WO2023246851A1

WO2023246851A1 - Portable in-vitro blood cell gene editing or modifying system

Info

Publication number: WO2023246851A1
Application number: PCT/CN2023/101640
Authority: WO
Inventors: 林欣; 袁建龙; 邓小红; 马文博; 王嘉盛
Original assignee: 北京昌平实验室
Priority date: 2022-06-24
Filing date: 2023-06-21
Publication date: 2023-12-28

Abstract

A portable in-vitro blood cell gene editing or modifying system, comprising: a fluid inlet (111) and a fluid outlet (112); and processing units (1200, 1201, 1202, 1203), the processing units (1200, 1201, 1202, 1203) being configured to perform gene editing or gene modification on a target cell in blood input through the fluid inlet (111) by means of gene delivery, and being further configured to remove non-therapeutic substances in the processed blood, and the processing units (1200, 1201, 1202, 1203) being in fluid connection between the fluid inlet (111) and the fluid outlet (112).

Description

Portable in vitro blood cell gene editing or modification system

Technical field

This application relates to extracorporeal blood cell therapy instruments and systems, in particular to extracorporeal blood cell gene editing or gene modification systems for treating diseases based on gene editing or gene modification technology.

Background technique

Currently, cell therapy products based on gene editing or gene modification technology are required to be prepared in cell preparation factories that meet certified clean standards, such as CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) ), T-Cell Receptor T-Cell Immunotherapy (TCR-T), synthetic T-Cell Antigen Receptor T-Cell Immunotherapy (Synthetic T-Cell Antigen Receptor T-Cell Immunotherapy , STAR-T) and other different types of cell therapy products.

However, the preparation process of these cell products has a long cycle, high consumption and large investment. Taking CAR-T as an example, the current mainstream process is that after blood collection in the hospital, it is transported to a GMP-level preparation workshop for the isolation of peripheral blood mononuclear lymphocytes (PBMC), the purification of T cells, and the isolation of T cells. Activation and CAR gene transduction, subsequent amplification and culture, concentration filling and cryopreservation. After quality release, it is transported to the hospital via cold chain for reinfusion to the patient. The entire process is complicated and has many environmental exposure links, which leads to increased quality control and further increases preparation costs. Therefore, there is an urgent need for an integrated rapid cell preparation system that no longer carries out complicated technological processes, but directly conducts rapid gene transduction of blood cells to achieve the preparation of genetically modified cell products. These blood cells can include T cells, B cells, NK cells, macrophages, monocytes, innate lymphoid cells, etc. This system does not need to go through complicated separation, activation and other steps. Instead, it collects PBMCs in vitro and directly performs real-time, online, fast, fully closed gene modification or gene delivery on PBMC cells, thereby achieving rapid CAR-T and CAR-NK The preparation of cell products such as TCR-T and TCR-T greatly shortens the preparation time of these cell products and reduces the preparation cost of gene-edited cell products.

Contents of the invention

In response to the above problems, this application aims to propose a novel genetically engineered cell therapy system, including collection of patient cells, PBMC cleaning device, reagent injection equipment, cleaning of cells after gene editing, and reinfusion of edited cells. Supporting equipment, as well as methods for isolating PBMCs, transducing PBMC cells, cleaning PBMCs after transduction, and diluting and reinjecting new genetically modified lymphocyte products performed on this system, such as CAR-T, CAR-NK, TCR-T, etc. can be produced using this system. Compared with traditional preparation methods in cell factories, there is no need to isolate and Purification, activation, amplification and other steps only require one transfection step to complete the preparation of these cell products, thus shortening the preparation cycle of genetically modified or gene-edited cell products, thereby shortening the treatment cycle and ensuring that patients can receive treatment in a timely manner. cells, thereby improving the patient's chance of survival.

According to one aspect of the present application, an extracorporeal blood cell therapy instrument is provided, which has a host computer, and the host computer includes:

case;

a fluid inlet and a fluid outlet provided in the housing;

a first processing unit located inside the housing, the first processing unit being configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet through gene delivery; and

a second processing unit located inside the housing, the second processing unit being configured to remove substances that do not require treatment in the blood processed by the first processing unit,

The first processing unit and the second processing unit are in turn fluidly connected in series between the fluid inlet and the fluid outlet.

Optionally, the first processing unit includes a container for containing liquid, the fluid inlet is fluidly connected to the container, the container is fluidly connected to the second processing unit via a pipeline, and the second A processing unit is in fluid connection with the fluid outlet.

Optionally, the first processing unit at least includes a first injection module and a second injection module that are independent of each other, and the first injection module stores a first reagent that can be selectively injected into the container, so The second injection module stores a second reagent that can be selectively injected into the container.

Optionally, the first reagent includes a buffer, and the second reagent includes a gene delivery reagent.

Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells.

Optionally, at least one of the injection modules is detachably installed within the housing.

Optionally, the second treatment unit includes a filter, a first recovery end located downstream of the filter, and a second recovery end located upstream of the filter.

Optionally, the first processing unit further includes a shaking mechanism by means of which the container can be selectively shaken.

Optionally, a first switch for selective on-off is provided at the first recovery end, and a second switch for selective on-off is provided at the second recovery end.

Optionally, a recovery module is fluidly connected to the first recovery end via a pipeline, so that when the first switch is in a conductive state, water can be collected from the second processing unit, especially from the filter. The downstream recovery liquid; and/or, a pipeline is configured to fluidly connect the container with the second recovery end, so that when the second switch is in a conductive state, the It is possible to recover liquid from the second treatment unit, in particular from upstream of the filter.

Optionally, the second treatment unit further includes a waste liquid outlet located downstream of the filter, so that the waste liquid filtered by the filter can be discharged from the second treatment unit; The output end of the filter is different from the second recovery end, so that the suspension intercepted by the filter can be discharged from the second treatment unit.

Optionally, a third switch capable of selectively switching on and off is provided at the waste liquid outlet end, and a fourth switch capable of selectively switching on and off is provided at the output end.

Optionally, the fluid outlet is fluidly connected to the output end, so that the suspension trapped by the filter can be discharged into the fluid outlet when the fourth switch is in a conductive state.

Optionally, the container includes a fluid outlet end, and a pipeline fluidly connecting the container to the second processing unit is connected to the fluid outlet end.

Optionally, a fifth switch that is selectively on and off is provided at the fluid outlet end, so that the liquid in the container can pass through the fluid outlet end only when the fifth switch is in a conductive state. to the second processing unit.

Optionally, the filter is a molecular sieve, a centrifugal filter device, a magnetic screening filter device, a chromatography column, or a filter membrane.

Optionally, the filter is configured such that target cells intended to be transduced or transfected are trapped upstream of the filter.

Optionally, at least when the fifth switch is in the off state, blood can be input into the first treatment unit, in particular the container of the first treatment unit, through the fluid inlet.

Optionally, a sorting module is provided upstream of the first processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is connected to the first processing unit through a pipeline. Fluidically connected, the sorting module is configured to exclude substances other than the target cells from the blood before the blood is introduced into the first processing unit.

Optionally, the sorting module can use physical means or biological means to exclude substances other than the target cells from the blood.

Optionally, the extracorporeal blood cell therapy instrument further includes a movable base and a pillar provided on the base, the pillar being configured to support the housing.

Optionally, the extracorporeal blood cell therapy instrument further includes a display for displaying data monitored during the operation of the extracorporeal blood cell therapy instrument; and an input device for setting operating parameters of the extracorporeal blood cell therapy instrument. .

Optionally, the reagents for gene delivery include viral vectors or non-viral vectors.

Optionally, the undesirable substance includes a gene delivery agent.

Alternatively, the target cells include, but are not limited to, T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

According to another aspect of the present application, an extracorporeal blood cell therapy instrument is provided, which has a host computer, and the host computer includes:

case;

a fluid inlet and a fluid outlet provided in the housing;

A processing unit located inside the housing, the processing unit is configured to perform gene editing or gene modification on the target cells in the blood input through the fluid inlet through gene delivery, and is also configured to perform gene editing or gene modification in pairs after the treatment Remove substances from the blood that do not require treatment,

The processing unit is fluidly connected between the fluid inlet and the fluid outlet.

Optionally, the processing unit includes a container for containing liquid, and the processing unit further includes at least a first injection module and a second injection module that are independent of each other, and the first injection module stores a liquid that can be selectively injected. to the first reagent in the container, and the second injection module stores a second reagent that can be selectively injected into the container.

Optionally, a filter is provided in the container, and the container includes a fluid input end fluidly connected to the fluid inlet; and a fluid output end fluidly connected to the fluid outlet, the fluid input end and the fluid output is located upstream of the filter.

Optionally, the first injection module and/or the second injection module are configured to selectively inject respective reagents into the container upstream of the filter.

Optionally, the first injection module and/or the second injection module are configured to selectively inject respective reagents into the container downstream of the filter.

Optionally, a first switch that is selectively on and off is provided at the fluid output end, so that the liquid in the container can be discharged through the fluid outlet when the first switch is in a conductive state.

Optionally, the container further includes a waste liquid outlet end and a recovery end located downstream of the filter and independent of each other, a second switch capable of selectively switching on and off is provided at the recovery end, and the A third switch capable of selectively switching on and off is provided at the waste liquid outlet end.

Optionally, a recovery module is fluidly connected to the recovery end via a pipeline, so that liquid can be recovered from the container, especially from downstream of the filter, when the second switch is in a conductive state.

Optionally, the treatment unit further includes a pressure difference generating device to selectively generate a pressure difference between upstream and downstream of the filter to urge liquid to flow through the filter.

Optionally, the processing unit further includes a shaking mechanism by means of which the container can be selectively shaken.

Optionally, a sorting module is provided upstream of the processing unit, the inlet of the sorting module is fluidly connected to the fluid inlet, and the outlet of the sorting module is fluidly connected to the processing unit via a pipeline, so The sorting module is configured to exclude substances other than the target cells from the blood before the blood is introduced into the processing unit.

Optionally, when the first switch, the second switch, and the third switch are all in an off state, blood is input into the container via the fluid inlet.

Optionally, after blood is input into the container, buffer is selectively injected into the container via the first injection module to ensure that there is at least enough to dilute the blood upstream of the filter amount of liquid; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

Optionally, the third switch is in a conductive state, so that at least a part of the liquid located downstream of the filter can be discharged through the waste liquid outlet end.

Optionally, when the first switch, the second switch, and the third switch are all in an off state, the transduction or infection reagent is selectively injected into the In the container, an amount of liquid sufficient to immerse the target cells intended to be transduced or transfected is present upstream of the filter; simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container.

Optionally, after a predetermined period of time since the transduction or infection reagent is injected, the second switch is in a conductive state, so that at least a part of the liquid located downstream of the filter can pass through The recovery end is output to the recovery module.

Optionally, after the liquid is recovered, when the first switch, the second switch, and the third switch are all in a disconnected state, the buffer liquid is used to flush the remaining liquid in the container.

Optionally, flushing is implemented as follows:

is selectively injected into the container via the first injection module to ensure that a predetermined amount of liquid is present upstream of the filter, and simultaneously or subsequently, the shaking mechanism is activated to selectively shake the container;

Then, the third switch is turned on, so that at least a part of the liquid located downstream of the filter can be discharged through the waste liquid outlet end.

Optionally, the flushing is completed multiple times.

Optionally, after the last flush is completed, the first switch is in a conductive state, so that the filter is At least a portion of the swimming liquid can be discharged via the fluid outlet.

Optionally, the undesirable substance includes a gene delivery agent.

According to another aspect of the present application, a portable in vitro blood cell gene editing or modification system is provided, which includes:

A first processing unit configured to perform gene editing or gene modification on target cells in the blood input to the system through gene delivery; and

a second processing unit configured to remove substances that do not require treatment in the blood treated by the first processing unit,

The first processing unit and the second processing unit are sequentially fluidly connected in series.

Optionally, the first processing unit includes a container for containing liquid, and the container is fluidly connected to the second processing unit via a pipeline.

Optionally, at least one of the injection modules is detachably mounted.

Optionally, a recovery module is fluidly connected to the first recovery end via a pipeline, so that when the first switch is in the conducting state The liquid can be recovered from the second treatment unit, especially from the downstream of the filter, in a pass-through state; and/or, a pipeline is configured to fluidly connect the container with the second recovery end, so as to This allows liquid to be recovered from the second processing unit, especially from upstream of the filter, when the second switch is in a conductive state.

Optionally, the portable extracorporeal blood cell therapy system further includes a movable base and a support provided on the base.

Optionally, the portable extracorporeal blood cell gene editing or modification system further includes a display for displaying data monitored during the operation of the extracorporeal blood cell therapy instrument; and an input device for setting the extracorporeal blood cell therapy instrument operating parameters.

Optionally, the undesirable substance includes a gene delivery agent.

Alternatively, the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

According to another aspect of the present application, a portable in vitro blood cell gene editing or modification system is also provided, which includes:

fluid inlet and fluid outlet;

A processing unit configured to perform gene editing or gene modification on target cells in the blood input through the fluid inlet through gene delivery, and further configured to perform gene editing or gene modification on unwanted cells in the processed blood. substances are removed,

Optionally, at least one of the injection modules is detachably mounted.

Optionally, flushing is implemented as follows:

Optionally, the flushing is completed multiple times.

Optionally, after the last flush is completed, the first switch is in a conductive state, so that at least a part of the liquid upstream of the filter can be discharged through the fluid outlet.

Optionally, the portable in vitro blood cell gene editing or modification system further includes a movable base and a support provided on the base.

Optionally, the undesirable substance includes a gene delivery agent.

According to another aspect of the present application, there is also provided an application for blood treatment using the aforementioned extracorporeal blood cell therapy device or the aforementioned portable extracorporeal blood cell gene editing or modification system.

fluid inlet and fluid outlet;

A processing unit configured to perform gene editing or gene modification on resting or non-activated target cells in the blood input through the fluid inlet through gene delivery, and is also configured to pair the treated blood Remove substances in cells that do not require treatment,

Optionally, the processing unit includes a container for containing liquid, and the processing unit further includes at least a first injection module and a second injection module that are independent of each other, and the first injection module stores a liquid that can be selectively injected. to the first reagent in the container, and the second injection module stores a second reagent that can be selectively injected into the container, so that the blood input into the container does not leave the container. In this case, the target cells in the blood are gene edited or genetically modified through gene delivery and the non-treatable substances in the treated blood are removed.

Optionally, the gene delivery reagent is used to achieve transduction or transfection of target cells under conditions that are not separated or purified or activated or amplified.

Optionally, a filter is provided in the container, and the container includes a fluid input end fluidly connected to the fluid inlet; and a fluid output end fluidly connected to the fluid outlet, the fluid input end and the fluid output is located at the upstream of the above filter.

Optionally, the filter is a molecular sieve, a magnetic screening filter device, a chromatography column, or a filter membrane.

Optionally, the processing unit is a separation device that uses density gradient centrifugation to separate PBMCs, and the container of the processing unit includes a first container that can selectively rotate around a rotation axis.

Optionally, the container of the processing unit further includes a second container that is independent of the first container. Ficoll cell separation liquid is injected into the first container and as the first container rotates, the Ficoll cell separation liquid is relatively Different liquid component layers are generated in the radial direction of the rotation axis, the liquid containing the substance requiring treatment is drawn into the second container, and the second reagent is injected into the second container.

Optionally, the axis of rotation is the axis of rotation of the container itself.

Optionally, before the second reagent is injected into the first container, the Ficoll cell separation solution is injected into the first container and as the first container rotates, relative to the rotation axis Different liquid components are stratified in the radial direction, leaving only the substances in the blood that require treatment within the first container to mix with the subsequently injected second agent.

Optionally, the non-viral vectors include synthetic vectors or biological vectors; and/or the viral vectors include retroviruses or modifications or mutants thereof, lentiviruses or modifications or mutants thereof, adenoviruses or variants thereof. Modified form or mutant, or adeno-associated virus or modified form or mutant thereof.

Alternatively, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle.

Optionally, the portable in vitro blood cell gene editing or modification system has a host, and the host has a housing, so The processing unit, the first injection module, the second injection module and the sorting module are arranged in the housing.

Optionally, the gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMC.

Optionally, the time for transducing or transfecting the target cells using the gene delivery reagent under conditions that are not separated or purified or activated or amplified is 1 to 5 hours.

According to another aspect of the present application, an in vitro blood cell treatment method is provided, including:

An apheresis machine is used to collect blood from the patient's body;

Inject blood into the portable extracorporeal blood cell gene editing or modification system or extracorporeal blood cell therapy device, so that the injected blood can be injected without leaving the portable extracorporeal blood cell gene editing or modification system or extracorporeal blood cell therapy device. The target cells in the blood undergo gene editing or gene modification through gene delivery and are also configured to remove substances in the treated blood that do not require treatment;

The treated fluid is returned to the patient.

Optionally, the portable extracorporeal blood cell gene editing or modification system or extracorporeal blood cell therapy device includes a container for holding liquid to receive the injected blood,

The first reagent can be selectively injected into the container, and/or the second reagent can be selectively injected into the container, so that the blood input into the container does not leave the container. In this case, the target cells in the blood are gene edited or genetically modified through gene delivery and the non-treatable substances in the treated blood are removed.

Optionally, a container capable of selective rotation around the rotation axis is used as the container of the processing unit.

Optionally, before the second reagent is injected into the container, the Ficoll cell separation liquid is injected into the container and as the container rotates, different oscillations are produced in a radial direction relative to the rotation axis. The liquid components are separated into layers, leaving only those substances in the blood that require treatment within the container to mix with the subsequently injected second agent.

Alternatively, the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle. Alternatively, the target cells include, but are not limited to, T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.

According to another aspect of the present application, a method for in vitro gene editing or modification of blood cells is also provided, including:

Collect blood from the patient;

Under closed conditions, the resting or inactivated target cells in the collected blood are gene-edited or modified through gene delivery, and are also configured to remove non-treatable substances from the treated blood cells in pairs. ;

The treated fluid is returned to the patient.

Optionally, a first reagent and a second reagent are respectively injected into the collected blood, the first reagent includes a buffer solution, and the second reagent includes a gene delivery reagent.

Optionally, before injecting the second reagent, density gradient centrifugation is used to separate PBMCs in the blood, and the second reagent is injected into the separated PBMC liquid.

Using the above technical means of this application, the extracorporeal blood cell therapy instrument can directly input and collect the patient's blood from the scene, and directly perform gene editing of target cells in the blood at the treatment site or hospital bed. Compared with traditional GMP cell preparation factories, The treatment cycle is significantly shortened. In addition, since the various components inside the extracorporeal blood cell therapy device can be packaged in a manner that meets medical cleanliness requirements during manufacturing, the possibility of any accidental infection during the treatment process is avoided. More importantly, since the extracorporeal blood cell therapy device can be used at the treatment site, the attending doctor can change the treatment plan at any time according to the patient's condition changes and use the extracorporeal blood cell therapy device to implement the changed treatment plan, which improves the patient's survival rate. .

Description of the drawings

The principles and various aspects of the present application will be more fully understood from the following detailed description in conjunction with the following drawings. It should be noted that the proportions of the drawings may be different for the purpose of clear explanation, but this will not affect the understanding of the present application. In the attached picture:

Figure 1 is a perspective view schematically showing an embodiment of an extracorporeal blood cell therapy device according to the present application;

Figure 2 is a system block diagram schematically showing an embodiment of a host computer for an extracorporeal blood cell therapy device;

Figure 3 is a system block diagram, schematically showing another embodiment of a host used to implement an extracorporeal blood cell therapy instrument;

Figure 4 is a system block diagram schematically showing another embodiment of a host for implementing an extracorporeal blood cell therapy instrument; and

Figure 5 is a system block diagram, schematically showing another embodiment of a host used to implement an extracorporeal blood cell therapy device;

Figure 6 is a system block diagram, schematically showing another embodiment of a host used to implement an extracorporeal blood cell therapy device;

Figure 7A schematically shows a view of a centrifuge device according to an example of the present application;

Figure 7B schematically shows a view of a centrifuge device according to another example of the present application;

Figures 8A to 8D respectively provide transfection data indicator diagrams of blood processed using an example of the in vitro blood cell treatment protocol of the present application; and

Figure 9 is a diagram of experimental results, schematically showing the killing function of the transduced CAR-T cells after rapid CAR gene transduction of blood cells using an example of the in vitro blood cell treatment protocol of the present application.

Detailed ways

In the various drawings of this application, structurally identical or functionally similar features are designated by the same reference numerals.

Figure 1 schematically illustrates an embodiment of an extracorporeal blood cell therapy apparatus 100. The extracorporeal blood cell therapy instrument 100 includes a main body having a housing 110 . For example, the housing 110 of the host computer may be generally cubic or any other suitable shape. According to this application, the extracorporeal blood cell therapy device mainly uses gene editing/gene modification technology to process the patient's blood extracorporeally and infuse it back into the patient's body. Therefore, the housing 110 of the host can accommodate those processing units and devices described in detail below that are required for implementing in vitro processing of patient blood using gene editing/gene modification technology, as well as other items that may be needed but are not used in this application. devices not described in the instructions. The housing 110 is provided with a fluid inlet 111 and a fluid outlet 112, which are respectively used to input the patient's blood and output the blood processed by the extracorporeal blood cell therapy apparatus 100. For example, the fluid inlet 111 can be fluidly connected with the output end of a blood collection machine (not shown) equipped in the medical office, and the fluid outlet 112 can be fluidly connected with a device (not shown) for supplying blood to the patient's body. In the context of this application, the term "fluid connection" refers to a fluid-tight connection between two associated features, for example via medical hoses and/or pipes (not shown). Realized; optionally, this connection can also be a detachable connection. The position of the fluid inlet 111 and the fluid outlet 112 on the housing 110 of the host is suitable for convenient medical applications. For example, they can be disposed on the same surface of the housing 110 of the host or on different surfaces.

For example, a display screen 141 may be provided on the surface of the housing 110 of the host computer for displaying data collected and/or processed by the extracorporeal blood cell therapy apparatus 100 . Suitable input devices 142 such as keyboards can also be provided on the surface of the casing 110 of the host, so that parameters of relevant processing units, devices, and/or devices accommodated in the casing 110 of the host can be set as needed. In alternative embodiments, input device 142 may be omitted and display 141 provided as a touch display.

For example, the extracorporeal blood cell therapy instrument 100 may further include a base 180 for contacting the ground. At the same time, pillars 170 are provided on the top surface of the base 180 for supporting the housing 110 of the host machine. In addition, a plurality of rollers 181 can be provided on the bottom surface of the base 180, such as four rollers 181 (only three of them are shown in the figure), which are respectively located around the periphery of the base 180, so that the extracorporeal blood cell therapy instrument 100 can Move easily. In addition, a cable 190 with a plug that is electrically connected to a power supply device (not shown) provided in the casing 110 of the host can extend from the casing 110 of the host, wherein the power supply device can be a relevant processing unit, Provide power for device, and/or device operation. For example, the cable 190 can be connected to a dedicated power supply socket in a medical room, so as to provide power for normal operation of the extracorporeal blood cell therapy apparatus 100 through the power supply device.

It should be clear to those skilled in the art that in alternative embodiments, the base 180 and/or the supports 170 can also be omitted, so that the housing 110 of the main body of the extracorporeal blood cell therapy apparatus 100 can be directly placed on the table in the treatment room. For example, in the case of the base 180 and the support 170, the extracorporeal blood cell therapy apparatus 100 can be configured as a portable therapy apparatus in a manner similar to the appearance of a computer host or a server host.

The relevant processing units, devices, and/or devices contained in the housing 110 of the host constitute an extracorporeal blood cell therapy instrument. 100 main components of the host. FIG. 2 schematically illustrates an embodiment of the main body of the extracorporeal blood cell therapy apparatus 100. According to the embodiment shown in FIG. 2 , the extracorporeal blood cell therapy instrument 100 or more specifically its host computer includes a first processing unit 120 , a second processing unit 130 , and a control device 140 that are provided or integrated in the housing 110 of the host computer. .

The first processing unit 120 may include, for example, a container 123 for containing liquid, a first injection module 121 and a second injection module 122 . The container 123 has a fluid inlet port 123a, which is in fluid connection with the fluid inlet 111, and fluid inlet ports 123b and 123c, which are in fluid connection with the first injection module 121 and the second injection module 122, respectively. Furthermore, the container 123 also has a fluid outlet port 123d, which is fluidly connected to the second processing unit 130.

The control device 140 may include, for example, a computer processing unit, a memory, a data storage unit, and the like. For example, the computer processing unit can take the form of a single-chip microcomputer, an embedded computer chip, a computer, etc., and is used to call and execute the program stored in the memory, and during the execution of the program, the data obtained or processed by the control device 140 can be Stored in the data storage unit for subsequent recall. The control device 140 is operatively connected to the first injection module 121 and the second injection module 122 . In the context of this application, the term "a control device operatively connected to a feature" means that the control device can be connected to the feature or a sub-feature within the feature and operate the feature or a sub-feature within the feature. Take control. Taking the first processing unit 120 as an example, the control device 140 being operatively connected to the first processing unit 120 means that the control device 140 can connect the first injection module 121 and the second injection module 122 via corresponding control lines (not shown). And the operation of its liquid injection mechanism (as described later) is controlled so that the corresponding liquid can be quantitatively injected into the container 123 as needed. In addition, the control device 140 is also operatively connected to the display screen 141 and the input device 142.

The first processing unit 120 and the second processing unit 130 are fluidly connected between the fluid inlet 111 and the fluid outlet 112 . The first processing unit 120 is configured to perform gene editing or gene modification through gene delivery on the target cells in the blood input through the fluid inlet 111 . For example, the reagents for gene delivery include viral vectors or non-viral vectors. For another example, the target cells include, but are not limited to, T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells. The gene delivery reagent is configured to achieve transduction or transfection of target cells. In the context of this application, the term "transduction" refers to the introduction of foreign genes into eukaryotic cells or prokaryotic cells through specific vectors, such as recombinant viral vectors, thereby causing the corresponding gene recombination of the cells or the expression or play of the foreign genes as needed. Functional process; the term "transfection" refers to the process in which a recombinant viral vector invades a recipient cell, causing gene recombination or gene expression or function in the recipient cell. Here, vectors or viral vectors refer to self-replicating DNA molecules or liquids or fluids containing such DNA molecules that transfer DNA fragments (target genes) to recipient cells in genetic engineering recombinant DNA technology. For example, commonly used vectors include non-viral vectors and viral vectors. Non-viral vectors include synthetic vectors represented by lipid nanoparticles (LNP), lipid polyplexes (LPP), etc., and biological carriers represented by extracellular vesicles. Viral vectors include retroviruses or modifications or mutants thereof, lentiviruses or modifications or mutants thereof, adenovirus or modifications thereof or Mutants, or adeno-associated viruses (AAV) or modifications or mutants thereof, etc., also include nucleic acid vectors such as plasmids and phages. In the technical solution of this application, the vectors or viral vectors used include wild-type or mutant or viral vectors modified by chemical or biological methods. At the same time, the vector containing the vector or viral vector is screened to improve The transduction or transfection efficiency of the vector or viral vector into the recipient cells.

The fluid outlet end 123d of the container 123 is located at the lowest point of the container 123 in the direction of gravity, and the fluid inlet ends 123a, 123b, 123c are a certain distance higher than the fluid outlet end 123d. It is advisable not to affect subsequent processing of blood cells. In this way, the blood cells transported through the fluid inlet 111 can be discharged into the container 123 through the fluid inlet end 123a. After undergoing certain processing, they can be discharged to the second processing unit 130 by gravity through the fluid outlet end 123d. During the processing , liquid or fluid can be discharged from the first and second injection modules 121 and 122 into the container 123 through the respective fluid inlet ports 123b and 123c as needed. In addition, the container 123 may also be provided with an exhaust port (not shown), thereby facilitating the exhaust gas generated during the treatment process to be discharged from the container 123 to the outside world.

Each injection module 121 or 122 may, for example, include a compartment and a liquid injection mechanism known to those skilled in the art such as an electric injector or a metering device for metering out the liquid or fluid contained in the compartment independently of each other via the fluid inlet port 123b or 123c. Pump. According to embodiments of the present application, the containers and/or compartments described above and/or below may be configured in any medically compliant liquid or fluid containing form such as a can, bag, bottle, etc. The container 123 and/or the injection modules or at least one of them is detachably arranged within the first processing unit 120 and/or the compartments are detachably arranged, for example in the form of, for example, cans, bags, bottles, etc. In the injection module 121 or 122, they can be easily replaced or replenished with injection liquid according to actual needs during medical treatment.

Each compartment of the first and second injection modules 121 and 122 can be loaded with different processing reagents as needed, so that the corresponding processing reagents can be injected into the container. For example, the compartment of the first injection module 121 may be configured to be able to load a buffer, and the compartment of the second injection module 122 may be configured to be able to load a gene delivery reagent for transducing or transfecting target cells. In the context of this application, gene delivery reagents mainly refer to reagents, including viruses (genetically modified or non-modified), that help transduce foreign genes or genes of interest into target cells such as T cells, B cells, NK cells or other types of cells. Vectors such as lentiviral vectors, adeno-associated virus vectors, plasmid vectors, lipid nanoparticles (LNP), cationic lipid complexes (LPX), lipid polypolymers (LPP), inorganic nanoparticles (INP) and other reagents; Or non-viral vectors, such as transposons, etc. For example, a buffer may be used to dilute blood supplied into container 123 via fluid inlet 111 or alternatively may be used to flush container interior 123 before or after each treatment. For example, the buffer solution can be physiological saline, culture solution, nutrient solution, culture medium, etc., or other liquids that can be used in the application of this application. A switch K10 can be provided at the fluid outlet end 123d, and whether the liquid in the container 123 enters the second processing unit 130 through the fluid outlet end 123d can be controlled by turning the switch K10 on or off. For example, the switch K10 may take any suitable form such as an electromagnetic switch valve, and the control device 140 is operatively connected to the switch K10, Therefore, under the control of the control device 140, the switch K10 can be selectively turned on and off.

The second processing unit 130 may include, for example, a filter 131, which can be implemented by membrane filter, molecular sieve, centrifugal filtration, chromatography, etc. In the embodiment shown in FIG. 2 , the second processing unit 130 is introduced using a filter membrane as an example. However, those skilled in the art should know that if other methods are used to implement the filter, the principles of the present application are still applicable. For example, by selecting a suitable pore size of the filter membrane as the filter 131, it is possible to ensure that selected eukaryotic cells or prokaryotic cells or receptor cells in the blood or blood-related cells or other types of cell suspensions can be intercepted by the filter membrane of the filter 131 of the second processing unit 130 . Therefore, the second treatment unit 130 includes an input end 130a located upstream of the filter 131, which is fluidly connected to the fluid outlet end 123d of the container 123 through the pipeline L10; and a waste liquid outlet end 130b located downstream of the filter 131, which is used to filter the The final waste liquid is discharged from the second treatment unit 130; and an output end 130c is also located upstream of the filter 131 but downstream of the input end 130a, which is used to discharge the suspension trapped by the filter 131. The second processing unit 130 is configured to remove substances that do not require treatment in the blood processed by the first processing unit. For example, the substances that do not require treatment include excess gene delivery reagents or other substances that do not require treatment.

In alternative or additional embodiments, the second processing unit 130 may include a first recovery end 130d downstream of the filter 131 and a second recovery end 130e upstream of the filter 131 . Switches K20, K30, K40, and K50 are respectively provided at the waste liquid outlet end 130b, the first recovery end 130d, the output end 130c, and the second recovery end 130e. For example, the switches K20, K30, K40, K50 can be constructed in a similar manner to K10, and the control device 140 is operatively connected to the switches K20, K30, K40, K50, so that under the control of the control device 140, the switches K20, K30, K40 and K50 can be selectively switched on and off. Furthermore, according to said alternative or additional embodiment, the first treatment unit 120 further comprises a recovery module 125, the third injection module being provided with a compartment for storing liquid. The first recovery end 130d is fluidly connected to the recovery module 125, in particular its compartment, via line L20. The second recovery end 130e is connected to the container 123 via the pipeline L30, so that the suspension trapped by the filter 131 during filtration can be returned to the container 123 via the pipeline L30. In an alternative embodiment, line L30 may also return directly upstream of fluid inlet port 123a so that returned liquid may be discharged into container 123 via fluid inlet port 123a.

In addition, the first processing unit 120 may further include a shaking mechanism 124 . The shaking mechanism 124 can be physically or mechanically connected to the container 123, thereby causing the container 123 to shake to a certain extent as needed. In this way, the buffer or carrier that helps to be injected into the container 123 can be repeatedly mixed with the blood injected into the container 123 through the fluid inlet 111, thereby improving the efficiency of dilution, transduction or transfection. It should be clear to those skilled in the art that although the container 123 is equipped with a shaking mechanism 124, the fluid connections of the inlet end 123a, 123b, 123c, and the outlet end 123d of the container 123 will not be adversely affected during the process of the container 123 being shaken. . The shaking mechanism 124 can be implemented in any manner known to those skilled in the art. As just an example, the shaking mechanism 124 may include a link structure that is physically or mechanically connected to the container 123 and a motor that drives the link structure to reciprocate. The control device 140 is operatively connected to the shaking mechanism 124, especially its motor, so that under the control of the control device 140, the shaking mechanism 124 can cause the container 123 to shake to a certain extent. In an alternative embodiment, rocking mechanism 124 may be independently controlled to operate.

The first processing unit 120 and the second processing unit 130 are fluidly connected to each other via the pipeline L10. The second processing unit 130 is configured to remove carriers and/or viruses from the liquid treated by the first processing unit 120 (for example, it is discharged via the line L10).

It will be clear to those skilled in the art that a pumping device can be provided in pipelines L10 and/or L20 and/or L30 or optionally any required pipelines (as described above or below), so that the movement of liquid can be independent of Gravity is achieved by the operation of the pumping device, and the control device 140 is operatively connected to the pumping device to control its operation.

The arrangement of the recovery end 130d allows the filtrate to be recovered and reused as needed, which is particularly important for expensive or costly carrier recovery. The following is a brief description of the process of using the extracorporeal blood cell therapy apparatus 100 to treat blood according to FIG. 2 . In the following process description, related control programs can be implemented as coded programs and stored in the memory for the control device 140 to call and execute. It should be clear that the process described below is only illustrative but not limiting. That is to say, those skilled in the art can think of using the extracorporeal blood cell therapy apparatus 100 to perform other feasible operations after reading the description of this application, which is also consistent with this application. purpose of the application and falls within the scope of this application.

First, the collected blood or blood-related cells or other types of cell suspensions are injected into the container 123 of the first processing unit 120 through the fluid inlet 111 . At this time, the switch K10 is in an off state, and the first injection module 121 starts to inject a certain amount of buffer solution into the container 123. At the same time, the shaking mechanism 124 operates to cause the container 123 to shake, ensuring that blood or blood-related cells or other types of The cell suspension and buffer are mixed evenly so that the components in the buffer, such as gene vectors or viral vectors, can fully contact the cells. Then, the switch K10 is turned on, and the mixed liquid is input to the second processing unit 130 via the pipeline L10. At this time, the switches K30, K40, and K50 of the second processing unit 130 are in the off state, and the switch K20 is in the on state. Therefore, the filtrate that has passed through the filter 131 (located downstream of the filter 131) is discharged from the second treatment unit 130 through the waste liquid outlet end 130b. By appropriately selecting the pore size of the filter membrane of the filter 131 , substances of smaller size that do not require treatment can be filtered out from the blood or blood-related cells or other types of cell suspensions. The eukaryotic cells or recipient cells that need to be transduced or transfected are trapped in the remaining suspension upstream of the filter 131 . At this time, the switches K20, K30, K40 of the second processing unit 130 and the switch K10 of the first processing unit 120 are in an off state, and for example, by activating the pumping device related to the pipeline L30, the second processing unit 130 The remaining suspension is returned to container 123 via line L30. It should be clear to those skilled in the art that the fluid inlet ends 123a, 123b, 123c of the container 123 and the fluid connection port of the pipeline L30 and the container 123 are positioned higher than the liquid level of the container 123 during transduction or transfection processing, or in other words, It is better if it does not affect the transduction or transfection processing in the container 123.

In order to fully dilute the blood and eliminate smaller non-treatable substances, the above steps can be repeated multiple times. Next, the second injection module 122 is started to inject a certain amount of reagents for transduction or transfection into the container 123 (the switch 10 is in an off state), and at the same time, the shaking mechanism 124 operates to cause the container 123 to shake, thereby ensuring The reagents are thoroughly mixed with the liquid in container 123 . Then, you can wait for a certain period of time to allow the eukaryotic cells or recipient cells in the liquid in the container 123 to be fully transduced or transfected. For example, the time can be say 2 hours or more. During this period, the shaking mechanism 124 can be selectively operated to facilitate the improvement of transduction or transfection efficiency.

Various sensors can be installed in the container 123 and/or in the pipeline, such as pressure sensors, temperature sensors, etc., and the control device 140 can be operatively connected to these sensors to determine whether the transduction or transfection process is based on the data measured by the sensors. Normal or monitor the operating status of the treatment device 100. Optionally, the measured data can be displayed on the display 141 for monitoring by the user.

After the transduction or transfection has been completed (for example, judged by the passage of a predetermined time or the monitoring of the operating status), the control device 140 instructs the switch K10 to be in a conductive state, so that the liquid that has been transduced or transfected is transferred from the first A processing unit 120 is input to the second processing unit 130 via pipeline L10.

Because reagents used for transduction or transfection are expensive, they need to be recycled. In this way, at least when the liquid that has been transduced or transfected is input to the second processing unit 130 for the first time (or in other words, the switch K10 is in the conductive state for the first time after the first processing unit 120 has completed the transduction or transfection processing. (on state), the switches K20, K40, and K50 of the second processing unit 130 are in an off state and the switch K30 is in an on state, so that the filtrate after the input liquid is filtered by the filter 131 still contains a high concentration of The vector is transduced or transfected, and the filtrate can then be collected into the recovery module 125 via line L20, so that it can be used again after further purification.

Then, the switches K30, K40, and K50 are in the off state, and the switches K10 and K20 are in the on state, and the first injection module 121 is started to inject the buffer into the container 123 and enter the second processing unit 130 , thereby further flushing the suspension trapped by the filter 131, and the filtrate is discharged through the waste liquid outlet end 130b. After the flushing has been carried out multiple times, the startup of the first injection module 121 can be stopped and the switches K20, K30, K50 are in the off state and the switch K40 is in the on state, so that the final suspension can pass through the output end 130c from Fluid outlet 112 output.

In an alternative embodiment, during the flushing process, the pipeline L30 can be used to return the suspension trapped by the filter 131 to the container 123 and the buffer solution can be injected again and the container 123 can be shaken while the switch 10 is in the off state. Finally, the switch is turned on and then filtered by the filter 131 of the second processing unit 130, thus ensuring a better flushing effect.

In the extracorporeal blood cell therapy device 100 of the present application, any part involving fluid connection is configured to comply with the mandatory national medical and health regulations. Compared with traditional GMP cell preparation factories that require the same high medical cleanliness, manufacturing costs The applied extracorporeal blood cell therapy apparatus 100 obviously achieves lower cost. In addition, the input blood is only processed inside the extracorporeal blood cell therapy device 100. There is no need to connect the various processes of the traditional GMP cell preparation factory, which greatly eliminates the possibility of accidental blood infection. More importantly, because the extracorporeal blood cell therapy device 100 of the present application can be used directly at the treatment site where the patient is located, the attending doctor can perform targeted and purposeful treatment by changing the therapeutic reagents or carriers according to the patient's condition. The treatment plan can be changed in a timely manner on site, which is conducive to the patient's recovery.

In alternative or additional embodiments, the first processing unit 120 of the extracorporeal blood cell therapy apparatus 100 can also be configured to have third, fourth, fifth or more injection modules, and each injection module stores different information in advance as needed. transduction or transfection reagents, so that the treatment plan can be easily switched according to changes in the patient's condition or different patients.

FIG. 3 schematically illustrates a host of an extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application. The first processing unit 1200 is provided or integrated in the housing 110 of the host.

The first processing unit 1200 may include, for example, a container 1230 for containing liquid, a first injection module 121 and a second injection module 122 . Here, the first injection module and the second injection module are denoted by the same reference numerals as the first injection module 121 and the second injection module 122 shown in FIG. 2 to mean the first injection module of the embodiment shown in FIG. 3 and the second injection module can be arranged in the manner described for the embodiment directed by Figure 2 or corresponding alternatives or additions. Therefore, the description of the first injection module 121 and the second injection module 122 in FIG. 3 may refer to the description of FIG. 2 .

The container 1230 has a fluid inlet port 1230a, which is in fluid connection with the fluid inlet 111; and fluid inlet ports 1230b and 1230c, which are in fluid connection with the first injection module 121 and the second injection module 122, respectively. A filter 1310 is provided inside the container 1230 . For example, the filter 1310, similar to the filter 131, can also be provided in the form of a filter membrane. Fluid input port 1230a, fluid inlet ports 1230b, and 1230c are located upstream of filter 1310. In addition, the container 1230 also has a fluid output end 1230d, which can be fluidly connected to the fluid outlet 112 via a pipeline (not shown). A switch K100 may be provided at the fluid output end 1230d, which is configured to control whether the liquid in the container 1230 can be output through the fluid outlet 112 by turning the switch K10 on or off.

A waste liquid outlet port 1230e and a recovery port 1230f are provided at the bottom of the container 1230. The waste liquid outlet end 1230e and the recovery end 1230f are located downstream of the filter 1310. The recovery end 1230f is fluidly connected to the recovery module 125 via the pipeline L100. For example, the recovery module 125 can adopt the same configuration as the recovery module in the embodiment shown in FIG. 2 . Switches K200 and K300 can be respectively provided at the waste liquid outlet end 1230e and the recovery end 1230f, so that their on and off can respectively allow or prohibit the liquid in the container 1230, especially the liquid downstream of the filter 1310, from the waste liquid outlet end 1230e and the recovery end. 1230f discharge.

According to embodiments of the present application, whether for the embodiment shown in FIG. 2 or the embodiment shown in FIG. 3 or the embodiments described below, the output end 130c or 1230d located upstream of the filter 131 or the filter 1310 may set just right Higher than the filter 131 or 1310, so that a sufficient amount of the trapped suspension after filtration can be output through the output end. According to a preferred embodiment, the portion of the container 1230 downstream of the filter 1310 may be designed to gradually taper toward its bottom, for example, generally in the form of a bell mouth (large end on top and small end on the bottom). In this way, it can be ensured that the filtrate filtered by the filter 1310 can be discharged more quickly.

In order to improve the filtration efficiency, the first treatment unit may also include a pressure difference generating device 126. For example, the pressure difference generating device 126 may be a negative pressure generating device located downstream of the filter 1310 (as shown in the figure) or located upstream of the filter 1310. positive pressure generating device (not shown). When necessary, the pressure difference generating device 126 operates to generate a pressure difference from the upstream of the filter 1310 to the downstream of the filter 1310, prompting the liquid to flow through the filter 1310 as quickly as possible, thereby shortening the time required for filtration.

Similar to the embodiment shown in FIG. 2 , the host also includes a control device 140 provided or integrated within the housing 110 of the host. In addition, the first processing unit 1200 may further include a shaking mechanism 124 . The description of the control device 140 and the rocking mechanism 124 may be included in the description of the embodiment of FIG. 2 . In the embodiment shown in FIG. 3 , the control device 140 is operatively connected to the first processing unit 1200 , in particular to the switches K100 , K200 , K300 and the pressure difference generating device 126 of the first processing unit 1200 .

It should be clear that various sensors, such as pressure sensors, temperature sensors, etc., can be provided in the container 1230 and/or in the pipelines connected to the container, and the control device 140 can be operatively connected to these sensors to thereby measure data based on the sensors. To determine whether the transduction or transfection process is normal or to monitor the operating status of the treatment device 100.

The following schematically describes the use process of the extracorporeal blood cell therapy apparatus 100 using the host machine shown in FIG. 3 . In the following process description, related control programs can be implemented as coded programs and stored in the memory for the control device 140 to call and execute. It should be clear that the process described below is only illustrative but not limiting. That is to say, those skilled in the art can think of using the extracorporeal blood cell therapy apparatus 100 to perform other feasible operations after reading the description of this application, which is also consistent with this application. purpose of the application and falls within the scope of this application.

First, the collected patient's blood is injected into the container 1230 of the first processing unit 1200 via the fluid inlet 111 . At this time, switches K100, K200, and K300 are in the off state. Simultaneously or subsequently, the first injection module 121 starts to inject a certain amount of buffer solution into the container 1230, and at the same time, the shaking mechanism 124 operates to cause the container 1230 to shake, ensuring that the blood and the buffer solution are evenly mixed, thereby achieving a suitable dilution effect. The amount of buffer injected should be such that the final mixing level is at a certain height above the filter 1310 to ensure dilution of the blood. That is, the amount of liquid injected should ensure that there is a sufficient amount of liquid upstream of the filter 1310 to submerge the target cells.

Then, with the switch K200 in the on state, the part of the mixed liquid (waste liquid) located downstream of the filter 1310 will be discharged from the container 1230 through the waste liquid outlet end 1230e, and the part of the mixed liquid located upstream of the filter 1310 (that is, filtered The portion trapped by the container 1310 is still inside the container 1230. Then, make switch K200 in the off state and continue The first injection module 121 is started to inject a certain amount of buffer solution into the container 1230, and at the same time, the shaking mechanism 124 is operated to cause the container 1230 to shake. Then, the switch K200 is turned on again, and the waste liquid is discharged from the container 1230 through the waste liquid outlet end 1230e. For example, by appropriately selecting the filter membrane pore size of the filter 1310, this dilution and flushing process can be repeated multiple times, thereby filtering out smaller substances in the blood that do not require treatment.

Next, after the smaller substances in the blood that do not require treatment have been properly removed, the switches K100, K200, and K300 are all in an off state. The second injection module 122 is activated to inject a certain amount of reagents for transduction or transfection into the container 1230 , while the shaking mechanism 124 operates to cause the container 123 to shake, thereby ensuring that the reagents are fully mixed with the liquid in the container 1230 . Then, wait for a certain period of time to allow the eukaryotic cells or recipient cells in the liquid in the container 123 to be fully transduced or transfected. For example, the time can be say 2 hours or more. During this period, the shaking mechanism 124 can be selectively operated to facilitate the improvement of transduction or transfection efficiency.

In the embodiment shown in Figure 3, the process of transduction or transfection is completed in a container 1230 provided with a filter 1310, so the amount of reagents injected for transduction or transfection should ensure that the final The liquid level is a certain height above the filter 1310, so that the selected eukaryotic cells or recipient cells are sufficiently immersed in the liquid mixed with the reagent.

After the transduction or transfection has been completed, the control device 140 instructs the switch K300 to be in a conductive state, so that the liquid downstream of the filter 1310 can enter the pipeline L100 through the recovery end 1230f and be collected into the recovery module 125, so that subsequently It can be used again after further purification. In an alternative example of the present application, the recovery module 125 can also be configured similarly to the second injection module 122 and have its own liquid injection mechanism such as an electric syringe or metering pump to be able to quantitatively inject liquid into the container 1230, so that the recovered The liquid (mainly composed of reagents used for transduction or transfection) can be reused for subsequent transduction or transfection.

Next, the switches K100, K200, and K300 are turned off. Simultaneously or subsequently, the first injection module 121 starts to inject a certain amount of buffer into the container 1230, and at the same time the shaking mechanism 124 operates to shake the container 1230 to ensure that the suspension upstream of the filter 1310 is evenly mixed with the buffer. Subsequently, the switch K200 is turned on, and the liquid downstream of the filter 1310 is discharged from the container 1230 . This process can be repeated multiple times to complete washing of transduced or transfected cells.

After the flushing is completed, there is still a suspension upstream of the filter 131, which contains cells that have been transduced or transfected and completed flushing. At this time, the switch K100 can be made to be in the conductive state and the switches K200 and K300 can be in the disconnected state, so that the suspension can be discharged from the container 1230 via the fluid outlet port 123d, and thereby can be output from the fluid outlet 112.

The embodiment shown in FIG. 3 further simplifies the host design of the extracorporeal blood cell therapy instrument 100, thereby ensuring a smaller instrument volume, while reducing the design of fluid joints, and further reducing the manufacturing cost in achieving medical cleanliness.

FIG. 4 schematically illustrates the main body of the extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application. As shown in the picture The first processing unit 1201 is provided or integrated in the housing 110 of the host shown in FIG. 4 . Except for the first processing unit 1201 , the remaining parts of the embodiment shown in FIG. 4 (those features having the same reference numerals as in FIG. 3 ) may be referred to the description of FIG. 3 . Most of the features of the first processing unit 1201 are the same as those of the first processing unit 1200, so the description of those features with the same reference numerals may refer to the embodiment shown in FIG. 3 . The difference between the first processing unit 1201 and the first processing unit 1200 is that the fluid inlet ports 1230b and 1230c fluidly connected to the first injection module 121 and the second injection module 122 are arranged in the filter container 1230 of the first processing unit 1201. downstream of the container 1310; in addition, switches K500 and K400 are respectively provided at the fluid inlet ends 1230b and 1230c, so that the switching of the switches can control whether the first injection module 121 and the second injection module 122 are allowed to be injected into the container 1230 and in When the switch is turned off, the liquid in the container 1230 is prevented from flowing back into the injection module. It should be clear that in the embodiments of the present application, the fluid inlet end and/or the switch provided at the fluid inlet end may be one-way conductive, that is to say, the liquid can only be allowed to flow even when the switch is in a conductive state. One-way input into the container prevents liquid in the container from flowing into the related injection module.

According to the embodiment shown in Figure 4, the first injection module 121 and the second injection module 122 inject the corresponding liquid into the container 1230 from downstream of the filter 1310. The advantage of this is that, for example, when injecting a buffer, due to There are already some cells trapped in the pores of the filter membrane of the filter 1310 due to their size that cannot pass through, so immersing the filter membrane from the downstream liquid helps to avoid the possibility of damage to the target cells caused by direct impact of the injected liquid.

When the host is operated as shown in FIG. 4 , first, the collected patient's blood is injected into the container 1230 of the first processing unit 1201 through the fluid inlet 111 . At this time, switches K100, K200, K300, K400, and K500 are in the off state. Then, the switch 500 is in the on state and the first injection module 121 starts to inject a certain amount of buffer solution into the container 1230, and again makes the switch 500 in the off state, and at the same time, the shaking mechanism 124 operates to cause the container 1230 to shake, Make sure the blood is mixed well with the buffer to achieve proper infection and/or conduction. The amount of buffer injected should be such that the final mixed liquid level is at a certain height above the filter 1310 to ensure dilution of the blood.

Then, with the switch 500 in the off state and the switch K200 in the on state, the part of the mixed liquid located downstream of the filter 1310 (waste liquid) will be discharged from the container 1230 through the waste liquid outlet end 1230e, and the mixed liquid will The portion located upstream of filter 1310 (ie, retained by filter 1310) remains within container 1230. Then, make the switch K200 be in the off state and make the switch 500 be in the on state, continue to start the first injection module 121 to inject a certain amount of buffer solution into the container 1230, and make the switch 500 be in the off state again, At the same time, the shaking mechanism 124 operates to cause the container 1230 to shake. Then, the switch K200 is turned on again, and the waste liquid is discharged from the container 1230 through the waste liquid outlet end 1230e. For example, by appropriately selecting the filter membrane pore size of the filter 1310, this dilution and flushing process can be repeated multiple times, thereby filtering out smaller substances in the blood that do not require treatment.

Next, after the blood has been suitably removed from the smaller non-treatable substances, the switches K100, K200, K300, and K500 are all in the disconnected state and make the switch K400 in the conducting state. The second injection module 122 is activated to inject a certain amount of reagents for transduction or transfection into the container 1230, and again makes the switch 400 in the off state, while the shaking mechanism 124 operates to cause the container 123 to shake, thereby ensuring The reagents are thoroughly mixed with the liquid in container 1230. Then, wait for a certain period of time to allow the eukaryotic cells or recipient cells in the liquid in the container 123 to be fully transduced or transfected. For example, the time can be say 2 hours or more. During this period, the shaking mechanism 124 can be selectively operated to facilitate the improvement of transduction or transfection efficiency.

After the transduction or transfection has been completed, the control device 140 instructs the switch K300 to be in a conductive state, so that the liquid downstream of the filter 1310 can enter the pipeline L100 through the recovery end 1230f and be collected into the recovery module 125, so that subsequently It can be used again after further purification. In the embodiment of the present application, the recovery module 125 can also be configured similarly to the second injection module 122, and have its own liquid injection mechanism such as an electric syringe or metering pump to be able to quantitatively inject liquid into the container 1230, so that the recovered The liquid (mainly composed of reagents used for transduction or transfection) can be reused for subsequent transduction or transfection.

Then, the switches K100, K200, K300, and K400 are in the off state and the switch K500 is in the on state. The first injection module 121 starts to inject a certain amount of buffer solution into the container 1230, and again makes the switch 500 in the on state. In the disconnected state, the shaking mechanism 124 is operated at the same time to cause the container 1230 to shake, ensuring that the suspension and the buffer upstream of the filter 1310 are evenly mixed. Subsequently, the switch K200 is turned on, and the liquid downstream of the filter 1310 is discharged from the container 1230 . This process can be repeated multiple times to complete washing of transduced or transfected cells.

After the flushing is completed, there is still a suspension upstream of the filter 1310, which contains cells that have been transduced or transfected and have completed flushing. At this time, the switch K100 can be made to be in a conductive state and the switches K200, K300, K400, K500 are in a disconnected state, so that the suspension can be discharged from the container 1230 via the fluid outlet port 123d, and thus can be output from the fluid outlet 112.

Regarding the embodiment shown in FIG. 4 , those skilled in the art should know that only one fluid access port in the container 1230 of the first injection module 121 or the second injection module 122 can be provided upstream of the filter 1310 .

FIG. 5 schematically illustrates the main body of the extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application. According to this embodiment, the host includes a first processing unit 1202 and a sorting module 127 located upstream of the first processing unit 1202, wherein an inlet of the sorting module 127 is fluidly connected to the fluid inlet 111, and the sorting module 127 The outlet is fluidly connected to the first processing unit 1202 via pipeline L80. Here, the first processing unit 1202 can be implemented by using the first processing unit 120, 1200 or 1201 introduced in Figure 2, 3 or 4 or their corresponding modifications, so a detailed description will not be given here. According to the embodiment shown in FIG. 5 , the function of the sorting module 127 is to screen out cells of interest for treatment in advance using physical means or biological means, and use the screened cell suspension as the input liquid of the first processing unit 1202 , and further improve High transduction or transfection efficiency, and ultimately improve the operating efficiency of the entire instrument. For example, for physical means, specific screening devices such as chromatography columns, filters, or molecular sieves can be used to first enrich and screen cells that meet size requirements and need treatment as the input liquid of the first processing unit 1202. Of course, those skilled in the art should know that specific biological reagents can also be selected in the sorting module 127 to implement cell screening by biological means. On the premise of biological means screening, an injection unit that can inject biological screening reagents for cell screening by biological means into the container can alternatively be added to the first processing unit 1202, so that the biological screening reagents are first injected into the container before diluting the blood for the first time. In the container, cells that do not need to be processed are removed from the blood in the container and specific cells that need to be processed are retained. For example, according to the present application, the sorting module 127 can be in the form of a chromatography column for special molecular markers or chelation, or other biological or physical methods available in the art, so that after collecting the patient's PBMC from the apheresis machine, After washing, these cells enter the sorting device so that specific lymphocytes such as T cells, B cells, NK cells, etc. can be sorted out, and then the corresponding functional units of the extracorporeal blood cell therapy device 100, such as the first processing unit 1202, are used to pass through Gene editing or gene modification via gene delivery.

In the embodiments of the present application, the target cells to be treated, such as T cells, B cells, NK cells, etc., can be transduced or transfected without undergoing the separation or purification or activation or amplification processes that must be performed in the prior art. It significantly shortens the time for gene editing or gene modification and significantly improves the efficiency of patient treatment.

FIG. 6 schematically shows a schematic diagram of the main body of the extracorporeal blood cell therapy apparatus 100 according to another embodiment of the present application. A first processing unit 1203 is provided or integrated in the housing 110 of the host. For example, the first processing unit 1203 may include a separation device that uses density gradient centrifugation to separate PBMCs (as described below). The host also includes a cell separation fluid injection module 123, which can be fluidly connected to the first processing unit 1203 in a manner similar to the first injection module 121 or the second injection module 122 described above, and can be controlled by the control device 140. The cell separation liquid is selectively injected into the first processing unit 1203. In addition, other features shown in FIG. 6 such as fluid inlet 111, fluid outlet 112, first injection module 121, second injection module 122, shaking mechanism 124, recovery module 125, pressure difference generating device 126, control device 140, display Relevant contents of the screen 141, the input device 142, etc. may refer to the description of the embodiments described above or below.

It should be clear to those skilled in the art that the use of density gradient centrifugation to separate PBMCs may include Percoll density gradient centrifugation and Ficoll density gradient centrifugation. The following examples of the specification mainly introduce the use of Ficoll density gradient centrifugal separation in the technical solution of the present application, but do not exclude the application of Percoll density gradient centrifugal separation in the technical solution of the present application.

According to an example of the present application, as shown in FIG. 7A , the first processing unit 1203 may include a container 12031 for containing liquid. For example, the container 12031 has a rotation axis to define a rotation axis O, which is substantially perpendicular to the ground after the extracorporeal blood cell therapy apparatus 100 is stabilized. For example, the rotation axis of the container can be driven via a drive device not shown in the figure. (for example, including a motor) is selectively driven to rotate under the control of the control device 140, such as forward rotation, reverse rotation, and/or forward and reverse rotation at a certain frequency for a predetermined time. According to an alternative or additional embodiment of the present application, as shown in Figure 6, the first processing unit 1203 may also include an independent container 12131, for example as a culture container or culture chamber. For example, transduction and/or transfection and/or washing and/or dilution of PBMCs can be accomplished separately within the independent container 12131. For another example, the independent container 12131 can be used as a temporary storage container to temporarily store PBMC to be transduced and/or transfected and/or cleaned and/or diluted, and to ensure the transduction and/or transfected and/or cleaned of PBMC. And/or the dilution can be completed only within the container 12031, for example, the container can be flushed with a suitable liquid such as physiological saline before each of the above-mentioned treatments on the container 12031 or 12131. Shaking mechanism 124 may be configured to independently rock container 12031 or 12131, respectively.

According to an example of the present application, a connecting pipe 12032 may be provided on the top of the container 12031. The connecting pipe 12032 can connect the top of the container 12031 in a manner that is not affected by the rotation of the container 12031, so that the multiple passages (hidden in the figure) provided in the connecting pipe 12032 can have a position relative to the rotation axis within the container 12031 as needed. O openings at different radial positions, the opposite corresponding openings of these passages can respectively connect the fluid inlet 111, the fluid outlet 112, the first injection module 121, the second injection module 122, the cell separation liquid injection module 123, and the recovery module 125 , and container12131. The cell separation solution injection module 123 is configured to inject Ficoll cell separation solution into the container 12031 as needed. In addition, the liquid can be drawn from the container 12031 into the container 12131 or from the container 12131 into the container 12031 via the connecting pipe 12032 as needed. When an independent container 12131 is provided, the first injection module 121, the second injection module 122, and the cell separation liquid injection module 123 can also be configured to be in fluid communication with the container 12131. The method of fluid communication can refer to the method of connecting to the container 12031. set up.

According to a non-limiting example, after the collected patient's blood is injected into the container 12031 of the first processing unit 1203 through the fluid inlet 111, the first injection module 121 starts to inject a certain amount of buffer solution into the container 12031 by shaking. The mechanism 124 operates and/or makes the container 12031 rotate forward and backward at a certain frequency for a predetermined time to ensure that the blood and the buffer are evenly mixed, thereby achieving appropriate infection and/or transmission effects. Then, the Ficoll cell separation solution is injected into the container 12031, and the container 12031 is rotated around the rotation axis O at a certain speed, so that the different components in the mixed solution are caused by the different densities of the liquid components along the radial direction of the rotation axis O. Corresponding radial delamination occurs. Then, these liquid radially layered components are extracted using different passages arranged relative to the rotation axis O in the container 12031. For example, some of the extracted liquid components can be sucked into the recovery module 125 for reuse, and some of the extracted liquid components can be The container 12031 is drained directly as waste liquid, so that only substances in the blood that require treatment are ultimately left in the container 12031; alternatively and/or additionally, the liquid components to be transduced or transfected can be drawn into the container 12131. For example, if it is expected that the transduction or transfection process is performed in the container 12031 (for example, the container 12031 is used as a culture chamber), it can be configured to first draw the liquid component to be transduced or transfected into the container 12131, and then first inject Module 121 may be configured to inject a certain amount of buffer into Enter the container 12031 to rinse the remaining liquid. Finally, the liquid component in the container 12131 is drawn back into the container 12031. For another example, if it is desired that the transduction or transfection process be performed in the container 12131 alone, the reagents for transduction or transfection can be directly injected into the container 12131 via the second injection module 122, and the container 12131 serves as a culture chamber at this time. use.

Next, the second injection module 122 is started to inject a certain amount of reagents for transduction or transfection into the container 12031 or 12131, again by operating the shaking mechanism 124 and/or by causing the container 12031 to rotate forward and reverse at a certain frequency. The predetermined time ensures that the reagent is fully mixed with the liquid in the container 12031 or 12131. Then, wait for a certain period of time to allow the eukaryotic cells or recipient cells in the liquid in the container 12031 or 12131 to be fully transduced or transfected. For example, the time can be say 2 hours or more. During this period, the shaking mechanism 124 and/or the container 12031 can be selectively operated to facilitate the improvement of transduction or transfection efficiency. After the transduction or transfection has been completed, for example, the liquid components after the transfection or transduction can be diluted with a buffer, especially the PBMC, and the transfection or transduction can be further removed by centrifugation depending on the density difference of the different components. Impurity components in the final PBMC liquid. For example, the liquid component after transduction or transfection can be located in the container 12031 or drawn from the container 12131 into the container 12031, and then a certain amount of buffer solution is selectively injected into the container 12031 through the first injection module 121, and The container 12031 is started to rotate, so that different components in the liquid are in different radial stratification positions according to different densities. Then, different passages arranged relative to the rotation axis O in the container 12031 are used to extract the radially stratified liquids. Components, such as beneficial liquid components (eg, desired post-transduction or transfection fluid components), may be collected through fluid outlet 112 for subsequent therapeutic use, and other liquid components may be discharged as waste.

According to another alternative example of the present application, as shown in FIG. 7B , the container 12031 may also be configured such that the liquid components are layered with each other along the direction of gravity height due to the difference in density of the components in the liquid after rotational separation. In this case, for example, a selective on-off switch (such as the above-mentioned first switch K100, second switch K200, and third switch K300) can be provided at the bottom of the container 12031, thereby controlling the switch to turn on and off the force due to gravity. Liquids in different layers can be collected under the action. For example, depending on the composition, these collected liquids can be directly discarded as waste liquid or used as final therapeutic substances or re-injected into the container 12031 or 12131 for further dilution or transduction or transfection. It should be clear to those skilled in the art that the sorting module 127 shown in Figure 5 can also be configured for the first processing unit 1203 shown in Figure 6 .

According to an embodiment of the present application, the containers 1230, 12031, and 12131 can be configured with heating or insulating devices to ensure that appropriate temperatures are maintained when PBMC cell culture is performed.

An example of a method for transducing PBMC with GFP or CAR genes using the device of the present application is described below. Compared with the existing technology, one of the advantages of this application is that the target cells in the blood to be processed can be transduced or transfected under conditions that are not separated or purified or activated or amplified, thereby significantly saving corresponding costs. The blood treatment time can achieve rapid pathological treatment and significantly shorten the patient's disease treatment time. First, fresh anticoagulated whole blood or apheresis can be obtained via the fluid inlet 111 is injected into the first processing unit 1203, such as into the container 12031, and then a certain amount of buffer solution, such as physiological saline, is injected into the container 12031 through the first injection module 121, thereby diluting the blood to reduce the blood viscosity. spare. For example, the volume of buffer injected: the volume of blood to be diluted = 1:1. During this process, the container 12031 may, for example, be configured to alternately rotate forward and backward at a certain rotation speed to ensure more uniform mixing.

Then, a certain amount of Ficoll cell separation fluid can be injected into the container 12031 via the cell separation fluid injection module 123 . For example, the injection ratio can be the volume of Ficoll cell separation solution: the volume of the aforementioned diluted blood = 1:2.

Next, the container 12031 is rotated at room temperature for a certain period of time, such as 20 minutes to 30 minutes, to generate a centrifugal force of 800g. After or at the same time as the centrifugation process, the separated PBMC layer (i.e., the white film layer) is centrifuged, and then the collected PBMC is used as Liquid components to be transduced or transfected can be stored or temporarily stored in container 12131. Depending on the volume of blood injected via fluid inlet 111, the liquid volume of PBMC collected may vary, for example. In one method example of this application, a liquid, such as 5 ml of PBMC, can be collected and stored in container 12131. For example, the above sub-process of diluting, centrifuging, and collecting PBMCs can be repeated 1, 2, or more times to maximize the extraction of PBMCs to be processed from the blood.

Next, PBMC are transduced or transfected with the GFP or CAR gene. For example, the transduction or transfection process can be completed within container 12031 or within container 12131. For example, a buffer such as culture medium can be injected into the container 12031 or 12131 via the first injection unit 121, so that the cell concentration is adjusted to 1*10 ⁶ to 1*10 ⁸ cells/ml. Then, the LNP-wrapped GFP-mRNA or CAR-mRNA or the genetically modified/modified AAV virus can be injected into the container 12031 or 12131 through the second injection unit 122, and incubated for 1 to 5 hours or other suitable time for transfer. guide.

Then, the above-mentioned transduced or transfected liquid is placed in the container 12031 (if the transduction or transfection is completed in the container 12131, the transduced or transfected liquid can be drawn into the container 12031) and 5 to 15 times the volume of buffer such as physiological saline or culture medium or culture medium, and the container 12031 is rotated at room temperature in a manner that generates a centrifugal force of 800g for a certain time, such as 20 minutes to 30 minutes, after which the transduction is drawn in a specific centrifugal layer or transfected PBMC for subsequent treatment. For example, the process of adding a buffer and using centrifugal layering to absorb the transduced or transfected PBMC (which can be regarded as a washing-centrifugation process) can be repeated multiple times, such as 2 to 3 times.

In view of the above-mentioned PBMC transduction or transfection test results, the inventor of the present application used GFP protein expression to detect the transduction or transfection efficiency. The process is as follows.

Adjust the cell concentration in the transduced or transfected PBMC liquid after the above washing and centrifugation treatment to 1*10 ⁶ to 5*10 ⁶ cells/ml by adding a buffer (such as FBS medium), and continue culturing. Thereafter, after 24 hours and 36 hours of culture, cells were collected for CD3 staining, and T cell-specific GFP fluorescent protein expression was detected using flow cytometry.

In addition, based on the above-mentioned PBMC transduction or transfection test results, the inventor of the present application tested the killing function of CAR-T cells after transduction or transfection. The process is as follows.

a) Place the transduced or transfected PBMC liquid (such as anti-CD19-CAR-mRNA transfected cell liquid) after the above washing and centrifugation treatment into container 12031 and rotate it at a speed of 1500 rpm for a certain period of time Centrifugation is performed, for example, for 5 minutes. Then, discard the supernatant, retain the cells, and add buffer so that the cell concentration is approximately 2*10 ⁶ cells/ml.

b) At the same time, obtain a Raji cell line that stably expresses luciferase enzyme. After the Raji-luciferase cells are removed by centrifugation (speed: 1500 rpm, for example, 5 minutes), adjust the cell concentration to 1*10 ⁶ cells/ml.

c) From the cells obtained in steps a) and b) above, take 100 microliters of transduced or transfected PBMC plus 100 microliters according to the effect-to-target ratio of cell number = 1:1, 2:1, 1:2. Microliter of Raji-luciferase (2:1); 50 microliter of transduced or transfected PBMC plus 100 microliter of Raji-luciferase (1:1); 25 microliter of transduced or transfected PBMC plus 100 μl of Raji-luciferase (1:2), set in duplicate wells. The cells were seeded in a 96-well plate, and culture medium was added to 200 μl/well, and culture was continued for 24 hours.

d) Centrifuge (1500 rpm, for example, 5 minutes), discard the supernatant, collect all cells into a V-shaped hole, add 100 ml of lysis solution, let stand at room temperature for 10 to 15 minutes, and then centrifuge (3500 rpm) (for example, 20 minutes), take 20 μl of the supernatant into the 8-row/round-bottom 96well plate. Add 50 μl/well of substrate and measure the OD value immediately.

e) Calculation: (1-test/NC)%=tumor lysis (cytotoxicity). Figures 8A to 8D respectively show the observation results after using LNP to deliver genes, removing impurities by centrifugation, and performing transfection in container 12031 before culturing cells.

Using the simple and rapid CAR-T preparation method under closed conditions of the present invention, it has been found that LNPs under different optimized conditions can be removed after 5 hours of transfection, and the transfection efficiency of T cells in PBMC can be improved by culturing for 24 hours. Hours later, whether using FBS culture conditions or the donor's own human serum culture conditions, GFP protein was expressed, indicating that LNP-wrapped mRNA has a certain transfection efficiency for T cells within a short period of 5 hours. The transfection expression efficiency could reach about 5% (Fig. 8A, Fig. 8B); after another 36 hours of culture, the transfection expression efficiency could reach about 10% (Fig. 8C, Fig. 8D).

After testing the transfection efficiency, the inventor also tested the killing function of CAR-T on the PBMC cells transfected with the CAR gene. The results showed that the CAR-T produced using this closed system had a transfection efficiency of about 5%. When the effective-target ratio is 1:1, it can be seen that the killing efficiency of target cells can reach >30%. When the effective-target ratio is 2:1, the killing efficiency of target cells can reach >90%. This result shows that In this closed system, the CAR-T cells generated using this new method with simplified steps have strong killing activity against targeted tumor cells (Figure 9).

The extracorporeal blood cell therapy instrument 100 of the present application can construct a closed condition that facilitates extracorporeal blood cell processing in a portable manner, and construct an extracorporeal blood treatment method under such a closed condition that meets medical standards. This method includes, for example:

Collect blood from the patient;

Under closed conditions, gene editing or gene modification is performed on the target cells in the collected blood through gene delivery and is also configured to remove substances in the treated blood that do not require treatment;

The treated fluid is returned to the patient.

It should be noted that the "closed conditions" mentioned in this application may refer to conditions in which no manual operations are involved and the processing process meets medical standards. Those skilled in the art should be aware that the "closed conditions" are not limited to those that can be achieved by the extracorporeal blood cell therapy device that has been introduced. Other portable centrifugation equipment that meets medical standards and specifications can also be configured to implement the method of the present application by being connected to a specific container. closed conditions.

Although in the embodiments that have been described, the filter is implemented in the form of a membrane. However, those skilled in the art should understand that when other filtering methods are used, the embodiments described in this application can be modified accordingly, and the purpose of this application can still be achieved.

For example, in a possible modification to the embodiment shown in Figure 2, the filter 131 can be replaced by a centrifugal filter device. It is clear to those skilled in the art that centrifugal filtration refers to using centrifugal force as a driving force to add the liquid to be filtered (in this case, a suspension containing blood) into a perforated drum with filter media (such as filter mesh, filter cloth, etc.) liquid), and then the cells to be treated can be trapped on the filter medium by selecting an appropriate pore size, and the remaining liquid (unwanted liquid) is discharged through the filter medium, ultimately achieving the retention of the cells to be treated.

As another example, in another possible modification to the embodiment shown in FIG. 2 , the filter 131 can be replaced with a chromatography column filter device. Chromatography columns mainly use fillers of different pore sizes to adsorb and filter particles of different sizes (here, cells) in a targeted manner.

As another example, in another possible modification to the embodiment shown in FIG. 2 , the filter 131 can be replaced with a magnetic screening filter device. This magnetic screening filtration device mainly uses antibody magnetic beads to specifically label target cells, and then the cells labeled by the magnetic beads are adsorbed by the magnetic device for enrichment and recovery after transduction or transfection. For example, in this modification, a unit for injecting specific antibody magnetic beads can be set up, and the filter can be set as a magnetic adsorption device, such as a strong magnetic holder or a chromatography column with magnetic characteristics to replace the filter and set it in the second treatment within the unit. When used, the antibody magnetic beads are selectively injected after diluting the blood, and the target cells will be specifically labeled by the antibody magnetic beads; then, after the transduction or transfection is completed, these labeled cells are then removed via a magnetic adsorption device. The target cells undergo enrichment collection and are ultimately discharged from the second treatment unit after flushing.

In the embodiments of the present application, the extracorporeal blood cell therapy device can also adopt any other suitable form. For example, on the premise of complying with medical and health requirements, the corresponding module can be directly connected with medical hoses or pipes and/or data cables. Connected to form a portable in vitro blood cell gene editing or modification system. For example, in the case of a system, blood can directly The blood is directly input into the first processing unit 120, and the processed blood may be output from the second processing unit 130.

Although specific embodiments of the present application are described in detail herein, they are given for purposes of explanation only and should not be construed as limiting the scope of the application. In addition, it should be clear to those skilled in the art that the embodiments described in this specification can be used in combination with each other. Various substitutions, changes and modifications may be devised without departing from the spirit and scope of the present application.

Claims

A portable in vitro blood cell gene editing or modification system, which includes:

Fluid inlet (111) and fluid outlet (112);

Processing unit (1200, 1201, 1202, 1203) configured to pass resting or non-activated target cells in the blood input through the fluid inlet (111) The method of gene delivery carries out gene editing or gene modification and is also configured to remove substances that are not required for treatment in the treated blood cells.

The processing unit (1200, 1201, 1202, 1203) is fluidly connected between the fluid inlet (111) and the fluid outlet (112).
The portable extracorporeal blood cell gene editing or modification system according to claim 1, characterized in that the processing unit (1200, 1201, 1202, 1203) includes a container (1230, 12031) for holding liquid, and the processing unit (1200, 1201, 1202, 1203) The unit (1200, 1201, 1202, 1203) also includes at least a first injection module (121) and a second injection module (122) that are independent of each other. The first injection module (121) stores information that can be selectively injected into The first reagent in the container, the second injection module (122) stores a second reagent that can be selectively injected into the container (1230, 12031), so that the blood in the container is infused The target cells in the blood are gene edited or genetically modified through gene delivery without leaving the container, and non-treatable substances in the treated blood are removed.
The portable in vitro blood cell gene editing or modification system according to claim 2, wherein the first reagent includes a buffer, and the second reagent includes a gene delivery reagent.
The portable in vitro blood cell gene editing or modification system according to claim 3, characterized in that the gene delivery reagent is used to transform target cells under conditions that are not separated or purified or activated or amplified. induction or transfection.
The portable extracorporeal blood cell gene editing or modification system according to claim 4, characterized in that a filter (1310) is provided in the container (1230), and the container (1230) includes a fluid input end (1230a) , which is fluidly connected to the fluid inlet; and a fluid output end (1230d), which is fluidly connected to the fluid outlet, the fluid input end (1230a) and the fluid output end (1230d) are located in the filter (1230d) 1310) upstream.
The portable extracorporeal blood cell gene editing or modification system according to claim 5, characterized in that the first injection module (121) and/or the second injection module (122) are configured to be able to operate on the filter. (1310) Upstream selectively injects respective reagents into the container (1230).
The portable extracorporeal blood cell gene editing or modification system according to claim 5, characterized in that the first injection module (121) and/or the second injection module (122) are configured to be able to operate on the filter. (1310) The respective reagents are selectively injected downstream into the container (1230).
The portable in vitro blood cell gene editing or modification system according to claim 6 or 7, characterized in that the filter (1310) is a molecular sieve, a magnetic screening filter device, a chromatography column, or a filter membrane.
The portable in vitro blood cell gene editing or modification system according to claim 4, wherein the processing unit is a separation device that uses density gradient centrifugation to separate PBMC, and the container of the processing unit includes a container capable of rotating around a rotation axis ( O) Selectively rotated first container (12031).
The portable extracorporeal blood cell gene editing or modification system according to claim 9, wherein the rotation axis (O) is the rotation axis of the first container (12031) itself.
The portable extracorporeal blood cell gene editing or modification system according to claim 10, characterized in that before the second reagent is injected into the first container, the cell separation liquid is injected into the first container and then As the first container rotates, different liquid compositions are stratified in the radial direction relative to the rotation axis (O), leaving only the substances in the blood that need treatment in the first container for subsequent injection. of the second reagent mix.
The portable extracorporeal blood cell gene editing or modification system according to claim 10, characterized in that the container of the processing unit further includes a second container (12131) independent of the first container (12031), a cell separation liquid is injected into the first container and as the first container rotates, different liquid component stratification is generated in the radial direction relative to the rotation axis (O), and the liquid containing the substance requiring treatment is drawn into the second container (12131), and the second reagent is injected into the second container (12131).
The portable in vitro blood cell gene editing or modification system according to claim 12, characterized in that: Agents for gene delivery include viral vectors or non-viral vectors.
The portable in vitro blood cell gene editing or modification system according to claim 13, wherein the non-viral vector includes a synthetic vector or a biological vector; and/or the viral vector includes a retrovirus or a modified body thereof; or Mutant, lentivirus or modifications or mutants thereof, adenovirus or modifications or mutants thereof, or adeno-associated virus or modifications or mutants thereof.
The portable in vitro blood cell gene editing or modification system according to claim 14, wherein the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), so The biological carrier is an extracellular vesicle.
The portable in vitro blood cell gene editing or modification system according to claim 15, wherein the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells. cell.
The portable in vitro blood cell gene editing or modification system according to claim 16, characterized in that a sorting module (127) is provided upstream of the processing unit, and the inlet of the sorting module (127) is connected to the fluid inlet. (111) Fluid connection, the outlet of the sorting module (127) is fluidly connected to the processing unit via a pipeline (L80), the sorting module (127) is configured to before blood is input into the processing unit Materials other than the target cells are eliminated from the blood.
The portable extracorporeal blood cell gene editing or modification system according to claim 17, characterized in that the sorting module (127) can use physical means or biological means to exclude substances other than the target cells from the blood.
The portable extracorporeal blood cell gene editing or modification system according to claim 18, characterized in that the portable extracorporeal blood cell gene editing or modification system has a host, the host has a housing (110), the processing unit, The first injection module (121), the second injection module (122) and the sorting module (127) are arranged in the housing (110).
The portable in vitro blood cell gene editing or modification system according to claim 19, characterized in that: Reagents for gene delivery include CAR genes to transduce or transfect T cells in PBMCs.
The portable extracorporeal blood cell gene editing or modification system according to claim 20, characterized in that the gene delivery reagent is used to transduce target cells under conditions that are not separated or purified or activated or amplified, or Transfection time is 1 to 5 hours.
A method for in vitro gene editing or modification of blood cells, including:

Collect blood from the patient;

Under closed conditions, the resting or inactivated target cells in the collected blood are gene-edited or modified through gene delivery, and are also configured to remove non-treatable substances from the treated blood cells in pairs. ;

The treated fluid is returned to the patient.
The method of claim 22, wherein a first reagent and a second reagent are respectively injected into the collected blood, the first reagent includes a buffer, and the second reagent includes a gene delivery reagent.
The method of claim 23, wherein the gene delivery reagent is used to transduce or transfect target cells under conditions that are not separated or purified, or activated or amplified.
The method of claim 24, wherein before injecting the second reagent, PBMCs in the blood are separated by density gradient centrifugation, and the second reagent is injected into the separated PBMC liquid.
The method of claim 25, wherein the gene delivery reagent includes a viral vector or a non-viral vector.
The method according to claim 26, wherein the non-viral vector includes a synthetic vector or a biological vector; and/or the viral vector includes a retrovirus or a modification or mutant thereof, a lentivirus or a modification thereof variants or mutants, adenovirus or modifications or mutants thereof, or adeno-associated virus or modifications or mutants thereof.
The method of claim 27, wherein the synthetic carrier is a lipid nanoparticle (LNP) or a lipid polyplex (LPP), and the biological carrier is an extracellular vesicle. .
The method of claim 28, wherein the target cells include but are not limited to T cells, B cells, NK cells, macrophages, monocytes, or innate lymphoid cells.
The method of claim 29, wherein the gene delivery reagent includes a CAR gene to transduce or transfect T cells in PBMC.
The method according to claim 30, characterized in that the time for transducing or transfecting target cells using the gene delivery reagent under conditions that are not separated or purified or activated or amplified is 1 to 5 Hour.