Adoptive immune cell, and preparation method therefor and use thereof

Abstract

Provided are an adoptive immune cell, and a preparation method therefor and the use thereof. The adoptive immune cell have a reprogramming factor expression system introduced therein, and can express one or more of an Oct4 transcription factor, a Sox2 transcription factor, a Klf4 transcription factor, a cMyc transcription factor, a Glis1 transcription factor and a Lin28 transcription factor. By means of reversing aging, the adoptive immune cell has an improved quantity and quality when cultured in vitro and has a prolonged survival time in vivo, and thus the effect of adoptive cell therapy is improved.

Description

Adoptive immune cells, preparation method and application thereof Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to adoptive immune cells, a preparation method and application thereof. Background Art

[0002] Adoptive Cell Therapy (ACT) is an emerging cancer treatment method that aims to fight cancer by enhancing the patient's own immune system response. It involves collecting, expanding, and modifying immune cells from the patient, giving them better capabilities, and then re-infusing them into the patient to enhance their anti-cancer effects.

[0003] Currently, adoptive immune cell therapy has the following problems: in the background art, it is difficult to further improve the quantity and quality of adoptive immune cells when they are cultured in vitro; in the background art, adoptive immune cells cannot exist in the body for a long time due to aging; thus, the effect of adoptive immune cell therapy is affected.

[0004] Summary of the Invention

[0005] In view of the above problems, the embodiments of the present application provide an adoptive immune cell, a preparation method and application thereof to solve the above technical problems.

[0006] In a first aspect, an embodiment of the present application provides an adoptive immune cell, which includes a reprogramming factor expression system, and the reprogramming factor expression system includes one or more of a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a cMyc transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0007] Optionally, the adoptive immune cells are chimeric antigen receptor T cells targeting ROR1;

[0008] Alternatively, the adoptive immune cells are CD4 T cells in which the CCR5 encoding gene is knocked out.

[0009] Optionally, the reprogramming factor expression system includes mRNA encoding Oct4 transcription factor, mRNA encoding Sox2 transcription factor, mRNA encoding Klf4 transcription factor, mRNA encoding Glis1 transcription factor, and mRNA encoding Lin28 transcription factor.

[0010] Optionally, the reprogramming factor expression system also includes mRNA encoding a mutant replicase of the genus Alphavirus, which produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

[0011] Optionally, some or all of the nucleotides in the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, the mRNA encoding Lin28 transcription factor, and the mRNA encoding the mutant alphavirus replicase are chemically modified to improve the stability of the mRNA in vivo; the chemical modification includes replacing the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor with N1-methylpseudouridine, pseudouracil, or methyluracil. 100% of the uracil in the mRNA, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, the mRNA encoding the Lin28 transcription factor and the mRNA encoding the mutant alphavirus replicase, and 100% of the cytosine in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor and the mRNA encoding the Lin28 transcription factor are replaced by 5-methylcytosine or 5-hydroxymethylcytosine.

[0012] In a second aspect, the present invention provides a method for preparing adoptive immune cells, comprising:

[0013] Providing chimeric antigen receptor CAR-ROR1 encoding genes, which include ROR1-targeting antibody encoding genes, transmembrane domain encoding genes, intracellular domain encoding genes, detection genes, rapamycin resistance genes, and inducible self-destruct genes;

[0014] Inserting the chimeric antigen receptor CAR-ROR1 encoding gene into a vector to obtain a CAR-ROR1 recombinant vector;

[0015] The CAR-ROR1 recombinant vector and the reprogramming factor expression system are transfected into PBMC immune cells to obtain the chimeric antigen receptor T cells targeting ROR1.

[0016] Optionally, it also includes:

[0017] The transfected PBMC immune cells were cultured in T cell activation medium;

[0018] Alternatively, the nucleotide sequence of the CAR-ROR1 recombinant vector is shown in SEQ ID NO.1.

[0019] In a third aspect, the present invention provides a method for preparing adoptive immune cells, comprising:

[0020] Providing an sgRNA for knocking out the CCR5 coding gene and constructing a recombinant vector containing the sgRNA;

[0021] The recombinant vector of the sgRNA and the reprogramming factor expression system are transfected into PBMC immune cells to obtain the CD4 T cells with the CCR5 encoding gene knocked out.

[0022] Optionally, it also includes:

[0023] The transfected PBMC immune cells were cultured in T cell activation medium;

[0024] Alternatively, the nucleotide sequence of the recombinant vector containing the sgRNA is shown as SEQ ID NO.2.

[0025] In a fourth aspect, the embodiments of the present application provide the use of the above-mentioned adoptive immune cells in the preparation of tumor treatment drugs or AIDS treatment drugs.

[0026] The adoptive immune cells, preparation methods, and uses provided in the embodiments of the present application are characterized by the introduction of a reprogramming factor expression system into the adoptive immune cells, which are capable of expressing one or more of the Oct4 transcription factor, Sox2 transcription factor, Klf4 transcription factor, cMyc transcription factor, Glis1 transcription factor, and Lin28 transcription factor. By reversing aging, the number and quality of the adoptive immune cells are greatly improved when cultured in vitro; and the survival time of the adoptive immune cells in vivo is prolonged, thereby improving the effect of adoptive immune cell therapy.

[0027] These and other aspects of the present application will become more readily apparent from the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 shows the results of the natural killer cell immune ability experiment in Example 2 of the present application.

[0029] FIG2 shows the results of the macrophage phagocytic ability experiment in Example 3 of the present application.

[0030] FIG3 shows a schematic diagram of the preparation principle of the control group in Example 4 of the present application.

[0031] FIG4 shows a schematic diagram of the preparation principle of the experimental group in Example 4 of the present application.

[0032] FIG5 shows a comparison of the cell numbers in the experimental group and the control group in Example 4 of the present application.

[0033] FIG6 shows the T cell ratio results of the experimental group in Example 4 of the present application.

[0034] FIG7 shows a diagram of the metabolic phenotype results of Example 5 of the present application.

[0035] FIG8 shows the results of the in vitro tumor killing experiment of Example 5 of the present application.

[0036] FIG9 shows the results of the in vivo tumor killing experiment of Example 5 of the present application.

[0037] Figure 10 shows a schematic structural diagram of the CAR-ROR1 recombinant vector in Example 4 of the present application.

[0038] Figure 11 shows a schematic structural diagram of the recombinant vector containing the sgRNA in Example 6 of the present application.

[0039] FIG12 shows a graph of the experimental results of Example 6 of the present application.

[0040] FIG13 shows a graph of the experimental results of Example 7 of the present application. DETAILED DESCRIPTION

[0041] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0042] In order to enable those skilled in the art to better understand the solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of this application.

[0043] In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second, etc., are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0044] Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0045] In the description of the embodiments of this application, words such as "example" or "for example" are used to indicate an example, illustration, or description. Any embodiment or design described as "for example" or "for example" in the embodiments of this application is not to be construed as being preferred or having more advantages than another embodiment or design. The use of words such as "example" or "for example" is intended to clearly present relative concepts.

[0046] In addition, in the embodiments of the present application, "plurality" refers to two or more. In view of this, in the embodiments of the present application, "plurality" can also be understood as "at least two". "At least one" can be understood as one or more, for example, one, two, or more. For example, "including at least one" means including one, two, or more, and does not limit which ones are included. For example, "including at least one of A, B, and C" means including A, B, C, A and B, A and C, B and C, or A, B, and C.

[0047] It should be noted that in the embodiments of the present application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / ", unless otherwise specified, generally indicates that the associated objects are in an "or" relationship.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] One embodiment of the present application provides an adoptive immune cell, which includes a reprogramming factor expression system, wherein the reprogramming factor expression system includes one or more of a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a cMyc transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0051] In this embodiment, a reprogramming factor expression system is introduced into the adoptive immune cells, and the adoptive immune cells are capable of expressing one or more of Oct4 transcription factor, Sox2 transcription factor, Klf4 transcription factor, cMyc transcription factor, Glis1 transcription factor and Lin28 transcription factor. By reversing aging, the number and quality of the adoptive immune cells are greatly improved when cultured in vitro; and the survival time of the adoptive immune cells in the body is prolonged; thereby improving the survival ability of the adoptive immune cells introduced into the body in the tumor microenvironment or other lesion microenvironment, and can inhibit the apoptosis of the adoptive immune cells and prolong the action time of the adoptive immune cells, thereby improving the tumor treatment effect of the corresponding immune cells in multiple aspects such as enhancing killing and reducing apoptosis.

[0052] As an embodiment, the above-mentioned adoptive immune cells can be selected from one or more of T cells, NK cells, NK T cells, macrophages, tumor infiltrating lymphocytes (TIL), CIK cells, CAR-T cells, CAR-NK cells, CAR-macrophages, TCR-T cells and CAR-CIK cells.

[0053] In some embodiments, the adoptive immune cells can be the patient's autologous T cells, T cells derived from pluripotent stem cells, T cell precursor cells or stem cells. The pluripotent stem cells can include embryonic stem cells or induced pluripotent stem cells.

[0054] As an embodiment, the reprogramming factor expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0055] In this embodiment, the formed OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28) has a good epigenetic reprogramming effect, can promote the initiation of epigenetic reprogramming of adoptive immune cells, and does not produce cytotoxicity.

[0056] As an embodiment, each of the above-mentioned nucleic acid molecules can be DNA. The expression system includes a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Sox2 transcription factor, a DNA nucleic acid molecule encoding a Klf4 transcription factor, a DNA nucleic acid molecule encoding a Glis1 transcription factor, and a DNA nucleic acid molecule encoding a Lin28 transcription factor.

[0057] As an embodiment, the expression system can be an expression cassette. In this embodiment, the expression cassette contains a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0058] As an embodiment, the expression system can be an adeno-associated virus expression vector or a lentivirus expression vector.

[0059] As an embodiment, the expression system can be a recombinant vector.

[0060] In some embodiments, the expression system is a recombinant vector with puromycin resistance.

[0061] As an embodiment, each of the above-mentioned nucleic acid molecules can be RNA. The reprogramming factor expression system includes mRNA encoding Oct4 transcription factor, mRNA encoding Sox2 transcription factor, mRNA encoding Klf4 transcription factor, mRNA encoding Glis1 transcription factor, and mRNA encoding Lin28 transcription factor.

[0062] In some embodiments, the ratio of the molar amount of the mRNA encoding Oct4 transcription factor to the sum of the molar amounts of the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, and the mRNA encoding Lin28 transcription factor is 2 to 8:1.

[0063] In this embodiment, by controlling the ratio of each transcription factor, the adoptive immune cells can be driven to undergo partial reprogramming more efficiently, the aging of the adoptive immune cells can be reversed, and the metabolic phenotype of the adoptive immune cells can be changed, thereby promoting the proliferation of the adoptive immune cells and enhancing the immune capacity of the adoptive immune cells, thereby improving both the quantity and quality of the adoptive immune cells.

[0064] In some embodiments, the reprogramming factor expression system also includes an mRNA encoding a mutant alphavirus replicase, which produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

[0065] In this embodiment, the replication of each of the above-mentioned transcription factors can be achieved by encoding an RNA-dependent RNA polymerase using mRNA encoding a mutant alphavirus replicase.

[0066] In some embodiments, the reprogramming factor expression system includes 7 to 8 molar parts of an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0067] In some embodiments, the reprogramming factor expression system includes 7 to 8 molar parts of an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 3 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0068] In some embodiments, the ratio of the molar amount of the mRNA encoding the mutant alphavirus replicase to the sum of the molar amounts of the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, and the mRNA encoding the Lin28 transcription factor is 2 to 8:1.

[0069] In some embodiments, the mRNA encoding the mutant alphavirus replicase may not be provided, and the replication of the above-mentioned transcription factors can be achieved by RNA polymerase in adoptive immune cells.

[0070] In some embodiments, some or all of the nucleotides in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, the mRNA encoding the Lin28 transcription factor, and the mRNA encoding the mutant alphavirus replicase are chemically modified to improve the stability of the mRNA in vivo; the chemical modification includes replacing the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor with N1-methylpseudouridine, pseudouracil, or methyluracil. 100% of the uracil in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, the mRNA encoding the Lin28 transcription factor and the mRNA encoding the mutant alphavirus replicase, and 100% of the cytosine in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor and the mRNA encoding the Lin28 transcription factor are replaced by 5-methylcytosine or 5-hydroxymethylcytosine.

[0071] As an embodiment, the adoptive immune cells are CAR-T cells, specifically chimeric antigen receptor T cells targeting ROR1.

[0072] The adoptive immune cells of this embodiment can be prepared according to the following steps:

[0073] S11, providing a chimeric antigen receptor CAR-ROR1 encoding gene, which includes a ROR1-targeting antibody encoding gene, a transmembrane domain encoding gene, an intracellular domain encoding gene, a detection gene, a rapamycin resistance gene, and an inducible self-destruct gene;

[0074] Among them, the antibody targeting ROR1 is used to bind to the antigen and exert the antigen binding effect. For example, a fully human anti-ROR1 scFV can be selected; the intracellular domain is used to carry out an immune response and exert an immune effect. For example, it can include a fragment from CD8a and / or a fragment from 4-1BB, and can further include a signal peptide of CD8a; the detection gene is used for detection, and the detection gene can be a reporter gene, an immune cell-specific gene, a selection marker, an enhancer sequence or a WRPE sequence; the rapamycin resistance gene can tolerate rapamycin treatment and can expand the population of CAR-T cell treatment; the inducible self-destruction gene can be, for example, Caspase9.

[0075] S12, inserting the chimeric antigen receptor CAR-ROR1 encoding gene into a vector to obtain a CAR-ROR1 recombinant vector;

[0076] S13, transfecting PBMC immune cells with the CAR-ROR1 recombinant vector and the reprogramming factor expression system to obtain the chimeric antigen receptor T cells targeting ROR1.

[0077] Among them, the CAR-ROR1 recombinant vector can include the piggybac transposon, and an auxiliary vector encoding the piggybac transposase is also required. The CAR-ROR1 recombinant vector, the auxiliary vector encoding the piggybac transposase, and the reprogramming factor expression system are nuclear transfected to achieve gene introduction. The PBMC immune cells after nuclear transfection are cultured in T cell activation medium to obtain chimeric antigen receptor T cells targeting ROR1.

[0078] In some embodiments, the nucleotide sequence of the CAR-ROR1 recombinant vector is shown in SEQ ID NO. 1, and the structure is shown in FIG10 .

[0079] In some embodiments, the T cell activation medium is prepared as follows:

[0080] Step 1, preparing an anti-CD3 antibody / anti-CD28 antibody coated plate;

[0081] Step 2, adding PBMC immune cells and IL-15 to T cell culture medium to obtain PBMC cell fluid;

[0082] Step 3: Inoculate PBMC cell fluid in an anti-CD3 antibody / anti-CD28 antibody coated plate, collect the PBMC cell fluid in the anti-CD3 antibody / anti-CD28 antibody coated plate after a preset culture time, centrifuge the collected PBMC cell fluid, collect the supernatant, and filter the supernatant with a sterile filter to obtain a T cell activation medium.

[0083] In some embodiments, step 1 may specifically include: diluting the anti-CD3 antibody or anti-CD28 antibody with PBS to form an antibody solution with a final antibody concentration of 1 mg / ml, adding 0.5 ml / well to a 24-well uncoated cell culture plate (Falcon), and placing the cell culture plate in a 37°C incubator for 4 hours, aspirating the antibody solution, and washing once with 1 ml of PBS.

[0084] In some embodiments, step 2 may specifically include: separating PBMC immune cells from peripheral blood, adding them to TexMACS medium, and then adding IL-15 (Miltenyi Biotec) to obtain PBMC cell fluid, wherein the concentration of IL-15 in the PBMC cell fluid is 5 ng / ml, and the cell concentration of PBMC immune cells in the PBMC cell fluid is 5×10 5 pcs / ml.

[0085] In some embodiments, step 3 may specifically include: inoculating 2 ml of PBMC cell fluid per well on an anti-CD3 antibody / anti-CD28 antibody-coated plate, collecting the PBMC cell fluid after 2 days, collecting the fluid three times in total, mixing the mixture, placing it in a low-temperature centrifuge, centrifuging it at 1500 rpm for 10 minutes, removing cell residues and debris, and filtering it with a 0.22 micron sterile filter to obtain a T cell activation medium.

[0086] As an embodiment, the adoptive immune cells are CD4 T cells in which the CCR5 encoding gene is knocked out.

[0087] The adoptive immune cells of this embodiment can be prepared according to the following steps:

[0088] S21, providing sgRNA for knocking out the CCR5 encoding gene and constructing a recombinant vector containing the sgRNA;

[0089] Among them, the nucleotide sequence of the sgRNA used to knock out the CCR5 encoding gene is shown in SEQ ID NO.11 and SEQ ID NO.19. The recombinant vector containing the sgRNA also includes the Cas9 encoding gene. The recombinant vector containing the sgRNA can be a Piggybac transposon plasmid, and an auxiliary vector encoding the Piggybac transposase is also required.

[0090] S22, transfecting PBMC immune cells with a recombinant vector containing sgRNA and a reprogramming factor expression system to obtain CD4 T cells with CCR5 coding gene knockout;

[0091] Among them, when the recombinant vector containing sgRNA is a Piggybac transposon plasmid, the recombinant vector containing sgRNA, the reprogramming factor expression system and the auxiliary vector encoding the Piggybac transposase are added.

[0092] In some embodiments, the nucleotide sequence of the recombinant vector containing the sgRNA is shown in SEQ ID NO. 2, and the structure is shown in FIG11 .

[0093] As an embodiment, the adoptive immune cells can be chimeric antigen receptor T cells targeting CD19, chimeric antigen receptor T cells targeting CD20, chimeric antigen receptor T cells targeting GD2, chimeric antigen receptor T cells targeting CD22, chimeric antigen receptor T cells targeting CD30, chimeric antigen receptor T cells targeting CD44, chimeric antigen receptor T cells targeting CEA, chimeric antigen receptor T cells targeting Her2, chimeric antigen receptor T cells targeting MUC1, chimeric antigen receptor T cells targeting IL13 receptor α, chimeric antigen receptor T cells targeting immunoglobulin light chain, chimeric antigen receptor T cells targeting PSMA antigen or chimeric antigen receptor T cells targeting VEGF receptor 2.

[0094] As an embodiment, the adoptive immune cells are CAR-T cells, which can target the GM-CSF (granulocyte monocyte colony-stimulating factor) receptor expressed by leukemic blood stem cells / precursor cells. The CAR-T cells can be used as drugs to treat myeloproliferative neoplasms, myelodysplastic / myeloproliferative neoplasms, myelodysplastic syndrome, and acute myeloid leukemia.

[0095] As an embodiment, the administration route of the adoptive immune cells of this embodiment is not particularly limited, including intravenous injection, intraarterial injection, portal vein injection, intradermal injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. It is not limited to systemic administration and can also be administered locally, including to target tissues and organs. In addition to single administration, multiple administrations can also be performed continuously or regularly.

[0096] As an embodiment, the reprogramming factor expression system can also use viral methods such as lentivirus, retrovirus, AAV, Sendai virus to deliver reprogramming factors, and also include non-viral methods such as transposon method to introduce reprogramming factors.

[0097] Example 1: Construction of reprogramming factor expression system

[0098] A) Preparation of mRNA encoding mutant alphavirus replicase

[0099] Step 1: Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) was used to synthesize the mutant replicase DNA coding sequence (a DNA nucleic acid molecule encoding the mutant replicase of the alphavirus genus and without a polyadenylation sequence). After successful synthesis, the mutant replicase DNA coding sequence was cloned into the pcDNA3.3 vector plasmid for industrial production.

[0100] 1.1 Mutant replicase DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO. 9), mutant replicase coding sequence, and 3' untranslated region DNA sequence (SEQ ID NO. 10). The mutant replicase coding sequence is divided into four DNA fragments: the nsP1 region fragment (SEQ ID NO. 3), the nsP2 region fragment (SEQ ID NO. 4), the nsP3 region fragment (SEQ ID NO. 5), and the nsP4 region fragment (SEQ ID NO. 6). All four DNA fragments are modified fragments with high GC content. The four DNA fragments were ordered directly from IDT Corporation in the United States as gblocks.

[0101] Specifically, the steps include: assembling a Gibson reaction according to Table 1, reacting at 50° C. for 60 minutes in a PCR instrument, and obtaining a PCR product.

[0102] Table 1 Gibson reaction system

[0103] 1.2 Transform the PCR product into One Shot™ TOP10 chemically competent E. coli cells, including the following steps:

[0104] Dilute the Gibson reaction system (PCR product) with nuclease-free water at a ratio of 1:5, add 12 μL of nuclease-free water and 3 μL of Gibson reaction system, mix well, and react on ice;

[0105] Add 1 μL of the dilution to One Shot™ TOP10 chemically competent E. coli cells and mix. Incubate the transformation mixture on ice for 20–30 minutes.

[0106] Incubate the cells at 42°C for 30 seconds without shaking;

[0107] Immediately transfer the reaction tube to ice and incubate on ice for 2 minutes;

[0108] 450 μL of room temperature SOC culture medium (Life Technology, USA) was added;

[0109] Shake at 300 rpm at 37°C for 1 hour;

[0110] Take 100 μL and apply it to a bacterial culture plate (100 μg / mL ampicillin or 50 μg / mL kanamycin.);

[0111] The cells were incubated at 37°C overnight, bacterial clones were selected, and the cells were shaken at 37°C. The double-mutant replicase sequence plasmid containing the correct sequence was selected by first-generation sequencing.

[0112] Step 2: Add the poly-(a) tail of mRNA by PCR to obtain the DNA synthesis template of replicase mRNA

[0113] The poly-(a) tail consists of 120 adenosine nucleotides.

[0114] Prepare PCR master mix according to Table 2 (total volume 200 μL, 25 μL for each of eight reactions);

[0115] Table 2 Composition of PCR premix

[0116] PCR reaction was performed according to the reaction conditions shown in Table 3;

[0117] Table 3 PCR reaction conditions

[0118] Check the quality of PCR products by gel electrophoresis;

[0119] The PCR product was recovered by gel excision (QIAquick PCR purification kit, Qiagen, cat. no. 28106), and the final concentration of the tail template was adjusted to 100 ng / μL, which was used as a template for DNA synthesis of replicase mRNA encoding in vitro transcription.

[0120] Step 3: In vitro transcription synthesis of replicase encoding mRNA

[0121] 1. Assemble the mRNA cap structure and nucleotide mixture according to Table 4:

[0122] The cap structure was 3′-O-Me-m7G(5′)ppp(5′)G RNA cap analog (New England Biolabs, cat. no. S1411S), -Methylcytidine-5′-triphosphate (Me-CTP; Trilink, cat. no. N1014), N1-methyl-pseudo-UTP (Trilink, cat. no. N1019), and other components were from the MEGAscript T7 kit (Ambion, cat. no. AM1334).

[0123] Table 4 mRNA cap structure and nucleotide mixture

[0124] 2. Assemble the in vitro transcription system encoding replicase mRNA according to Table 5:

[0125] Table 5 In vitro transcription system of replicase encoding mRNA

[0126] 3. Place the reaction in a PCR instrument and incubate at 37°C for 3 to 6 hours.

[0127] 4. Add 2 μL of Turbo DNase (from MEGAscript T7 kit, Ambion, cat. no. AM1334) to each sample.

[0128] 5. Mix gently and incubate at 37°C for 15 minutes.

[0129] 6. Purify the reaction treated with DNase and RNAseIII using the MEGAclear kit (Ambion, cat. no. AM1908); elute the modified mRNA with a total of 100 μL of elution buffer (elute twice with 50 μL of elution buffer).

[0130] 7. Treat the purified modified mRNA with Antarctic phosphatase (New England Biolabs, cat. no. M0289S).

[0131] 8. To each sample (~100 μL), add 11 μL of 10× phosphatase buffer, followed by 2 μL of phosphatase; gently mix the samples and incubate at 37°C for 0.5-1 h.

[0132] 9. After elution, measure the concentration of the modified replicase-encoding mRNA in a NanoDrop spectrophotometer. The expected total yield should be ~50 μg (30-70 μg range; a 100 μL elution volume from a 40 μL IVT reaction is 300-700 ng / μL). Adjust the concentration to 100 ng / μL by adding elution buffer or TE buffer (pH 7.0), or purify by FPLC.

[0133] B) Preparation of mRNA encoding each transcription factor

[0134] The synthesis steps of mRNA encoding transcription factors are similar to those of mRNA encoding replicase, including the following steps:

[0135] Step 1: Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) was used to synthesize the specifically modified target factor DNA coding sequence (the nucleic acid molecule encoding the transcription factor mRNA does not contain a polyadenylation sequence);

[0136] Among them, the specifically modified target factor DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO.9), replicase 5' end specific DNA sequence (SEQ ID NO.7), target factor DNA coding sequence (see Table 6), replicase 3' end specific DNA sequence (SEQ ID NO.8), 3' untranslated region DNA sequence (SEQ ID NO.10).

[0137] Step 2: Adding a poly-(a) tail of mRNA to the specifically modified target factor DNA coding sequence by PCR to obtain a DNA synthesis template encoding the transcription factor mRNA;

[0138] Step 3: In vitro transcription to synthesize mRNA encoding transcription factors.

[0139] The five transcription factor encoding mRNAs shown in Table 6 were synthesized according to the above method.

[0140] Table 6 DNA synthesis templates for different mRNA encoding transcription factors

[0141] C) The molar ratio of mRNA encoding the mutant alphavirus replicase, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor is 7:3:1:1:1:1.

[0142] Example 2: Experiment on improving the immune ability of natural killer cells using the reprogramming factor expression system

[0143] Human natural killer cells were purchased from Stem Cells Inc, and tumor cells K562 were obtained from ATCC. They were cultured in RPMI-1640 with 15% fetal bovine serum at 37 degrees Celsius in a 5% CO 2 incubator.

[0144] Reprogrammed cell group (partial reprogramming enhanced NK+K562): The reprogramming factor expression system of Example 1 was transfected into human natural killer cells, and human natural killer cells and tumor cells were added to the same well at a cell number of 1:1 and co-cultured. After culturing for 4 hours, PE staining was performed, and CD56 antibody (Abcam) was used to label human natural killer cells. PE-positive cells were killed cells, and flow cytometry was used for quantification to determine the immune ability of human natural killer cells.

[0145] Comparative group 1 (no partial reprogramming to enhance NK+K562): Untransfected human natural killer cells and tumor cells were added to the same well at a cell number of 1:1 and co-cultured. After 4 hours of culture, PE staining was performed and CD56 antibody (Abcam) was used to label human natural killer cells. PE-positive cells were killed cells. Flow cytometry was used for quantification to determine the immune capacity of human natural killer cells.

[0146] Comparative group 2 (K562 only): Tumor cells were added to the wells and cultured for 4 hours before PE staining. PE-positive cells were killed cells, and the cells were quantified by flow cytometry.

[0147] Experimental results and analysis:

[0148] As shown in Figure 1, OSKGL-driven partial reprogramming can enhance the ability of human natural killer cells (NK cells) to kill tumor cells K562. In Figure 1:

[0149] A: Experimental process of human natural killer cells killing tumor cells K562.

[0150] B: PE staining of NK cell marker CD56-negative cells indicates the proportion of K562 cells killed by NK cells, indicating that under the condition of 1:1 effector cells and target cells, the NK cell killing rate against K562 after partial reprogramming increased from 13.9% in non-partially reprogrammed cells to 79.4%, a 5.7-fold increase.

[0151] C: Quantitative results of flow cytometric evaluation of human natural killer cells killing tumor cells K562 suggest that partial reprogramming can significantly increase the ability of human natural killer cells to kill tumor cells by nearly 6 times.

[0152] Example 3: Experiment on improving macrophage phagocytic ability by using reprogramming factor expression system

[0153] Human macrophages were purchased from Stem Cells Inc, and porcine PBMC cells were from ATCC. They were cultured in RPMI-1640 with 15% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0154] Reprogrammed cell groups (partially reprogrammed human macrophages + porcine PBMCs; partially reprogrammed enhanced wild-type porcine PBMCs): The reprogramming factor expression system of Example 1 was transfected into human macrophages, and human macrophages were labeled with CD14 antibody (Abcam). After PBMCs were labeled with CFSE (Abcam) dye, human macrophages and porcine PBMCs were added to the same well at a cell number of 1:1 and co-cultured. After 4 hours of culture, the CD14 and CFSE double-positive cells were human macrophages that phagocytosed porcine PBMC cells, and the phagocytic ability of human macrophages was determined by flow cytometry.

[0155] Comparative group 1 (human macrophages + porcine PBMCs; wild-type porcine PBMCs without partial reprogramming enhancement): Untransfected human macrophages were labeled with CD14 antibody (Abcam), and PBMCs were labeled with CFSE (Abcam) dye. Human macrophages and porcine PBMCs were then added to the same well at a 1:1 ratio and co-cultured. After 4 hours of culture, the CD14 and CFSE double-positive cells, which are human macrophages, phagocytosed porcine PBMCs. The phagocytic ability of human macrophages was determined by flow cytometry.

[0156] Comparative group 2 (human macrophages + human PBMCs; human PBMCs): Untransfected human macrophages were labeled with CD14 antibody (Abcam), and PBMCs were labeled with CFSE (Abcam) dye. Human macrophages and human PBMCs were then added to the same well at a ratio of 1:1 and co-cultured. After 4 hours of culture, the CD14 and CFSE double-positive cells became human macrophages and phagocytosed human PBMCs. The phagocytic ability of human macrophages was determined by flow cytometry.

[0157] Experimental results and analysis:

[0158] As shown in Figure 2, OSKGL-driven partial reprogramming can enhance the phagocytic capacity of human macrophages.

[0159] A: Experimental process of human macrophages phagocytizing pig peripheral blood cells (PBMCs). Human macrophages are labeled with CD14, and pig PBMCs are labeled with CFSE. CD14 and CFSE double-positive cells are determined to be macrophages phagocytizing pig PBMCs.

[0160] B: The results of flow cytometric analysis after macrophages phagocytosed porcine PBMCs. According to the rule that CD14 and CFSE double-positive cells are macrophages phagocytosing porcine PBMCs, partial reprogramming increased the phagocytic capacity of human macrophages from 25.4% to 75%, an increase of nearly 3 times.

[0161] C: Quantitative results of flow cytometric evaluation of human macrophages phagocytosing porcine peripheral blood cells (PBMCs), indicating that partial reprogramming can significantly increase the phagocytic capacity of human macrophages by nearly 3 times.

[0162] Example 4: Preparation of chimeric antigen receptor T cells targeting ROR1

[0163] (1) Preparation of T cell activation medium:

[0164] 1. Preparation of anti-CD3 antibody (Miltenyi Biotec) / anti-CD28 antibody-coated (sensitized) plates: Anti-CD3 and anti-CD28 antibodies were diluted in PBS to a final concentration of 1 mg / ml. 0.5 ml / well was added to a 24-well uncoated cell culture plate (Falcon). The cell culture plate was incubated at 37°C for 4 hours. The antibody-diluted PBS was aspirated and the plate was washed once with 1 ml of PBS.

[0165] 2. PBMCs were separated from peripheral blood and prepared with TexMACS medium + 5 ng / ml IL-15 (Miltenyi Biotec) to a cell concentration of 5×10 5 / ml. 2 ml of the PBMCs were seeded per well on anti-CD3 antibody / anti-CD28 antibody-coated cell plates.

[0166] 3. Collect the cell culture medium 3 times every 2 days, mix and centrifuge at 1500 rpm for 10 minutes to remove cell debris and debris, and filter through a 0.22 μm sterile filter to form T cell activation medium for later use.

[0167] (2) Control group: Transposon-specific chimeric antigen receptor T cell preparation plan

[0168] Preparation steps for the transposon ROR1CAR vector (as shown in SEQ ID NO. 1). The vector CAR sequence includes at least a target-specific extracellular domain (for antigen binding), a transmembrane domain, and an intracellular domain (for immune response), detection genes (reporter genes, cell or tissue-specific genes, selection markers, enhancer sequences, WRPE sequences, etc.), a rapamycin resistance gene (tolerate rapamycin treatment and expand the CAR T treatment population), and an inducible autodestruction gene (e.g., one that can induce Caspase 9). The target-specific extracellular domain can contain an scFv fragment of an anti-target monoclonal antibody. The specific sequence can be seen in Figure 10. In this example, a fully human anti-ROR1 scFV with transmembrane domains derived from CD8a, 4-1BB, and a CD8a signal peptide was used. We used SnapGene software and exported the specific sequence, which was then customized by a corresponding biotechnology company such as SystemBio. This resulted in a ROR1 transposon and transposon enzyme vector for CAR T preparation.

[0169] 1. On day 0, PBMCs were isolated from peripheral blood and counted, with a mean of 1×10 7 Monocytes + 15 μg Piggbac-CAR-ROR1 vector + 15 μg Piggybac transposase helper vector were introduced by nuclear transfection using a 4D Nucleofector (Lonza) and cultured in TexMACS medium (IL7 concentration was 10 ng / ml, IL15 concentration was 5 ng / ml) on a 24-well plate coated with anti-CD3 antibody / anti-CD28 antibody for 48 hours.

[0170] 2. On day 3, the cells were transferred to G-Rex10 and cultured in MACS medium (IL7 concentration was 10 ng / ml, IL15 concentration was 5 ng / ml), and cultured every 3 days until day 14.

[0171] The preparation principle of the control group is shown in Figure 3.

[0172] (3) Experimental group: Transient mRNA expression of reprogramming factor OSKGL transposon-specific chimeric antigen receptor T cell preparation plan

[0173] 1. On day 0, PBMCs were isolated from peripheral blood and counted, with a mean of 1×10 7 Monocytes + 15 μg Piggbac-CAR-ROR1 vector (as shown in SEQ ID NO.1, designed by the inventors and customized by the biological company SystemBio), + 15 μg Piggybac transposase helper vector (SystemBio) + 200 μg of OSKGL mRNA complex prepared in Example 1 were introduced by nuclear transfection using a 4D Nucleofector (Lonza). After gene introduction, the cells were cultured in the above-mentioned T cell activation medium (IL7 concentration was 10 ng / ml, IL15 concentration was 5 ng / ml) in a 24-well plate coated with anti-CD3 antibody / anti-CD28 antibody for 48 hours.

[0174] 2. On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 concentration of 10 ng / ml, IL15 concentration of 5 ng / ml) every 3 days until day 14.

[0175] The preparation principle of the experimental group is shown in Figure 4.

[0176] (4) Gene transfer efficiency and specific chimeric antigen ROR1 receptor T cell count: After the culture was completed, the specific chimeric antigen receptor T cell count and gene transfer efficiency (specific chimeric antigen ROR1 receptor T cell count / total live T cell count) were calculated by flow cytometry and compared between the control group and the experimental group.

[0177] The transposon method in this embodiment is one of the non-viral gene introduction methods. Transposons are short genes that have been preserved during the evolutionary process and cause gene transposition, such as Piggybac, Sleeping Beatuy, Frog Prince, Tol1, and Tol2 transposons.

[0178] Experimental results and analysis:

[0179] As shown in Figure 5, the number of CAR T cells obtained by the chimeric antigen receptor T cell (CAR T cell) preparation method in the experimental group (the present invention) was compared with the chimeric antigen receptor T cells in the control group (the prior art). The gene introduction rate was promoted (increased by nearly 8 times), and T cell proliferation was promoted, thereby significantly increasing the chimeric antigen receptor T cell yield by 4 to 5 times.

[0180] As shown in FIG6 , the proportion of ROR1-positive cells among the CD3-positive T cells obtained by the chimeric antigen receptor T cell (CAR T cell) preparation method in the experimental group.

[0181] Example 5: Specific chimeric antigen receptor T cell activity evaluation experiment

[0182] 1. Hippocampal energy meter to measure the metabolic phenotype of T cells with specific chimeric antigen ROR1 receptor:

[0183] The chimeric antigen receptor T cell samples obtained in step (2) of Example 4 (control group of Example 4) and the chimeric antigen receptor T cell samples obtained in step (3) of Example 4 (experimental group of Example 4) were collected respectively: the T cell samples were collected and washed to remove the residues in the culture medium; and the T cell metabolic parameters were measured:

[0184] a. Treated T cells were evenly distributed into the Seahorse measurement microplate for metabolic assay.

[0185] b. Insert the Seahorse measurement 24-well plate into the Seahorse instrument preheated to the appropriate temperature.

[0186] c. Set the experimental parameters in the Seahorse instrument software according to the instructions for the Agilent T Cell Metabolism Characterization Kit (No. 103771) to monitor cellular metabolic activity, including oxygen consumption rate (OCR) and acidification rate (ECAR). OCR reflects the level of oxidative phosphorylation, while ECAR reflects the level of glycolysis.

[0187] f. After the measurement is completed, the software will generate a data report, including the cell metabolic activity curve, mean value, and standard deviation, and input the data into Graph Pad 9.0 software for data analysis and output.

[0188] 2. Co-culture experiment with ROR1-positive metastatic ovarian cancer cells: The specific chimeric antigen ROR1 receptor T cells (1×10 5 ) and ROR1-positive metastatic ovarian cancer cells (5×10 5 ) The cells were co-cultured in RPMI1640 medium supplemented with 10% fetal bovine serum for 7 days, and ROR1 antibody (Abcam, ab280197) was used to count the cells using counting microspheres and flow cytometry;

[0189] The specific chimeric antigen ROR1 receptor T cells (1×10 5 ) and ROR1-positive metastatic ovarian cancer cells (5×10 5 ) were co-cultured in RPMI1640 medium containing 10% fetal bovine serum for 7 days, and ROR1 antibody (Abcam, ab280197) was used and cell counting was performed using counting microspheres and flow cytometry.

[0190] 3. Evaluation based on tumor-bearing mice: A mouse tumor model was used in which ROR1-overexpressing SKOV3ROR1 cells were transduced to express firefly luciferase. 1×10 6 SKOV3ROR1 cells were injected intraperitoneally into NODPrkdc scid IL2rg em1 / Smoc (Nanjing Model Organisms Center) mice, and then injected intraperitoneally with 1×10 7 ROR1 chimeric antigen receptor T cells (experimental group of Example 4) were used to evaluate the size of the tumor based on the intensity and range of firefly luciferase; a mouse tumor model was used in which ROR1-overexpressing SKOV3ROR1 cells were transduced to express firefly luciferase. 1×10 6 SKOV3ROR1 cells were injected intraperitoneally into NODPrkdc scid IL2rg em1 / Smoc (Nanjing Model Organisms Center) mice, and then injected intraperitoneally with 1×10 7 ROR1 chimeric antigen receptor T cells (control group in Example 4) were used to evaluate the size of the tumor based on the intensity and range of firefly luciferin.

[0191] Experimental results and analysis:

[0192] As shown in Figure 7, the hippocampus energy meter provides a detailed description of the metabolism of partially reprogrammed chimeric antigen receptor T cells (CAR T cells) in the experimental group (partially reprogrammed CAR T), indicating that CAR T cells prepared by the partial reprogramming method require less resting oxygen consumption, a higher proportion of oxidized ATP, and a higher respiratory reserve than T cells prepared by the control group (conventional CAR T).

[0193] As shown in Figure 8 , the results of the tumor cytotoxicity experiment of partially reprogrammed chimeric antigen receptor T cells in the experimental group (partially reprogrammed CAR T) were obtained. ROR1-positive metastatic ovarian cancer cells were co-cultured in vitro at a 1:1 ratio. The flow cytometry results and tumor residual rate decreased from 87.1% (control group, conventional CAR T) to 9.2% (experimental group, partially reprogrammed CAR T).

[0194] As shown in Figure 9, in the tumor toxicity experiment of partially reprogrammed chimeric antigen receptor T cells in the experimental group (enhanced PORCAR T), chimeric antigen receptor T cells and ovarian tumor cells SKOV3 were injected intraperitoneally at a ratio of 10:1 in a tumor-bearing mouse model. The intensity of fluorescein was used to indicate the size of the tumor to assess the tumor size and quantify the in vivo tumor toxicity of partially reprogrammed chimeric antigen receptor T cells. The results showed that compared with the chimeric antigen receptor T cells prepared in the control group (ordinary PORCAR T), the partially reprogrammed chimeric antigen receptor T cells in the experimental group (enhanced PORCAR T) showed very strong tumor inhibition and killing effects. Compared with the chimeric antigen receptor T cells prepared in the control group (ordinary PORCAR T), the killing ability was at least 4 times higher.

[0195] Example 6: Preparation of CD4 T cells with CCR5 encoding gene knockout

[0196] Transposon plasmid preparation: Cas9 and double CCR5 knockout gRNA (as shown in SEQ ID NO.11 and SEQ ID NO.19) were designed using Macvector software to form the Piggybac transposon plasmid sequence, which was then submitted to a corresponding biological company (e.g., SystemBio) for preparation of the specific vector.

[0197] Control group:

[0198] 1. On day 0, PBMCs were isolated from peripheral blood and counted, with a mean of 1×10 7Monocytes + 15 μg Piggbac-CAS9-CCR5 vector (as shown in SEQ ID NO.2) + 15 μg Piggybac transposase helper vector were introduced by nucleofection using 4D Nucleofector (Lonza), and then cultured in TexMACS medium + 10 ng / ml IL7 + 5 ng / ml IL15 on anti-CD3 antibody / anti-CD28 antibody-coated 24-well plates for 48 hours.

[0199] 2. On day 3, cells were transferred to G-Rex10 (Wilson Wolf) and cultured in MACS medium + 10 ng / ml IL7 + 5 ng / ml IL15, and cultured every 3 days until day 14.

[0200] Experimental group: transient mRNA expression of reprogramming factor OSKGL transposon CCR5 gene knockout T cell preparation plan:

[0201] 1. On day 0, PBMCs were isolated from peripheral blood and counted, with a mean of 1×10 7 Monocytes + 15 μg Piggbac-CCR5CAS91 vector (as shown in SEQ ID NO. 2, designed by the inventors and customized by the biological company SystemBio), + 15 μg Piggybac transposase helper vector (SystemBio) + 200 μg of the OSKGL mRNA complex prepared in Example 1 were introduced by nucleofection using a 4D Nucleofector (Lonza). After gene introduction, the cells were cultured in the above-mentioned T cell activation medium (IL7 concentration was 10 ng / ml, IL15 concentration was 5 ng / ml) on a 24-well plate coated with anti-CD3 antibody / anti-CD28 antibody for 48 hours.

[0202] 2. On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 concentration of 10 ng / ml, IL15 concentration of 5 ng / ml) every 3 days until day 14.

[0203] Gene transfer efficiency and CCR5 knockout T cell count: After the culture was completed, the CCR5 knockout efficiency was calculated by flow cytometry analysis and compared between the control group culture and the experimental group culture method.

[0204] Experimental results and analysis:

[0205] As shown in Figure 12, the preparation method of CCR5 knockout T cells shows that compared with the control group (existing technology), the partial reprogramming of the experimental group (technology of the present invention) can increase the number of CCR5 knockout cells by more than 3 times, with no (low) CCR5 protein expression.

[0206] Example 7: Adoptive immune cell experiments constructed with different ratios of transcription factor mRNA

[0207] This embodiment is divided into four groups:

[0208] Experimental group: The reprogramming factor expression system prepared in Example 1 was used (OSKGL ratio 3:1:1:1:1); on day 0, PBMCs were isolated from peripheral blood of volunteers and counted, with a mean of 1×10 7 Monocytes and 200 μg of the reprogramming factor expression system prepared in Example 1 were added by nuclear transfection using a 4D Nucleofector (Lonza). The cells were then cultured for 48 hours in a 24-well plate coated with anti-CD3 / anti-CD28 antibodies in the aforementioned T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). The cells were cultured every 3 days until day 14.

[0209] Comparative group 1: The following blank expression system was used: the molar ratio of mRNA encoding the mutant alphavirus replicase to mRNA encoding the firefly fluorescent protein was 7:3; on day 0, PBMCs were isolated from the peripheral blood of volunteers and counted, with a mean of 1×10 7 Monocytes and 200 μg of the blank expression system were transfected by nucleofection using a 4D Nucleofector (Lonza). The cells were then cultured for 48 hours in a 24-well plate coated with anti-CD3 / anti-CD28 antibodies in the aforementioned T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). The cells were cultured every 3 days until day 14.

[0210] Comparative group 2: The following first expression system was used: the molar ratio of mRNA encoding the mutant alphavirus replicase, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor was 7:1:1:1:1:1; the molar ratio of mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly fluorescent protein was 7:3; on day 0, PBMCs were isolated from the peripheral blood of volunteers and counted, with a concentration of 1×10 7Monocytes and 200 μg of the first expression system were transfected by nucleofection using a 4D Nucleofector (Lonza). The cells were then cultured for 48 hours in a 24-well plate coated with anti-CD3 / anti-CD28 antibodies in the aforementioned T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). The culture was continued every 3 days until day 14.

[0211] Comparative group 3: The following second expression system was used: the molar ratio of mRNA encoding the mutant alphavirus replicase, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor was 7:0.5:1:1:1:1; the molar ratio of mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly fluorescent protein was 7:3; on day 0, PBMCs were isolated from the peripheral blood of volunteers and counted, with a concentration of 1×10 7 Monocytes and 200 μg of the second expression system were transfected by nucleofection using a 4D Nucleofector (Lonza). The cells were then cultured for 48 hours in a 24-well plate coated with anti-CD3 / anti-CD28 antibodies in the aforementioned T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). On day 3, the cells were transferred to G-Rex10 (Wilson Wolf) and cultured in T cell activation medium (IL7 at 10 ng / ml and IL15 at 5 ng / ml). The culture was continued every 3 days until day 14.

[0212] The above experimental group, control group 1, control group 2, and control group 3 were respectively reversed for epigenetic age determination. After the above culture, the genomic DNA of the corresponding cells was collected, and the epigenetic age of the corresponding volunteer T cells was calculated according to the Horvath epigenetic age calculation method (https: / / horvath.genetics.ucla.edu / html / dnamage / ).

[0213] Experimental results and analysis:

[0214] As shown in Figure 13, as shown in 13-A, compared with control group 1 (mRNA reporter gene treatment group), the experimental group (OSKGL mRNA ratio 3:1:1:1:1) can significantly reduce the epigenetic age of volunteers A, volunteer B, and volunteer C; and for volunteer C, as shown in 13-B, compared with control group 2 (OSKGL mRNA ratio 1:1:1:1:1) and control group 3 (OSKGL mRNA ratio 0.5:1:1:1:1), the experimental group (OSKGLmRNA ratio 3:1:1:1:1) can significantly reduce the epigenetic age of T cells.

[0215] The above is only an implementation method of the present application. It should be pointed out that for ordinary technicians in this field, improvements can be made without departing from the creative concept of the present application, but these all fall within the scope of protection of the present application.

Claims

1. An adoptive immune cell, characterized in that: The adoptive immune cells include a reprogramming factor expression system, which includes one or more of a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a cMyc transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

2. The adoptive immune cell according to claim 1, characterized in that The adoptive immune cells are chimeric antigen receptor T cells targeting ROR1; Alternatively, the adoptive immune cells are CD4 T cells in which the CCR5 encoding gene is knocked out.

3. The adoptive immune cell according to claim 1, characterized in that The reprogramming factor expression system includes mRNA encoding Oct4 transcription factor, mRNA encoding Sox2 transcription factor, mRNA encoding Klf4 transcription factor, mRNA encoding Glis1 transcription factor and mRNA encoding Lin28 transcription factor.

4. The adoptive immune cell according to claim 3, characterized in that The reprogramming factor expression system also includes mRNA encoding a mutant replicase of the genus Alphavirus, which produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

5. The adoptive immune cell according to claim 4, characterized in that Part or all of the nucleotides in the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, the mRNA encoding Lin28 transcription factor, and the mRNA encoding the mutant replicase of the alphavirus are chemically modified to improve the stability of the mRNA in vivo; the chemical modification includes replacing 100% of the uracil in the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, the mRNA encoding Lin28 transcription factor, and the mRNA encoding the mutant replicase of the alphavirus with N1-methylpseudouridine, pseudouracil, or methyluracil, and replacing 100% of the uracil in the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, the mRNA encoding Lin28 transcription factor, and the mRNA encoding the mutant replicase of the alphavirus with 5-methylcytosine or 5-methylcytosine. 100% of the cytosine in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, and the mRNA encoding the Lin28 transcription factor were substituted with hydroxymethylcytosine.

6. A method for preparing adoptive immune cells, characterized in that: include: Providing a chimeric antigen receptor CAR-ROR1 encoding gene, which includes an antibody encoding gene targeting ROR1, a transmembrane domain encoding gene, an intracellular domain encoding gene, a detection gene, a rapamycin resistance gene, and an inducible self-destruct gene; Inserting the chimeric antigen receptor CAR-ROR1 encoding gene into a vector to obtain a CAR-ROR1 recombinant vector; The CAR-ROR1 recombinant vector and the reprogramming factor expression system are transfected into PBMC immune cells to obtain the chimeric antigen receptor T cells targeting ROR1.

7. The method for preparing adoptive immune cells according to claim 6, characterized in that: Also includes: The transfected PBMC immune cells were cultured in T cell activation medium; Alternatively, the nucleotide sequence of the CAR-ROR1 recombinant vector is as shown in SEQ ID NO.

1.

8. A method for preparing adoptive immune cells, characterized in that: include: Providing an sgRNA for knocking out a CCR5 coding gene, and constructing a recombinant vector containing the sgRNA; The recombinant vector of the sgRNA and the reprogramming factor expression system are transfected into PBMC immune cells to obtain the CD4T cells with the CCR5 encoding gene knocked out.

9. The method for preparing adoptive immune cells according to claim 8, characterized in that: Also includes: The transfected PBMC immune cells were cultured in T cell activation medium; Or, the nucleotide sequence of the recombinant vector containing the sgRNA is shown as SEQ ID NO.

2.

10. Use of the adoptive immune cells according to any one of claims 1 to 5 in the preparation of a drug for treating tumors or AIDS.

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