Nucleic acid modified biological cells with amplification dependent gene expression

Abstract

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

Description

[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 nucleic acid modified cells. Technical Field

[0002] The present invention relates generally to the field of cell or molecular biology, and more particularly to the field of cell therapy. More specifically, the present invention relates to therapeutically effective biological cells produced by genetic engineering. Background Art

[0003] Cell therapy uses living cells that are injected, transplanted or implanted into a patient’s body to achieve the effects of a drug. For example, in immunotherapy, T cells that fight cancer cells through cell-mediated immunity or stem cells that regenerate diseased tissue are transplanted.

[0004] Cells carrying chimeric antigen receptors (CARs) are a core tool in cell therapy approaches. CARs enable engineered T cells to eliminate cells displaying the antigen targeted by the CAR. Autologous CAR T cells have been successfully used to treat B-cell malignancies, with four approved products targeting CD19 and one targeting the B-cell maturation antigen. Several other CAR T-cell products are in development, and more than 100 clinical trials are currently underway for hematologic malignancies and solid tumors. CAR T cells can also be used to combat autoimmune diseases and viral infections.

[0005] Despite the initial success of CAR T cells, several challenges continue to hinder their widespread clinical application. Primary resistance occurs in 10%-20%, and relapses are also common, with an incidence of 30%-50%. Mechanisms of resistance within the infused T cells include failure of in vivo expansion, early exhaustion, poor persistence, and insufficient adaptability. The tumor itself may also lead to resistance in the form of target antigen loss, downregulation, or mutation. CAR T cell-mediated toxicity in the form of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) ranges from mild to severe, resulting in a 1.5% mortality rate. Prolonged and / or severe neutropenia and B cell aplasia may lead to fatal infections.

[0006] Currently approved CAR T cells represent a heterogeneous population of clones with varying efficacy. This heterogeneity has been attributed to (1) lentiviral vector-based production leading to accidental genomic integration and (2) phenotypic heterogeneity of apheresis products.

[0007] Effective CAR T cell clones can proliferate, eliminate targeted cells, not become exhausted, and persist in the body for months to a year. However, hyperactive CAR T cell clones—those that proliferate too much and do not become exhausted—can cause toxicity.

[0008] Several T-cell modulation strategies are being tested to reduce T-cell-mediated resistance and toxicity and to expand the range of targetable diseases. These modulation strategies are based on the current understanding of why some CAR T-cell clones are more effective than others.

[0009] One of these regulation strategies involves the use of so-called small interfering RNA (siRNA). For example, the use of siRNA in cell therapy to regulate the activity of cells is disclosed in WO2015 / 084897. Alternatively, so-called small hairpin RNA (shRNA) is used for this purpose in cell therapy, as disclosed, for example, in WO2020 / 206248, WO2019 / 138354, WO2015 / 136001 or WO2011 / 059836.

[0010] However, with current technologies, the effectiveness of cell therapy is insufficient, and in particular, the problem of toxicity has not been satisfactorily addressed.

[0011] In this context, the object of the present invention is to provide a biological cell that can be used in the context of cell therapy without causing the problems described for current cell therapy, in particular toxicity. In particular, the present invention aims to provide such a biological cell that allows temporal control of the expression of a target gene or protein, such that expression occurs at the beginning of an amplification phase or late in an amplification phase. Summary of the invention

[0012] The object of the present invention is achieved by providing a nucleic acid-modified biological cell, the nucleic acid-modified biological cell comprising:

[0013] - Its genome contains

[0014] (i) a nucleic acid comprising a first expressible nucleotide sequence encoding a target,

[0015] (ii) a recombinant nucleic acid comprising a second expressible nucleotide sequence configured to modulate the expression and / or function of the first nucleotide sequence, and

[0016] - a nucleic acid not contained in its genome, said nucleic acid comprising a third nucleotide sequence configured to inhibit the expression and / or function of said second nucleotide sequence.

[0017] The object is also achieved by a method for regulating the expression of a first nucleotide sequence encoding a target in a nucleic acid-modified cell, the method comprising:

[0018] 1) providing a biological cell, wherein the biological cell comprises a first expressible nucleotide sequence encoding a target protein integrated into the genome of the biological cell,

[0019] 2) integrating a recombinant nucleic acid into the genome of the biological cell, wherein the recombinant nucleic acid comprises a second expressible nucleotide sequence configured to regulate the expression and / or function of the first nucleotide sequence,

[0020] 3) introducing a nucleic acid comprising a third nucleotide sequence outside the genome of the biological cell, wherein the third nucleotide sequence is configured to inhibit the expression and / or function of the second nucleotide sequence.

[0021] According to the present invention, "biological cells" include eukaryotic cells, such as animal cells (eg mammalian cells or human cells) and plant cells, and prokaryotic cells (eg bacterial cells).

[0022] According to the present invention, "nucleic acid modified" means that the cell and / or its nucleic acid (such as DNA or RNA) is modified by genetic engineering techniques (such as nucleic acid recombination), but it can also be modified by introducing artificial non-recombinant nucleic acid molecules into the cell. Therefore, it can also refer to "genetically modified" biological cells synonymously.

[0023] The biological cell according to the invention has a genome, i.e. heritable genetic information, usually in the form of DNA. All genetic information that is part of the genome of a biological cell is replicated and transferred to daughter cells during proliferation or cell division. Therefore, the genome and the genetic information contained therein are not lost by proliferation, i.e. they are "independent of proliferation". The genome of cells from higher organisms (such as eukaryotes) is usually formed by chromosomes. Extrachromosomal elements, such as episomes, can be introduced by biotechnological means and also constitute part of the cell genome.

[0024] According to the present invention, a "nucleotide sequence" is a nucleotide sequence of a nucleic acid, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It may be composed of natural and / or chemically modified nucleotides.

[0025] According to the present invention, the genome of the biological cell comprises a first nucleotide sequence that is expressible and encodes a target (e.g., a target protein or an untranslated target RNA or a non-protein target, respectively). In a preferred embodiment, the first protein is encoded by a gene represented by the first expressible nucleotide sequence.

[0026] According to the present invention, the genome of the biological cell comprises a second nucleotide sequence that has become part of the cell genome or integrated into the cell genome. The integration can be achieved by nucleic acid recombination or genetic engineering methods, respectively. In a preferred embodiment, the second nucleotide sequence has been integrated into the chromosome of the cell, or introduced into the cell on an extrachromosomal element (such as an episome).

[0027] "Recombinant DNA" refers to artificial DNA formed by laboratory methods of genetic recombination, such as molecular cloning, which brings together genetic material from multiple sources to produce sequences not found in the genome. DNA or genomes that occur in nature and result from natural recombination events are not called recombinant DNA.

[0028] "Expressible" means that the first and second nucleotide sequences can be read by the cell structure, preferably mediated by an upstream promoter, and can be converted into nucleic acid transcripts (such as mRNA), and in a preferred embodiment, can be translated into a target protein.

[0029] Cell according to the present invention also comprises the 3rd nucleotide sequence that is not a part of genome.By method well known to those skilled in the art, for example conjugate-mediated delivery, electroporation or lipid nanoparticles are introduced into cells.Because the 3rd nucleotide sequence is not a part of genome or is not integrated into the genome, the 3rd nucleotide sequence will not be replicated during cell proliferation, nor will it be passed to daughter cells.Therefore, the 3rd nucleotide sequence is " diluted " by the cell division event in daughter cells, and is lost during proliferation.Therefore, the 3rd nucleotide sequence is " proliferation-dependent ".

[0030] The third nucleotide sequence has a structure capable of inhibiting expression of the second nucleotide sequence, for example, in embodiments, by steric interaction (e.g., by annealing of complementary sequence fragments) or enzymatic cleavage (chaptalized) interaction between the third sequence and the second sequence, preventing the latter from being read and expressed.

[0031] The inventors have recognized that in the modified biological cells according to the present invention, the expression of the first nucleotide sequence or target can be controlled so that it is expressed over time at the beginning of the proliferation or amplification phase (e.g., at the beginning of cell therapy) or later in the proliferation / amplification phase or at the end of the proliferation / amplification phase. As part of the genome, the first nucleotide sequence and the second nucleotide sequence are replicated during mitosis, and the number of copies in the daughter cells remains unchanged. Their expression remains insensitive to cell proliferation. The third nucleotide sequence is present in the biological cell at a certain concentration and is diluted with each cell division. The duration of the effect can be set by adjusting the initial concentration used. A combination of a second nucleotide sequence and a third nucleotide sequence with opposite effects is introduced into the cell. As the third nucleotide sequence is diluted during the proliferation process, the second nucleotide sequence will be released from its blockade and begin to exert its effect, such as expressing a protein or downregulating a target.

[0032] Therefore, the cells according to the present invention create optimal conditions for use in cell therapy. By utilizing the dilution effect acting on the third nucleotide sequence, it is possible to achieve a target, such as a specific cell gene, at the beginning of the therapeutic amplification burst, before the active induction of proliferation, in the cells according to the present invention, specifically expressed, and thereafter inhibited, or at the end of the therapeutic amplification burst, after the active induction of proliferation, specifically expressed, and inhibited before this. This timely control and / or limited expression of the target significantly reduces the toxicity and side effects often observed in the current state of the art of cell therapy, such as cytokine release syndrome in CAR-T cells, or graft-versus-host disease in donor lymphocyte infusion. It can also enhance treatment-related proliferation in a safe manner, or enrich treatment-beneficial phenotypes in a specific time window of the amplification burst, thereby enhancing the efficacy of cell therapy.

[0033] The biological cells according to the present invention allow the use of effective, non-toxic cell clones that can reach and persist, never exceeding the upper limit of the cell therapy window. The inventors have found that the timing of the regulation strategy is crucial to ensure that the therapeutic window is maintained. In an embodiment of the present invention, proliferation and tissue penetration should be accelerated and exhaustion should be suppressed in the early stage to enable the therapeutic window to be reached. Proliferation should be slowed down in the later stage to flatten the curve and prevent overactivation. Therefore, ideally, the cell regulation strategy should be sensitive to the expansion stage. The present invention meets these and other needs.

[0034] In an embodiment of the present invention, the second expressible nucleotide sequence is configured to inhibit the expression of the first nucleotide sequence.

[0035] This measure creates constructive conditions for enabling strong expression of the target at the beginning of the proliferation phase and for inhibiting such expression of the target during the proliferation process. Thus, the second nucleotide sequence itself is inhibited by the third nucleotide sequence at the beginning. After several replication cycles, the third nucleotide sequence is gradually diluted and loses its inhibitory effect on the second nucleotide sequence. Then, the second nucleotide sequence can increasingly exert its inhibitory effect on the target, so that after several replication cycles and cell divisions, the target can be completely inhibited.

[0036] In another embodiment of the biological cell according to the invention, the second expressible nucleotide sequence is integrated into the chromosome and / or episome of the biological cell.

[0037] This measure ensures that the second nucleotide sequence is or becomes part of the genome of the biological cell and is passed on to daughter cells at each cell division. Episomes are plasmids that form extrachromosomal elements that remain part of the eukaryotic genome without integration. Episomes control this by replicating with the rest of the genome and subsequently associating with metaphase chromosomes during mitosis. Unlike standard plasmids, episomes are not degraded and are not epigenetically silenced within the eukaryotic nucleus.

[0038] In yet another embodiment of the present invention, the nucleic acid comprising the third nucleotide sequence is an oligonucleotide.

[0039] This measure creates constructive conditions for the temporary presence of the third nucleotide sequence in the cell. In the present invention, an oligonucleotide is understood to be an oligomer constructed of several nucleotides (DNA or RNA), typically 7 to 30 nucleotides, sometimes 7 to 10 nucleotides. It should be understood that the specific number of nucleotides may vary depending on the application. In addition, the oligonucleotide may include natural nucleotides and / or chemically modified nucleotides. In the present invention, the stability of the oligonucleotide can be additionally affected by the type of chemical modification, thereby affecting the duration of dilution or "thinning", especially when RNA is used. Generally, stability is regulated by the modification of uridine nucleotides and cytidine nucleotides. Examples of chemically modified uridines include pseudouridine (Ψ), 2-thiouridine, 5-methyluridine, 5-iodouridine (idouridine), 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. Chemical modifications also include modifications that affect the sugar and phosphate backbones, such as 2'-fluoro, 2'-O-methyl, 2-methoxyethyl, LNA, phosphorothioate, vinylphosphonate, and the like.

[0040] In another embodiment of the present invention, the second expressible nucleotide sequence and the third nucleotide sequence encode regulatory RNA.

[0041] This measure advantageously enables the type of oligonucleotides that can be particularly well and easily designed as expression inhibitors of the second nucleotide sequence.Regulatory RNAs are a group of RNA molecules that do not encode proteins but can perform a variety of cellular functions, which include inhibition of the expression and / or function of another nucleotide sequence.

[0042] In yet another embodiment of the present 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, transposon.

[0043] These measures advantageously provide the second expressible nucleotide sequence in a form such that it can be integrated into the genome of a cell of the organism and, when expressed, regulates the expression of the first nucleotide sequence, for example by RNA interference.

[0044] In a further embodiment of the present invention, the third nucleotide sequence encodes an RNA selected from the group consisting of: siRNA (small interfering RNA), ASO (antisense oligonucleotide), miRNA (micro RNA), AMO (anti-miRNA), aptamer.

[0045] This measure has the advantage that such an oligonucleotide comprising a third nucleotide sequence is provided which allows efficient inhibition of the expression of the second nucleotide sequence, for example by hybridization, RNA interference, RNA silencing and similar mechanisms.

[0046] In another preferred embodiment of step (3) of the method according to the present invention, the nucleic acid comprising a third nucleotide sequence is introduced into the biological cell by a method selected from the group consisting of: electroporation, lipid conjugation delivery, aptamer conjugation delivery, lipid nanoparticle delivery, viral vector delivery, antibody conjugation delivery, small molecule conjugation delivery, peptide conjugation delivery, and extracellular vesicle delivery.

[0047] The advantage of this approach is that the oligonucleotides are introduced into biological cells using a proven and efficient method currently available in the art.

[0048] In yet another embodiment of step (2) of the method according to the invention, the recombinant nucleic acid comprising a second expressible nucleotide sequence is introduced into the biological cell by an integrating virus and / or an episomal virus, thereby adding the recombinant nucleic acid as part of the genome.

[0049] This embodiment uses mature recombinant biology methods to allow the second nucleotide sequence to be safely and reliably integrated into the genome of the biological cell.

[0050] In another embodiment of the present invention, the biological cell is an immune cell.

[0051] With this further development, such cells are nucleic acid-modified according to the invention and are particularly suitable as tools for cell therapy.

[0052] In yet another embodiment of the present invention, the biological cell or immune cell is selected from the group consisting of: T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocyte infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, tumor infiltrating macrophages, hematopoietic stem cells, including allogeneic cells listed above.

[0053] The above cells are particularly suitable and effective for cell therapy. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a modified receptor that is conferred or transplanted to an immune effector cell (eg, a human T cell) with specificity for an antigen. The chimeric antigen receptor comprises at least an extracellular ligand binding domain or portion, a transmembrane domain, and an intracellular domain comprising one or more signaling domains and / or co-stimulatory domains. Therefore, "CAR T cells" refer to T cells that have been genetically engineered to produce the artificial T cell receptor. These receptors are chimeric because they combine both antigen binding and T cell activation functions into a single receptor.

[0054] T cells can be genetically modified to express a CAR construct that enables T cells to recognize and target surface markers on so-called "cancer stem cells". In the prior art, this usually leads to the problem that these surface markers are not specifically expressed on cancer cells, so these "traditional" CAR T cells also recognize and target non-tumor cells. This means that targeting "cancer stem cells" will have serious side effects. These side effects severely limit the use of these CAR T cells. According to the inventor's discovery, these CAR T cells can be genetically modified to express another CAR construct in addition to the first CAR construct described, because CAR T cells can express a variety of CAR constructs. This second CAR construct can target genes that specifically recognize proteins that are specifically expressed on cancer cells as much as possible. CAR T cells can use this CAR construct to find tumors. This is where the present invention comes into play. The "first" CAR construct used by CAR T cells to identify the so-called "cancer stem cells" is downregulated using an oligonucleotide with a third nucleotide sequence at the beginning of the expansion phase. Therefore, CAR T cells only recognize tumor cells and prevent side effects. Once the CAR T cells recognize and amplify the tumor through the permanently expressed "second" CAR construct, the oligonucleotide will be diluted. As a result, the CART cells of the present invention located in the tumor thus proliferate, expressing the CAR constructs that can recognize and target "cancer stem cells". Therefore, the present invention makes it possible to fight "cancer stem cells" with almost no side effects.

[0055] In another embodiment of the present invention, the first expressible nucleotide sequence encoding the 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, TIM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, Siglec7 / 9.

[0056] The advantage of this approach is that it addresses such target genes that should preferably be downregulated during the early expansion phase of cells, especially CAR T cells.

[0057] In yet another embodiment of the present invention, the first expressible nucleotide sequence encoding the 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, CAR constructs targeting tumor microenvironment, IL-15, IL-7 and CAR constructs targeting cancer stem cell markers.

[0058] On the other hand, this approach has the advantage that those target genes that should preferably be downregulated during the late expansion phase of cells, in particular CAR T cells, are already addressed.

[0059] Another subject matter of the present invention relates to a nucleic acid-modified biological cell according to any 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, alloimmunization (rejection or graft-versus-host disease), viral infections, bacterial infections.

[0060] The features, properties and advantages disclosed for the cells or the regulation methods according to the invention apply likewise to the uses according to the invention.

[0061] Yet another subject matter of the present invention relates to a method for treating an organism in need thereof by administering a cell of the organism modified with a nucleic acid according to the invention.

[0062] The features, properties and advantages disclosed for the cells or the regulatory methods according to the invention apply equally to the methods of treatment according to the invention.

[0063] According to the present invention, "living being" relates to any animal or vegetable being, in particular mammals, including humans.

[0064] In one embodiment of the method of treatment according to the invention, the organism suffers from a disease selected from the group consisting of cancer, autoimmunity, viral infection.

[0065] This measure has the advantage that the invention can be used specifically for the treatment of those diseases which are particularly suitable for cell therapy.

[0066] It is understood that the features mentioned above and those yet to be mentioned below can be used not only in the combination specified in each case but also in other combinations or in isolation, without departing from the scope of the present invention.

[0067] The present invention will now be described and explained in more detail by reference to the following non-limiting examples and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Regulation of CAR T cell expansion within specific time windows.

[0069] Figure 2 Proliferation sensitivity inhibition—early stage.

[0070] Figure 3 Proliferation sensitivity inhibition - delayed.

[0071] Fig. 4 The duration of silencing is dose-dependent in rapidly dividing T lymphoblasts.

[0072] Figure 5 Silencing persisted for at least 13 days in activated T cells.

[0073] Figure 6 Increased repression of the luciferase transgene in Jurkat T lymphoblastoid cells. DETAILED DESCRIPTION

[0074] Example

[0075] 1. Overview

[0076] CAR T cells—the need for acceleration

[0077] Chimeric antigen receptors (CARs) enable engineered T cells to eliminate cells displaying the antigen targeted by the CAR. Autologous CAR T cells have been successfully used to treat B-cell malignancies, with four approved products targeting CD19 and one targeting the B-cell maturation antigen. Several other CAR T-cell products are in development, with more than 100 clinical trials currently underway for hematologic malignancies and solid tumors. CAR T cells may also be used to combat autoimmune diseases and viral infections.

[0078] Despite the initial success of CAR T cells, several challenges continue to hinder their widespread clinical application. Primary resistance occurs in 10%-20%, and relapses are also common, with an incidence of 30%-50%. Mechanisms of resistance within the infused T cells include failure of in vivo expansion, early exhaustion, poor persistence, and insufficient adaptability. The tumor itself may also lead to resistance in the form of target antigen loss, downregulation, or mutation. CAR T cell-mediated toxicity in the form of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) ranges from mild to severe, resulting in a 1.5% mortality rate. Prolonged and / or severe neutropenia and B cell aplasia may lead to fatal infections.

[0079] Several T-cell modulation strategies are being tested to reduce T-cell-mediated resistance and toxicity and to expand the range of targetable diseases. These modulation strategies are based on the current understanding of why some CAR T-cell clones are more effective than others.

[0080] Currently approved CAR T cells represent a heterogeneous population of clones with varying efficacy. This heterogeneity has been attributed to (1) lentiviral vector-based production leading to accidental genomic integration11 and (2) phenotypic heterogeneity of apheresis products.

[0081] Precise genomic insertion of the CAR construct via CRISPR or transient CAR expression via mRNA avoids the problem of accidental integration induced by lentiviruses.

[0082] Highly efficient CAR T cell clones are able to proliferate, eliminate targeted cells, not become exhausted, and persist in the body for months to a year. However, highly active CAR T cell clones that proliferate too much and do not become exhausted can cause toxicity. Therefore, the inventors propose that effective, non-toxic CAR T cell clones can reach and persist, never exceeding their upper therapeutic window ( Figure 1 ).

[0083] The timing of the regulatory strategy is critical to ensure that this therapeutic window is maintained. Proliferation and tissue penetration should be accelerated and exhaustion suppressed in the early stages to achieve the therapeutic window. Proliferation should be slowed in the later stages to flatten the curve and prevent overactivation ( Figure 1 ). Therefore, ideally, CAR T-cell modulation strategies should be sensitive to the expansion phase.

[0084] RNA-treated CAR T cells

[0085] Regulatory RNAs are able to program phenotypes by fine-tuning transcription, splicing, translation, and mRNA lifespan. Therefore, RNA therapy can be used for rational phenotypic design of CAR T cells.

[0086] RNA therapeutics include a variety of reversible and titratable sequence-specific inhibitors. Inhibition may depend on cell proliferation.

[0087] RNA interference directs short double-stranded RNA (miRNA or siRNA) against complementary mRNA to induce mRNA degradation. Antisense oligonucleotides (ASOs) show different mechanisms of action, cleaving pre-mRNA and regulating translation and splicing. Four approved siRNAs and three approved ASOs demonstrate the potential of RNA therapeutics to modulate clinically relevant phenotypes.

[0088] RNA therapy is being explored to enhance CAR T cells. Several studies have demonstrated the feasibility and utility of RNA interference or ASO-mediated silencing in primary T cells and CAR T cells. siRNA can be effectively delivered to T cells ex vivo using electroporation, lipid-conjugated siRNA, aptamer-conjugated siRNA, lipid nanoparticles, or viral vectors (shRNA). Three clinical trials are currently testing the clinical utility of RNA-modified CAR T cells. When modulating complex systems such as CAR T cells, a combination of shRNAs against multiple targets may be advantageous.

[0089] Proliferation-sensitive expression control

[0090] The inventors proposed a combination of RNA therapeutics and genetic engineering strategies to achieve proliferation-sensitive silencing in CAR T cells.

[0091] RNA therapeutics such as siRNA, artificial miRNA, ASOs, and anti-miRs are chemically synthesized oligonucleotides that are introduced exogenously into cells. However, genetic engineering can also be used to introduce the same therapeutic mechanisms into cells. For example, siRNA and miRNA can be encoded as short hairpins (shRNA) and delivered via integrating or episomal viruses. Viral delivery may also introduce engineered small nucleolar RNAs that target pre-mRNAs for degradation of ref and miRNA sponges or decoys to antagonize miRNA function.

[0092] One major difference between RNA therapeutics and genetic engineering is the timeline: the effects of RNA therapeutics are temporary, while the effects of genetic engineering are permanent.

[0093] The duration of action of RNA therapeutics depends on (i) their metabolic stability, and (ii) the rate of cell proliferation. Extensive chemical modifications have enhanced the metabolic stability of RNA therapeutics and extended their duration of action to 6 months in nondividing patient hepatocytes. However, the intracellular concentration of RNA therapeutics halves with each cell division, and the duration of action in rapidly dividing cells remains largely unknown. In the case of siRNA, 50 to 3000 times more siRNA can be delivered to cells than is required for silencing, so a dilution effect is expected after 5 to 12 cell divisions. Indeed, we have observed silencing durations of 4 to 30 divisions in primary immune cells and immune cell lines.

[0094] The combination of RNA therapeutics and genetic engineering strategies achieved three levels of sensitivity to cell proliferation:

[0095] (1) Insensitivity to proliferation: Genetic engineering tools (i.e., shRNA, snRNA, miRNA sponges) can be expressed from sequences integrated into the genome or from episomes. Both chromosomal and extrachromosomal episomes replicate during mitosis, and the copy number in daughter cells remains unchanged. Therefore, the expression of genetic engineering tools depends only on the promoter used and remains insensitive to cell proliferation.

[0096] (2) Proliferation-sensitive inhibition: RNA therapeutics (i.e., siRNA, artificial miRNA, ASO, anti-miR) enter cells at a certain concentration and are diluted with each cell division. The duration of action can be set by adjusting the initial concentration used ( Figure 2 ).

[0097] (3) Proliferation-sensitive expression: A combination of an RNA therapeutic and a genetic engineering tool with an opposing effect (i.e., anti-miR-shRNA, miRNA-miRNA sponge, siRNA-mRNA) is delivered to the cell. The RNA therapeutic blocks the action of the genetic engineering tool. As the RNA therapeutic is diluted, the genetic engineering tool is released from this blockade and begins to exert its effect—i.e., expressing a protein or downregulating its target ( Figure 3 ).

[0098] Specific interventions within the selected time window

[0099] Toxicity Control

[0100] The timing of T cell regulatory strategies may help distinguish between toxicity and antitumor activity, two mechanisms that otherwise overlap.

[0101] IL-1 and IL-6 are optimal targets for delayed proliferation-sensitive inhibition. Although IL-1 / IL-6 inhibition is a common toxicity control strategy, it also impairs the therapeutic activity of T cells. However, delayed inhibition of IL-1 / IL-6 allows chemokine production throughout the CAR T cell proliferation required for treatment and only inhibits the effect excessively. Delayed inhibition of the IL-1 / IL-6 axis may 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 CD40L54, CD6955, LAG356 (responsible for contact-dependent activation of macrophages).

[0102] Lower expression levels of CAR constructs or reduced numbers of CAR T cells may result in reduced toxicity but unchanged therapeutic activity. CAR expression levels can be controlled by precise genetic integration sites or by pre- or post-treatment treatments with RNA therapeutics. Delayed silencing of CAR constructs may be able to specifically inhibit overactivation.

[0103] Proliferation-sensitive inhibition may be superior to genetically engineered on- and off-switches (e.g., lenalidomide-, asunaprevir-, or rimiducid-gated switches that have been successfully used in CAR T cells in the preclinical setting) in terms of toxicity control because ProsIT prevents toxicity, whereas on / off switches only reduce toxicity that has already occurred.

[0104] Phenotypic optimization

[0105] Successful T cell therapies display a phenotype that enables potent antitumor activity with limited toxicity. RNA-based modifications may enable initial enrichment of this successful phenotype while allowing cells to differentiate and exhaust in a delayed manner to ensure effective antitumor function and a healthy lifespan. T cells with a less differentiated degree, such as the memory phenotype, are associated with better antitumor outcomes. The scarcity of such cells makes it difficult to rely on apheresis as a supply of memory T cells for CAR T cell production. Therefore, several strategies have been pursued to induce a memory phenotype, including small molecule inhibitors and interleukins—concepts that have entered clinical trials (NCT02652910, NCT01087294).

[0106] However, some of these therapeutic approaches have been found to limit the ability of memory T cells to differentiate into effector T cells, thereby impairing the antitumor effect.1 Therefore, after an initial enrichment of the memory phenotype (ex vivo production), the cells should be able to differentiate into the effector phenotype (in patients).

[0107] Memory T cells express TCF1, BCL-6, BMI1, FOXO1, KLF2, LEF1, TCF7, IL2RA, CD27, TNF, CCR7, SELL, CD62L. Initial exogenous expression of these genes may be able to reprogram cells to a memory phenotype. However, loss of expression (by delayed inhibition) should occur similar to natural cell differentiation. Inhibition of BRD4 and p300, TET2, miR-15 / 16, or miR-150 resulted in a memory phenotype and superior antitumor activity. Overexpression of miR-143 or downregulation of the miR-143 target GLUT1 by siRNA also promoted the formation of memory T cells. These interventions can be scheduled during the ex vivo production phase (early inhibition) to improve antitumor activity.

[0108] 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, IFNG, and may express the exhaustion markers PD1, TIM3, LAG3, TIGIT, CTLA4, and miR 146a. Early inhibition of these genes may be able to eliminate effector cells from the CAR T cell product and block early exhaustion. However, re-expression of effector genes should be allowed to allow natural cell differentiation and successful anti-tumor effects (early inhibition only). miR-17-92, miR-155 promote the persistence of effector T cells. Ideally, these genes should be expressed in a delayed manner, paralleling the differentiation of T cells to the effector phenotype.

[0109] Whether the above genes are only associated with or drive the memory / effector phenotype remains to be elucidated. Phenotype-driving genes would be ideal targets for RNA preprogramming of CAR T cells. RNAi or CRISPRi screening of CAR T cells and integration site analysis of approved CAR T cell products may yield evidence-based, less mechanism-based target identification.

[0110] CAR T cell enhancement in solid tumors

[0111] Infiltration promotion, microenvironmental antagonism, and precise timing of expression of antigen recognition genes may enhance the hitherto unsatisfactory activity of CAR T cells targeting solid tumors.

[0112] infiltration

[0113] Infiltration as well as proliferation can be enhanced by inhibiting inhibitory receptors on CAR T cells, such as PD1, TIM3, and LAG3 or CX3CR1. In fact, the combined silencing of these factors by shRNA is a concept that has entered the clinical trial stage. Overexpression of miRNA targeting the above receptors has also been shown to be beneficial. Early inhibition of these factors can improve safety by controlling proliferation in a delayed manner.

[0114] CAR T cells designed to co-express IL-7 and CCL19 or IL-7 and CL21 (chemokines that are important for establishing T cell zones in lymphoid organs) show better infiltration and higher anti-solid tumor activity than conventional CAR T cells (clinical trial NCT03778346). However, overexpression of IL-7, CCL19, and CCL21 may be oncogenic. Early inhibition of the above inhibitors may improve safety by controlling proliferation in a delayed manner.

[0115] Identification of receptors for highly abundant chemokines in solid tumors (CCR2 62, CCR5 63) or inhibition of their inhibitors appear to be promising strategies to promote infiltration. However, CCR5 may mediate non-classical apoptotic pathways within tumors, leading to (CAR) T cell death. Therefore, CCR5 is an ideal candidate for delayed silencing.

[0116] Microenvironment

[0117] Several genetic engineering strategies exist to overcome the tumor microenvironment. Tumor vasculature may be targeted via CAR. CAR T cells may express matrix-degrading enzymes. Immunosuppressive solute receptors may be inhibited via dominant-negative knock-in, shRNA, or CRISRP. Alternatively, immunosuppressive solutes may be targeted via CAR T cells. Immunosuppressive cell types, such as Tregs, tumor-associated macrophages, myeloid-derived suppressor cells, or cancer-associated fibroblasts, may also be targeted via CAR T cells.

[0118] In general, tumor microenvironment degradation should ideally be a local response, so once CAR T cells are docked with the tumor, they should ideally exert tumor microenvironment inhibitory or degradative activity. Therefore, tumor microenvironment targeting should occur in a delayed manner.

[0119] Armed CAR T cells

[0120] CAR T cells can 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, and IL-23 achieve higher efficacy against solid tumors. However, the constitutive expression of these cytokines can have severe toxicity. Therefore, cytokines should only be produced when CAR T cells dock with tumors and proliferate locally. Early inhibition / delayed expression can achieve this goal and enhance the safety of armed CAR T cells.

[0121] Overexpression of miR-155 has also been shown to enhance T cell activity against solid tumors. The artificial miRNAs described here may be able to prevent toxicity caused by T cell overactivation.

[0122] Antigen recognition

[0123] Cancer stem cell targeting has been explored as a strategy to prevent relapse and overcome tumor-associated antigen heterogeneity. However, cancer stem cell markers are often not tumor-specific, leading to severe on-target 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 various tissues. Delayed expression of cancer stem cell-specific CARs combined with early expression of tumor-associated marker-specific CARs may improve efficacy and safety.

[0124] Allogeneic CAR T cells

[0125] “Off-the-shelf” allogeneic CAR T cells could be a game changer in the field, shortening production length and improving initial T cell fitness. However, allogeneic T cells are often rejected by the host immune system or induce graft-versus-host disease.

[0126] Genetic engineering strategies to overcome allorejection include MHC I knockout (β2-microglobulin locus). However, MHC-deficient CAR T cells are susceptible to NK cell-mediated destruction. This NK-mediated “missing-self” response can be avoided by expressing NK inhibitory receptors such as HLA-E or HLA-G or Siglec7 / 9. Class II MHC molecules can also be knocked out by targeting CIITA. A promising approach is to target the 4-1BB receptor on activated allogeneic immune lymphocytes by expressing defense receptors on allogeneic CAR T cells. However, completely blocking alloimmunity in allogeneic T cells eliminates important proliferation controls and may lead to overactivation. Therefore, allogeneic rejection elimination strategies may be limited to the early proliferation stage to improve safety.

[0127] T cell receptor deficiency may circumvent allogeneic CAR T cell-induced GvHD. One strategy is to knock out the constant α chain of the T cell receptor. Alternatively, shRNA-mediated TCR silencing has been successfully used in adoptive T cell therapy. Another strategy is to exchange the TCR for a CAR construct via CRISPR. However, the T cell receptor is thought to be involved in low-level “tonic” signaling that is important for T cell maintenance and effector function. Therefore, the T cell receptor is a promising target for delayed silencing.

[0128] 2. Materials and Methods

[0129] siRNA

[0130] siRNAs were fully modified with 2'-fluoro, 2'-OMe, and phosphorothioate as described previously. Lipid conjugates included cholesterol and divalent myristic acid and were synthesized as described previously.

[0131] Cell culture

[0132] Jurkat cells were passaged twice a week and cultured at 1 million cells / ml in RPMI supplemented with 10% FBS and 1% P / S.

[0133] Jurkat cells expressing NanoLuc were obtained from Christian Seitz, University of Tübingen, and cultured as described above.

[0134] Generation of activated T cells

[0135] Buffy coats from healthy blood donors were obtained from the Blood Donation Center of the University Hospital of Tübingen. Peripheral blood mononuclear cells were obtained from the buffy coats by Ficoll density centrifugation. 1.5 million cells were plated per 24 well in 1 ml of RPMI supplemented with 10% FBS, 1% P / S and 25 mM HEPES. MACSiBeads (Miltenyi Human T Cell Activation / Expansion Kit) were added at a cell to bead ratio of 2:1. Cells were incubated with beads for 3 days, after which the beads were removed by centrifugation and the activated cells were plated at a density of 1.5 million / ml. The medium was now supplemented with IL-7 and IL-15.

[0136] siRNA treatment

[0137] siRNAs were added to cells at the final concentrations indicated in the figures (0.5, 1, 2, or 4 µM). Cells were cultured as described above. At each passage, cells were counted, harvested for downstream mRNA quantification, and the remaining cells were subcultured at the above density. This process was repeated for each passage.

[0138] mRNA quantification

[0139] mRNA was quantified using the QuantiGene Singleplex assay (Invitrogen) following the manufacturer's protocol.

[0140] Luminescence measurement

[0141] Jurkat cells stably expressing Nanoluc were plated at a density of 1 million cells / ml in phenol red-free RPMI supplemented with 10% FBS and 1% P / S in 96-well plates. Cells were treated with different concentrations of siRNA targeting NanoLuc or 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 transferred back to the incubator. On day 7, the same procedure was repeated. However, before returning to the incubator, the cells were passaged and replated at a density of 1 million cells / ml. The procedure was repeated on the following days as implied on the figures.

[0142] 3. Results

[0143] To demonstrate proliferation-sensitive inhibition, we treated Jurkat cells (a human T lymphoblastoid cell line) and primary human T cells with siRNA targeting PPIB and JAK1 as well as a non-targeting control (NTC). Cells were passaged in the presence of siRNA and maintained in culture for 30 days. At each passage, samples were taken to quantify mRNA levels.

[0144] Jurkat T lymphoblasts were treated with different concentrations of cholesterol-conjugated siRNA targeting JAK1, divalent myristic acid-conjugated siRNA targeting PPIB, or cholesterol-conjugated siRNA targeting PP1B. Cells were passaged at a ratio of 1:3, twice a week. Samples were collected at each passage, cells were lysed, and mRNA expression was analyzed using QuantiGene assay. Target mRNA expression was normalized to housekeeping genes (HPRT) and untreated controls. The results of this experiment are shown in Figure 4. Depending on the target mRNA, the initial siRNA dose, and the identity of the lipid conjugate on the siRNA, the inventors observed that the duration of silencing (at least 50%) was 14 to 30 days.

[0145] Inhibition of proliferation sensitivity can also be shown in human primary T cells. Activated T cells generated from PBMCs were treated with siRNA targeting PPIB or JAK1 and subcultured in the presence of IL-7 and IL-15. siRNA was not removed from the culture.

[0146] Activated T cells were treated with 2 µM of divalent myristic acid- or cholesterol-conjugated siRNA targeting JAK1 or PPIB. Cells were passaged at each doubling time. At each passage, samples were collected, cells were lysed, and mRNA expression was analyzed using the QuantiGene assay following the manufacturer's protocol. Target mRNA expression (PPIB) was normalized to a housekeeping gene (HPRT) and an untreated control. Results can be obtained from Figure 5 The inventors observed very effective silencing, which lasted for at least 13 days.

[0147] To demonstrate proliferation-sensitive expression, Jurkat T lymphoblastoid cells stably expressing the NanoLuc transgene were treated with varying concentrations of siRNA targeting NanoLuc, and luminescence was measured twice a week.

[0148] Jurkat cells stably expressing NanoLuc were treated with cholesterol-conjugated 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 The inventors observed increased inhibition of luciferase upon cell proliferation.

Claims

1. A nucleic acid-modified biological cell, the nucleic acid-modified biological cell comprising - Its genome contains (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 nucleotide sequence, and - a nucleic acid not contained in its genome, said nucleic acid comprising a third nucleotide sequence configured to inhibit the expression and / or function of said second nucleotide sequence.

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

3. The nucleic acid-modified biological cell according to claim 1 or 2, wherein the second expressible nucleotide sequence is integrated into the chromosome of the biological cell.

4. The nucleic acid-modified biological cell according to any one of the preceding claims, wherein the second expressible nucleotide sequence is integrated into an episome of the biological cell.

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

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

7. The nucleic acid-modified biological cell according to any 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 transcript, transposon.

8. The nucleic acid modified biological cell according to any 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 (micro RNA), AMO (anti-miRNA), aptamer.

9. The nucleic acid-modified biological cell according to any one of the preceding claims, wherein the biological cell is an immune cell.

10. A nucleic acid-modified biological cell according to any of the preceding claims, wherein the immune cell is selected from the group consisting of: T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocyte infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, tumor infiltrating macrophages, hematopoietic stem cells, including allogeneic cells listed above.

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

12. A nucleic acid-modified biological cell according to any 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, TIM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, Siglec7 / 9.

13. A nucleic acid-modified biological cell according to any of the preceding claims, wherein the first expressible nucleotide sequence encoding the 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, let7, TGFβ, IL-12, IL-18, IL-23, T cell receptor, miR-27a, IL-7, CCL19, CCL21, CAR constructs targeting the tumor microenvironment, IL-15, IL-7, and CAR constructs targeting cancer stem cell markers.

14. A nucleic acid-modified biological cell according to any of the preceding claims for use in medicine, preferably for 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, bacterial infections.

15. A method for regulating expression of a first nucleotide sequence encoding a target in a nucleic acid-modified cell, comprising: 1) providing a biological cell, wherein the biological cell comprises a first expressible nucleotide sequence encoding a target protein integrated into the genome of the biological cell, 2) integrating a recombinant nucleic acid into the genome of the biological cell, wherein the recombinant nucleic acid comprises a second expressible nucleotide sequence configured to regulate the expression and / or function of the first nucleotide sequence, 3) introducing a nucleic acid comprising a third nucleotide sequence outside the genome of the biological cell, wherein the third nucleotide sequence is configured to inhibit the expression and / or function of the second nucleotide sequence.

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

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

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

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

21. The method 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).

22. The method according to 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 (micro RNA), 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 by a method selected from the group consisting of: electroporation, lipid conjugated delivery, aptamer conjugated delivery, lipid nanoparticle delivery, viral vector delivery, antibody conjugated delivery, small molecule conjugated delivery, peptide conjugated delivery, extracellular vesicle delivery.

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

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

26. The method according to any one of the preceding claims, wherein the biological cell is selected from the group consisting of: T cells, CAR T cells, tumor infiltrating lymphocytes, donor lymphocyte infusion, NK cells, CAR NK cells, B cells, dendritic cells, macrophages, tumor infiltrating macrophages.

27. The method according to any one of the preceding claims, wherein the first expressible nucleotide sequence encoding the 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, TIM3, LAG3, TIGIT, CTLA4, miR-146a, miR-155, MHC-I, MHC-II, miR-17-92, CX3CR1, HLA-E, HLA-G, Siglec7 / 9.

28. The method according to any of the preceding claims, wherein the first expressible nucleotide sequence encoding the 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, CAR constructs targeting the tumor microenvironment, IL-15, IL-7.

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

30. The method of claim 29, wherein the organism suffers from a disease selected from the group consisting of cancer, autoimmunity, viral infection.

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