Nucleic acid modified biological cells exhibiting proliferation-dependent gene expression

Abstract

The present invention relates to nucleic acid modified biological cells and methods for regulating the expression of a nucleotide sequence encoding a target in a nucleic acid modified cell.

Description

[Technical field]

[0001] The present invention relates to nucleic acid modified biological cells and methods for regulating the expression of a nucleotide sequence encoding a target in a nucleic acid modified cell.

[0002] The present invention relates generally to the field of cell or molecular biology, and in particular to the field of cell therapy. More specifically, the present invention relates to therapeutically active biological cells produced using genetic engineering techniques. [Background technology]

[0003] Cell therapy uses living cells that are injected, transplanted or implanted into a patient to achieve a medical effect, for example, T cells capable of fighting cancer cells through cellular immunity are transplanted during immunotherapy, or stem cells are transplanted to regenerate diseased tissue.

[0004] Cells bearing chimeric antigen receptors (CARs) are important tools in cell therapy. CARs allow engineered T cells to eliminate cells that present the antigen targeted by the CAR. Autologous CAR T cells have been successfully used to treat B-cell malignancies, with four products targeted to CD19 and one targeted to a B-cell maturation antigen approved. Several other CAR T-cell products are in development, with over 100 clinical trials currently underway for hematological malignancies and solid tumors. CAR T cells can also be used to treat autoimmune diseases and viral infections.

[0005] Despite the initial success of CAR T cells, several issues have hindered their widespread clinical application. CAR T cell therapy is often associated with primary resistance in 10-20% of patients, with relapse occurring at a rate of 30-50%. Mechanisms of resistance observed in infused T cells include poor in vivo proliferation, early exhaustion, low persistence, and poor compatibility. The tumor itself may also contribute to resistance in the form of loss, downregulation, or mutation of target antigens. In addition, CAR T cells may exhibit toxicity in the form of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). ICANS can develop in a range of severity from mild to severe, with a mortality rate of 1.5%. Prolonged and / or severe neutropenia and fatal infections due to B-cell aplasia may also occur.

[0006] Currently approved CAR T cells are a heterogeneous clonal population with variable efficacy across each clone, due to (1) inadvertent genomic integration during lentiviral vector production and (2) phenotypic heterogeneity of apheresis preparations.

[0007] Effective CAR T cell clones are capable of proliferation, eliminating target cells, and persistence in vivo for months up to a year without exhaustion, whereas overly aggressive CAR T cell clones that do not become exhausted and show excessive proliferation cause toxicity.

[0008] To reduce T cell-mediated resistance and toxicity and expand the range of targetable diseases, several T cell modulation strategies are being explored based on the current knowledge that certain CAR T cell clones are more effective than others.

[0009] One of such regulation methods utilizes so-called small interfering RNA (siRNA).The method of using siRNA to regulate the activity of cells in cell therapy is disclosed, for example, in WO2015 / 084897.As an alternative, the so-called small hairpin RNA (shRNA) is utilized in cell therapy for the same purpose, and such methods are disclosed, for example, in WO2020 / 206248, WO2019 / 138354, WO2015 / 136001 or WO2011 / 059836.

[0010] However, the problems of poor efficacy and, in particular, toxicity of cell therapies are inadequately addressed by current technology.

[0011] In this situation, the present invention aims to provide biological cells that can be used in cell therapy without causing the problems reported in current cell therapies, particularly toxicity, and specifically, to provide biological cells that can be temporally regulated to regulate the expression of target genes or proteins at the beginning or late stage of the proliferation phase. Summary of the Invention [Means for solving the problem]

[0012] The object of the present invention is a living cell modified with a nucleic acid, (i) a nucleic acid comprising a first expressible nucleotide sequence encoding a target; (ii) a recombinant nucleic acid comprising a second expressible nucleotide sequence configured to modulate the expression and / or function of the first expressible nucleotide sequence. in its genome, a nucleic acid comprising a third nucleotide sequence configured to inhibit expression and / or function of the second expressible nucleotide sequence outside the genome; This is accomplished by providing a living cell.

[0013] The object of the present invention is a method for modulating the expression of a first nucleotide sequence encoding a target in a cell modified with a nucleic acid, comprising the steps of: 1) providing a biological cell having integrated into its genome a first expressible nucleotide sequence encoding a target protein; 2) integrating into the genome of said biological cell a recombinant nucleic acid comprising a second expressible nucleotide sequence configured to regulate the expression and / or function of the first expressible nucleotide sequence; and 3) exogenously introducing into said biological cell a nucleic acid comprising a third nucleotide sequence configured to inhibit expression and / or function of a second expressible nucleotide sequence. This can also be achieved by a method comprising: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] According to the present invention, a "biological cell" includes eukaryotic cells, such as animal cells (eg, mammalian or human cells) and plant cells, and prokaryotic cells (eg, bacterial cells).

[0015] According to the present invention, "nucleic acid modified" means that a cell and / or its nucleic acid (e.g., DNA or RNA) has been modified by genetic engineering techniques, such as nucleic acid recombination methods, for example, by the introduction of non-recombinant artificial nucleic acid molecules into the cell. Thus, the term can be used interchangeably with "genetically modified" living cells.

[0016] According to the invention, a living cell has a genome, i.e., it has genetic information, usually in the form of heritable DNA. All genetic information that is part of the genome of a living cell is replicated during proliferation or cell division and passed on to descendent cells. Thus, the genome and the genetic information contained therein are not lost through proliferation, i.e., are "proliferation independent". The genome of cells of higher organisms, such as eukaryotes, is usually composed of chromosomes. Extrachromosomal elements, such as episomes, can be introduced by biotechnological techniques and likewise constitute part of the genome of the cell.

[0017] According to the present invention, a "nucleotide sequence" is a nucleotide sequence consisting of a nucleic acid, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleotide sequence may be composed of naturally occurring nucleotides and / or chemically modified nucleotides.

[0018] According to the invention, the genome of the biological cell comprises a first expressible nucleotide sequence encoding a target, for example a target protein, a non-translated target RNA, or a non-protein target. In a preferred embodiment, the first protein is encoded by a gene represented by the first expressible nucleotide sequence.

[0019] According to the invention, the genome of said living cell comprises a second nucleotide sequence, which is part of the genome of said living cell or is integrated into the genome of said living cell. Such integration can be achieved by nucleic acid recombination methods or genetic engineering methods. In a preferred embodiment, the second nucleotide sequence is integrated into a chromosome of said cell or is introduced into said cell via an extrachromosomal element, e.g. an episome.

[0020] "Recombinant DNA" means man-made DNA formed by laboratory recombinant methods (e.g., molecular cloning) to bring together genetic material from multiple sources to produce sequences not found in a genome. DNA or genomes that occur in nature and have been produced by natural recombination events are not called recombinant DNA.

[0021] By "expressible" it is meant that the first and second nucleotide sequences can be read by a cellular structure, preferably via an upstream promoter, and converted into a nucleic acid transcript, such as an mRNA, which in a preferred embodiment can be translated into a target protein.

[0022] The cell according to the present invention further comprises a third nucleotide sequence that is not part of the genome. The third nucleotide sequence is introduced into the cell by a method known to those skilled in the art, such as delivery by some modified molecule, electroporation, lipid nanoparticles, etc. The third nucleotide sequence is not part of the genome, and is not integrated into the genome, so it is not replicated during cell proliferation and is not inherited by progeny cells. Thus, the third nucleotide sequence is "diluted" during cell division into progeny cells and is lost as the cell proliferates. In other words, the third nucleotide sequence is "proliferation dependent".

[0023] The third nucleotide sequence has a structure capable of inhibiting expression of the second nucleotide sequence, for example, in one embodiment, the third nucleotide sequence prevents reading and expression of the second nucleotide sequence due to steric interactions between the third nucleotide sequence and the second nucleotide sequence (e.g., due to annealing between complementary sequence segments) or due to enzyme-catalyzed interactions.

[0024] The inventors have found that in modified biological cells according to the invention, the first nucleotide sequence or the target can be controlled to be expressed over time at the beginning of the growth or expansion phase (e.g. at the beginning of cell therapy) or over time at the end of the later growth / expansion phase. The first and second nucleotide sequences, which are part of the genome, are replicated during mitosis, so that the copy number in daughter cells does not change. Thus, the expression of the first and second nucleotide sequences remains insensitive to cell proliferation. The third nucleotide sequence is present in the biological cells at a specific concentration and is diluted with each cell division. The duration of its effect can be set by adjusting the initial concentration used. A combination of the second and third nucleotide sequences, which have antagonistic effects on each other, is introduced into the cells. As the third nucleotide sequence is diluted during the growth process, the second nucleotide sequence is released from the blocking state and begins to exert its effect, for example, expressing a protein or downregulating a target.

[0025] The cells according to the present invention can be optimized for use in cell therapy in this way. By utilizing the dilution effect acting on the third nucleotide sequence, it is possible to express a target, such as a specific intracellular gene, exclusively in the cells of the present invention at the beginning of the therapeutic burst of proliferation before the proliferation is induced by activity, and then inhibit the expression of the target, or inhibit the expression of the target in advance and express the target exclusively at the end of the therapeutic burst of proliferation after the proliferation is induced by activity. This time-controlled and / or limited expression of the target can significantly reduce the toxicity and side effects frequently observed in current cell therapies, such as cytokine release syndrome caused by CAR-T cells and graft-versus-host disease caused by donor-derived lymphocyte infusion. Furthermore, such a method can enhance the efficacy of cell therapy by safely enhancing therapeutically relevant proliferation or enriching therapeutically beneficial phenotypes in specific time windows of the burst of proliferation.

[0026] The biological cells of the present invention can utilize a cell therapeutic window in which effective and non-toxic cell clones can reach and persist within, but not exceed, an upper limit. The inventors have found that the timing of the modulation method is crucial to ensure that this therapeutic window is maintained. In one embodiment of the present invention, in order to reach the therapeutic window, early stages should accelerate proliferation and tissue migration and inhibit exhaustion. Later stages should slow proliferation to flatten the growth curve and prevent overactivation. Ideally, therefore, the cell modulation method should be sensitive to this proliferation phase. The present invention meets these and other needs.

[0027] In one embodiment of the invention, the second expressible nucleotide sequence is configured to inhibit expression of the first expressible nucleotide sequence.

[0028] By this means, a favorable condition can be created that allows the target to be strongly expressed at the beginning of the growth phase and the expression of this target to be inhibited during the growth process. Thus, in the initial stage, the second nucleotide sequence itself is inhibited by the third nucleotide sequence. After several replication cycles, the third nucleotide sequence is gradually diluted and loses its inhibitory effect on the second nucleotide sequence. Then, the inhibitory effect of the second nucleotide sequence on the target gradually increases, and after several replication cycles and cell divisions, it can completely inhibit the target.

[0029] In another embodiment of the biological cell according to the invention, the second expressible nucleotide sequence is integrated into a chromosome and / or an episome of said biological cell.

[0030] By this means, the second nucleotide sequence forms or becomes part of the genome of the living cell, thereby ensuring that the second nucleotide sequence is passed on to descendant cells with each cell division. Episomes are plasmids that form extrachromosomal elements that remain in the cell as part of the eukaryotic genome without being integrated into it. Episomes can remain in the cell as part of the eukaryotic genome without being integrated into it by binding to metaphase chromosomes after they are replicated in synchronous with the rest of the genome during mitosis. Episomes do not degrade, unlike standard plasmids, and are not silenced by epigenetic mechanisms in the eukaryotic nucleus.

[0031] In yet another embodiment of the invention, said nucleic acid comprising a third nucleotide sequence is an oligonucleotide.

[0032] By this means, beneficial conditions can be created that allow the third nucleotide sequence to be temporarily present in the cell. In the present specification, an "oligonucleotide" is understood to be an oligomer composed of several nucleotides (DNA or RNA), usually 7 to 30 nucleotides, and optionally 7 to 10 nucleotides. The specific number of nucleotides varies depending on the application. Furthermore, the oligonucleotide may contain natural nucleotides and / or chemically modified nucleotides. In the present specification, there may be an additional effect on the stability of the oligonucleotide, i.e., on the time required for dilution or "dilution", depending on the type of chemical modification, especially when using RNA. Usually, the stability is regulated by modification of uridine and cytidine nucleotides. Examples of chemically modified uridines include pseudouridine (Ψ), 2-thiouridine, 5-methyluridine, 5-iodouridine, 4-thiouridine, 5-bromouridine, 2'-methyl-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, and 2'-fluoro-2'-deoxyuridine. Examples of chemically modified cytidines include 5-methylcytidine (m5C), 3-methylcytidine, 2-thiocytidine, 2'-methyl-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-bromocytidine, and 2'-azido-2'-deoxycytidine. Additionally, chemical modifications include those that affect the sugar and phosphate backbones, such as 2'-fluoro, 2'-O-methyl, 2-methoxyethyl, LNA, phosphorothioate, vinylphosphonate, and many others.

[0033] In another embodiment of the invention, the second expressible nucleotide sequence and the third nucleotide sequence encode a regulatory RNA.

[0034] This approach has the advantage that it is possible to obtain a class of oligonucleotides that are particularly good and easily designable as inhibitors of the expression of a second nucleotide sequence. Regulatory RNAs are a class of RNA molecules that do not code for proteins but can be responsible for a number of cellular functions, including the inhibition of the expression and / or function of another nucleotide sequence.

[0035] In yet another embodiment of the invention, the second expressible nucleotide sequence encodes an RNA selected from the group consisting of shRNA (small / short hairpin RNA), snRNA (small nuclear RNA), miRNA (microRNA), miRNA sponge, piRNA, lncRNA (long non-coding RNA), guide RNA, mRNA (messenger RNA), antisense transcript, and transposon.

[0036] This has the advantage that the second expressible nucleotide sequence can be provided in a form that can be integrated into the genome of a biological cell and that, when expressed, is capable of regulating expression of the first nucleotide sequence, for example via RNA interference.

[0037] In yet another embodiment of the invention, the third nucleotide sequence encodes an RNA selected from the group consisting of siRNA (small interfering RNA), ASO (antisense oligonucleotide), miRNA (microRNA), AMO (anti-miRNA), and an aptamer.

[0038] This approach has the advantage of providing an oligonucleotide comprising a third nucleotide sequence that is capable of effectively inhibiting expression of a second nucleotide sequence, for example through hybridization, RNA interference, RNA silencing or similar mechanisms.

[0039] In another preferred embodiment, in the step (3) of the method of the present invention, the nucleic acid comprising a third nucleotide sequence is introduced into the biological cell via a method selected from the group consisting of electroporation, delivery by lipid modification, delivery by aptamer modification, delivery by lipid nanoparticles, delivery by viral vector, delivery by antibody modification, delivery by small molecule modification, delivery by peptide modification, and delivery by extracellular vesicles.

[0040] This approach has the advantage of utilizing methods that have been demonstrated in the art to be effective for introducing oligonucleotides into living cells.

[0041] In yet another embodiment, in step (2) of the method of the present invention, the recombinant nucleic acid comprising a second expressible nucleotide sequence is introduced into the living cell via a genome-integrating virus and / or an episomal virus, thereby adding the recombinant nucleic acid as part of the genome.

[0042] This embodiment employs established recombinant biology methods that allow the safe and reliable integration of a second nucleotide sequence into the genome of a living cell.

[0043] In another embodiment of the invention, the biological cell is an immune cell.

[0044] With this further development, immune cells which are particularly suitable as a tool for cell therapy can be modified with nucleic acids according to the invention.

[0045] In yet another embodiment of the invention, said biological cells or said immune cells are selected from the group consisting of T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocytes for infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, tumor infiltrating macrophages, hematopoietic stem cells, and cells of the same type.

[0046] Said immune cells are particularly suitable and effective for use in cell therapy. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant receptor that confers or transfers specificity to an effector immune cell (e.g., human T cell) for an antigen. A chimeric antigen receptor comprises at least an extracellular ligand-binding domain or portion, a transmembrane domain, and an intracellular domain that comprises one or more signaling and / or costimulatory domains. Thus, "CAR T cell" refers to a T cell that has been genetically engineered to generate this artificial T cell receptor. A chimeric antigen receptor is chimeric because it combines antigen-binding and T cell activation functions in a single receptor.

[0047] T cells can be genetically modified to express a CAR construct that can recognize and target surface markers on so-called "cancer stem cells". In the prior art, such surface markers are not exclusively expressed on cancer cells, and therefore such "conventional" CAR T cells often have the problem of recognizing and targeting cells other than tumor cells. This means that targeting "cancer stem cells" is accompanied by severe side effects. These side effects severely limit the use of CAR T cells. According to the findings of the inventors, CAR T cells can express multiple CAR constructs, and therefore, in addition to the first CAR construct described herein, CAR T cells can also be genetically modified to express another CAR construct. This second CAR construct can target a gene that recognizes as specifically as possible a protein that is exclusively expressed on cancer cells. CAR T cells can use this CAR construct to find the tumor. This can be achieved by the present invention. The "first" CAR construct utilized by the CAR T cells to recognize so-called "cancer stem cells" is downregulated at the beginning of the proliferation phase using an oligonucleotide containing a third nucleotide sequence. In this way, the CAR T cells only recognize tumor cells and side effects are prevented. If the CAR T cells recognize the tumor using the permanently expressed "second" CAR construct, the CAR T cells will proliferate and the oligonucleotide containing the third nucleotide sequence will be diluted. As a result, the CAR T cells of the present invention that are localized to the tumor and proliferate there express a CAR construct that can recognize and target the "cancer stem cells". In this way, the present invention makes it possible to fight "cancer stem cells" with few side effects.

[0048] In another embodiment of the invention, the first expressible nucleotide sequence encoding a target protein is a gene selected from the group consisting of BRD4, p300, TET2, DNMT3, miR-15 / 16, miR-150, GLUT1, TOX, Blimp-1, NR4A, BATF, IRF4, NFAT, KLRG1, EOMES, TBX2, PRDM1, GZMA, HZMB, PRF1, IFNG, PD1, TlM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, and Siglec7 / 9.

[0049] This approach has the advantage that it allows addressing target genes that are preferably downregulated early in the proliferation phase of the cells, in particular in the proliferation phase of CAR T cells.

[0050] In yet another embodiment of the invention, the first expressible nucleotide sequence encoding a target protein is a gene selected from the group consisting of IL-1, IL-6, GM-CSFm, CD40L, LAG3, CD69, TCF1, BCL-6, BMI1, FOXO1, KLF2, LEF1, TCF7, IL2RA, CD27, TNF, CCR7, SELL, CD62L, miR-143, CCR5, CCR2, Iet7, TGFβ, IL-12, IL-18, IL-23, T cell receptor, miR-27a, IL-7, CCL19, CCL21, a CAR construct targeting the tumor microenvironment, IL-15, IL-7, and a CAR construct targeting a cancer stem cell marker.

[0051] This approach has the advantage that target genes that are preferably downregulated during the later expansion phase of the cells, in particular during the later expansion phase of CAR T cells, can be addressed.

[0052] Another subject of the present invention relates to a biological cell modified with a nucleic acid according to any one of the preceding claims for use in medicine, preferably for use in the treatment and / or prevention of a disease selected from the group consisting of cancer, autoimmune diseases, alloimmunity (rejection or graft-versus-host disease), viral infections and bacterial infections.

[0053] The features, properties and advantages disclosed for the cells or the regulatory methods of the invention apply analogously to the uses of the invention.

[0054] Yet another subject of the invention relates to a method for treating an organism in need of treatment by administering biological cells modified with a nucleic acid according to the invention.

[0055] The features, properties and advantages disclosed for the cellular or regulatory methods of the invention apply equally to the therapeutic methods of the invention.

[0056] According to the present invention, an "organism" relates to any animal or plant, and in particular to a mammal, including a human.

[0057] In one embodiment of the therapeutic method of the invention, the organism is afflicted with a disease selected from the group consisting of cancer, an autoimmune disease, and a viral infection.

[0058] This approach has the advantage that the invention can be used specifically for the treatment of those diseases for which cell therapy is particularly suitable.

[0059] The features mentioned above and those to be described below can be used not only in the combinations shown in the respective embodiments, but also in other combinations or alone without departing from the scope of the present invention. [Brief description of the drawings]

[0060] The present invention will now be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Figure 1] 1 shows the regulation of CAR T cell proliferation in a specific time frame. [Diagram 2] Showing inhibition of early proliferation sensitivity. [Diagram 3] Delayed inhibition of proliferation sensitivity is shown. [Figure 4] Figure 2 shows that silencing persisted in a dose-dependent manner in rapidly dividing T lymphoblasts. [Diagram 5] It is shown that silencing persisted for at least 13 days in activated T cells. [Figure 6] 1 shows increased inhibition of the luciferase transgene in Jurkat T lymphoblasts. EXAMPLES

[0061] 1. Overview CAR T Cells – The Need to Speed ​​Up Chimeric antigen receptors (CARs) allow engineered T cells to eliminate cells that present the antigen targeted by the CAR. Autologous CAR T cells have been successfully used to treat B cell malignancies, with four products targeted to CD19 and one targeted to a B cell maturation antigen approved. Several other CAR T cell products are in development, with over 100 clinical trials currently underway for hematological malignancies and solid tumors. CAR T cells can also be used to treat autoimmune diseases and viral infections.

[0062] Despite the initial success of CAR T cells, several issues have hindered their widespread clinical application. CAR T cell therapy is often associated with primary resistance in 10-20% of patients, with relapse occurring at a rate of 30-50%. Mechanisms of resistance observed in infused T cells include poor in vivo proliferation, early exhaustion, low persistence, and poor compatibility. The tumor itself may also contribute to resistance in the form of loss, downregulation, or mutation of target antigens. In addition, CAR T cells may exhibit toxicity in the form of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). ICANS can develop in a range of severity from mild to severe, with a mortality rate of 1.5%. Prolonged and / or severe neutropenia and fatal infections due to B-cell aplasia may also occur.

[0063] To reduce T cell-mediated resistance and toxicity and expand the range of targetable diseases, several T cell modulation strategies are being explored based on the current knowledge that certain CAR T cell clones are more effective than others.

[0064] Currently approved CAR T cells are heterogeneous clonal populations, and the efficacy of each clone varies. This heterogeneity is due to (1) accidental genomic integration during production using lentiviral vectors; 11 and (2) due to the phenotypic heterogeneity of apheresis products.

[0065] Accidental integration by lentivirus can be avoided by precisely inserting the CAR construct into the genome using CRISPR or by using mRNA to transiently express the CAR.

[0066] Effective CAR T cell clones are capable of proliferation, eliminating target cells, and persisting in vivo for months to up to a year without exhaustion. On the other hand, overly aggressive CAR T cell clones that do not become exhausted and show excessive proliferation cause toxicity. In light of this, we propose a therapeutic window where non-toxic and effective CAR T cell clones can reach and persist within, but not exceed, an upper limit (Figure 1).

[0067] To ensure that this therapeutic window is maintained, the timing of the modulation strategy is crucial: early on, proliferation and tissue homing must be accelerated and exhaustion inhibited to reach the therapeutic window, while later on, proliferation must be slowed to flatten the growth curve and prevent excessive activation (Figure 1). Ideally, therefore, the modulation strategy for CAR T cells should be sensitive to this proliferation phase.

[0068] RNA-treated CAR T cells Regulatory RNAs can program phenotypes by fine-tuning the transcription, splicing, translation, and life span of mRNAs, therefore RNA therapeutics can be used for rational phenotypic engineering, i.e., rational phenotypic engineering of CAR T cells.

[0069] RNA therapeutics include various sequence-specific inhibitors that are reversible and have tunable expression levels, and the inhibitory effect may be dependent on cell proliferation.

[0070] RNA interference involves pairing of short double-stranded RNA (miRNA or siRNA) to complementary mRNA to induce mRNA degradation. Antisense oligonucleotides (ASOs) regulate translation and splicing by cleaving pre-mRNA through various mechanisms of action. Four approved siRNAs and three approved ASOs have been demonstrated to be effective RNA therapeutics for clinically relevant phenotype modulation.

[0071] RNA therapeutics are being investigated for enhancing CAR T cells. Some studies have shown that RNA interference silencing or ASO-mediated silencing is available and beneficial in primary T cells and CAR T cells. siRNA can be efficiently delivered to T cells ex vivo using electroporation, lipid-modified siRNA, aptamer-modified siRNA, lipid nanoparticles or viral vectors (shRNA). Currently, three clinical trials are investigating the clinical utility of RNA-modified CAR T cells. When trying to modulate a complex system such as CAR T cells, it may be advantageous to combine shRNAs against multiple targets.

[0072] Regulation of proliferation sensitivity We propose to combine RNA therapeutics with genetic engineering approaches to achieve proliferation-sensitive silencing in CAR T cells.

[0073] RNA therapeutics (e.g., siRNA, artificial miRNA, ASO, and anti-miR) are chemically synthesized oligonucleotides that are exogenously introduced into cells. On the other hand, similar therapeutic mechanisms can also be introduced into cells by utilizing genetic engineering methods. For example, siRNA and miRNA can be encoded into short hairpin (shRNA) and delivered by genome-integrated or episomal viruses. Furthermore, recombinant small nucleolar RNAs that target pre-mRNAs can be introduced by viral delivery to degrade ref and miRNA sponges or RNA decoys to antagonize the function of miRNAs.

[0074] The major difference between RNA therapeutics and genetic engineering approaches is the time course: the effects of RNA therapeutics are temporary, whereas the effects of genetic engineering approaches are permanent.

[0075] The duration of effect of an RNA therapeutic depends on (i) its metabolic stability and (ii) the proliferation rate of the cells. Extensive chemical modifications of the RNA therapeutic can enhance the metabolic stability of the RNA therapeutic in non-dividing liver cells of patients, and extend the duration of its effect up to 6 months. However, the intracellular concentration of the RNA therapeutic is halved with each cell division, and in rapidly dividing cells, the duration of its effect is almost unknown. In the case of siRNA, 50-3,000 times the amount of siRNA required for silencing may be delivered to the cells, resulting in a dilution effect expected after 5-12 cell divisions. Indeed, the inventors observed that silencing persisted after 4-30 cell divisions in primary immune cells and immune cell lines.

[0076] By combining RNA therapeutics with genetic engineering methods, three levels of sensitivity to cell proliferation can be established: (1) Proliferation insensitive: Genetic engineering tools (i.e. shRNA, snRNA, miRNA sponges) may be expressed from genomic integrated sequences or episomes. Both chromosomal and extrachromosomal episomes are replicated during mitosis, with no change in copy number in daughter cells. Thus, expression of the genetic engineering tool depends only on the promoter used and remains insensitive to cell proliferation. (2) Inhibition of proliferation sensitivity: RNA therapeutics (i.e., siRNA, artificial miRNA, ASO, anti-miR) enter cells at a specific concentration and are diluted with each cell division. The duration of the effect can be set by adjusting the initial concentration used (Figure 2). (3) Expression of proliferation sensitivity: A combination of RNA therapeutics and genetic engineering tools that have antagonistic effects on each other (i.e., a combination of anti-miR and shRNA, a combination of miRNA and miRNA sponge, or a combination of siRNA and mRNA) is delivered to cells. The RNA therapeutic blocks the effect of the genetic engineering tool. As the RNA therapeutic is diluted, the genetic engineering tool is released from the state blocked by the RNA therapeutic and begins to exert its effect, i.e., expressing a protein or downregulating the target (Figure 3).

[0077] Specific interventions in selected time frames Toxicity Control Although toxicity and antitumor activity are usually observed mechanisms of overlap, the timing of T cell modulating methods may allow for differentiation between toxicity and antitumor activity.

[0078] IL-1 and IL-6 are ideal targets for delayed inhibition of proliferation sensitivity. IL-1 / IL-6 inhibition is a common toxicity management strategy, but it also impairs the therapeutic activity of T cells. However, delayed inhibition of IL-1 / IL-6 still allows the production of chemokines in the proliferation of CAR T cells required for therapy, and only the excessive effects are inhibited by delayed inhibition of IL-1 / IL-6. Delayed inhibition of the IL-1 / IL-6 axis can be achieved by (1) delayed expression of IL-1 / IL-6 inhibitors, (2) delayed silencing of GM-CSF (responsible for humoral activation of IL-1 / IL-6 producing macrophages), or (3) delayed silencing of CD40L (responsible for contact-dependent activation of macrophages). 54 , CD69 55 , LAG3 56 This is believed to be achievable through delayed silencing of the gene.

[0079] Reducing the expression of the CAR construct or reducing the number of CAR T cells would reduce toxicity but not affect therapeutic activity. The expression of the CAR could be controlled by precise integration into the gene site or by pre- or post-treatment with RNA therapeutics. Delayed silencing of the CAR construct would allow specific inhibition of excessive activation.

[0080] Blocking proliferation sensitivity may be superior to engineered on / off switches to control toxicity (e.g., those using lenalidomide, asunaprevir, or rimiducide, which have been successfully used in CAR T cells in preclinical trials) because blockade of proliferation sensitivity can prevent toxicity, whereas engineered on / off switches can only shorten the duration of toxicity.

[0081] Phenotype optimization Successful T cell therapy exhibits a phenotype that exerts strong antitumor activity and limited toxicity. RNA-based modifications promote cell differentiation while enriching for phenotypes that are likely to be therapeutically successful at an early stage, but then exhaust the cells at a delayed stage, allowing for both effective antitumor function and a healthy life. The lower degree of differentiation of T cells, such as memory phenotype, correlates with better antitumor efficacy. However, the memory phenotype is scarce, making it difficult to rely on apheresis as a source of memory T cells for CAR T cell manufacturing. With this in mind, several methods, including small molecule inhibitors and interleukins, have been explored to induce the memory phenotype. This concept has been advanced to the clinical trial stage (NCT02652910, NCT01087294). However, some of these treatments were found to limit the differentiation ability of memory T cells into effector T cells, compromising their antitumor efficacy. Therefore, one should first enrich for the memory phenotype (ex vivo production) before differentiating the cells (in the patient) towards the effector phenotype.

[0082] Memory T cells express TCF1, BCL-6, BMI1, FOXO1, KLF2, LEF1, TCF7, IL2RA, CD27, TNF, CCR7, SELL, and CD62L. First, exogenous expression of these genes may reprogram cells to a memory phenotype. However, loss of expression (delayed inhibition) may occur, similar to natural cell differentiation. Inhibition of BRD4 and p300, TET2, miR-15 / 16, or miR-150 induced the memory phenotype and improved antitumor activity. Moreover, overexpression of miR-143 or downregulation of GLUT1, a target of miR-143, with siRNA promoted memory T cell formation. These interventions may be timed to ex vivo production (early inhibition) to improve antitumor activity.

[0083] The effector T cell phenotype is characterized by the expression of TOX, Blimp-1, NR4A, BATF, IRF4, NFAT, KLRG1, EOMES, TBX2, PRDM1, GZMA, HZMB, PRF1, and IFNG, and may express exhaustion markers PD1, TIM3, LAG3, TIGIT, and CTLA4 by miR-146a. By inhibiting these genes at an early stage, it may be possible to eliminate effector cells from the CAR T cell product and prevent early exhaustion. However, re-expression of effector genes should be possible for spontaneous differentiation of the cells and achievement of antitumor effects (i.e., only inhibiting at the early stage). Effector T cell persistence is promoted by miR-17-92 or miR-155. Ideally, these miR genes should be expressed in parallel with the differentiation of T cells into the effector phenotype with a delayed expression.

[0084] Whether the above genes are correlated with or induce only memory or effector phenotypes remains to be elucidated. Genes that induce these phenotypes would be ideal targets for pre-programming RNA in CAR T cells. Targets based on science, not mechanism of action, may be found through RNAi or CRISPRi screening of CAR T cells and analysis of integration sites of approved CAR T cell products.

[0085] Enhancement of CAR T cells in solid tumors Suboptimal activity of CAR T cells targeting solid tumors may be enhanced by precisely timing gene expression that can promote invasion, resist the microenvironment, and recognize antigens.

[0086] infiltration Invasion and proliferation could be enhanced by inhibiting inhibitory receptors on CAR T cells, such as PD1, TIM3, LAG3, and CX3CR1. In fact, the concept of combining shRNA-mediated silencing of these factors has already entered the clinical trial stage. Overexpression of miRNAs targeting such inhibitory receptors has proven beneficial. If we could inhibit these factors at an early stage and suppress proliferation in a delayed manner, safety could be enhanced.

[0087] CAR T cells engineered to co-express the combination of IL-7 and CCL19 or the combination of IL-7 and CCL21, which are important chemokines for establishing T cell areas in lymphoid organs, show better infiltration and higher activity against solid tumors than conventional CAR T cells (clinical trial number: NCT03778346). However, overexpression of IL-7, CCL19 and CCL21 may be carcinogenic. If we can inhibit the above inhibitory factors at an early stage and suppress proliferation in a delayed manner, we can enhance safety.

[0088] The receptor that recognizes chemokines abundant in solid tumors (CCR2 62 , CCR5 63 Overexpression of CCR5 and inhibition of their inhibitory factors appears to be promising ways to promote invasion. On the other hand, CCR5 can induce non-classical apoptotic pathways in tumors that kill (CAR) T cells. Therefore, CCR5 is an ideal candidate for delayed silencing.

[0089] microenvironment Some genetic engineering methods aim to overcome the tumor microenvironment. Tumor vasculature may be targeted via CAR. CAR T cells may express matrix degrading enzymes. Immunosuppressive soluble receptors may be inhibited via dominant negative knock-in, shRNA or CRISPR. Alternatively, immunosuppressive soluble factors may be targeted via CAR T cells. In addition, immunosuppressive cells such as Tregs, tumor associated macrophages, bone marrow derived immunosuppressive cells, and cancer associated fibroblasts may be targeted via CAR T cells.

[0090] In general, tumor microenvironment degradation is ideally a localized response, and therefore CAR T cells should bind to the tumor before exerting their inhibitory or degradative activity on the tumor microenvironment, so targeting of the tumor microenvironment should be delayed.

[0091] Armed CAR T cells CAR T cells may be "armed" to produce proinflammatory cytokines that are normally expressed by other cell types. CAR T cells expressing IL-12, IL-18, IL-15, IL-7, TGFβ or IL-23 show higher efficacy against solid tumors. However, constitutive expression of these cytokines may show strong toxicity. Therefore, cytokine production should occur only when CAR T cells bind to the tumor and proliferate locally. Early inhibition and delayed expression may achieve such goals and improve the safety of armed CAR T cells.

[0092] Overexpression of miR-155 has also been shown to enhance T cell activity against solid tumors. The artificial miRNAs described herein may be able to prevent toxicity due to overactivation of T cells.

[0093] Antigen Recognition Targeting cancer stem cells has been explored as a means to prevent recurrence and overcome the heterogeneity of tumor-associated antigens. However, cancer stem cell markers are often not tumor specific and cause severe on-target and off-tumor toxicity. CD133, the most commonly used cancer stem cell marker, is also expressed in hematopoietic stem cells, human embryonic stem cells, and epithelial cells of some tissues. Efficacy and safety may be improved by expressing a CAR specific for a tumor-associated marker at an early stage and then expressing a CAR specific for cancer stem cells at a delayed stage.

[0094] Allogeneic CAR T cells The development of "off-the-shelf" allogeneic CAR T cells would be a game changer in the field by shortening manufacturing times and improving early-stage T cell compatibility, but allogeneic T cells are often rejected via the host immune system and can induce graft-versus-host disease.

[0095] A genetic engineering approach that can overcome allorejection is knockout of MHC I (β2-microglobulin locus). However, MHC-deficient CAR T cells are vulnerable to NK cell-mediated destruction. This NK cell-mediated “missing self” response may be avoided by expressing receptors that inhibit NK cells, such as HLA-E, HLA-G, and Siglec7 / 9. Class II MHC molecules may be knocked out by targeting CIITA. A promising approach is to target the 4-1BB receptor on activated alloimmune lymphocytes by expressing protective receptors on allogeneic CAR T cells. However, completely blocking alloimmunity in allogeneic T cells eliminates important growth controls and may lead to excessive activation. Therefore, methods to eliminate allorejection may be limited to the early proliferation phase only to improve safety.

[0096] It is possible to avoid GvHD induced by allogeneic CAR T cells by deleting the T cell receptor. One method of deleting the T cell receptor is to knock out the constant region of the α chain of the T cell receptor. Alternatively, silencing the TCR with shRNA has been successful in adoptive T cell therapy. Yet another method is to replace the TCR with a CAR construct by CRISPR. However, the T cell receptor is thought to be involved in weak "tonic" signaling, which is important for T cell maintenance and effector function. Therefore, the T cell receptor is a promising target for delayed silencing.

[0097] 2. Materials and Methods siRNA Each siRNA was modified with 2'-fluoro, 2'-OMe, and phosphorothioate as previously described. The lipid-modified siRNA contained cholesterol and divalent myristic acid and was synthesized as previously described.

[0098] cell culture Jurkat cells were passaged twice weekly and cultured at a density of 1 million cells / ml in RPMI medium supplemented with 10% FBS and 1% penicillin / streptomycin.

[0099] Jurkat cells expressing NanoLuc were obtained from Dr. Christian Seitz, University of Tübingen, and cultured similarly to the method described above.

[0100] Generation of activated T cells Buffy coats of healthy blood donors were obtained from the Blood Donation Centre of the University Hospital Tübingen. Peripheral blood mononuclear cells were obtained from the buffy coats by Ficoll density gradient centrifugation. Cells were seeded at a density of 1.5 million cells / well in 24-well plates in 1 ml of RPMI medium supplemented with 10% FBS, 1% penicillin / streptomycin and 25 mM HEPES. MACSiBeads were added at a cell to bead ratio of 2:1 (Miltenyi Human T Cell Activation / Proliferation Kit). After incubating the cells with beads for 3 days, the beads were removed by centrifugation and the activated cells were seeded at a density of 1.5 million cells / ml. IL-7 and IL-15 were then added to the medium.

[0101] Treatment with siRNA Each siRNA was added to the cells at the final concentration (0.5 μM, 1 μM, 2 μM or 4 μM) indicated in each graph. The cells were cultured as described above. At each passage, the cells were counted, a portion of the cells was collected for the next mRNA quantification assay, and the remaining cells were subcultured at the density described above. This procedure was repeated at each passage.

[0102] Quantification of mRNA Quantification of mRNA was performed using the QuantiGene Singleplex assay (Invitrogen) according to the manufacturer's protocol.

[0103] Luminescence measurement Jurkat cells stably expressing Nanoluc were seeded in 96-well plates at a density of 1 million cells / ml in phenol red-free RPMI medium supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were treated with various concentrations of siRNA targeting NanoLuc or a non-targeting control. On day 3, furimazine (ChemShuttle) was added to the cells at a concentration of 1 μM and luminescence was measured using a Tecan MPlex plate reader. The cells were then returned to the incubator. On day 7, the same procedure was repeated, except that before returning to the incubator, the cells were passaged and reseeded at a density of 1 million cells / ml. The following day, the same procedure was repeated as shown in the graph.

[0104] 3.Results To demonstrate inhibition of proliferation sensitivity, Jurkat cells (a human T lymphoblastoid cell line) or primary human T cells were treated with siRNA targeting PPIB or JAK1, or a non-targeting control (NTC). Cells were passaged in the presence of each siRNA and continued in culture for 30 days. Samples were taken at each passage and mRNA levels were quantified.

[0105] Jurkat T lymphoblasts were treated with various concentrations of cholesterol-modified siRNA targeting JAK1, bivalent myristic acid-modified siRNA targeting PPIB, or cholesterol-modified siRNA targeting PPIB. Cells were passaged twice a week at a ratio of 1:3. At each passage, samples were taken, cells were lysed, and mRNA expression was analyzed using the QuantiGene assay. Target mRNA expression levels were normalized to a housekeeping gene (HPRT) and to untreated controls. The results of this experiment are shown in Figure 4. Sustained silencing (at least 50%) was observed for 14 to 30 days, depending on the target mRNA, the initial dose of siRNA, and the type of lipid modification on the siRNA.

[0106] A similar inhibition of proliferation sensitivity was observed in primary human T cells. Activated T cells prepared from PBMCs were treated with siRNA targeting PPIB or JAK1 and passaged in the presence of IL-7 and IL-15 without removing the siRNA from the cultures.

[0107] Activated T cells were treated with bivalent myristic acid-modified siRNA targeting JAK1 or PPIB or cholesterol-modified siRNA targeting JAK1 or PPIB at 2 μM each. Cells were passaged at every doubling. Samples were taken at every passage, cells were lysed and mRNA expression was analyzed using QuantiGene assay according to the manufacturer's protocol. Expression levels of target mRNA (PPIB) were normalized to a housekeeping gene (HPRT) and untreated control. The results are shown in Figure 5. Highly efficient silencing was observed, which lasted for at least 13 days.

[0108] To demonstrate expression of proliferation sensitivity, Jurkat T lymphoblasts stably expressing the NanoLuc transgene were treated with various concentrations of siRNA targeting NanoLuc and luminescence was measured twice weekly.

[0109] Jurkat cells stably expressing NanoLuc were treated with cholesterol-modified siRNA targeting NanoLuc. The cells were passaged twice a week, and luminescence was measured at each passage. The results of this experiment are shown in Figure 6. It was observed that luciferase, which increased with cell proliferation, was inhibited.

Claims

1. 1. A biological cell modified with a nucleic acid, comprising: (i) a nucleic acid comprising a first expressible nucleotide sequence encoding a target; (ii) a recombinant nucleic acid comprising a second expressible nucleotide sequence configured to regulate the expression and / or function of the first expressible nucleotide sequence. in its genome, a nucleic acid outside the genome that includes a third nucleotide sequence configured to inhibit expression and / or function of the second expressible nucleotide sequence; Living cells.

2. A nucleic acid modified biological cell, wherein a second expressible nucleotide sequence is configured to inhibit expression of a first expressible nucleotide sequence.

3. 3. A biological cell modified with the nucleic acid of claim 1 or 2, wherein the second expressible nucleotide sequence is integrated into a chromosome of the biological cell.

4. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the second expressible nucleotide sequence is integrated into an episome of said biological cell.

5. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprising the third nucleotide sequence is an oligonucleotide.

6. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the second expressible nucleotide sequence and the third nucleotide sequence encode a regulatory RNA.

7. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the second expressible nucleotide sequence encodes an RNA selected from the group consisting of shRNA (small hairpin RNA), snRNA (small nuclear RNA), miRNA (microRNA), miRNA sponge, piRNA, lncRNA (long non-coding RNA), guide RNA, mRNA (messenger RNA), antisense transcripts and transposons.

8. A biological cell modified with the nucleic acid of any one of the preceding claims, wherein the third nucleotide sequence encodes an RNA selected from the group consisting of siRNA (small interfering RNA), ASO (antisense oligonucleotide), miRNA (microRNA), AMO (anti-miRNA), and aptamer.

9. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the biological cell is an immune cell.

10. 2. The nucleic acid modified biological cell of any one of the preceding claims, wherein said immune cells are selected from the group consisting of T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocytes for infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, tumor infiltrating macrophages, hematopoietic stem cells, and the like.

11. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the target encoded by the first expressible nucleotide sequence is a protein.

12. 2. A biological cell modified with a nucleic acid according to any one of the preceding claims, wherein the first expressible nucleotide sequence encoding the target is a gene selected from the group consisting of BRD4, p300, TET2, DNMT3, miR-15 / 16, miR-150, GLUT1, TOX, Blimp-1, NR4A, BATF, IRF4, NFAT, KLRG1, EOMES, TBX2, PRDM1, GZMA, HZMB, PRF1, IFNG, PD1, TlM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, and Siglec7 / 9.

13. 10. The nucleic acid modified biological cell of any one of the preceding claims, wherein the first expressible nucleotide sequence encoding a target is a gene selected from the group consisting of IL-1, IL-6, GM-CSF, CD40L, LAG3, CD69, TCF1, BCL-6, BMI1, FOXO1, KLF2, LEF1, TCF7, IL2RA, CD27, TNF, CCR7, SELL, CD62L, miR-143, CCR5, CCR2, Iet7, TGFβ, IL-12, IL-18, IL-23, T cell receptor, miR-27a, IL-7, CCL19, CCL21, a CAR construct targeting the tumor microenvironment, IL-15, IL-7, and a CAR construct targeting a cancer stem cell marker.

14. 10. A biological cell modified with a nucleic acid according to any one of the preceding claims for use in medicine, preferably for use in the treatment and / or prevention of a disease selected from the group consisting of cancer, autoimmune diseases, alloimmunity (rejection or graft versus host disease), viral infections and bacterial infections.

15. 1. A method for modulating expression of a first nucleotide sequence encoding a target in a nucleic acid modified cell, comprising: 1) providing a biological cell having a first expressible nucleotide sequence encoding a target protein integrated into its genome; 2) integrating into the genome of said biological cell a recombinant nucleic acid comprising a second expressible nucleotide sequence configured to regulate the expression and / or function of the first expressible nucleotide sequence; and 3) introducing into said biological cell from an exogenous nucleic acid comprising a third nucleotide sequence configured to inhibit expression and / or function of a second expressible nucleotide sequence. The method includes:

16. 16. The method of claim 15, wherein the second expressible nucleotide sequence is configured to inhibit expression of the first expressible nucleotide sequence.

17. The method of claim 15 or 16, wherein in step (2), the second expressible nucleotide sequence is integrated into a chromosome of the biological cell.

18. 2. The method of any one of the preceding claims, wherein in step (2), the second expressible nucleotide sequence is integrated into an episome of the biological cell.

19. 2. The method of any one of the preceding claims, wherein the nucleic acid comprising the third nucleotide sequence in step (3) is an oligonucleotide.

20. 10. The method of any one of the preceding claims, wherein the second expressible nucleotide sequence and the third nucleotide sequence encode a regulatory RNA.

21. 2. The method of any one of the preceding claims, wherein the second expressible nucleotide sequence encodes an RNA selected from the group consisting of shRNA (small hairpin RNA), snRNA (small nuclear RNA), miRNA (microRNA), miRNA sponge, piRNA, lncRNA (long non-coding RNA), guide RNA, and mRNA (messenger RNA).

22. 2. The method of any one of the preceding claims, wherein the third nucleotide sequence encodes an RNA selected from the group consisting of siRNA (small interfering RNA), ASO (antisense oligonucleotide), miRNA (microRNA), and AMO (anti-miRNA).

23. The method according to any one of the preceding claims, wherein in step (3), the nucleic acid comprising a third nucleotide sequence is introduced into the biological cell via a method selected from the group consisting of electroporation, delivery by lipid modification, delivery by aptamer modification, delivery by lipid nanoparticles, delivery by viral vectors, delivery by antibody modification, delivery by small molecule modification, delivery by peptide modification, and delivery by extracellular vesicles.

24. 2. The method of any one of the preceding claims, wherein in step (2), the recombinant nucleic acid comprising a second expressible nucleotide sequence is added as part of the genome by being introduced into the living cell via a genome-integrating virus and / or an episomal virus.

25. 2. The method of any one of the preceding claims, wherein the biological cell is an immune cell.

26. 2. The method of any one of the preceding claims, wherein the biological cells are selected from the group consisting of T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocytes for infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, and tumor infiltrating macrophages.

27. 2. The method of any one of the preceding claims, wherein the first expressible nucleotide sequence encoding a target protein is a gene selected from the group consisting of BRD4, p300, TET2, DNMT3, miR-15 / 16, miR-150, GLUT1, TOX, Blimp-1, NR4A, BATF, IRF4, NFAT, KLRG1, EOMES, TBX2, PRDM1, GZMA, HZMB, PRF1, IFNG, PD1, TlM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, and Siglec7 / 9.

28. 10. The method of any one of the preceding claims, wherein the first expressible nucleotide sequence encoding a target protein is a gene selected from the group consisting of IL-1, IL-6, GM-CSFm, CD40L, LAG3, CD69, TCF1, BCL-6, BMI1, FOXO1, KLF2, LEF1, TCF7, IL2RA, CD27, TNF, CCR7, SELL, CD62L, miR-143, CCR5, CCR2, Iet7, TGFβ, IL-12, IL-18, IL-23, T cell receptor, miR-27a, IL-7, CCL19, CCL21, a CAR construct targeting the tumor microenvironment, IL-15, and IL-7.

29. A method of treating an organism in need of treatment by administering a biological cell modified with a nucleic acid according to any one of claims 1 to 14.

30. 30. The method of claim 29, wherein the organism is afflicted with a disease selected from the group consisting of cancer, an autoimmune disease, and a viral infection.

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