such as non-adherent cells, has proven to be very difficult using traditional methods, particularly when used on a scale.

Disclosure of Invention

The present subject matter provides a cellular engineering platform that scales with technologies for carrier-free delivery of payloads/cargo compounds and compositions into non-adherent cells. The platform can realize rapid transfer of a large number of cells. For example, some embodiments of the platform may be delivered 10 at a time for diagnostic and therapeutic use⁷To 10⁹One or more cells. The platform can be a closed system, can realize sterile transfection, can transfer mRNA and RNP (ribonucleoprotein particles) to primary T cells, is convenient to use, can be transferred repeatedly, and the like.

By performing the following steps: by providing a population of non-adherent cells and contacting the population of cells with a volume of aqueous solution comprising a payload and an alcohol at a concentration greater than 2% (v/v), the platform of the invention can effect delivery of the payload to the plasma membrane of the non-adherent cells. For example, the alcohol comprises ethanol, e.g., greater than 5% ethanol. In some embodiments, the aqueous solution comprises 5-30% ethanol, for example 12% or 25% ethanol. Other combinations are also possible.

In one aspect, disclosed herein is a method comprising filling a chamber of a cell engineering platform with a mixture of cells and a first medium, and draining the first medium from the chamber to pass the mixture through a filter to deposit the cells on the filter. The cell engineering platform can include a chamber, a lid disposed at a first end of the chamber, a base disposed at a second end of the chamber, and a filter holder disposed within the chamber.

One or more of the following features may be included in any feasible combination. The method can include spraying a delivery solution containing an osmotic agent and a payload onto the cells deposited on the filter. The method may include applying a stop solution in the chamber. The method may also further comprise filling the chamber with a second medium to resuspend the cells from the filter. The discharged first culture medium can be reused as a second culture medium. The method may include agitating the chamber. The method may include extracting resuspension cells from the chamber. Filling of the chamber may be accomplished automatically by a pump and controller. The method may include culturing the cells in the chamber. The first medium may be expelled from the chamber by providing positive pressure to the chamber. Alternatively or additionally, the first culture medium may be drained from the chamber by gravity. In addition, a stop solution may be applied to wash the cells. Filling the chamber with the second medium may be performed as a cell washing process, a cell concentration changing process and/or a cell culture medium changing process.

In another aspect, a system includes a housing configured to receive a filter plate, the filter plate including an aperture therein; a differential pressure applicator configured to apply different pressures into the bore; a delivery solution applicator configured to deliver the atomized delivery solution into the well; a stop solution applicator configured to deliver a stop solution to the well; and a medium applicator configured to deliver the medium into the well.

One or more of the following features may be included in any feasible combination. The housing may include a chamber, a cover disposed at a first end of the chamber, and a base disposed at a second end of the chamber. The filter plate may be disposed within the chamber. The differential pressure applicator may be connected to a lid of the housing and include a showerhead having a plurality of apertures at an end of the showerhead. The stop solution applicator may deliver the stop solution into the well through a septum disposed in the housing cover. The medium applicator may deliver the medium into the well through a septum disposed in the housing cover or through a port of the chamber. The housing may be configured as an inclined filter plate. The delivery solution applicator may include a mechanical arm and a spray head, the mechanical arm being magnetically coupled to the spray head. The system may include a soft, resilient barrier surrounding the filter plate and the showerhead. A soft elastomeric barrier separates the mechanical arm and the spray head. The mechanical arm is configured to transport the spray head to a plurality of locations on the filter plate. The stop solution applicator and the medium applicator are integrated as a port within a housing, and the housing forms a container. The vessel includes a spent media outlet for collecting spent media.

The system may further include a soft elastomeric barrier enclosing the filter pad and the delivery solution applicator within the housing, the soft elastomeric barrier and the housing forming the bioreactor. The housing may include a filter plate base configured to tilt, rotate, and/or vibrate the filter plate.

The delivery solution applicator may include a robotic arm and a spray head, the spray head being a disposable device. The filter plate is sized to receive 10⁷More than one T cell. The system may be configured to automatically: providing cells to a filter plate in a culture medium; removing the culture medium to form a cell monolayer on top of the filter plate; applying an aerosolized delivery solution on the cell monolayer; culturing the cells; applying a stop solution to the cultured monolayer of cells; providing a new culture medium to the cell monolayer; the filter plate is tilted, vibrated and/or rotated to resuspend the cells in fresh media. The system is configured to repeatedly apply the nebulized delivery solution, the culturing, and the administration stop solution.

The delivery solution applicator may include an atomizer. The delivery solution applicator may be configured to deliver 10-300 microliters of delivery solution per actuation. The system may also further include a temperature control system configured to control the temperature of the delivery solution and/or the plate (including wells). The delivery solution can include an aqueous solution including a payload and an alcohol at a concentration greater than 2% (v/v). The alcohol comprises ethanol. The aqueous solution may comprise greater than 5% ethanol. The aqueous solution may comprise 5-30% ethanol. The aqueous solution may comprise 12% or 25% ethanol. The aqueous solution may comprise 12.5-500mM KCl (potassium chloride). The aqueous solution may comprise 10⁶mM KCl。

The system may further comprise a filter plate, and the well may be configured to contain a plurality of non-adherent cells. The non-adherent cells may comprise peripheral blood mononuclear cells. The non-adherent cells may comprise immune cells. The non-adherent cells may comprise T lymphocytes. The payload may include, among other things, messenger ribonucleic acid (mRNA). The mRNA may encode a gene editing composition. The gene editing composition can reduce the expression of PD-1 (programmed cell death protein 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 may be a monolayer.

In yet another aspect, a system may include a chamber; a cover disposed at the first end of the chamber; a base disposed at the second end of the chamber; and a filter holder disposed within the chamber.

One or more of the following features may be included in any feasible combination. The filter holder may include a plurality of holes arranged in a predetermined pattern. The filter holder may comprise a plurality of targets, wherein the plurality of wells are arranged to allow deposition of cells on the filter at areas corresponding to the plurality of wells. The system may also include a gasket disposed between the filter holder and the base. The system may also include a filter holder insert positioned within the filter holder to receive the filter between the filter holder and the filter holder insert. The filter holder insert may include a concave top surface. The filter holder insert may include a plurality of openings corresponding to the plurality of targets. The filter holder may comprise three targets. The filter holder may include seven targets. The filter holder may comprise 19 targets. The plurality of targets may be arranged in a square pattern, a rectangular pattern, a triangular pattern, or a linear pattern.

The system may also include a controller configured to operate at least one of the pump, the valve, the heating element, the cooling element, and the agitation device. The pump may be a peristaltic pump or a volumetric pump. The cap may also include a pressure port and an aperture. The base may include a port for receiving media or discharging media. The cap may further comprise a septum. What is needed isThe pressure port may include a showerhead including a plurality of apertures at an end thereof. A 0.2 micron filter may be connected to the pressure port. The system may also include a pinch valve configured to open and close a pressure port to atmosphere through the 0.2 micron filter. The chamber of the system may include a port for extracting the processed cells. The system can handle the size of more than 1x10⁹The T cell of (1).

The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims.

Drawings

Fig. 1 shows an exemplary embodiment of a process involving cell engineering.

Fig. 2 illustrates an exemplary embodiment of a cellular engineering platform for carrier-free payload delivery across a cell membrane.

FIG. 3 shows an exemplary embodiment of the cell engineering platform of FIG. 2, a portion of which is broken away for illustrative purposes.

FIGS. 4A-4C show a lid of an exemplary embodiment of the cell engineering platform.

Fig. 5 shows a base according to an exemplary embodiment of a unit engineering platform.

FIG. 6 shows a filter holder in an exemplary embodiment of the cell engineering platform of the present invention.

FIGS. 7A-7C show various configurations of filter holder inserts in exemplary embodiments of the cell engineering platform of the present invention.

FIGS. 8A-8C illustrate various configurations of a gasket in exemplary embodiments of the cell engineering platform of the present invention.

FIGS. 9A-9C show a flow chart for the delivery of payload across a cell membrane without a carrier.

FIG. 10 shows an exemplary embodiment of a cell engineering platform.

Fig. 11A-11C show an exemplary embodiment of an atomizer for a spraying process. Fig. 11D shows an exemplary image after the cell deposition process, and fig. 11E-11G show schematic diagrams of mass flow regulation of the operation of the nebulizer.

Fig. 12 is a computer-aided design (CAD) drawing showing an exemplary embodiment for mounting three jets.

Fig. 13 shows a CAD drawing of only one showerhead.

Fig. 14A-14F show an exemplary embodiment of a display chamber assembly.

Figure 15A shows an image of an exemplary embodiment of a cell engineering platform with a support system and mounting frame, and figure 15B shows an image of the platform after chamber removal.

Fig. 16 is a computer-aided design (CAD) diagram showing an exemplary clinical cell engineering platform for cell therapy.

Fig. 17 is an enlarged view of a portion of the platform.

Fig. 18 is a CAD drawing showing an embodiment of a filter plate and filter.

Fig. 19 is a CAD drawing showing a cross-sectional view of an exemplary platform and filter plate rotation.

FIGS. 20A-20C show a series of pictures showing the various components of an exemplary embodiment of the platform.

FIG. 21 is a functional block diagram of an exemplary platform.

FIG. 22 is a schematic diagram showing the disposable and reusable portions of an exemplary platform.

FIG. 23 is a table showing some experimental performance capabilities of an exemplary platform.

FIG. 24 is a table illustrating compatible techniques that may be integrated with the exemplary platform of the present invention.

FIG. 25 is a flow chart showing exemplary cell engineering process steps.

Fig. 26 is a flow diagram showing that this exemplary platform can address cell engineering issues encountered during the production of adoptive cell therapies.

Figure 27 is a series of diagrams showing other exemplary bioreactor designs used in the present platform.

Fig. 28 is an image of kymeriah (tisageneleclecel) approved by the U.S. food and drug administration (US FDA) for the treatment of Acute Lymphoblastic Leukemia (ALL) in children and young adults, the latter using human own T cells to combat cancer.

FIG. 29 is a process flow diagram illustrating a sub-process of some aspects of the inventive subject matter.

FIG. 30 shows an exemplary operating cycle of an exemplary platform, similar to that shown in FIG. 29.

Figure 31 shows a comparison of processing time and complexity to isolate and activate 100 million cells. Peripheral Blood Mononuclear Cells (PBMCs), a cluster of differentiated 3 cells (CD3+) and Pan T cells were compared.

FIG. 32 shows the results of comparing the treatment starting materials (e.g., cells and activating reagents) to the number of cycles, number of cells, number of cryovials, and number of cryobags

FIG. 33 illustrates another exemplary platform of the present subject matter.

FIG. 34 is a CAD drawing showing an embodiment of a disposable bioreactor.

Figure 35 shows a filter plate container with addition of stop solution.

Figure 36 shows the filter plate vessel with fresh media added.

FIG. 37 shows the inclination of the bioreactor during operation.

Figure 38 shows the filter plate container during "decanting" of the cell culture medium, wherein the container (e.g., reactor) is tilted, rotated, and vibrated to facilitate removal of cells from the culture medium.

Fig. 39 shows an embodiment of a membrane scaffold.

Fig. 40 shows an exemplary system. This is an intermediate system in which up to 5x10 can be loaded⁷And (4) cells. The system and clinical system (full scale, 1x 10)⁹One or more cells) have the same characteristics but do not include a translational jet system. Instead, the system utilizes a single showerhead or a multi-headed array of showerheads.

Like reference symbols in the various drawings indicate like elements.

Detailed description of the preferred embodiments

Despite some advances, delivery of certain particles and/or molecules to cells remains a challenge. Factors such as the size or charge of the molecule can limit and/or prevent entry of the molecule into the cell. In particular, transmission across cell membranes can be complex due to the molecules and/or membranes of the cell. The cell membrane or plasma membrane is a semi-permeable biological membrane that acts as a selective barrier. The cell membrane regulates the internal chemical composition of the cell. For example, the selective molecule can only passively diffuse through the cell membrane to the cell membrane. Hydrophobic small molecules (e.g. O)₂、CO₂And N₂) And uncharged small polar molecules (e.g., H)₂O and glycerol) can passively diffuse to the cell membrane. Larger, uncharged polar molecules (e.g., amino acids, glucose, and nucleotides) and ions (e.g., H)⁺、Na⁺、K⁺And Cl^-) Cannot passively spread across the cell membrane.

Fig. 1 illustrates an exemplary embodiment of a treatment/process involving cell engineering. Referring to fig. 1, cells can be extracted from a patient, isolated (e.g., concentrated or enriched), and then processed using cell engineering methods. The cells engineered may be expanded and returned to the patient. For trans-cell membrane delivery, a method using a viral vector can be used. However, viral vector-based methods often require costly and complex processes, provide limited accessibility, and produce variable and inconsistent results. Electroporation-based methods may also be used. However, electroporation-based methods generally result in higher cell damage and provide poor cell recovery and cell function.

It is an object of the present invention to provide a vector-free delivery method to address the cost and complexity challenges of cell engineering techniques. In order to provide a reliable and consistent method of cell therapy, the present subject matter provides a cell engineering method and platform for delivering a compound or mixture of compounds (e.g., a payload) across a cell membrane by contacting the cell with a delivery solution (e.g., a vehicle) containing the payload and an agent that reversibly permeates or lyses the cell membrane. Specifically, the system supplies the cells into a suspension, forms a monolayer of the cells by draining the suspension, uses (e.g., sprays) the delivery solution to permeate the cells, and the payload is transported through the permeated cell membrane.

Using some embodiments of the platform of the present invention, additional cell engineering processes can also be performed before and/or after the carrier-free payload transfer process, which significantly increases productivity and simplifies the overall process. Furthermore, both non-viral and viral transfection methods can be performed within a single platform. Thus, the exemplary embodiments of the platform may provide better down-scaling than other methods or systems, and may process 10 in a single process¹¹Or more cells.

In some embodiments, the platform for payload delivery across cell membranes may be used for vector-free payload delivery, viral payload delivery, or a combination of vector-free and viral payload delivery. For example, cells can be collected on a filter substrate by draining the cell-containing medium through a filter, after which a solution comprising a viral payload can be delivered to the collected cells, thereby effecting delivery of the viral payload in the platform. Alternatively or additionally, the vector-free viral payload delivery process may be performed on the same cells within a single device, which may reduce processing steps, time, and/or cost, increase cell throughput, viability, and effectiveness of treatment, and reduce cell contamination that may occur when cells are transported between multiple devices. Additional aspects of viral payload delivery are discussed in U.S. provisional patent application No. 62/855241, attorney docket No. 048831-520P01US, entitled "method of viral delivery to a cell population and viral production" filed on 31/5/2019, which is hereby incorporated by reference in its entirety.

Furthermore, some embodiments of the inventive subject matter can provide a cellular engineering platform that can combine payloads/cargoThe carrier-free delivery techniques of the objects and compositions are scaled to non-adherent cells. This example platform can enable the delivery of large numbers of cells quickly. For example, some embodiments of the platform may deliver 10 in a single use⁷To 10⁹One or more cells. The platform can be a closed system, can perform sterile transfection, can transfer mRNA and RNP to primary T cells, is convenient to use, can be repeatedly transferred, and the like. In addition, Human Embryonic Kidney (HEK) cells can be treated using the platform of the invention.

This platform allows for the delivery of a payload across the cytoplasmic membrane of a non-adherent cell by: providing a population of non-adherent cells and contacting the population of cells with a volume of an aqueous solution comprising a payload and an alcohol at a concentration greater than 2% (v/v). For example, the alcohol comprises ethanol, e.g., greater than 5% ethanol. In some embodiments, the aqueous solution comprises 5-30% ethanol, such as 12% or 25% ethanol. Other ingredients are also possible.

The present subject matter also provides a platform that can automate the process of delivery of carrier-free and/or viral payloads and allow the process to be performed on a variety of scales. When cells are manually loaded onto and/or unloaded from the platform, the throughput of the system is limited, thereby presenting difficulties in the application of clinical procedures/treatments. Depending on the operator and/or the various environmental parameters, contamination and inconsistent process problems may exist. By process automation, the carrierless payload transport process can be performed more consistently, contamination concerns can be significantly reduced, and therefore the system can be more easily scaled. In the following, exemplary embodiments of the platform will be described to perform a bearer-less payload transfer process with automated processing.

Example 1

Fig. 2 shows an exemplary embodiment of a cell engineering platform 100 for carrier-free payload delivery across a cell membrane, and for illustration purposes, partially exploded components of the exemplary embodiment of the cell engineering platform 100 of fig. 2 are shown in fig. 3. Referring to fig. 2 and 3, the platform 100 for cell engineering includes a chamber 110, a cover 120 disposed at a first end of the chamber 110, a base 130 disposed at a second end of the chamber 110, a filter holder 140, and a filter holder insert 150. The platform may include a gasket 160 disposed between the base 130 and the filter holder 140. The platform 100 may also include a controller.

The chamber 110 is surrounded by chamber walls. To provide optical viewing capabilities, the chamber walls may comprise (e.g., be made of) a transparent plastic material, such as polypropylene, acrylic, polycarbonate, and the like. The chamber wall may have the overall shape of a cylindrical housing, with top and bottom surfaces open, and the diameter and height of the chamber 110 may be determined based on the application and system requirements. For example, the inner diameter of the chamber 110 may be 110 mm. Alternatively or additionally, chamber 110 may include a flat surface to facilitate monitoring and/or controlling the transfection process using an optical imaging device. Although the chamber 110 is shown as a cylindrical housing, the invention is not so limited and the chamber 100 may have various shapes, e.g., square, rectangular, and triangular, as well as other configurations, such as those in which cell targets are arranged in a linear configuration.

Fig. 4A-4C are pictures showing the cover 120. Fig. 4A shows the top surface of the cover 120, fig. 4B shows the bottom surface of the cover 120, and fig. 4C shows the side surface of the cover 120. A lid 120 is disposed on top of the cell engineering platform 100 to seal the chamber 110 of the platform 100. For sealing, the cap 120 may include an o-ring 121. The cap 120 may include a pressure port 122. Pressure port 122 allows chamber 110 to be connected to a pressure source. The pressure source may provide positive or negative pressure to the chamber 110 of the platform 100. Referring to fig. 4A and 4C, the pressure source may be connected to the top of the pressure port 122, which is located above or outside the lid 120. The bottom of the pressure port 122 (below or inside the lid 120) may include a showerhead including a plurality of holes for distributing fluid over the plurality of holes for more uniform supply of pressure into the chamber 110. As shown in fig. 4C, the plurality of apertures of the showerhead may be substantially sideways to prevent the gas flow (e.g., gas or liquid) conveyed through the apertures from interfering with (e.g., purging) cells deposited on the filter.

Alternatively or additionally, pressure port 122 may allow chamber 110 to be connected to (e.g., exposed to) ambient pressure. In addition, pressure port 122 may allow the chamber to be connected to ambient pressure through a filter. For example, the filter may be a 0.2 micron filter. The filter may also be a nanoparticle filter and/or a High Efficiency Particulate Air (HEPA) filter. When the pressure port 122 is connected to ambient pressure through a filter, the chamber 110 may drain due to gravity rather than the pressure source. A valve may be provided upstream of the filter in order to open or close the pressure port. For example, the valve may be a pinch valve.

The lid 120 may comprise (e.g., consist of) a stainless steel material, such as SS316, a plastic material, and the like. The cap 120 may also include at least one aperture 123 to accommodate a nebulizer, sensor, etc. The sensor may include one or more of a temperature sensor, a humidity sensor, and a pressure sensor. When the atomizer is mounted in the at least one hole 123, the atomizer may be mounted by an orientation adjustment means, such as a universal joint, to allow adjustment of the orientation of the atomizer. In some embodiments, the cover 120 may include at least one mounting hole 124 to receive a post 170 to secure the cover 120 to the post 170 and support the platform 100.

Fig. 5 shows the base 130 of the platform 100. The base 130 is disposed at the bottom of the chamber 110 and mounts the membrane holder 140. The base 130 may include an oil drain 131. Alternatively or additionally, the base 130 can include one or more vacuum ports, conduits connected to a syringe (for resuspending cells in a medium to provide an opportunity for resuspension at different concentrations or under different media), heating elements, temperature sensors (e.g., PT100 RTD, thermocouple, etc.), and vibrating devices to provide agitation during the process. The base 130 may include an aperture 132 to receive a post 170 mounted thereon.

Fig. 6 shows a filter holder 140. The filter holder 140 may be made of acetal, aluminum, or the like. The filter holder 140 is disposed at the bottom of the chamber 110 and fixed to the base 130. The filter holder 140 may receive at least one filter on an upper surface thereof. The filter holder 140 can include a target configured to allow cells to be deposited on the filter. The filter holder 140 may include a plurality of targets. The filter holder 140 may include three targets. At a location corresponding to each target, the filter holder 140 may include a plurality of holes 141 to allow cell suspension media to be delivered through the holes 141. When positive pressure is applied to the chamber 110 through the pressure port 122 of the lid 120, the cell suspension culture may be discharged through the plurality of holes 141 and the base 130 while the cells are collected on the filter surface, thereby forming a monolayer of cells. Alternatively or additionally, draining may be performed by gravity draining the media by opening a valve connected to the pressure port 122 of the 0.2 micro filter to communicate with ambient pressure. The plurality of holes 141 may be arranged to have a predetermined pattern. For example, the apertures 141 may be aligned around the outer diameter of the filter and/or along multiple radial directions of the filter. Filter holder 140 may also include an o-ring 142 to provide a seal between filter holder 140 and chamber 110.

Fig. 7A-7C show various configurations of filter holder insert 150. Filter holder insert 150 is disposed on the bottom of chamber 110 and the upper surface of filter holder 140 to secure (e.g., secure or clamp) the filter between filter holder 140 and filter holder insert 150. As shown in fig. 7A-7C, filter holder insert 150 may have various configurations. For example, the filter holder insert 150 can have a configuration that includes three targets (fig. 7A), one target (fig. 7B), or an open interior portion (fig. 7C). Filter holder insert 150 with three goals (fig. 7A) promotes cell distribution and cell recovery only within filtration zone 151. When smaller numbers of cells are required, a filter holder insert 150 with one target (FIG. 7B) can be used. As shown in fig. 2, filter holder insert 150 may include a concave shaped upper surface to allow cells to be deposited on the filter to more effectively receive the spray plume. The concave shape also prevents the filter from bulging near the center during filling and draining and resuspending the cells. The configuration of the targets is not limited to the illustrated configuration, and may also have more than three targets, for example, 7 to 19 or more targets. The plurality of targets may be arranged in a square, rectangular, triangular, and/or linear configuration.

In some embodiments, platform 100 can include gasket 160, such as a rubber gasket, to provide a seal between base 130 and the bottom surface of filter holder 140. The seal between the base 130 and the filter holder 140 prevents cell culture medium from flowing between the base 130 and the filter holder 140, allowing the entire cell culture medium to flow through the filter, thereby improving cell collection efficiency. As shown in fig. 8A-8C, the gasket 160 can have various shapes corresponding to filter configurations. Fig. 8C illustrates a configuration of a gasket 160 suitable for use with the open interior structure shown in fig. 7C. Fig. 8A and 8B show a gasket 160 to be used with the three target configurations shown in fig. 7A.

In some embodiments, the platform 100 includes a pump, such as a peristaltic pump or a volumetric pump, and a valve for automatically controlling the flow of fluid. The pump may be controlled by a controller. The controller may also control the atomizer, the temperature/pressure sensor, the heating element of the base, and the vibrating device. For nebulizer control, a Human Machine Interface (HMI) (e.g., ohilon NB3Q-TW01B 5 inch HMI) and Programmable Logic Controller (PLC) (e.g., ohilon NX1 NX1P29024DT1 PLC) hardware platform and a 3 channel rotary controller Printed Circuit Board (PCB) may be included in the controller. The controller may include a plurality of modules that are responsible for controlling the various elements of the platform. The controller may also include a processor configured to execute program instructions to perform the vector-free payload transfer process, operate the input/output device for the user interface, and operate the communication module to connect the platform to the network.

Referring to fig. 9A, fig. 9A depicts an exemplary method of carrier-free payload delivery across a cell membrane. In operation, the target cells may be mixed in a medium of a specific concentration. For example, about 6000 million cells may be mixed in about 60 milliliters of culture medium. The prepared cell-containing culture medium may be introduced into the chamber via a disposable tubing set and/or a sterile needle/cannula (step S11). The cell-containing medium may be supplied to the chamber through a port in the septum or lid (e.g., a septum for receiving a plastic welded tube). The addition process may be performed manually or automatically using a pump (e.g., a peristaltic or volumetric pump) and a controller. After the cell-containing medium is added to the chamber, the chamber is sealed by a shut-off valve. Similarly, the valve operation may be performed manually, or may be performed automatically using, for example, a solenoid valve and a controller.

After the valve is closed and the chamber is sealed, the culture medium is discharged through the filter, thereby depositing target cells (e.g., T cells) on the surface of the filter (step S12). For example, positive pressure may be supplied to the chamber through a central pressure port of the lid. After the medium is evacuated from the chamber, vacuum pressure may be provided to the chamber to relieve the positive pressure remaining in the chamber. It may or may not be necessary to remove the remaining positive pressure with vacuum pressure. In some embodiments, positive and vacuum pressures may be alternately provided to the chamber during media evacuation to adjust/rearrange cell deposition on the filter. The media may also be discharged by opening a valve (e.g., pinch valve), exposing a pressure port of the cap to ambient pressure, and discharging the media by gravity. A filter (e.g., a 0.2 micron filter) may be provided at the pressure port to prevent foreign particles from entering the chamber.

Fig. 11D shows an exemplary image of the cell deposition pattern after step S12. Fig. 11D is an image taken using colored beads that mimic cell behavior. Due to the plurality of holes 141 in the filter holder 140, the cells are deposited in a pattern substantially corresponding to the pattern of the plurality of holes 141. For step 12, a filter of appropriate pore size can be selected for efficient cell deposition. The filter membrane can facilitate the filtration of liquid while retaining cells and not affecting cell viability. The pores obtained by the track etch technique have the highest yield (e.g., minimal impact on activity, minimal loss of cells in the filtrate). To avoid deformation during filtration and to promote uniform cell distribution on the target surface, a drain pan with a thicker membrane than the filter may be provided under the filter. For example, the filter may be 9 μm to 30 μm thick and the drain pan may be 80 μm thick. During filtration, when the liquid (e.g., cell culture medium) suspending the cells is removed, the cells are uniformly distributed only in the filtration region corresponding to the target site, with minimal loss in the non-filtration region.

Thus, the cell deposition pattern can be controlled and limited to a specific area where cells are deposited as a monolayer on the filter. Herein, a "monolayer" of cells may refer to cells that are distributed substantially horizontally and form one or more vertical cell layers. Monolayers can include one cell layer, one to two cell layers, one to three cell layers, one to five cell layers, or more. The number of cell layers is not limited thereto, and in some embodiments, a monolayer may refer to any number of cell layers.

Subsequently, a delivery solution containing a cell permeabilizing agent and a payload (e.g., cargo) is sprayed through the nebulizer (step S13). The controller may control the amount and duration of the spray. For example, the delivery solution may be sprayed for about 300 milliseconds. For spray delivery solutions, the cargo may be introduced into the spray head via the microvilli or injected via the resealable injection port.

After the delivery solution is sprayed, a stop solution is introduced through the disposable tubing set and/or the sterile plastic needle/cannula (step S14). The stop solution may be supplied to the chamber through a port in the septum or cap. The stop solution may be supplied manually or automatically using a pump and controller. The desired amount of stop solution is introduced into the chamber. For example, about 10mL of stop solution may be introduced in about 20 seconds. However, step S14 is not limited to stopping the solution application, and may also provide a cell washing process depending on the composition of the applied solution.

After the stop solution is introduced, the cells are resuspended (step S15). For resuspension, approximately 60mL of media can be introduced via syringe or pump, which can be fresh media or used media previously removed from the incubator. Fresh medium may be fed into the chamber through a port in the septum or cap. Alternatively or additionally, new medium may be fed into the chamber from below the filter through the drain hole. The new media may be supplied to the chamber by injecting the new media into the chamber at a positive pressure, or by applying a vacuum pressure to the chamber (e.g., through the pressure port 122), thereby allowing the chamber to absorb the new media. For example, the duration of the resuspension step can be about 3 seconds to about 1 minute. In some embodiments, to improve the resuspension, various methods can be used during or after the resuspension process, such as platform tilt, agitation (e.g., platform vibration), and the like. However, step S15 is not limited to the resuspension step, and may also provide a cell concentration changing process, a cell washing process, and/or a cell culture medium changing process. The cell concentration changing process, the cell washing process, and/or the cell culture medium changing process may also be performed in steps S11 and S12 by refilling the chamber after step S12 and repeating the draining/refilling process as many times as necessary.

After the cells are resuspended in culture medium, the engineered cells are harvested for further processing (step S16). Alternatively or additionally, the engineered cells may be cultured in a chamber prior to collection and/or further processing. The platform may be rinsed or washed after subsequent procedural processing. Alternatively or additionally, the entire chamber or a portion of the chamber may be made as a disposable unit, which may be disposed of after use and replaced with a new unit.

Fig. 9B and 9C show an exemplary process flow with exemplary process parameters. However, the process parameters are not limited to those shown in fig. 9B and 9C, and the process parameters, such as the number (volume) of culture medium, the number of cells, the concentration, the duration of each step, may vary depending on the application. Referring to FIG. 9B, the first step may be to mix 6X10 in 30ml of medium⁷And (S21). By applying a positive pressure of 20mbar, the medium can be removed (step S22), which takes 40 to 60 seconds. In step S23, 100 μ Ι _ of delivery solution can be delivered to target 1 over 420ms, and then the nebulizer can be moved and the delivery solution can be repeated on targets 2 and 3. After delivery, the cells may be cultured for 30 seconds (step S24), and 1mL of the stop solution may be delivered to each target for 30 seconds of culture (step S25). To resuspend the cells, fresh medium can be supplied from a pump at the bottom. When the loop insert is used, 75mL of medium may be used (step S261), and when 3 target inserts are used, 50mL of medium may be used (step S262).In step S27, the resuspended cells may be washed 15 times (i.e., 5 times per target). Finally, the engineered cells may be transferred to T75 flasks and stored in an incubator (step S28).

Referring to FIG. 9C, 6X 107 to 8X 107 cells may be mixed in 30ml of the medium (step S31). The medium may be removed by gravity (step S32), which takes 40 to 60 seconds. In step S33, 60-100 μ Ι _ of the delivery solution can be delivered to target 1 over 420ms, and then the nebulizer can be moved and the delivery solution can be repeated on targets 2 and 3. After the transfer, the cells were cultured for 30 seconds (step S34), and 5mL of the stop solution was delivered to each target for 30 seconds of culture (step S35). To resuspend the cells, the medium can be supplied from a pump at the bottom. When the loop insert is used, 75mL of the medium can be used (step S36). A step of waiting for 10 minutes may be added (step S37). In step S38, the resuspended cells may be washed 15 times (i.e., 5 times per target). Finally, the engineered cells may be transferred to T75 flasks and stored in an incubator (step S39).

Fig. 10 shows an exemplary embodiment of a cell engineering platform, and fig. 11A-11C show an exemplary embodiment of an atomizer 1100 for a spray coating process. Referring to fig. 11A-11C, a nebulizer 1100 includes a liquid orifice plate 1101 and a gas orifice plate 1102 on a lower surface thereof (fig. 11A). On the upper surface of the atomizer 1100, a liquid pipe inlet 1103 and an air pipe inlet 1104 may be formed (fig. 11B). Accordingly, the liquid orifice plate 1101 is connected to the reservoir by a liquid orifice plate inlet 1103 and the gas orifice plate 1102 is connected to the gas reservoir by an air tube inlet 1104, as shown in FIG. 11C.

The gas (e.g., air) flow rate may be controlled by a mass flow controller or a volumetric flow controller. By using a mass/volume flow controller, a constant amount (e.g., constant mass and/or constant volume) of gas can be supplied by the nebulizer 1100, thereby ensuring that the amount of liquid to be ejected is constant and the droplet size remains consistent regardless of variations in pressure, temperature, humidity, on/off timing, etc. within the chamber. Mass/volume flow regulation also allows for more accurate and reproducible dosing of the nebulized solution into the cells. By actively controlling the mass/volume flow of the driving gas, the spray plume is less sensitive to changes in plume density, angle, velocity, vortex formation, droplet size, deposition area, etc., resulting in more repeatable and stable transfection performance.

Fig. 11E shows an embodiment of a pneumatic circuit driving gas mass/volume flow regulation. Drive gas may be supplied from a gas cylinder 1110, through a gas supply 1111, a control valve 1112, an orifice plate 1113, and a back pressure regulator 1114. The gas may then be controlled by mass/volume flow controller 1115 prior to delivery to atomizer 1116 via gas line 1117. The solution to be sprayed may be supplied by a pump 1118. The pump 1118 may be a screw pump, a positive displacement pump, a pneumatic jet pump, or the like. Thus, the solution may be sprayed through the atomizer 1116 and delivered into the chamber 1119. Fig. 11F and 11G show control and flow path embodiments for spray operation. FIG. 11F shows the path of one nozzle, and FIG. 11G shows the path of three nozzles.

Fig. 12 is a computer-aided design (CAD) diagram illustrating an exemplary embodiment in which three nozzles 1200 are installed. Fig. 13 shows a CAD drawing of one of the showerheads 1200. Referring to fig. 13, the spray head 1200 includes a cover of nozzles 1203, partial liquid reservoir 1201 and partial liquid reservoir 1206 supported on a cylindrical base plate 1202. Threaded holes 1204 allow for securing the showerhead to the chamber. The cylindrical base plate 1202 includes a valve 1205, such as a pinch valve, for activating and/or deactivating fluid flow from the reservoir.

Fig. 14A-14F illustrate an exemplary embodiment of a disposable chamber assembly. Referring to fig. 14B, an exemplary disposable chamber assembly 1400 may include a chamber wall 1401, e.g., made of polycarbonate, a seam welded lid module 1402, a substrate module with welded membrane 1403, a nebulizer mounting hole 1404, a cell port 1407, and a three-way valve 1408. To operate the nebulizer, payload and delivery solution may be supplied through the culture base port 1405 and compressed air may be supplied through the air pressure port 1406. Fig. 14C shows a side view, fig. 14D shows a cross-sectional view, and fig. 14E shows a front view of the exemplary disposable chamber assembly. Fig. 14F shows another exemplary disposable chamber assembled from five targets and five spray heads.

Fig. 15A shows an exemplary embodiment of a cell engineering platform with a support system and mounting rack, while fig. 15B shows the platform with the chamber removed.

The exemplary embodiment described in example 1 section can be from about 10 in one transfection⁷Transfection of individual cells to approximately 10⁹A single cell or a plurality of cells. The platform allows for the sustained delivery of cargo, such as mRNA, to T cells. The system can be enclosed in a biological safety cabinet for sterile operation, and can also be used as a closed system for any environment. Platform operation may be performed manually or automatically. For automated operation, the fluid handling system may be automatically controlled by a controller and control software. The platform can be configured as a multi-purpose system that can be reused after cleaning. In some embodiments, the platform may be configured as a single use system that includes a disposable component such as a disposable chamber unit. In some embodiments, the platform can be used for vector-free payload delivery across cell membranes and/or viral payload delivery across cell membranes.

Due to process automation, the cell engineering platform can perform the carrier-free payload transfer process more stably, contamination can be minimized, and thus the system can be scaled. Therefore, the cell engineering platform can provide a reliable carrier-free delivery method, and reduce the cost and complexity of the cell engineering technology.

Example 2

Fig. 16 is a computer-aided design (CAD) diagram illustrating another embodiment of a clinical cell engineering platform for cell therapy, and fig. 17 is an enlarged view of a portion of the platform. The platform 1700 includes a robotic arm 1701 with a spray head, a soft elastomeric dome 1702 surrounding a filter base 1703, and a spray head. The platform also includes media, reagents, cargo and waste ports, thus realizing a closed system.

The robotic arm 1701 may carry a spray head to various locations within the elastomeric dome 1702 to spray a solution onto the filter base 1703 to form a monolayer of cells. In addition, the filter base 1703 may be tilted and rotated to soak the cells in the reagent.

Fig. 18 is a CAD drawing illustrating a filter plate and filter embodiment. To achieve delivery, a cell body may be formed on the filter plate. A positive pressure is applied to the container. During positive pressure, pinch valves in the input and output directions may be closed. Depending on the degree of activation, the filter can be etch traced and used once. The filter holder comprises a pattern of holes designed to completely and quickly remove the culture medium. During operation, spraying may be carried out immediately after monolayer formation.

Fig. 19 is a CAD drawing illustrating an exemplary platform cross-sectional view and filter plate rotation. Platform 1700 is shaped to collect waste media under the filter plate. Platform 1700 may include a soft elastic dome 1702, an annular port 1704 for media and stop solution, a filter base 1705, a spent media chamber 1706, and a tilt/rotate unit 1707. The filter base 1705 may be molded from polysulfone and when the cells are pressed out of the culture medium, a template may be formed. During operation, the device can be tilted and rotated to remove cells suspended in fresh medium.

Figures 20A-20C are a series of pictures showing the components of an exemplary embodiment of the platform. Figure 20A shows a robotic arm embodiment, figure 20B shows a safety housing embodiment for receiving a platform, and figure 20C shows a filter tray embodiment. The filter disks may be polycarbonate track etch (PCTE) filters of 293mm diameter.

FIG. 21 is a functional block diagram of an exemplary platform. The platform includes modules that apply fresh media, stop solution, cells in the media, and cargo in the transfer solution to the main bioreactor. In addition, the platform may include a 0.2 micron to atmospheric size filter. The platform can utilize the input and output of waste and engineered cells to perform the delivery protocol.

FIG. 22 is a diagram illustrating the disposable and reusable portions of an embodiment platform. The robotic arm may be magnetically coupled to the encapsulated filter cartridge, which may comprise a disposable filter cartridge that maintains a local environment between the spray head and the filter base. The disposable tubing can carry fluids into and out of the bioreactor. The cargo may be introduced into the transfer solution by syringe or other means.

FIG. 23 is a table illustrating some exemplary functions of an exemplary platform. In some embodiments, system inputs may include cells, culture media, molecular cargo, stop solutions, and air and/or gases. When the carrier-free payload transmission is carried out, relevant regulation and standard operation are carried out according to the operation scheme. Relevant standard operations include current production automation management Practice (cGAMP), ASTM E2500, ISO 14791,21 CFR 211.68, and 21 CFR 1271.160[ d ]).

In some embodiments, the cells may comprise adherent cells or non-adherent cells. Adherent cells may 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) cells or immortalized cells (e.g., cell lines). In preferred embodiments, 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 include primary cells and immortalized cells (e.g., cells of a cell line). Exemplary non-adherent/suspension cells include primary Hematopoietic Stem Cells (HSCs), T cells (e.g., (differentiation group 3) CD3+ cells, (differentiation group 4) CD4+ cells, (differentiation group 8) 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. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In certain aspects, a CD8+ subpopulation of CD3+ T cells is used. CD8+ T cells can be purified from a PBMC population by positive isolation using anti-CD 8 microspheres.

In embodiments, the cells are cultured in standard cell culture media, such as whole RPMI (ross wel park memorial institute medium), heat-inactivated Fetal Bovine Serum (FBS), e.g., about 10% v/v penicillin streptomycin and L-glutamine, using RPMI base media. In some embodiments, the standard medium may be supplemented with a cytokine, such as interleukin-2 (IL-2) (200U/ml).

In embodiments, the concentration of the cytokine may be from about 10U/ml to about 500U/ml. In other embodiments, the concentration of the cytokine may be about 50U/ml, about 100U/ml, about 200U/ml, about 300U/ml, about 400U/ml, or about 500U/ml. The cytokine concentration may be about 200U/ml.

In some embodiments, a cytocompatible medium is used in the delivery method. For example, Prime XV (Irvine Scientific) and X-Vivo (Lonza) are serum-free and animal component-free media that can be used. ). In some embodiments, the cell culture medium is supplemented with a higher concentration of cytokines. For example, cytokines may include interleukin-2 (IL-2) to increase proliferation rates (Tumeh P, et al, J Immunother 2010.33(6):759-768 and Besser MJ, et al, Cytotherapy 2009.11:206-217). In other embodiments, the culture medium may comprise Immucult^TMXF expansion medium (StemCell Technologies). Like Prime XV, it is also a serum-free, xeno-free T cell culture medium. In some embodiments, TexMACS (Miltenyi Biotech) may be used as an alternative serum-free medium for T cell culture. CTS-OpTsizer-T cell expansion SFM can also be used as serum-free medium for T cell culture.

In embodiments, the molecular cargo (e.g., payload) may include gene editing tools, small chemical molecules, peptides or proteins, or nucleic acids. The payload may include a messenger ribonucleic acid (mRNA). The mRNA may encode a gene editing composition. The mRNA can encode a chimeric antigen receptor.

In other embodiments, the molecular cargo (e.g., payload) to be delivered may include a composition that edits genomic DNA (i.e., a gene editing tool). For example, a gene editing composition may include a compound or complex that is capable of cleaving, nicking, splicing, rearranging, translocating, recombining, or otherwise altering genomic DNA. Alternatively or additionally, the gene editing composition may comprise the following compounds: (i) may include gene editing complexes that cleave, nick, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA; or (ii) the compound can be processed or altered to a compound contained in a cleaved, nicked gene editing complex that splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. In various embodiments, a gene-editing composition comprises (a) a gene-editing protein; (b) an RNA molecule; and/or (c) one or more of Ribonucleoproteins (RNPs).

In some embodiments, the gene-editing composition comprises a gene-editing protein, and the gene-editing protein is a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein can be a fusion protein that binds the homing endonuclease to the modular DNA-binding domain of talens (megatals). For example, megaTAL can be delivered as a protein, or mRNA encoding the megaTAL protein is delivered into a cell.

In embodiments, the molecular cargo (e.g., payload) may include small chemical molecules. The small molecule chemical molecule may be less than 1000 Da. The chemical molecule may compriseRed CMXRos, disodium propyliodide, methotrexate and/or DAPI (4', 6-diamino-2-phenylindole).

In embodiments, the molecular cargo may be a peptide. The peptide may be 5000 Da. The peptides may include ecasside (Ecallantide) under the trade name Kalbitor, a 60 amino acid polypeptide for the treatment of hereditary angioedema and prevention of blood loss during cardiothoracic surgery, liraglutide (Victoza, a trade name for the treatment of type II diabetes, Saxenda for the treatment of obesity) and Icatibant (a peptidomimetic preparation for the treatment of acute episodes of hereditary angioedema). The length of the small interfering ribonucleic acid (siRNA) molecule is about 20-25 base pairs, or about 10000-15000 Da. The siRNA molecule can reduce the expression of any gene product, e.g., knockdown of a clinically relevant target gene or gene expression of a model gene, e.g., glyceraldehyde-3 phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC, cyclophilin B siRNA, and/or lamin siRNA. Protein therapies may include peptides, enzymes, structural proteins, receptors, cellular or circulating proteins or fragments thereof. The protein or polypeptide is about 100-500000Da, such as 1000-150000 Da. The protein may include any therapeutic, diagnostic or research protein or peptide, such as beta-lactoglobulin, ovalbumin, Bovine Serum Albumin (BSA) and/or horseradish peroxidase. In other examples, the protein may include a cancer specific apoptosis protein, such as tumor necrosis factor-related apoptosis-inducing protein (TRAIL).

The molecular weight of the antibody is typically about 150000 Da. The antibody can include an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc antibody, and/or an anti-Raf antibody. The antibody may include a Green Fluorescent Protein (GFP) plasmid and a GLuc plasmid. The DNA molecule may be greater than 5000000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, e.g., an antibody to ritumumab tiuxetin, tomomab-CD 3, tositumamab, a human antibody, or a humanized mouse (or other species of origin). In other embodiments, the antibody may be a chimeric monoclonal antibody, e.g., abciximab, basiliximab, cetuximab, infliximab, or rituximab. In other embodiments, the antibody may be a humanized monoclonal antibody, for example, alemtuzumab, bevacizumab, polyethylene glycol conjugated certuzumab (certolizumab-pegol), daclizumab (daclizumab), gentuzumab-ozogamicin, trastuzumab (trastuzumab), tosituzumab (tocilizumab), yiprilimumab (Ipilimumab), or Panitumab. The antibody may comprise an antibody fragment, for example, abamectin, alfisepril, alexipt, or etanercept. The present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineering single chain antibody molecules; or a chimeric molecule, e.g., an antibody comprising the binding specificity of one antibody, e.g., an antibody of mouse origin, and the remainder of another antibody, e.g., an antibody of human origin.

The molecular cargo (e.g., payload) can include a therapeutic agent. The therapeutic agent, e.g., drug or active agent "refers to any compound used for therapeutic or diagnostic purposes, which term is understood to mean any compound administered to a patient for the treatment of a disease. Thus, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. Therapeutic agents described in U.S. patent No.7667004 (incorporated herein by reference) entitled "humanized antibodies to vascular endothelial growth factor" may be used in the methods described herein. The therapeutic agent can include at least one of cisplatin, aspirin, a statin (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylflunuride hydrochloride, and phenazopyridine hydrochloride), and fluoxetine. The payload may include a diagnostic agent. The diagnostic agent may include a detectable marker or label, such as at least one of methylene blue, patent blue V, and indocyanine green. The payload may include a fluorescent molecule. The payload may include detectable nanoparticles. The nanoparticle may comprise a quantum dot.

In some embodiments, the cargo may comprise linear DNA or DNA plasmids.

In some embodiments, after the delivering, the cells are cultured before adding the stop solution. In an embodiment, the stop solution comprises Phosphate Buffered Saline (PBS). The concentration of phosphate buffer may be about 0.5X PBS.

In certain embodiments, the delivery solution may be formulated by a GMP (manufacturing quality control) supplier and may be provided in a 10ml aluminum foil bottle. The individual Drug Master File (DMF) is archived in preparation for delivery solutions. The stability test and analysis certificate (C of a) may be provided by a vendor with current production management specifications (cGMPs).

FIG. 24 is a table illustrating compatible technologies with which an exemplary platform may be integrated.For example, various selection/isolation/concentration kits may be used. In addition, various activation and stimulation techniques may be used, for example, monoclonal antibodies (mAbs) against CD3 (cluster of differentiation 3) Muromonab-CD3(OKT3) and interleukin 2(IL-2), CD 3/cluster of differentiation 28(CD28)Transact^TMMicrobeads, viral peptides, Artificial Antigen Presenting Cells (AAPC), expamers^TMOr ImmunoCult^TMHuman CD2/CD3/CD 28T cell activator. In an example, a variety of amplification and culture media (breathable static bags, amplifiable bags, breathable rapid amplification, Xuri) may be used^TMCell expansion System W25, Xuri^TMThe cell expansion system W5 is provided with a cell expansion system,) In the examples, a variety of formulations (a) may be used2991 Cell expert, Cell5, LOVO automated cell processing system and Sefia), cryopreservation (Mr^TM,VIA Freeze^TM Duo,VIA Freeze^TMQyad,Cryomed^TMAnd are and) And thawing buffer (VIA ThawCB1000, VIA thawcc 2,automatic thawing system andCFTZ transport and cell thawing systems.

FIG. 25 is a process flow diagram illustrating exemplary cell engineering process steps. An exemplary platform may be used between DynaMag CTS and Sefia/COBE 2991. In an embodiment, Dynabead isolation and activation takes approximately two days. After activation, the sample can be added to a WAVE bioreactor/G-rex (gas permeation rapid expansion) to generate immune cells, which are then further processed using DynaMag CTS (ThermoFisher Cat. No. 12102), e.g., for T cell isolation, Solupore Clinical, Sefia/COBE2991, and the output can be measured using a flow cytometer

Fig. 26 is a process flow diagram illustrating that the exemplary platform can address cell engineering issues in the production of adoptive cell therapies.

Example 3

FIG. 27 is a series of images illustrating another exemplary bioreactor design for use on a platform. The exemplary bioreactor processes 2x10⁷And achieves a wearable delivery capacity in terms of efficiency, viability and function. All absorption was done in the area of about 30mm in diameter. In this configuration, a single injection may achieve greater than about 60% efficiency. In some embodiments, the filter may be a polycarbonate track etch (PCTE) filter or a polyester track etch (PETE) filter having a nominal pore size of about 1 μm to 3 μm. A 1 μm to 3 μm filter may be used for T cell applications. In some embodiments, the nominal pore size of the filter can be from about 5 μm to about 10 μm or more. A5 μm to 10 μm filter can be used for HEK cells, typically ranging in size from 11 μm to 15 μm. The filter material and the filter pore size are not limited thereto, and may be selected according to the type of target cells, the size of target cells, the concentration of target cells, and the like.

Fig. 28 is an image of kymeriah (tisagenelecleel) approved by the U.S. food and drug administration (US FDA) for the treatment of children and young Acute Lymphoblastic Leukemia (ALL) patients, which uses human own T cells to combat cancer. In the examples, the basic unit of cell therapy is a single dose, which can vary from 10⁶From one cell to 10⁹The number of cells varied. In FIG. 28, CAR-T (chimeric antigen receptor T cells) treats Kimriah at doses up to 2.5X 10⁸The CAR + T cell of (1). In other embodiments, the Lonza nucleofector LV closure/sterility system is within about 10 minutesTransfection 10⁹And (4) cells. In an embodiment, the overall processing time may be less than about 1 hour and less than about 20 steps.

FIG. 29 is a process flow diagram illustrating a sub-process in accordance with certain aspects of the present subject matter. In step 1, T cells were loaded into the culture medium by peristaltic pump and 1/4 inch PVC tubing. This system can be filled with reagents, cells and gases. At step 2, the medium is removed by positive pressure. The culture was stopped and fresh medium was added. In certain embodiments, step 2 may be repeated multiple times to improve delivery. In the third step, the bioreactor is gently tilted to one side to "dump" the cells and media. And fourthly, gently shaking and rotating the bioreactor to vibrate the filter membrane to assist the cells to be resuspended and leave the filtration bioreactor. At step 5, the bioreactor is returned to the upright position and the process is restarted, for example, returning to step 2 for additional processing or continuing to pump the engineered cells in new media into the next unit process.

FIG. 30 illustrates an exemplary operating cycle of an exemplary platform, similar to that shown in FIG. 29. Example sub-flow execution times are illustrated with a total processing time of approximately 3.5 minutes.

For some exemplary embodiments, the starting material may comprise clusters of differentiated 3(CD3+) T cells or PBMC cells (peripheral blood mononuclear cells). Cells can be activated by a variety of methods, including Dynabeads (e.g., Dynabeads CTS (cell therapy system) microspheres), soluble CD3/CD28 (differentiated population 28) antibodies, or for T cell activation. T cell TransAct is a macromolecular nanomatrix that, in combination with humanized recombinant CD3 and CD28 agonists, ensures successful activation of resting T cells in a blood cell population (e.g., PBMCs or enriched T cell population) without involving CD4 (differentiation population 4) or CD8 (differentiation population 8).

Figure 31 illustrates a complexity comparison of isolating and activating 100 million cells. Peripheral Blood Mononuclear Cells (PBMCs), a cluster of differentiated 3 cells (CD3+) and Pan T cells were compared.

Fig. 32 shows a comparison of various processing methods for exemplary numbers of cells, such as activation reagents, cycle number, cell number, freezer bottle number, and freezer bag number.

In some embodiments, the system inputs may include cells, culture media, molecular cargo, stop solutions, and air and/or gases.

In some embodiments, the cells may comprise adherent cells or non-adherent cells. Adherent cells may 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) cells or immortalized cells (e.g., cell lines). In preferred embodiments, the population of cells comprises non-adherent cells, e.g., the% non-adherent cells in the population are at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99% or 100% non-adherent cells. Non-adherent cells primary cells and immortalized cells (e.g., cells of a cell line). Exemplary non-adherent/suspension cells include primary Hematopoietic Stem Cells (HSCs), T cells (e.g., (differentiation group 3) CD3+ cells, (differentiation group 4) CD4+ cells, (differentiation group 8) 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. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In certain aspects, a CD8+ subpopulation of CD3+ T cells is used. CD8+ T cells can be purified from a PBMC population by positive isolation using anti-CD 8 microspheres.

In embodiments, the cells are cultured in standard cell culture media, such as whole RPMI (ross wel park memorial institute medium) using RPMI base media, heat-inactivated Fetal Bovine Serum (FBS), e.g., about 10% v/v, penicillin streptomycin, and L-glutamine. In some embodiments, the standard medium may be supplemented with a cytokine, such as interleukin-2 (IL-2) (200U/ml).

In embodiments, the concentration of the cytokine may be from about 10U/ml to about 500U/ml. In other embodiments, the cytokine may be about 50U/ml, about 100U/ml, about 200U/ml, about 300U/ml, about 400U/ml, or about 500U/ml. The cytokine concentration may be about 200U/ml.

In some embodiments, a cytocompatible medium is used in the present delivery methods. For example, Prime XV (Irvine Scientific) and X-Vivo (Lonza) are serum-free and animal component-free media that can be used. In some embodiments, the cell culture medium is supplemented with a higher concentration of cytokines. For example, the cytokine may include interleukin-2 (IL-2) to increase proliferation rates (Tumeh P, et al, J Immunother 2010.33(6): 759-. Like Prime XV, it is also a serum-free, xeno-free T cell culture medium. In some embodiments, TexMACS (Miltenyi Biotech) may be used as an alternative serum-free medium for T cell culture.

In embodiments, the molecular cargo (e.g., payload) may include gene editing tools, small chemical molecules, peptides or proteins, or nucleic acids. The payload may include a messenger ribonucleic acid (mRNA). The mRNA may encode a gene editing component. The mRNA can encode a chimeric antigen receptor.

In other embodiments, the molecular cargo (e.g., payload) to be delivered may include a composition that edits genomic DNA (i.e., a gene editing tool). For example, a gene editing composition may include compounds or complexes that cleave, nick, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA. Alternatively or additionally, the gene editing composition may comprise the following compounds: (i) may include gene editing complexes that cut, nick, splice, rearrange, displace, recombine, or otherwise alter genomic DNA; or (ii) can be processed or altered to a compound contained in a cleaved, nicked gene editing complex, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA. In various embodiments, a gene-editing composition comprises (a) a gene-editing protein; (b) an RNA molecule; and/or (c) one or more of Ribonucleoproteins (RNPs).

In some embodiments, the gene-editing composition comprises a gene-editing protein, and the gene-editing protein is a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein can be a fusion protein that binds the homing endonuclease to the modular DNA-binding domain of talens (megatals). For example, megaTAL can be delivered as a protein, or mRNA encoding the megaTAL protein is delivered to the cell.

In embodiments, the molecular cargo (e.g., payload) may include small chemical molecules. The small molecule chemical molecule may be less than 1000 da. The chemical molecule may compriseRed CMXRos, disodium propyliodide, methotrexate and/or DAPI (4', 6-diamino-2-phenylindole).

In embodiments, the molecular cargo may be a peptide. The peptide has a molecular weight of about 5000 Da. The peptides may include ecasside (ecallantide) under the trade name Kalbitor, a 60 amino acid polypeptide used for the treatment of hereditary angioedema and for preventing blood loss during cardiothoracic surgery, liraglutide (trade name vicoza on the market for the treatment of type II diabetes, and Saxenda for the treatment of obesity) and Icatibant (Icatibant) (trade name fiazyer, a peptidomimetic preparation for the treatment of acute episodes of hereditary angioedema). The length of the small interfering ribonucleic acid (siRNA) molecule is about 20-25 base pairs, or about 10000-15000 da. The siRNA molecule can reduce the expression of any gene product, e.g., the gene expression of a clinically relevant target gene or model gene is reduced, e.g., glyceraldehyde-3 phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA FITC, cyclophilin B siRNA and/or lamin siRNA. Protein therapies may include peptides, enzymes, structural proteins, receptors, cellular or circulating proteins or fragments thereof. The protein or polypeptide is about 100-500000Da, such as 1000-150000 Da. The protein may include any therapeutic, diagnostic or research protein or peptide, such as beta-lactoglobulin, ovalbumin, Bovine Serum Albumin (BSA) and/or horseradish peroxidase. In other embodiments, the protein may comprise a cancer specific apoptosis protein, for example, tumor necrosis factor-related apoptosis-inducing protein (TRAIL).

The molecular mass of an antibody is generally about 150000 Da. The antibody can include an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc antibody, and/or an anti-Raf antibody. The antibody may include a Green Fluorescent Protein (GFP) plasmid and a GLuc plasmid. The DNA molecule may be greater than 5000000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, e.g., an ibrinomab-tiuxetin, a mumomab-CD 3, a tositumab, a human antibody, or a humanized mouse (or other species of origin) antibody. In other examples, the antibody may be a chimeric monoclonal antibody, e.g., abciximab, basiliximab, cetuximab, infliximab, or rituximab. In other embodiments, the antibody may be a humanized monoclonal antibody, for example, alemtuzumab, bevacizumab, polyethylene glycol conjugated certuzumab (certolizumab-pegol), daclizumab (daclizumab), gentuzumab-ozogamicin, trastuzumab (trastuzumab), tacitumumab (tocilizumab), Ipilimumab (Ipilimumab), or panitumumab. The antibody may comprise an antibody fragment, for example, abamectin, alfisepril, alexipt, or etanercept. The invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab)₂A fragment; engineering single chain antibody molecules; or a chimeric molecule, e.g., an antibody comprising the binding specificity of one antibody, e.g., an antibody of mouse origin, and the remainder of another antibody, e.g., an antibody of human origin.

The molecular cargo (e.g., payload) can include a therapeutic agent. A therapeutic agent, e.g., a drug or active agent "can mean any compound used for therapeutic or diagnostic purposes, which term can be understood to mean any compound administered to a patient for the treatment of a disease. Thus, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. Therapeutic agents described in U.S. patent No.7667004, entitled "humanized antibodies to vascular endothelial growth factor" (incorporated herein by reference) can be used in the methods described herein. The therapeutic agent can include at least one of cisplatin, aspirin, a statin (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylflunuride hydrochloride, and phenazopyridine hydrochloride), and fluoxetine. The payload may include a diagnostic reagent. The diagnostic agent may include a detectable label or marker, such as at least one of methylene blue, patent blue V, and indocyanine green. The payload may include a fluorescent molecule. The detectable payload comprises a nanoparticle. The nanoparticle may comprise a quantum dot.

In one embodiment, the cells are cultured after delivery prior to addition of the stop solution. In an embodiment, the stop solution comprises Phosphate Buffered Saline (PBS). The concentration of PBS was about 0.5 times PBS.

FIG. 33 shows another exemplary platform in accordance with the present subject matter. The closed system 3300 may include a bag or tank 3301, a pump 3302, an interface screen 3303, a cargo introduction module 3304, and an inclined bioreactor 3305.

FIG. 34 is a CAD drawing showing an exemplary disposable bioreactor. The bioreactor can be tilted 15 degrees from a normal angle, can be rotated, can be titrated simultaneously, can be shaken gently, and can comprise a vibrating element to vibrate the filter membrane. The cargo may be introduced through SMA 01. SMA01 is an atomizer, and in embodiments, can include any atomizer. In embodiments, the values when using a nebulizer relate to nebulization of a volume between about 10-300 μ l of cell-permeable solution. Exemplary atomizers are described in U.S. patent No. 5411208 or U.S. patent No. 6634572, which are incorporated herein by reference in their entirety. Other atomizers are commercially available, e.g. Duramist^TMAtomizers (Sigma-Aldrich GXARG1DM04-1EA), atomizers, Oneeb, series 2 inert concentric atomizers, or used with ICP-OES (Agilent technologies G8010-60293). In embodiments, the atomizer may be an ultrasonic atomizer, or a vibrating mesh atomizer. The input and output tubes may be welded or a Hospira spin-on spiral closure connector may be used. Other atomizers are commercially available, e.g. Duramist^TMNebulizer (Sigma-Aldrich GXARG1DM04-1EA), nebulizer, Oneeb, series 2 inert concentric type nebulizer, or use with ICP-OES (Ann)Jieren technologies G8010-60293). In embodiments, the atomizer may be an ultrasonic atomizer, or a vibrating mesh atomizer. The input and output tubes may be welded or a Hospira spin-on spiral closure connector may be used.

Figure 35 illustrates a filter pad container with stop solution added. The stop solution may be added through a series of holes (as shown) in the circumference of the filter plate container. The internal manifold of the vessel is designed to ensure equal flow to each orifice.

Figure 36 illustrates the filter plate container after addition of new media. After culturing, cell culture medium may be added thereto through the second set of circumferential wells. Addition of media can be performed at a second time (for mL volume).

FIG. 37 shows the inclination of the bioreactor during operation. The whole bioreactor is shown in the left figure, and the filter plate container is shown in the right figure. This system is a closed system. The bioreactor may be tilted 15 degrees from a normal angle (the bioreactor includes a filter plate container). The bioreactor may be rotated while being tilted. The bioreactor may be rocked gently and contain a vibrating element to vibrate the filter membrane.

Figure 38 illustrates a filter plate container during "decanting" of cell culture media, wherein the container (e.g., reactor) is tilted, rotated, and vibrated to facilitate removal of cells from the media. When containing cell culture media in which cells are suspended, the filter plate container can be tilted and poured off the membrane surface by tilting, rotating and vibrating as needed. For example, this method may remove cellular "cake" from the filter. Different vibration modes and offsets may be used.

The present subject matter can include a number of components, for example, accelerated product introduction, acoustics, additive manufacturing, adhesives, improved assembly, automation, device integration, digital prototyping, dynamic tuning, fluidics, human-machine interfaces, optical communications, optics, power electronics, precision injection molding, precision mechanics, printed electronics, sensors, software application design, wireless connectivity, and document documentation

FIG. 39 is a membrane stent embodiment. The example membrane stent may be implanted for use with the first example platform (described with reference to fig. 16-26) or the second example platform (described with reference to fig. 27-38). Controlling the cell deposition pattern on the filter membrane can be challenging when using positive pressure to form a monolayer on the filter plate. The ridge pore structure on the filter plate affects the sedimentation form of the suspended cells on the filter membrane. Cells are deposited on the filter, but are limited to where the ridges and pores are on the underlying membrane shelf (e.g., acting as a template). The location of the cell deposition and the deposition pattern in this region can be controlled by the membrane scaffold design. Furthermore, a membrane scaffold comprising discrete filtration regions like drains may be used to create a discrete cell monolayer on a continuous filtration membrane.

As shown in fig. 39, the membrane holder includes a single filtration zone. In this example, a 44mm polycarbonate track etch (PCTE) filter (denoted as "a" and indicated by the outer dashed circle) was placed on the membrane support (b). The membrane scaffold has ridges and holes located within a central diameter of 25mm (c). To illustrate the function of an exemplary design of a filtration unit, the membrane holder was inserted into the base of an Amicon stirred cell pressure filtration unit (not shown here). The Dynabeads suspension was added to the chamber and positive pressure was applied. Dynabeads (used as T cell models) were deposited only where the ridges and wells were located (indicated by the inner dashed circles).

Fig. 40 illustrates an embodiment of an intermediate system that can handle up to 5x107 units. The system has the same features as the clinical system (full size, e.g., each procedure can handle more than 5x 10)⁷Individual cells, e.g. 1x10⁸And 10⁹One or more cells), but the system does not include a translatable showerhead mechanism, but rather uses a single showerhead or a multi-showerhead array.

Although some variations have been described in detail above, other modifications or additions may be made. For example, design variations may include filter bases of other geometries, such as rectangular, square, or oval. In addition, filtration substrates having different topographies may include convex, concave, and textured surfaces having micro-or macro-features. In addition, target configurations including circular targets and annular targets are also contemplated. In embodiments, the modification or addition may optimize cell deposition under spray objectives.

The subject matter described herein provides a number of technical advantages. For example, the single use avoids the need for system sterilization, greatly reduces the risk of cross-contamination between patient samples, and makes the validation process simpler. In addition, the single use allows for temperature and humidity control within the bioreactor, resulting in a healthier cell population. Another advantage is that it can be used to transfer multiple products together through a common transfer process, with a single spray head or the ability to install multiple spray heads. Furthermore, the subject matter described herein is fast and simple, and gentle cell processing procedures are able to maintain cell health and enable engineering of native cell populations. Temperature control of the filter substrate also allows for better control of the transfer process and facilitates recovery of cells from the system. The combination of membrane scaffold design and nebulizer parameters enables the system to be optimized to deliver different cargo to different cell types.

Embodiments of delivery scheme

The present invention is based on the surprising discovery that a compound or mixture of compounds (composition) is delivered into the cytoplasm of a eukaryotic cell by contacting the compound to be delivered (e.g., a payload) with a solution comprising an agent that reversibly permeabilizes or lyses the cell membrane. Preferably, the solution is delivered to the cells in the form of a spray (e.g., water particles). (see, e.g., PCT/US2015/057247 and PCT/IB2016/001895, which are incorporated by reference in their entirety). For example, the cells are coated with a spray, but not soaked or submerged in a solution containing the delivery compound. Typical agents that permeate or lyse eukaryotic cell membranes include alcohols and detergents, such as ethanol and Triton X-100, respectively. Other exemplary detergents, such as surfactants, include polysorbate 20 (e.g., tween 20), 3- [ (3-cholamidopropyl) dimethylamino ] -1-propanesulfonic acid (CHAPS), 3- [ (3-cholamidopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid (CHAPSO), Sodium Dodecyl Sulfate (SDS), and octyl glucoside.

Examples of conditions for achieving coating of a population of cells include providing fine particle spray delivery, for example, conditions that do not include dripping or pipetting a large volume of solution onto the cells such that the large volume of cells is wetted or submerged by a volume of liquid. Thus, a mist or spray comprises a ratio of fluid volume to cell volume. Alternatively, the conditions include the ratio of the volume of the mist or spray to the area of exposed cells, e.g., the area of the cell membrane that is exposed when the cells are present as a confluent or substantially confluent layer, such as on the bottom of a tissue culture vessel (e.g., the pores of a tissue culture plate (e.g., a microtissue culture plate)).

"cargo" or "payload" refers to a compound or composition that enters the interior of a cell through the plasma membrane of the cell by way of an aqueous solution.

In one aspect, delivering a payload through a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of aqueous solution. The aqueous solution includes a payload and an alcohol content of greater than 2%. The volume of the aqueous solution may be a function of the exposed surface area of the cell population, or may be a function of the number of cells in the cell population.

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

One or more of the following features may be included in any feasible combination. The volume of solution to be delivered into the cells is a plurality of units, e.g. a spray, e.g. a plurality of droplets on a water particle. The volume is described relative to the exposed surface area of a single cell or relative to a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) population of cells. For example, the volume may be 6.0x10 per cell^-7Microlitre to 7.4x10 per cell^-4In the microliter range. The volume may be 4.9x10 per cell^-6Microlitre to 2.2x10 per cell^-3In the microliter range. The volume may be in the range of 9.3x10 per cell^-6Microlitre to 2.8x10 per cell^-5In the microliter range. What is needed isThe volume may be about 1.9x10 per cell^-5Microliter, or in the ten percent range. The volume may be at 6.0x10 per cell^-7Microlitre to 2.2x10 per cell^-3In the microliter range. The volume may be at 2.6x10 per square micron of exposed area^-9Microliter to 1.1x10 per square micron exposed area^-6In the microliter range. The volume may be at 5.3x10 per square micron of exposed area^-8Microliter to 1.6x10 per square micron exposed area^-7In the microliter range. The volume may be about 1.1x10 per square micron of exposed area^-7Microliter, by about is meant in the ten percent range.

Cell fusion refers to the cells contacting each other on a surface. For example, it may be expressed as an estimated (or calculated) percentage, e.g., 10% confluency means that 10% of the surface (e.g., tissue culture vessel) is covered by cells, 100% means that it is completely covered. For example, adherent cells grow in two dimensions on the surface of a tissue culture well, a petri dish or a culture flask. Non-adherent cells can be spun down, aspirated from the top of the cell population by vacuum or tissue culture medium, or removed by aspiration or removal of vacuum from the bottom of the vessel.

The aqueous solution is propelled by the gas to form a spray, thereby effecting contact of the cell population with a volume of the aqueous solution. The gas may include nitrogen, ambient air, or an inert gas. The spray may comprise discrete volume units having a size in the range of 1nm to 100 μm, for example 30-100 μm in diameter. The spray comprises discrete volume units of about 30-50 μm in diameter. In some embodiments, the spray comprises discrete volume units having a diameter of about 5 to 8 μm. The aqueous solution, with a total volume of 20. mu.l, can be delivered by spraying to about 1.9cm²For example, one well of a 24-well culture plate. A total volume of 10. mu.l of aqueous solution is delivered to a cell footprint of about 0.95cm2, for example, one well of a 48-well culture plate. Typically, the aqueous solution includes a payload to be delivered into the cell through the cell membrane, and the second volume is a buffer or culture medium that does not contain the payload. Alternatively, the second volume (buffer or medium) may also contain a payload. In some embodimentsThe aqueous solution includes a payload and an alcohol, and the second volume does not include an alcohol (and optionally does not include a payload). The population of cells may be contacted with the aqueous solution for 0.110 minutes before adding a second volume of buffer or culture medium to submerge or suspend the population of cells. The buffer or culture medium may be Phosphate Buffered Saline (PBS). The cell population may be contacted with the aqueous solution for 2 seconds to 5 minutes before adding the second volume of buffer or culture medium to submerge or suspend the cell population. The cell population can be contacted with the aqueous solution (e.g., containing the payload) for 30 seconds to 2 minutes to submerge or suspend the cell population before adding the second volume of buffer or culture medium (e.g., without the payload). The cell population can be contacted with the spray for about 1-2 minutes before adding the second volume of buffer or culture medium to submerge or suspend the cell population. During the time period between spraying the cells and adding buffer or culture medium, the cells are kept hydrated by the sprayed water layer.

The aqueous solution may comprise ethanol at a concentration of 5% to 30%. The aqueous solution may comprise 75% to 98% H₂One or more of O, 2% to 45% ethanol, 6 to 91mM sucrose, 2 to 500mM KCl, 2 to 35mM ammonium acetate, and 1 to 14mM (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) (HEPES). For example, the delivery solution contains 10⁶mM KCl and 25% ethanol.

The cell population may include adherent cells or non-adherent cells. The adherent cells may comprise at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neural cells, fibroblasts, Human Umbilical Vein (HUVEC) cells, Chinese Hamster Ovary (CHO) cells, and Human Embryonic Kidney (HEK) cells, or immortalized cells (e.g., cell lines). The cell population may also include adherent cells suspended in a culture medium. Adherent cells in suspension media may include Human Embryonic Kidney (HEK) cells or macrophages. In preferred embodiments, the population of cells includes non-adherent cells, e.g., a percentage of non-adherent cells in the population of cells is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% of non-adherent cells. Non-adherent cells primary cells and immortalized cells (e.g., cells of a cell line). Exemplary non-adherent/suspension cells include primary Hematopoietic Stem Cells (HSCs), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), Natural Killer (NK) cells, Cytokine Induced Killer (CIK) cells, human cord blood CD34+ cells, B cells, or cell lines, such as Jurkat T cell line.

The payload may include a small chemical molecule, peptide or protein, or nucleic acid. The small chemical molecules may have a molecular weight of less than 1000 Da. The chemical molecule may compriseRed CMXRos, propidium iodide, methotrexate and/or DAPI (4', 6-diamino-2-phenylindole). The peptide may have a molecular weight of about 5000 Da. The peptides may include ecasside (Ecallantide) under the trade name Kalbitor, a 60 amino acid polypeptide for the treatment of hereditary angioedema and prevention of blood loss during cardiothoracic surgery, liraglutide (marketed under the trade name Victoza for the treatment of type II diabetes, under the trade name Saxenda for the treatment of obesity) and Icatibant (peptidomimetic preparation for the treatment of acute episodes of hereditary angioedema). The length of the small interfering ribonucleic acid (siRNA) molecule is about 20-25 base pairs, or about 10000-15000 Da. The siRNA molecule can reduce the expression of any gene product, e.g., knockdown of a clinically relevant target gene or gene expression of a model gene, e.g., glyceraldehyde-3 phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC, cyclophilin B siRNA, and/or lamin siRNA. Protein therapies may include peptides, enzymes, structural proteins, receptors, cellular or circulating proteins or fragments thereof. The protein or polypeptide is about 100-500000Da, such as 1000-150000 Da. The protein may include any therapeutic, diagnostic or research protein or peptide, such as beta-lactoglobulin, ovalbumin, Bovine Serum Albumin (BSA) and/or horseradish peroxidase. In other examples, the protein may include a cancer specific apoptosis protein, such as tumor necrosis factor-related apoptosis-inducing protein (TRAIL).

The molecular mass of the antibody is typically about 150000 Da. The antibody can include an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc antibody, and/or an anti-Myc antibodyRaf antibodies. The antibody may include a Green Fluorescent Protein (GFP) plasmid, a GLuc plasmid, and a BATEM plasmid. The DNA molecular weight may be greater than 5000000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, e.g., an ibrinomab-tiuxetin, a mumomab-CD 3, a tositumab, a human antibody, or a humanized mouse (or other species of origin) antibody. In other embodiments, the antibody may be a chimeric monoclonal antibody, e.g., abciximab, basiliximab, cetuximab, infliximab, or rituximab. In still other embodiments, the antibody may be a humanized monoclonal antibody, for example, alemtuzumab, bevacizumab, polyethylene glycol conjugated certuzumab (certolizumab-pegol), daclizumab (daclizumab), gentuzumab ozogamicin, trastuzumab (trastuzumab), tacitumumab (tocilizumab), Ipilimumab (Ipilimumab), or panitumumab. The antibody may comprise an antibody fragment, for example, abamectin, alfisepril, alexipt, or etanercept. The invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab)₂A fragment; engineering single chain antibody molecules; or a chimeric molecule, e.g., an antibody comprising the binding specificity of one antibody, e.g., an antibody of mouse origin, and the remainder of another antibody, e.g., an antibody of human origin.

The molecular cargo (e.g., payload) can include a therapeutic agent. A therapeutic agent, e.g., "drug or active agent" can mean any compound used for therapeutic or diagnostic purposes, which term can be understood to mean any compound administered to a patient for the treatment of a disease. Thus, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. Therapeutic agents described in U.S. patent No.7667004, entitled "humanized antibodies to vascular endothelial growth factor" (incorporated herein by reference) can be used in the methods described herein. The therapeutic agent can include at least one of cisplatin, aspirin, a statin (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylflunuride hydrochloride, and phenazopyridine hydrochloride), and fluoxetine. The payload may include a diagnostic reagent. The diagnostic agent may include a detectable label or marker, such as at least one of methylene blue, patent blue V, and indocyanine green. The payload may include a fluorescent molecule. The detectable payload comprises a nanoparticle. The nanoparticle may comprise a quantum dot.

The population of non-adherent cells can be substantially confluent, e.g., greater than 75% confluent. Cell fusion refers to the cells contacting each other on a surface. For example, it may be expressed as an estimated (or calculated) percentage, e.g., 10% confluency means that 10% of the surface (e.g., tissue culture vessel) is covered by cells, 100% means that it is completely covered. For example, adherent cells grow in two dimensions on the surface of a tissue culture well, a petri dish or a culture flask. The non-adherent cells can be spun down, pulled from the top of the cell population by vacuum or tissue culture medium aspiration, or removed from the bottom of the vessel by aspiration or vacuum. The population of cells may form a monolayer of cells.

The alcohol may be selected from methanol, ethanol, isopropanol, butanol and benzyl alcohol. The salt is selected from NaCl, KCl, Na₂HPO₄、KH₂PO₄And C₂H₃O₂And (4) NH. In a preferred embodiment, the salt is KCl. The sugar may comprise sucrose. The buffer may include 4-2- (hydroxyethyl) -1-piperazine ethanesulfonic acid.

The present subject matter relates to a method of delivering molecules through a plasma membrane. The present subject matter may find application in the field of intracellular delivery and has been applied, for example, to the delivery of molecular biological and pharmacological therapeutic agents to a target site, such as a cell, tissue or organ. The method of the inventive subject matter includes introducing the molecule into an aqueous composition to form a matrix; atomizing the substrate into a spray; and contacting the substrate with the plasma membrane.

The present subject matter relates to a composition for delivery of molecules across a plasma membrane. The inventive subject matter finds utility in the field of intracellular delivery and finds application, for example, in the delivery of molecular biological and pharmacological therapeutic agents to a target site, such as a cell, tissue or organ. The compositions of the present subject matter include alcohol, salt, sugar, and/or buffer.

In some embodiments, presented herein is an infiltration technique that facilitates the delivery of molecules within cells independent of molecule and cell type. Nanoparticles, small molecules, nucleic acids, proteins and other molecules can be efficiently delivered in situ into suspension cells or adherent cells, including primary and stem cells, with low cytotoxicity, and the technology is compatible with high throughput and cell-based automated analysis.

An exemplary method described herein includes a payload, wherein the payload includes an alcohol. The term "alcohol" refers to a polyatomic organic compound that includes a hydroxyl (-OH) functional group attached to at least one carbon atom. The alcohol may be a monohydric alcohol and may include at least one carbon atom, such as methanol. The alcohol may include at least two carbon atoms (e.g., ethanol). In other aspects, the alcohol comprises at least three carbon atoms (e.g., isopropanol). The alcohol may include at least four carbon atoms (e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol). An exemplary payload can include no more than 50% (v/v) alcohol, more preferably, the payload includes 2-45% (v/v) alcohol, 5-40% alcohol, and 10-40% alcohol. The payload may comprise 20-30% (v/v) alcohol.

Most preferably, the payload delivery solution comprises 25% (v/v) alcohol. Alternatively, the payload may comprise 2-8% (v/v) alcohol, or 2% alcohol. The alcohol may comprise ethanol and the payload comprises 5, 10, 20, 25, 30 and up to 40% or 50% (v/v) ethanol, for example 27%. In exemplary methods the alcohol is methanol, and the payload can include 5%, 10%, 20%, 25%, 30%, or 40% (v/v) methanol. The payload may comprise 2-45% (v/v) methanol, 20-30% (v/v) or 25% (v/v) methanol. Preferably, the payload comprises 20-30% (v/v) methanol. Further alternatively, the alcohol is butanol and the payload comprises 2%, 4% or 8% (v/v) butanol.

In some aspects of the inventive subject matter, the payload is in a solution or buffer.

According to the inventive subject matter, the payload includes at least one salt. The salt is selected from NaCl, KCl and Na₂HPO₄、C₂H₃O₂NH₄And KH₂PO₄. For example, the concentration of KCl ranges between 2mM and 500 mM. In some preferred embodiments, the concentration is greater than 100mM, e.g., 106 mM.

According to exemplary methods of the present subject matter, the payload can include a sugar (e.g., sucrose or disaccharide). According to exemplary methods, the payload comprises less than 121mM sugar, 6-91mM sugar, or 26-39mM sugar. In addition, the payload includes 32mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload comprises 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8 or 89.6mM sucrose.

According to an exemplary method of the present subject matter, the payload can include a buffer (e.g., a weak acid or a weak base). The buffer may include a zwitterion. According to an exemplary method, the buffer is 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid. The payload can include less than 19mM buffer (e.g., 1-15mM, 4-6mM, or 5mM buffer). According to an exemplary method, the buffer is 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid and the payload comprises 1,2, 3, 4, 5, 10, 12, 14mM of 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid. Further preferably, the payload comprises 5mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid.

According to an exemplary method of the present subject matter, the payload includes ammonium acetate. The payload can include less than 46mM ammonium acetate (e.g., between 2-35mM, 10-15mM, or 25mM ammonium acetate). The payload may comprise 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8 or 33.6mM ammonium acetate.

The aqueous solution volume is driven by a gas, which may include compressed air (e.g., ambient air), and other embodiments may include inert gases such as helium, neon, and argon.

In certain aspects of the present subject matter, the cell population can include adherent cells (e.g., lung, kidney, immune cells, such as macrophages) or non-adherent cells (e.g., suspension cells).

In certain aspects of the inventive subject matter, the population of cells can be substantially confluent, and the substantial confluency can comprise greater than 75%. In a preferred embodiment, the population of cells may form a single monolayer.

According to an exemplary method, the payload to be transferred has an average molecular weight of up to 20,000,000 Da. In some embodiments, the average molecular weight of the payload to be transferred may be up to 2,000,000 Da. In some embodiments, the payload to be transferred may have an average molecular weight of up to 150,000 Da. In further embodiments, the payload to be delivered has an average molecular weight of up to 15000Da, 5000Da or 1000 Da.

The payload delivered through the plasma membrane of a cell may include small chemical molecules, peptides or proteins, polysaccharides or nucleic acids or nanoparticles. The small chemical molecule may be less than 1000Da, the peptide may have a molecular weight of about 5000Da, the siRNA may have a molecular weight of about 15000Da, the antibody may have a molecular weight of about 150,000Da, and the DNA may have a molecular weight greater than or equal to 5,000,000 Da. In a preferred embodiment, the payload comprises mRNA.

According to an exemplary method, the payload comprises 3.0-150.0 μ M of the molecule to be delivered, more preferably 6.6-150.0 μ M of the molecule to be delivered (e.g., 3.0, 3.3, 6.6, or 150.0 μ M of the molecule to be delivered). In some embodiments, the payload to be delivered has an average molecular weight of up to 15000Da, and the payload comprises the molecule to be delivered at 3.3 μ M.

According to an exemplary method, the payload to be delivered has an average molecular weight of up to 15000Da and the payload comprises molecules to be delivered at 6.6 μ M. In some embodiments, the payload to be delivered has an average molecular weight of up to 1000Da, and the payload comprises the molecule to be delivered at 150.0 μ M.

In accordance with a further aspect of the present subject matter, there is provided a method of delivering molecules of more than one molecular weight through a plasma membrane; the method comprises the following steps: introducing molecules of more than one molecular weight into the aqueous solution; the aqueous solution is contacted with the plasma membrane.

In some embodiments, the method comprises introducing a first molecule having a first molecular weight and a second molecule having a second molecular weight into the payload, wherein the first and second molecules may have different molecular weights, or wherein the first and second molecules may have the same molecular weight. According to an exemplary method, the first and second molecules may be different molecules.

In some embodiments, the payload to be delivered may include a therapeutic or diagnostic agent, including, for example, cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, imipramine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenyl fluoroether hydrochloride, and phenyl azole pyridine hydrochloride), and fluoxetine. Other therapeutic agents include antibacterial agents (aminocyclohexane (e.g., gentamicin, neomycin, streptomycin), penicillins (e.g., amoxicillin, ampicillin), glycopeptides (e.g., avoparcin, vancomycin), macrolides (e.g., erythromycin, tilmicosin, tylosin), quinolones (e.g., sarafloxacin, enrofloxacin), streptomycins (e.g., virginiamycin, quinolpristin, carbapenems, lipopeptides, oxazolidinones, cycloserines, ethambutol, ethoxyamide, isonicotinamide, p-aminosalicylic acid, and pyrazinamide). For example, cancer, infectious disease, hemophilia, anemia, multiple sclerosis, and hepatitis b or c.

Other exemplary active ingredients may include detectable labels or tags, for example, methylene blue, patent blue V, and indocyanine green.

The methods described herein can also include a payload that includes a detectable moiety or detectable nanoparticle (e.g., quantum dot). The detectable moiety may comprise a fluorescent molecule or a radioactive agent (e.g., 125I). When a fluorescent molecule is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, terephthalaldehyde and fluorescein. Such molecules may also be labeled with a fluorescent emitting metal (e.g., 152Eu) or other lanthanide. These metals can be attached to the molecule using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Such molecules can also be detected by coupling to a chemiluminescent compound. The presence of the chemiluminescent label molecule is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.

In other embodiments, the payload to be delivered may include a composition that edits the genomic DNA (i.e., a gene editing tool). For example, a gene editing composition may include compounds or complexes that cleave, nick, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA. Alternatively or additionally, the gene editing composition may comprise the following compounds: (i) a gene editing complex that can include cutting, nicking, splicing, rearranging, translocating, recombining, or otherwise altering genomic DNA; or (ii) a compound that can be treated or altered, the compound being included in a gene editing complex that cuts, nicks, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. In various embodiments, the gene-editing composition comprises one or more of (a) a gene-editing protein; (b) an RNA molecule; and/or (c) a ribonucleoside protein (RNP).

In some embodiments, the gene-editing composition comprises a gene-editing protein, and the gene-editing protein is a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein can be a fusion protein that binds the homing endonuclease to the modular DNA-binding domain of talens (megatals). For example, megaTAL can be delivered as a protein, or mRNA encoding the megaTAL protein is delivered to the cell.

In various embodiments, the gene editing composition comprises an RNA molecule, and the RNA molecule comprises a sgRNA, a crRNA, and/or a trace RNA.

In certain embodiments, the gene-editing composition comprises an RNP, and the RNP comprises a Cas protein and an sgRNA or a crRNA and a tracrRNA. Some aspects of the inventive subject matter are particularly useful for controlling the time and duration of presence of a particular gene-editing compound in a cell.

In various embodiments of the inventive subject matter, the gene-editing composition can be detected in the cell population or progeny thereof (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 contacting the cell population with the aqueous solution, or (b) less than 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 contacting the cell population with the aqueous solution.

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

Some aspects of the invention relate to a cell population that includes one gene-editing compound and another gene-editing compound is inserted into the cell population. For example, one component of RNP may be introduced into cells that express or already contain another component of RNP. For example, a cell in a population of cells, or progeny thereof, can comprise a sgRNA, a crRNA, and/or a tracrRNA. In some embodiments of the cell population, the cell population or a subsequent representation thereof expresses sgRNA, crRNA, and/or tracrRNA. Alternatively or additionally, the cells or progeny thereof in the population express the Cas protein.

Various embodiments of the inventive subject matter herein include Cas proteins. In some embodiments, the Cas protein is a Cas9 protein or a mutant thereof. Exemplary Cas proteins (including non-limiting examples of Cas9 and Cas9 mutants) are described herein.

In various aspects, the concentration of Cas9 protein is in the range of about 0.1 to about 25 micrograms. For example, the concentration of Cas9 protein may be about 1 microgram, about 5 microgram, about 10 microgram, about 15 microgram, or about 20 microgram. Alternatively, the concentration of Cas9 is in the range of about 10ng/μ L to about 300ng/μ L, for example in the range of about 10ng/μ L to about 200ng/μ L; or in the range of about 10 ng/. mu.L to about 100 ng/. mu.L, or in the range of about 10 ng/. mu.L to about 50 ng/. mu.L.

In certain embodiments, a 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 (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

In some aspects, the ratio of Cas9 protein to guide RNA can be 1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9, or 1: 10.

In embodiments, increasing the number of times a cell passes through the delivery process (or, alternatively, increasing the number of doses), the edit percentage may be increased; wherein, in some embodiments, the number of doses can include 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

In various embodiments, the first and second sgrnas or the first and second crRNA molecules collectively comprise a nucleic acid sequence that is complementary to a side chain target sequence of a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, wherein the gene, exon, intron, exon sequence, or genomic nucleic acid sequence is about 1,2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length or at least about 1,2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length. In some embodiments, the use of paired RNPs comprising first and second sgrnas or first and second crRNA molecules can be used to create polynucleotide molecules comprising genes, exons, introns, extrachromosomal sequences, or genomic nucleic acid sequences.

In certain embodiments, the nucleotide length of the target sequence of the 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. In some embodiments, the target sequence is 20 nucleotides or about 20 nucleotides in length.

In various embodiments, the first and second sgrnas or first and second crRNA molecules are complementary to extrachromosomal sequences flanking the sequences, which are also within the expression vector.

Various aspects of the inventive subject matter relate to the delivery of multiple components of a gene editing complex, wherein the multiple components are not complexed together. In some embodiments, the 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 or complexed to each other.

The present subject matter can achieve high gene editing efficiency while maintaining high cell viability. In some embodiments, after contacting with the aqueous solution, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cells or progeny thereof are genetically modified. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cell population or progeny thereof is viable upon contact with an aqueous solution.

In certain embodiments, the gene-editing composition induces a single-strand or double-strand break in DNA within the cell. In some embodiments, the gene editing composition further comprises a repair template polynucleotide. In various embodiments, the repair template comprises (a) a first side chain region comprising nucleotides of sequence complementary to about 40 to about 90 base pairs on one side of the single-or double-stranded break, and a second side chain region comprising nucleotides of sequence complementary to about 40 to about 90 base pairs on the other side of the single-or double-stranded break; or (b) a first side-chain region comprising nucleotides of 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-stranded break, and a second side-chain region comprising nucleotides of sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-or double-stranded break. Non-limiting instructions regarding gene editing (including repair templates) using CRISPR-Cas systems are described in Ran et al (2013) Nat protoc.2013 Nov; 2281 and 2308, the entire contents of which are hereby incorporated by reference. Embodiments involving repair of the template are not limited to embodiments including CRISPR-Cas systems.

In various embodiments of the inventive subject matter, a volume of aqueous solution is delivered to the cell population in the form of a spray. In some embodiments, the volume in each cell is 6.0x10^-7Microlitre to 7.4x10 per cell^-4Between microliters. In certain embodiments, the spray comprises colloids or sub-particles having a diameter of 10nm to 100 μm. In various embodiments, the volume is 2.6x10^-9Microliter/square micron exposed surface area and 1.1x10^-6Between microliters per square micron of exposed surface area.

In some embodiments, the RNP is about the size ofOr 10nm × 10nm × 5 nm. In various embodiments, the spray particles are sized to accommodate at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNPs per spray particle.

For example, a cell population can be contacted with a volume of aqueous solution by a gas to propel the aqueous solution to form a spray. In certain embodiments, the population of cells is contacted with the aqueous solution for 0.01-10 minutes (e.g., 0.1-10 minutes), and then a second volume of buffer or culture medium is added to submerge or suspend the population of cells.

In various embodiments, the population of cells includes at least one of primary cells or immortalized cells. For example, the cell population can include mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, Human Umbilical Vein (HUVEC) cells and Human Embryonic Kidney (HEK) cells, primary or immortalized Hematopoietic Stem Cells (HSCs), T cells, Natural Killer (NK) cells, Cytokine Induced Killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In certain aspects, a CD8+ subpopulation of CD3+ T cells is used. CD8+ T cells can be purified from a PBMC population by positive isolation using anti-CD 8 microspheres. In certain aspects, primary NK cells are isolated from PBMCs, and GFP mRNA is delivered by platform delivery techniques (i.e., 3% expression at 24 hours and 96% survival). In other aspects, an NK cell line, such as NK92, can be used.

Cell types also include cells that have been previously modified, such as T cells, NK cells, and MSCs, to enhance their therapeutic effect. For example: t cells or NK cells expressing a chimeric antigen receptor (CAR T cells, CAR NK cells, respectively); t cells expressing a modified T Cell Receptor (TCR); MSCs that have been modified virally or non-virally to overexpress therapeutic proteins that complement their intrinsic properties (e.g., Epo delivered using lentiviral vectors or BMP-2 delivered using AAV-6) (described in Park et al, Methods,2015 Aug; 84-16); MSCs pretreated with non-peptide drugs or magnetic nanoparticles to enhance therapeutic efficacy and external stability modulate targeting, respectively (Park et al, 2015); MSCs functionalized with targeting moieties are enhanced in their homing to the treatment site by enzymatic modification (e.g. fucosyltransferase), chemical coupling (e.g. chemically modifying SLeX on MSCs using N-hydroxysuccinimide (NHS)) or non-covalent interactions (e.g. engineering the cell surface with palmitoylated proteins, which act as hydrophobic anchors for subsequent antibody binding) (Park et al, 2015). For example, a T cell (e.g., a primary T cell or T cell line) modified to express a chimeric antigen receptor (CAR T cell) can be further treated according to the invention with a gene editing protein and/or a complex containing a specific guide nucleic acid for the CAR-encoding sequence to edit the gene encoding the CAR to reduce or stop expression of the CAR in the modified T cell.

Aspects of the invention relate to expression vector-free delivery of gene editing compounds and complexes to cells and tissues, for example, delivery of Cas-gRNA ribonucleoproteins for genome editing in primary human T cells, Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs). In some embodiments, mRNA encoding these proteins is delivered into the cell.

Various aspects of CRISPR-Cas systems are known in the art. Non-limiting aspects of the present system are as disclosed in U.S. patent No. 9023649 issued 5/2015, U.S. patent No. 9074199 issued 7/2015, U.S. patent No. 8697359 issued 4/15/2014, U.S. patent No. 8932814 issued 1/2015 13/2015, PCT international patent application No. WO 2015/071474 published 27/8/2005; cho et al, (2013) Nature Biotechnology Vol 31 No 3 pp 230-; and Jinek et al, (2012) Science Vol 337 No 6096 pp 816-821, all of which are incorporated herein by reference in their entirety.

Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 (also called Csn 7 and Csx 7), Cas7, Csy 7, Cse 7, Csc 7, Csa 7, Csn 7, Csm 7, Cmr 7, Csb 7, Csx 7, CsaX 7, csaf 7, Csx 7, Csf 7, Csx 36x 7, Csf 7, Csx 36f 7, Csf. These enzymes are known; for example, the amino acid sequence of the streptococcus pyogenes Cas9 protein can be found under accession number Q99ZW2 in the SwissProt database and accession number Q99ZW2.1 in the NCBI database. UniProt database accession numbers A0G4DEU5 and CDJ55032 provide another example of a Cas9 protein amino acid sequence. Another non-limiting example is the streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, e.g., Cas 9. In certain embodiments, the CRISPR enzyme is Cas9, and may be Cas9 from streptococcus pyogenes or streptococcus pneumoniae. In various embodiments, the CRISPR enzyme directs cleavage of one or both strands at a location of the target sequence (e.g., within the target sequence and/or within the complement of the target sequence). In some embodiments, the CRISPR enzyme cleaves one or both strands within about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs. In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising a target sequence. For example, substitution of aspartic acid to alanine in the RuvC i catalytic domain of Cas9 from streptococcus pyogenes converts Cas9 from a two-strand cleaving nuclease to nickase (cleaving single-strand). Other examples of mutations that make Cas 9a nickase include, but are not limited to, H840A, N854A, and N863A. In various aspects of the invention, the nickase can be used for genome editing by homologous recombination.

In certain embodiments, Cas9-nickase can be used in combination with a guide sequence, e.g., two guide sequences directed against the sense and antisense strands of a DNA target, respectively. This combination allows both lines to be cut and used to induce NHEJ.

As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to generate a mutant Cas9 that lacks substantially all DNA cleavage activity. The D10A mutation may bind to one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that lacks substantially all DNA cleavage activity. In certain embodiments, a CRISPR enzyme is considered to lack substantially all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less relative to its non-mutated form. Other mutations may be useful; if Cas9 or other CRISPR enzymes are from a species other than streptococcus pyogenes, mutations of the corresponding amino acids may produce similar effects.

In certain embodiments, the delivered protein (e.g., Cas protein or a variant thereof) may comprise a subcellular localization signal. For example, a Cas protein within an RNP may comprise a subcellular localization signal. Depending on the context, a fusion protein comprising, for example, Cas9 and a nuclear localization signal may be referred to herein as "Cas 9" without specifying that a nuclear localization signal is comprised. In some embodiments, the payload (e.g., RNP) comprises a fusion protein comprising a localization signal. For example, the fusion protein can comprise a nuclear localization signal, a nucleolar localization signal, or a mitochondrial targeting signal. These signals are known in the art and non-limiting examples are described in the following documents: kalderon et al, (1984) Cell 39(3 Pt 2): 499-509; makkerh et al, (1996) Curr biol.6(8): 1025-7; dingwall et al, (1991) Trends in Biochemical Sciences 16(12) 478-81; scott et al, (2011) BMC Bioinformatics 12:317(7 pages); omura T (1998) J biochem.123(6): 1010-6; rapaport D (2003) EMBO Rep.4(10): 948-52; and Brocard & Hartig (2006) Biochimica et Biophysica Acta (BBA) -Molecular Cell Research 1763(12): 1565-1573, each of which is herein incorporated by reference in its entirety. In various embodiments, the Cas protein may comprise more than one localization signal, e.g., 2, 3, 4, 5, or more nuclear localization signals. In some embodiments, the localization signal is at the N-terminus of the Cas protein, while in other embodiments, the localization signal is at the C-terminus of the Cas protein.

In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression of the coding sequence in a particular cell (e.g., a eukaryotic cell). The eukaryotic cell may be a cell of or derived from a particular organism, such as a mammalian cell including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a method of modifying a nucleic acid sequence by replacing at least one codon (e.g., about or more than 1,2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) in the native sequence with a codon that is more frequently or most frequently used in a gene of a host cell, thereby increasing expression in the host cell of interest while maintaining the native amino acid sequence. Different species show a particular preference for a particular codon for a particular amino acid. Codon bias (difference in codon usage between organisms) is usually related to the translation efficiency of messenger rna (mrna), which in turn depends on the nature of the codons being translated and the availability of specific transfer rna (trna) molecules. The predominance of selected tRNAs in cells generally reflects the codons most commonly used in peptide synthesis.

Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism. Codon usage tables are readily available, e.g., in the "codon usage database", which can be adjusted in many ways. See Nakamura, y., et al, "Codon usage taped from the interactive DNA sequences databases: status for the year 2000" nucleic acids res.28:292 (2000.) computer algorithms optimize specific Codon sequences for expression in a particular host cell, e.g., Gene form (Aptagen; Jacobus, Pa.) in some embodiments, one or more codons (e.g., 1,2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding a CRISPR enzyme correspond to the most commonly used codons for a particular amino acid.

In general, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and sequence-specifically bind the CRISPR complex directly to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using a suitable alignment algorithm. In some embodiments, the degree of complementarity is 100%. Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burrows-Wheeler transform-based algorithms (e.g., Burrows-Wheeler aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, elad (san and san diego, california), soap (applicable at soap. genomics. org. cn), and Maq (applicable at maq. sourceform. net.) in some embodiments, the guide sequence length is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more, in some embodiments, the ability of the guide sequence to specifically bind to a target sequence may be assessed by, for example, the CRISPR analysis of the target sequence by a guide sequence length of about 5, 10, 20, 25, 30, 35, 40, 45, 50, 75, or more, components of the CRISPR system (including the guide sequence to be tested) sufficient to form a CRISPR complex can be provided to a host cell having the corresponding target sequence, for example by transfection with a vector encoding the components of the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence, for example by the surfyor assay described herein. Similarly, cleavage of a polynucleotide sequence of interest can be assessed in vitro by providing the sequence of interest, components of the CRISPR complex (including the guide sequence to be tested and a control guide sequence different from the test guide sequence), and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions.

CRISPR Cas technology has facilitated the development of genome engineering for a variety of cell types. Recent studies have shown that providing Cas9-gRNA editing tools in the form of Ribonucleoproteins (RNPs) has several benefits over providing plasmids encoding Cas9 and gRNAs. Benefits include faster and more efficient editing, less off-target effects, and less toxicity. RNPs have been delivered by lipofection and electroporation, but these delivery methods still have limitations, particularly for certain clinically relevant cell types, including toxicity and inefficiency. Accordingly, there is a need to provide a vector-free (e.g., virus-free) method for delivering biologically relevant payloads (e.g., RNPs) across the plasma membrane and into cells. "cargo" or "payload" is a term used to describe a compound or composition that enters the interior of a cell through the plasma membrane of the cell by passing through an aqueous solution.

The present subject matter relates to a delivery technique that can accelerate the delivery of a wide range of payloads into cells with low toxicity. Genome editing can be achieved by delivering RNPs to cells using certain aspects of the present subject matter. Thereafter the level drops until Cas9 is no longer detected. The delivery technique itself does not have a deleterious effect on the activity or function of Jurkat and primary T cells. The subject matter of the invention is the gene editing of clinically relevant cell types by Cas9-RNPs with minimal toxicity.

Transient and direct delivery of CRISPR/Cas components, such as Cas and/or grnas, has advantages over expression vector-mediated delivery. For example, an amount of Cas, gRNA, or RNP can be added at a more precise timing and for a limited time than when using an expression vector. The components expressed from the vector may be produced in different quantities and at different times, which makes it difficult to achieve consistent gene editing without deviating from the targeted editing. In addition, preformed complexes (RNPs) of Cas and gRNAs cannot be delivered with the expression vector.

In one aspect, the present subject matter describes cells attached to a solid support (e.g., a strip, a polymer, a bead, or a nanoparticle). The scaffold or scaffolding may be a porous or non-porous solid scaffold. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbros and magnetite. For the purposes of the present subject matter, the nature of the carrier may be either soluble or insoluble to some extent. The stent material can have virtually any possible structural configuration. Thus, the scaffold configuration may be spherical, for example, in the bead type or conical, on the outer surface of a tube, such as the inner surface of a tube or tube. Alternatively, the surface may be flat, such as a sheet or test strip. Preferred scaffolds include polystyrene beads.

In other aspects, the solid support comprises a polymer to which the cells are chemically bound, immobilized, dispersed, or otherwise associated. The polymeric carrier may be a polymer network and may be in the form of beads (e.g., by suspension polymerization). Cells on such scaffolds can be sprayed with an effective load of the present invention containing an aqueous solution to deliver the desired compound to the cytoplasm of the scaffold. Exemplary scaffolding includes stents and other implantable medical devices or structures.

Other embodiments

In the foregoing description and claims, phrases such as "at least one" or "one or more" may appear after a conjunctive list of elements or features. The term "and/or" may also be present in a list of two or more elements or features. The phrase is intended to mean any element or feature listed individually or in any combination of any recited element or feature and any other recited element or feature unless otherwise implicitly or explicitly contradicted by context in which the phrase is used. For example, the phrases "at least one of a and B," one or more of a and B, "and" a and/or B "each mean" a alone, B alone, or a and B together. A similar interpretation may also mean that the list comprises three or more items. For example, the phrases "at least one of A, B and C", "one or more of A, B and C", and "A, B and/or C" each 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". Furthermore, the use of the term "based on" in the claims means "based at least in part on" such an unrecited feature or element is also permissible.

The subject matter described herein may be implemented in systems, apparatuses, methods, and/or articles of manufacture according to a desired configuration. The embodiments set forth in the foregoing description do not represent all embodiments consistent with the subject matter described herein. Rather, they are merely a few examples consistent with aspects related to the subject matter described herein. Although some variations have been described in detail above, other modifications or additions may be made. In particular, other features and/or variations may be provided in addition to those set forth herein. For example, the embodiments described above may refer to combinations or subcombinations of the various features disclosed above and/or combinations and subcombinations of several further features disclosed above. In addition, the logic flows depicted in the figures and/or described herein do not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other embodiments may be within the scope of the following claims.