Nucleic acid systems for specifically reprogramming B and T cells and uses thereof

Abstract

The present invention relates to a nucleic acid system, comprising a sensor component and a transducer / effector component, which allows specific reprogramming of B cells and T cells. The inventors have successfully demonstrated that upon binding of a target molecule to a dedicated sensor (targeting a given physiological signal), the transducer pNR4A1 is specifically activated and the effector therapeutic molecule under its control is expressed. The main advantage of this system is its complete reprogramming ability in terms of recognized signals and output functions, which can be applied to targeted diseases. Thus, the present invention can be applied to many pathologies, such as tumors, autoimmune diseases, graft rejection, allergies, neurological disorders, and infectious diseases.

Description

[Technical field]

[0001] Technical Field The present invention relates to the field of synthetic immunology, in particular to novel synthetic circuits that can be equipped into B cells or T cells by transduction with lentiviral vectors to reprogram the cells. The present invention further relates to the use of said synthetic circuits and lentiviral vectors in the treatment of tumors, immune disorders, transplant rejection, allergies, neurological disorders and infectious diseases.

[0002] Technical background The increase in techniques for genetic engineering has recently brought a new dimension to synthetic biology approaches. Synthetic biology relies on the design of genetic parts and biological blocks that are assembled in target cells to create novel genetic networks (Sedlmayer et al.). For example, synthetic biosensing circuits will be composed of sensor elements that bind signal molecules and transducer modules, which will lead to a specific cellular response. The input or "inducing signal" should be disease-specific, for example a specific antigen or a dysregulation of the microenvironment, and it should be recognized by a dedicated receptor. This recognition should trigger a signaling cascade and integration of information that may or may not lead to an output response from the reprogrammed cell. Such circuits are valuable and important from a therapeutic point of view, especially for apoptosis induction or regulated drug delivery in vivo.

[0003] This kind of approach is equipped with immune cells and represents a new field called "synthetic immunology", which shows great promise for the treatment of many diseases, since immune system cells play a key role in detecting and responding to pathological deviations (Roybal et al.). Genetic reprogramming of immune cells can endow them with several new capabilities by enhancing their intrinsic properties. Indeed, immune cells recognize pathological signals such as pathogens, autoantigens, or inflammation and trigger a response to restore homeostasis, but by adding novel receptors, their recognition ability can be enhanced to more accurately distinguish pathological environments from normal ones. Similarly, their effector functions can be acted upon, for example by forcing the expression of therapeutic cytokines.

[0004] Several characteristics of immune cells make them perfect candidates for synthetic biology approaches.

[0005] First, these cells freely migrate through the body, patrolling and infiltrating various tissues, and because of their global distribution in the body, immune cells can act as intermediaries and communicate with other cell types, thus modulating the immune response on a larger scale.

[0006] Second, they proliferate spontaneously when stimulated, and they can differentiate into memory cells and thus persist for a long period of time, which may be particularly useful in the event of disease relapse.

[0007] Moreover, they are involved in many diseases, such as cancer or autoimmune diseases, because they either fail to ensure their defense function or they contribute to the disease in a pathological way.

[0008] Finally, these cells can be easily harvested and modified. Most synthetic immunological approaches use T cells, the best known example being the successful chimeric antigen receptor (CAR) T cells, which have been developed specifically in cancer immunotherapy approaches (Chabannon et al.).

[0009] However, B cells also offer unique opportunities for synthetic immunology. To lay the groundwork, a first-in-man Phase I / IIa clinical trial was recently initiated in patients to evaluate the safety and tolerability of adoptively transferred donor B cells. B cells were produced under Good Manufacturing Practice (GMP) conditions, and their transfer was well tolerated without any acute adverse reactions during a 4-month follow-up period after adoptive transfer (Tittlbach et al.).

[0010] The immune system is involved in many diseases, such as autoimmune diseases (where the immune system attacks its own components) or cancer (where the immune system no longer recognizes cancer cells as abnormal).

[0011] Depending on the condition to be treated, it would be advantageous to be able to specifically either stimulate or suppress the immune response in an individual.

[0012] Immunostimulation or immune stimulation refers to the stimulation of the immune system by an external source. It aims to elicit an immune response, especially the production of antibodies, to enhance the body's ability to fight disease or as a preventative measure, to provide a protective effect, for example against microorganisms or tumors. Vaccines and synthetic peptides have been used successfully in the past as immune stimulants.

[0013] Conversely, when a disease or condition derives from an aberrant activity of the immune system, such as in the case of autoimmune diseases or allergies, it is essential to be able to suppress the immune response. Induction of deliberate immunosuppression can also be performed to protect the body against organ transplant rejection.

[0014] Current treatments do not allow the treatment of most autoimmune diseases or certain cancers that are resistant to conventional therapies. In fact, treatments of autoimmune diseases are based on global immunosuppression (especially via corticosteroids) and do not restore tolerance to "self" components. Therefore, they can only address the symptoms of the disease without treating the cause, and are therefore ineffective in preventing recurrence. Similarly, certain cancers are refractory to current treatments such as radiation therapy, chemotherapy, or immunotherapy, and new alternative options that have fewer side effects and act in a more targeted manner are needed.

[0015] Therefore, there is a need for safe and improved treatments for diseases involving the immune system, particularly treatments that allow patients to achieve complete remission with minimal side effects and prevent possible relapse of the disease.

[0016] There is also a need for treatments for diseases involving the immune system that allow for increased therapeutic capacity and patient-specific responses of B or T lymphocytes.

[0017] Furthermore, there is a need for treatments for diseases involving the immune system whose effects can be "turned on" or "turned off," but also increased or decreased in response to pathological signals.

[0018] There is also a need for a safe cell therapy system that can be easily adapted to treat a variety of diseases involving the immune system, such as tumors, immune disorders, transplant rejection, neurological disorders, allergies, and infectious diseases.

[0019] The present invention aims to satisfy all or part of these needs.

[0020] The present invention results from the inventors' development of a cell therapy system in the form of a nucleic acid-based system aimed at reprogramming in particular B lymphocytes or T cells ex vivo via vectors, such as lentiviral vectors, encoding therapeutic synthetic circuits, and then re-injecting them into the patient.

[0021] The nucleic acid system of the present invention encodes a sensor that targets a pathological ligand, particularly a pathological antigen, a transducer that is activated upon binding of the target pathological ligand, particularly a target pathological antigen, to the sensor, and a transducer / effector component that generates a therapeutic effector molecule upon activation of the transducer. Thus, recognition of a pathological ligand, particularly a pathological antigen, by the sensor will result in activation of the transducer and regulated expression of the therapeutic effector molecule.

[0022] The terms "effector molecule" and "effector protein" may be used synonymously herein. In particular, the effector protein is a therapeutic effector protein.

[0023] Said therapeutic effector protein is then secreted by the initiated blood cells, in particular by the initiated B lymphocytes (also called "initiated B cells"), which generate one or more therapeutic effector molecules after recognition of a pathological ligand, in particular after recognition of a pathological antigen. As a result, once the targeted physiological disorder is overcome, the target pathological ligand, in particular the pathological antigen, is usually no longer available and therefore no longer activates the nucleic acid system present in the initiated blood cells, in particular the initiated B cells, and the effector molecules generated upon activation of said nucleic acid system are no longer produced and secreted by said initiated cells, in particular by said initiated B cells. This switching of the nucleic acid system allows to avoid any systemic effects often found in current treatments (which are based on the ingestion or injection of drugs) and will increase the efficiency of the molecules, which will be more concentrated locally as a result of the "homing and proliferation" effect of the initiated cells.

[0024] The aim is to enhance the immunological properties of the cells of the patient's immune system, particularly B lymphocytes, to cure the targeted disease or targeted disorder.

[0025] One of the many advantages of this nucleic acid system is its complete reprogramming ability, which allows it to be adapted to treat various diseases by appropriate selection of sensor and / or effector molecules. Another major advantage of the nucleic acid system is its signal-specific regulation combined with memory and long-term therapy.

[0026] Thus, the present invention provides (i)(a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) optionally a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor; A sensor component comprising a sequence encoding (ii)(d) the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof; and (e) at least one effector protein of interest. A transducer / effector component comprising a sequence encoding The present invention relates to a nucleic acid system comprising:

[0027] In some embodiments, the transducer / effector component comprises (d) a functional fragment of the pNR4A1 promoter (which may be referred to herein as the "NR4A1 promoter"). In particular, the functional fragment of the NR4A1 promoter has a length of 200 bp to 2210 bp, in particular 500 bp to 2210 bp. In particular, the functional fragment of the NR4A1 promoter may have a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, and SEQ ID NO:23, in particular SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and preferably has the sequence of SEQ ID NO:5.

[0028] According to a further advantage, the extracellular ligand recognition domain (a), the transmembrane domain (b) and, if present, the signalling domain (c) form a B cell receptor, a T cell receptor, a chimeric immune receptor (CIR), such as a CAR-T cell receptor, a CAR-NK cell receptor, a B cell antibody receptor (BAR) or a chimeric autoantibody receptor T (CAAR-T) cell, in particular a B cell receptor.

[0029] In some embodiments, the extracellular ligand recognition domain (a) comprises at least one ligand-binding fragment that binds to a ligand of interest, in particular a ligand-binding fragment derived from an antibody, i.e. a ligand-binding fragment comprising the antigen-binding domain of an antibody, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment; an Fd fragment, a complementarity determining region (CDR)-containing fragment, an Fv fragment, a single-chain FV (scFV), a double-chain Fvs, and a single-chain (Fv)2 fragment; an antibody mimic such as an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a designed ankyrin repeat protein (DARPins), anticalin, an avimer, an fynomer, and a versabodies; an aptamer, and mixtures thereof.

[0030] Advantageously, the ligand of interest is selected from one or more tumor antigens, one or more autoantigens, one or more alloantigens, one or more viral antigens, one or more bacterial antigens, one or more allergen antigens, or one or more markers of neuropathic disorders.

[0031] In certain embodiments, the at least one effector protein of interest is an immunostimulatory or immunosuppressive protein. In particular, the at least one effector protein of interest is selected from proinflammatory cytokines, such as interleukin-18, gamma-interferon, and tumor necrosis factor; anti-inflammatory cytokines, such as interleukin-10 and interleukin-4; costimulatory molecules, such as CD80, CD86, and CD40; and inhibitory molecules, such as Fas ligand and Fas.

[0032] According to another object, the present disclosure relates to a vector comprising a nucleic acid system as described herein, in particular said vector being a retroviral vector, in particular a lentiviral vector.

[0033] The disclosure further provides a first vector comprising a nucleic acid-based sensor component as described herein; and A second vector comprising a nucleic acid-based transducer / effector component as described herein. The present invention relates to a kit comprising:

[0034] According to another object, the present disclosure relates to a cell, in particular a B cell or a T cell, transformed by a nucleic acid system, by a vector or by a kit as described herein.

[0035] The disclosure also relates to pharmaceutical compositions comprising the nucleic acid system, a vector containing the sensor component, transducer / effector component, or both of the nucleic acid system, a kit as described herein, or a cell, and a pharma- ceutically acceptable vehicle.

[0036] The disclosure also relates to methods for preventing and / or treating tumors, immune disorders, transplant rejection, allergies, neurological disorders, and / or infectious diseases, comprising at least one step of administering to an individual in need thereof a described nucleic acid system, a vector, a kit, a cell, or a pharmaceutical composition as described herein.

[0037] According to another embodiment, the disclosure relates to a nucleic acid system, a vector, a kit or a cell as described herein for its use in the prevention and / or treatment of tumors, immune disorders, transplant rejection, allergies, neurological disorders and / or infectious diseases.

[0038] The disclosure further relates to a nucleic acid system, a vector, a kit, or a cell as described herein for use as a medicament.

[0039] The above and other objects, features and advantages of the present invention will be explained in more detail with reference to the accompanying drawings, the following description and examples provided hereinafter. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows the isolation of small ectopic B cell receptor-inducible promoter constructs. (A) is a diagram of the lentiviral vector containing the pNR4A1(2204) fragment. (B) is a diagram of the NR4A1 reporter construct, containing putative binding domains for NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) transcription factors. Reporters of several sizes (2204, 1750, 1251 or 734 bp) were constructed. (C) shows the induction of each reporter construct by GFP (green fluorescent protein) after 24 hours of stimulation of the B cell receptor. From top to bottom: pNR4A1(734)-GFP, pNR4A1(1251)-GFP, pNR4A1(1750)-GFP, pNR4A1(2204)-GFP and control SFFV-GFP. BJAB cells were transduced with lentiviral vectors encoding reporter constructs and then stimulated with F(ab')2 IgM (2.5 μg / ml) or PMA (phorbol myristate acetate) (15 ng / ml) in combination with ionomycin (1 μM) for 24 h. Median GFP expression of GFP-positive cells after stimulation was normalized to unstimulated conditions. (D) Transduction efficiency of BJAB cells with reporter constructs was assessed by the proportion of GFP-positive cells after transduction for each construct and 4 days after transduction (n=4, two-way ANOVA and multiple comparison t-test with Sidak-Bonferroni correction (α=0.05)). [Diagram 2]Figure 2 shows the characterization and inducibility of the 734bp NR4A1 reporter construct. (A) Dose response of the 734bp NR4A1 reporter construct with increasing amounts of F(ab')2 IgM. BJAB cells were transduced with a lentiviral vector encoding the 734bp inducible reporter of the SFFV constitutive reporter and then stimulated with F(ab')2 IgM for 24 hours. Median GFP expression of DAPI-GFP positive cells compared to unstimulated cells was assessed (n=4). (B) Specificity of induction through the B cell receptor. BJAB cells were transduced with lentiviral vectors encoding reporter constructs (734 bp NR4A1 or SFFV as constitutive control) and then stimulated for 24 h with F(ab')2 IgM (2.5 μg / ml), F(ab)' IgG (2.5 μg / ml), LPS (lipopolysaccharide) (10 μg / ml), or CpG (10 μg / ml), or PMA (15 ng / ml) in combination with ionomycin (1 μM). Median GFP expression of stimulated GFP-positive cells was normalized by unstimulated conditions (n=4). [Diagram 3]Figure 3 shows the characterization of the kinetics of the 734bp inducible NR4A1 promoter. (A) Kinetics of promoter induction. BJAB cells were transduced with a lentiviral vector encoding TurboGFPdes under a 734bp inducible reporter or a SFFV constitutive reporter and then stimulated with PMA in combination with F(ab')2 IgM or ionomycin for 0-24 hours. The median TurboGFPdes expression in each condition (after the indicated stimulation time (h)) was evaluated and normalized by the median TurboGFPdes in unstimulated cells (n=3). (B) Kinetics of promoter extinction. BJAB cells were transduced with a lentiviral vector encoding TurboGFPdes under a 734bp inducible reporter or a SFFV constitutive reporter and then stimulated with PMA in combination with F(ab')2 IgM or ionomycin for 0, 1, 4, 8 and 24 hours. Cells were then washed three times and placed in culture medium. Median TurboGFPdes expression was assessed 1, 3, and 6 days after washing and normalized by median TurboGFPdes in unstimulated cells (n=3). (C) Reversibility of inducible promoter. Reversibility of pNR4A1(734bp)-responsive TurboGFPdes expression was assessed by culturing BJAB cells transduced with lentiviral vectors encoding TurboGFPdes under a 734bp inducible reporter or a SFFV constitutive reporter, alternating 8 hours of stimulation (gray) with PMA in combination with F(ab')2 IgM or ionomycin and 80 hours of rest (white) three times. In the PMA / ionomycin condition, only one stimulation was performed due to significant cell death. Median TurboGFPdes expression was assessed before and after stimulation and normalized by median TurboGFPdes in unstimulated cells (n=3). [Figure 4]Figure 4 shows the design and validation process of the sensor components. (A) BJAB cells (IgM positive, IgG negative) were transduced with lentiviral vectors encoding membrane-anchored B cell receptors that recognize either the sensor components, ovalbumin (FAM0-OVA) or the HbS (hepatitis B surface antigen) glycoprotein of the hepatitis B virus (FMA0-ADRI), and stained for IgG by Western blot (WB) 5 days later. (B) Specific recognition of ovalbumin by membrane-anchored B cell receptors directed to ovalbumin. BJAB cells were transduced with lentiviral vectors encoding membrane-anchored B cell receptors targeting either ovalbumin or hepatitis B surface antigen. Five days after transduction, the cells were incubated with fluorescent beads coated with ovalbumin for 24 hours. Fluorescent bead binding was assessed by flow cytometry. (C) Signal transduction and cell activation after binding of synthetic particulate antigen-ovalbumin. BJAB cells were transduced with tagged or untagged membrane B cell receptors recognizing hepatitis B surface antigen or ovalbumin, incubated with synthetic particulate antigen-ovalbumin for 24 h, and then stained with anti-CD86 or anti-HLA-DR antibodies. One representative overlay is presented with the median fluorescence intensity quantified from triplicate experiments for both markers (one-way ANOVA and multiple comparison test with Tukey correction, n=4). [Diagram 5]Figure 5 shows antigen-specific activation of the nucleic acid system. BJAB cells were transduced with lentiviral vectors encoding SFFV-TurboGFPdes or pNR4A1(734)-TurboGFPdes and one week later with lentiviral vectors encoding membrane-anchored B cell receptors directed against ovalbumin (FAM0-OVA) or against Hepatitis B surface antigen (FAM0-ADRI). The twice-transduced cells were then stimulated for 24 hours with either F(ab')2 directed against IgM or IgG, or PLA / ionomycin as control (A), or beads coated with the binding region of ovalbumin / spike RBD (ligand binding domain) with or without CD40L, after which TurboGFPdes was detected by flow cytometry. Two-way ANOVA with post hoc comparisons (n=5 / 6) with Tukey correction. ***: P value <0.001. [Figure 6] Figure 6 shows promoter inducibility in T cells. Jurkat cells were transduced with the 734 bp pNR4A1 reporter construct and then stimulated for 24 h with anti-CD3 and anti-CD28 antibodies (1 μg / ml) or beads (TransAct) or PMA (15 ng / ml) in combination with ionomycin (1 μM). (A) Induction of the reporter construct by GFP after 24 h of T cell receptor stimulation. (B) Median GFP expression of GFP-positive cells after stimulation was normalized by the unstimulated condition. Data are representative of four independent experiments (n=4, two-way ANOVA followed by multiple comparison t-test with Sidak-Bonferroni correction (α=0.05)). [Figure 7]FIG. 7 shows an all-in-one vector encoding a self-amplifying circuit. (A) shows the structure of the self-amplifying vector. The inducible pNR4A1-734 bp fragment drives both TurboGFPdes and FAM0-OVA sensor transgenes with a T2A sequence between them. (B) and (E) show the induction of effector expression by the self-amplifying vector in transduced BJAB cells after stimulation with F(ab')2 IgM or F(ab')2 IgG for 24 hours (B) or 48 hours (E). (C) and (F) show the induction of sensor expression by the self-amplifying vector in transduced BJAB cells after stimulation with F(ab')2 IgM or F(ab')2 IgG for 24 hours (C) or 48 hours (F). The fold change of sensor-encoding mRNA was assessed by reverse transcription quantitative PCR after stimulation. (D) and (G) Induction of effector expression by a self-amplifying vector in transduced BJAB cells stimulated with ovalbumin / spike RBD (receptor binding domain)-coated beads in the presence or absence of CD40L for 24 (D) or 48 (G) hours, after which TurboGFPdes fluorescence was detected by flow cytometry. Median expression of TurboGFPdes, normalized by the median of unstimulated cells, was assessed by flow cytometry. Two-way ANOVA with Tukey correction and post-hoc comparison test (n=5 / 6 for panels B, D, E, G, n=3 for panels C, F). Error bars indicate standard error. [Figure 8]FIG. 8 demonstrates higher basal levels of TurboGFPdes signal in B cells transduced with a self-amplifying vector. (A) shows leakage of TurboGFP expression in the absence of stimulation. The mean fluorescence intensity of TurboGFPdes was evaluated in BJAB cells transduced with a lentiviral vector encoding pNR4A1(734)-TurboGFPdes with or without the T2A-FAM0-OVA sequence. The mean fluorescence intensity was normalized by the vector copy number of transduced cells (n=3). (B) shows the half-life of TurboGFPdes alone or followed by the 16 amino acid T2A motif. Cycloheximide was added to the transduced cells and the mean fluorescence intensity of TurboGFPdes was evaluated every 40 min for 360 min. The half-life was then computed as the time to reach a mean fluorescence intensity equal to 0.5 times the initial mean fluorescence intensity (n=3). [Figure 9] Figure 9 shows the decay kinetics of self-amplifying vectors. BJAB cells were transduced with lentiviral vectors encoding TurboGFPdes alone or fused to the T2A-FAM0-OVA sequence followed by a 734 bp inducible reporter. Transduced cells were stimulated with F(ab')2 IgM (A) or F(ab')2 IgG (B) for 24 h, then washed three times and subsequently cultured. The median expression of TurboGFPdes was assessed on days 1, 3, 6 and 10 after washing and normalized by the median expression of TurboGFPdes in unstimulated cells (n=3).

[0041] Detailed Description definition The present invention relates to (i)(a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) optionally a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor; A sensor component comprising a sequence encoding (ii)(d) the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof; and (e) at least one effector protein of interest. A transducer / effector component comprising a sequence encoding The present invention relates to a nucleic acid system comprising:

[0042] As shown in the Examples section, the inventors have demonstrated the efficacy of the nucleic acid system, particularly in tumor models, which may be applied to many other pathologies, such as autoimmune diseases, transplant rejection, allergies, neurological disorders, and infectious diseases.

[0043] Indeed, the main advantage of the nucleic acid system of the present invention is its complete reprogramming ability in terms of recognized signals and output functions, which can be applied to targeted diseases toward the same goal of continuous sensing of disease-specific biomarkers and triggering physiological expression of therapeutic molecules in response in vivo.Therefore, the system of the present invention is highly promising for the long-term treatment of many diseases.

[0044] The inventors were also able to demonstrate that lentiviral vectors containing autoregulated constructs containing the nucleic acid system of the present invention allowed specific expression of effector proteins upon sensor stimulation. As further explained below, such all-in-one lentiviruses can increase the number of cells co-expressing all circuit components, thus reducing variability (i.e., cells partially modified with but not all circuit components are avoided).

[0045] Unless otherwise defined herein, units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers that delimit the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects of the disclosure. Thus, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0046] Throughout the specification and embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising", will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. The words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising", will be understood to mean the inclusion of a stated element(s) (such as the composition or method step in question), but not the exclusion of any other elements. The term "consisting of" means the inclusion of a stated element(s) to the exclusion of any additional elements. The term "consisting essentially of" also means the inclusion of a stated element(s) and possibly other element(s), where the other element(s) do not substantially affect the basic characteristic(s) of the disclosure. Various embodiments of the disclosure using the term "comprising" or its equivalents are understood to cover embodiments in which this term is replaced with "comprising only," "consisting of," or "consisting essentially of."

[0047] Whenever an embodiment is described herein using the word "comprising," it is understood that otherwise similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0048] The term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, "a" (or "an"), "one or more," and "at least one" may be used synonymously herein.

[0049] Furthermore, when "and / or" is used herein, it should be taken as a specific disclosure of each of the two specific features or components in the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0050] The term "approximately" or "about" is used herein to mean approximately, roughly, in the vicinity of, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated numerical value by, for example, a 10% upward or downward (higher or lower) variation. In some embodiments, the term indicates a deviation from the stated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the stated numerical value by ±10%. In some embodiments, "about" indicates a deviation from the stated numerical value by ±5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, "about" refers to deviation from the indicated numerical value by ±0.05%. In some embodiments, "about" refers to deviation from the indicated numerical value by ±0.01%.

[0051] The terms "significantly" or "substantially" within the disclosure used to qualify differences or changes, e.g., "significantly different from" or "substantially different from" with respect to a feature or parameter, are intended to mean that the observed changes or differences are discernible and / or that they have statistical significance. Conversely, the terms "significantly" or "substantially" used to qualify similarity or identity, e.g., "not significantly different from" or "substantially identical to" with respect to a feature or parameter, are intended to mean that any observed changes or differences are such that the property and function of the parameter or feature of interest is not substantially affected.

[0052] In the context of the present invention, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). Preferably, the nucleic acid is DNA. It should be understood that the term also includes equivalents, analogs of either RNA or DNA made from nucleotide analogs.

[0053] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that (i) is free of sequences that normally flank one or both sides of the nucleic acid in a genome, (ii) is incorporated into a vector or into the genomic DNA of an organism such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA, or (iii) is a cDNA, a genomic nucleic acid fragment, a fragment generated by polymerase chain reaction (PCR), or a restriction enzyme fragment. Additionally, an isolated nucleic acid can include an engineered nucleic acid, such as a recombinant DNA molecule, that is part of a hybrid or fusion nucleic acid.

[0054] Nucleic acids can include coding and / or non-coding sequences. Coding nucleic acids have a nucleotide sequence that can be transcribed into an RNA molecule and translated to produce a polypeptide. Non-coding nucleic acids are typically transcribed into RNA that cannot be translated.

[0055] All nucleic acid sequences defined in this application may be codon optimized. Codon optimization methods are known in the art and can be useful in an effort to achieve one or more of several goals. These goals include matching codon frequency in the target and host organisms, ensuring correct folding, biasing the GC content to increase mRNA stability or reduce secondary structures, minimizing tandem repeat codons or sequences that may impair gene assembly or expression, customizing transcriptional and translational control regions, inserting or removing protein trafficking sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, adding, removing or shuffling protein domains, inserting or deleting restriction enzyme sites, modifying ribosome binding sites and mRNA degradation sites, adjusting the translation rate to allow the various domains of the protein to fold correctly, or reducing or eliminating problematic secondary structures in the mRNA, or avoiding potential splice sites as described in Fallot et al. (2009) Nucleic Acids Res. 37:e134 or Resse et al. (1997) J. Comput. Biol. 4:31 1 -323. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, Calif.) and / or proprietary methods.

[0056] By "a sequence that is at least x% identical to a reference sequence," it is intended that the sequence differs from the reference sequence by each nucleotide change for each 100 amino acids of the reference sequence, up to 100-x amino acids. In particular, a sequence having at least 90% sequence identity to another sequence includes sequences having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity to another sequence.

[0057] Alignment and determination of percent identity can be performed manually or automatically using, for example, the Needle program, which is based on the Needleman and Wunsch algorithm as described in Needleman and Wunsch (1970) J. Mol Biol. 48:443-453, with, for example, the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62, gap open penalty: 10, and gap extension penalty: 0.5, end gap penalty: false, end gap open penalty=10, end gap extension penalty=0.5; and the following parameters for polynucleotide sequence comparison: comparison matrix: DNAFULL; gap open penalty=10, gap extension penalty=0.5, end gap penalty: false, end gap open penalty=10, end gap extension penalty=0.5.

[0058] In the context of the present invention, the term "immunoglobulin" relates to immunoglobulin superfamily proteins (which also include T cell receptors), preferably antigen receptors, such as antibodies, or B cell receptors (BCR). Immunoglobulins are characterized by structural domains, i.e. immunoglobulin domains, which have a characteristic immunoglobulin (Ig) fold (one or more). The term encompasses membrane-anchored immunoglobulins as well as secreted immunoglobulins. Membrane-anchored or membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the B cell receptor.

[0059] Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are mainly composed of immunoglobulin domains, including a VL domain (light chain variable domain), a CL domain (light chain constant domain), a VH domain (heavy chain variable domain), and a CH domain (heavy chain constant domain) CH1, optionally a hinge region, CH2, CH3, and optionally CH4.

[0060] There are five main classes of heavy chains (i.e. isotypes) that determine the functional activity of an antibody molecule: mu (μ) for IgM, delta (δ) for IgD, gamma (γ) for IgG, alpha (α) for IgA, and epsilon (ε) for IgE. In the context of the present invention, the immunoglobulin may be IgM, IgD, IgG, IgA, or IgE. Preferably, the immunoglobulin is an IgG. As is well known to those skilled in the art, the IgG isotype encompasses four subclasses: subclasses IgG1, IgG2, IgG3, and IgG4. In the context of the present invention, the immunoglobulin may be any IgG subclass. Preferably, the immunoglobulin is an IgG1. Unlike the heavy chains of secretory immunoglobulins, the heavy chains of membrane-anchored immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini.

[0061] In mammals, there are two types of light chains: lambda (λ) and kappa (κ). Both types of light chains can bind equally well to either class of heavy chain.

[0062] Immunoglobulin chains comprise variable and constant regions, the constant regions being substantially conserved among the different immunoglobulin isotypes, whereas the variable portions are highly diverse.

[0063] The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed within more conserved regions, called framework regions (FRs). Each of the VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental transfer, complement binding, and Fc receptor (FcR) binding.

[0064] As used herein, the term "constant region of an immunoglobulin heavy chain" preferably refers to a region of an immunoglobulin heavy chain composed of the CH1, optionally hinge region, CH2, CH3, and optionally CH4 domains, preferably including one or more, preferably all, of the linker candidates and / or hinge regions. It is particularly preferred that the constant region of an immunoglobulin heavy chain comprises one or more cysteine ​​residues capable of mediating association with another constant region of an immunoglobulin heavy chain via a disulfide bond.

[0065] The term "B cell receptor" or "BCR" as used herein refers to an antigen receptor in the plasma membrane of a B cell. B cell receptors are generally composed of a membrane-anchored antibody as defined above associated with a heterodimer of Ig-a and Ig-β, capable of signal transduction. Such B cell receptors are described, for example, in Janeway et al. (Immunobiology: The Immune System in Health and Disease. 5th edition. Janeway CA Jr, Travers P, Walport M, et al. New York: Garland Science; 2001). The loci encoding these genes are (i) gene ID: 3492 for IgH, (ii) gene ID: 50802 for Igκ, and (iii) gene ID: 3535 for Igλ.

[0066] The term "T cell receptor" or "TCR" as used herein refers to the antigen receptor in the plasma membrane of T cells, which is responsible for recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. It is generally composed of an α-chain and a β-chain, which contain immunoglobulin domains. When the T cell receptor binds its specific antigenic peptide presented on MHC (peptide / MHC) by antigen presenting cells (MHC-II for CD4+ T cells) or other cells (MHC-I for CD8+ cells), the T lymphocyte is activated when costimulatory signals are provided through interactions with CD80 / 86 and CD28, as well as differentiation signals (secretion of cytokines by the presenting cell). Depending on the cytokine signal and the subtype of T cell (CD4 or CD8), the output function can range from helping to activate other immune cells to direct cytotoxic activity against target cells. Such B cell receptors are described, for example, in Janeway et al. (Immunobiology: The Immune System in Health and Disease. 5th edition. Janeway CA Jr, Travers P, Walport M, et al. New York: Garland Science; 2001).

[0067] As used herein, the term "chimeric immune receptor" (CIR) encompasses tumor- or virus-specific ligands or antibodies fused to the signaling domains of either the T cell receptor or the Fc receptor of B cells. CIRs comprise an extracellular antigen-binding domain derived from an antigen-specific antibody or ligand linked to an intracellular signaling domain derived from either the CD3 ζ chain of the T cell receptor complex, the Fc receptor (FcR) γ chain of the tumor, or a more distal component of the T cell signaling pathway such as the syk molecule.Such chimeric immune receptors have been described, for example, in (i) Parvathaneni, Kalpana, et David W. Scott. ≪ Engineered FVIII-Expressing Cytotoxic T Cells Target and Kill FVIII-Specific B Cells in Vitro and in Vivo ≫. Blood Advances 2, no 18 (25 September 2018): 2332-40. https: / / doi.org / 10.1182 / bloodadvances.2018018556 and (ii) Sicard, Antoine, Megan K. Levings, et David W. Scott. ≪ Engineering Therapeutic T Cells to Suppress Alloimmune Responses Using TCR s, CAR s, or BAR s ≫. American Journal of Transplantation 18, no 6 (June 2018): 1305-11. https: / / doi.org / 10.1111 / ajt.14747, and (iii) Ellebrecht, Christoph T., Vijay G. Bhoj, Arben Nace, Eun Jung Choi, Xuming Mao, Michael Jeffrey Cho, Giovanni Di Zenzo, et al. ≪ Reengineering Chimeric Antigen Receptor T Cells for Targeted Therapy of Autoimmune Disease ≫. Science 353, no 6295 (8 July 2016): 179-84. Described at https: / / doi.org / 10.1126 / science.aaf6756.

[0068] CIRs include CAR-T cells, CAR-NK cells, B cell antibody receptor (BAR), and chimeric autoantibody receptor T (CAAR-T) cells.

[0069] The term "chimeric antigen receptor" or "CAR" as used herein has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide that contains an antibody antigen-binding domain (e.g., single-chain Fv) linked to a T cell receptor signaling domain. The characteristics of CARs include their ability to exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to selected targets in an MHC-unrestricted manner. MHC-unrestricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thus bypassing a major tumor evasion mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains. Chimeric antigen receptors, in particular, are described in Feins, Steven, Weimin Kong, Erik F. Williams, Michael C. Milone, et Joseph A. Fraietta. An Introduction to Chimeric Antigen Receptor (CAR) T-cell Immunotherapy for Human Cancer. American Journal of Hematology 94, no. o S1 (mai 2019): S3-9. Available at https: / / doi.org / 10.1002 / ajh.25418.

[0070] The term "CAR-NK cells" as used herein defines CAR engineered natural killer (CAR-NK) cells. Natural killer (NK) cells are considered specialized immune cells that can be genetically modified to obtain effector cells capable of adoptive cell treatment of cancer patients due to efficient recognition and lysis of malignant cells. However, there are mechanisms by which tumors evade immune surveillance and suppress the function of NK cells, such as tumor microenvironment and immune suppressive factors that prevent the expression of activating receptors and the interaction of NK cells with other cells, and also avenues of antigen evasion can trigger inhibitory NK cell receptors and suppress activating NK cell receptors. CAR-NK technology consists of a chimeric antigen receptor equipped natural killer cell that has the ability to recognize, target, and kill specific cells. It can provide the results of CAR T cell therapy without the high toxicity and risk of graft-versus-host disease. CAR NK cells are described, inter alia, in (i) Schmidt, Paula, Martin J. Raftery, et Gabriele Pecher. ≪ Engineering NK Cells for CAR Therapy-Recent Advances in Gene Transfer Methodology ≫. Frontiers in Immunology 11 (7 January 2021): 611163. https: / / doi.org / 10.3389 / fimmu.2020.611163, and (ii) Yilmaz, Ahmet, Hanwei Cui, Michael A. Caligiuri, et Jianhua Yu. ≪ Chimeric Antigen Receptor-Engineered Natural Killer Cells for Cancer Immunotherapy ≫. Journal of Hematology & Oncology 13, no 1 (7 December 2020): 168. https: / / doi.org / 10.1186 / s13045-020-00998-9.

[0071] A novel CIR approach has been developed to suppress or eliminate unwanted antibody-producing B cells in autoimmune diseases such as pemphigus vulgaris (PV) and hemophilia A. These BAR-T (B cell antibody receptor T cell) or CAAR-T (chimeric autoantibody receptor T cell) cells are similar to CAR-T cells, but express transmembrane and intracellular domains with specific antigens on their surface instead of classical single-chain Fv fragments. These BAR / CAAR cells capture autoreactive lymphoblasts by inducing recognition between antigens exposed on the surface of rhotoikotrienes and the B cell receptor. The binding between these two molecules activates the CIR, thus releasing the cytotoxic potential of the modified T cells to specifically eliminate antigen-specific B cells.

[0072] As used herein, "chimeric autoantibody receptor T (CAAR-T) cells" or "B cell antibody receptor" (BAR) are modified forms of CAR-T cells that identify cells secreting antibodies, such as autoreactive B cells. The CAAR-T cell construct consists of a specific antigen, a transmembrane domain, and an intracellular signaling domain with or without a costimulatory domain. CAAR-T cells recognize and bind target autoantibodies expressed on autoreactive cells via the specific antigen, and subsequently destroy them.Those skilled in the art may refer to (i) Parvathaneni, Kalpana, and David W. Scott. “Engineered FVIII-Expressing Cytotoxic T Cells Target and Kill FVIII-Specific B Cells in Vitro and in Vivo.” Blood Advances 2, no. 18 (25 September 2018): 2332-40. https: / / doi.org / 10.1182 / bloodadvances.2018018556, (ii) Sicard, Antoine, Megan K. Levings, and David W. Scott. “Engineering Therapeutic T Cells to Suppress Alloimmune Responses Using TCRs, CARs, or BARs.” American Journal of Transplantation 18, no. 6 (June 2018): 1305-11. https: / / doi.org / 10.1111 / ajt.14747, and (iii) Ellebrecht, Christoph T., Vijay G. Bhoj, Arben Nace, Eun Jung Choi, Xuming Mao, Michael Jeffrey Cho, Giovanni Di Zenzo, et al. “Reengineering Chimeric Antigen Receptor T Cells for Targeted Therapy of Autoimmune Disease.” Science 353, no. 6295 (8 July 2016): 179-84. https: / / doi.org / 10.1126 / science.aaf6756.

[0073] By "antibody of interest" is meant herein an immunoglobulin as defined above, comprising light and heavy chain variable domains, which determine its antigen specificity, and light and heavy chain constant domains, which are produced by a cell. Preferably, the amino acid sequence of the antibody of interest and / or the coding sequence of the antibody of interest and / or the gene sequence encoding the antibody of interest are known or can be determined by the skilled artisan.

[0074] The antibody of interest is preferably a monoclonal antibody. The antibody of interest may also be a chimeric antibody.

[0075] A "chimeric" antibody refers to an antibody made from components derived from at least two different sources. In certain embodiments, a chimeric antibody comprises a portion of an antibody derived from a first species fused to another molecule, e.g., a portion of an antibody derived from a second species. In certain such embodiments, a chimeric antibody comprises a portion of an antibody derived from a non-human animal fused to a portion of an antibody derived from a human. In certain such embodiments, a chimeric antibody comprises all or a portion of a variable region of an antibody derived from a non-human animal fused to a constant region of an antibody derived from a human.

[0076] In particular, the antibody of interest may be a humanized antibody. A "humanized" antibody refers to a non-human antibody that has been modified to match more closely (in amino acid sequence) with a human antibody. In certain embodiments, amino acid residues outside the antigen-binding residues of the variable regions of the non-human antibody are altered. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable regions have been replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or a non-human primate, having the desired specificity, affinity, and capacity. In certain embodiments, a humanized antibody is constructed by replacing all or a portion of the CDRs of a human antibody with all or a portion of the CDRs from another antibody, such as a non-human antibody, having the desired antigen-binding specificity. In certain embodiments, a humanized antibody comprises a variable region in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the framework regions (FRs) correspond to the FRs of a human antibody. In certain such embodiments, a humanized antibody further comprises a human antibody constant region (Fc). Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.

[0077] The antibody of interest may further be a human antibody.

[0078] The term "human antibody" refers to a monoclonal antibody that contains human antibody sequences and does not contain antibody sequences derived from animals other than humans. In certain embodiments, human antibodies may contain synthetic sequences not found in natural antibodies. The term is not limited by the method by which the antibody is produced. For example, in various embodiments, human antibodies can be produced in transgenic mice, by phage display, by human B lymphocytes, or by recombinant methods.

[0079] The term "B cell" as used herein refers to B lymphocytes. B cell precursors reside in the bone marrow, where immature B cells are produced. Very briefly, B cell development occurs through several stages, each stage representing a change in the genomic content at the antibody locus. In the heavy chain variable region of the genome, there are three segments, V, D and J, which are randomly combined in a process called VDJ rearrangement to generate a unique variable region in the immunoglobulin of each B cell. A similar rearrangement occurs in the light chain variable region, except that only two segments, V and J, are involved. After complete rearrangement, B cells reach the IgM<+> immature stage in the bone marrow. These immature B cells display membrane-anchored IgM, the B cell receptor, on their surface and migrate to the spleen, where they are called transitional B cells. Some of these cells differentiate into mature B lymphocytes. Mature B cells expressing B cell receptors on their surface circulate through the blood and lymphatic systems, fulfilling the role of immune surveillance. They do not produce secretory immunoglobulins until they become fully activated. Each B cell has a unique receptor protein that will bind to one specific antigen. Once a B cell encounters its antigen and receives additional signals from helper T cells, it can further differentiate into either plasma B cells or memory B cells, which express and secrete secretory immunoglobulins.

[0080] The term "T cell" as used herein refers to T lymphocytes. T lymphocytes are regulators or effectors of adaptive functions that serve as the primary effectors for cell-mediated immunity. They originate from the bone marrow and mature in the thymus. In the thymus, T cells expand and differentiate into helper T cells, regulatory T cells, or cytotoxic T cells, or become memory T cells. They are then sent to peripheral tissues or circulate in the blood or lymphatic system. Antigen specificity is dictated by the heterodimeric receptor of the T cell receptor, which is derived from the recombination of gene segments. CD4-positive helper T lymphocytes recognize foreign antigens presented in the context of MHC class II molecules. Different subclasses of helper T cells secrete unique subsets of cytokines that support functional activity. These cells can also directly interact with other immune cells to provide the "rescue signals" required to activate them. CD8 positive T lymphocytes, also called cytotoxic T cells (CTLs), recognize endogenous antigens presented in the context of MHC class I molecules. CTLs will directly kill target cells by inducing apoptosis via released preformed proteins.

[0081] Although, for clarity, certain features of the invention are described in the context of separate embodiments, it will be understood that these may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0082] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications described herein are incorporated by reference to disclose and describe the methods and / or materials to which the relevant publications are cited.

[0083] Listed herein below are lists of sources, ingredients, and components, whereby combinations and mixtures thereof are contemplated and within the scope of the present specification.

[0084] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitation were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitation were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical range were all expressly written herein. Every numerical range given throughout this specification will also include the minimum and maximum numerical values ​​given in the range.

[0085] Any list of items, e.g., a list of ingredients, is intended to be and should be interpreted as a Markush group. Thus, the list may be read and interpreted as an item "selected from the group consisting of" the list of items, "and combinations and mixtures thereof."

[0086] The present specification may refer to trade names for components, including various ingredients used in this disclosure. The inventors do not intend to be limited herein by materials under any particular trade name. Materials equivalent to those referenced by trade name (e.g., those obtained from different sources under different names or reference numbers) may be substituted and utilized in the description herein.

[0087] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation is not an admission that any of these documents form part of the common and general knowledge in the art.

[0088] Nucleic acid system The nucleic acid system according to the invention comprises: (i)(a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) optionally a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor; A sensor component comprising a sequence encoding (ii)(d) the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof; and (e) at least one effector protein of interest. A transducer / effector component comprising a sequence encoding Includes.

[0089] Sensor components The nucleic acid system of the present invention comprises a first sensor component.

[0090] A "sensor" as defined herein is a portion of a nucleic acid system that encodes a protein that contains at least one extracellular ligand recognition domain that will recognize a pathological ligand, such as a pathological antigen, which in some embodiments is a biomarker for a disease. Upon activation through binding to a targeting ligand, the extracellular ligand recognition domain will generate one or more signals that will then activate the effector components of the nucleic acid system. Thus, recognition of the targeting ligand by the extracellular recognition domain encoded by the sensor component will trigger a signaling cascade that will result in an output response from the initialized cell.

[0091] The sensor component comprises a sequence encoding (a) at least one extracellular ligand recognition domain; (b) a transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and optionally (c) a signaling domain that controls cellular activation of the B cell receptor or T cell receptor.

[0092] In summary, these domains form a cell surface receptor that targets a pathological ligand, such as a disease-specific antigen, and becomes activated upon binding to the pathological ligand, e.g., the disease-specific antigen.

[0093] Extracellular Ligand Recognition Domain As mentioned above, one of the many advantages of the nucleic acid system of the present invention is its ability to be adapted for the prevention and / or treatment of many types of conditions and diseases, which is possible in part due to the great flexibility in terms of the appropriate type of extracellular ligand recognition domain that can be selected to target disease-specific pathological ligands.

[0094] In certain embodiments, the extracellular ligand recognition domain comprises at least one ligand-binding fragment, in particular, the ligand-binding fragment is an antigen-binding domain.

[0095] In the context of the present invention, the term "antigen-binding domain" refers to any peptide, polypeptide, scaffold-based molecule, peptide-presenting molecule, or polypeptide-containing construct that can specifically bind to a particular antigen of interest. Antigen-binding domains include, for example, antibodies, single-chain antibodies, single-domain antibodies (e.g., VHH antibodies derived from camelids), peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins that contain the ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (e.g., DARPins (designed ankyrin repeat proteins), HEAT repeat proteins, ARM (armadillo) repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on natural repeat proteins), and aptamers or antigen-binding portions thereof.

[0096] The antigen-binding domain may be formed by a single peptide or protein albumin-binding domain (AbD) or by the combination of two antigen-binding domain subunits AbD1 and AbD2, the combination of these two subunits allowing specific interaction of the entire antigen-binding domain with the antigen of interest.

[0097] In particular embodiments, the antigen-binding domain of the present invention may be selected from antigen-binding domains of antibodies, such as Fab fragments, Fab' fragments, F(ab')2 fragments; Fd fragments, single domain antibodies (sdAbs), complementarity determining regions (CDRs), Fv fragments, single chain FVs (scFVs), double chain Fvs, and single chain (Fv)2; from antigen-binding domains of antibody mimetics, such as affibodies, affilins, affitins, adnectins, atrimers, evasins, designed ankyrin repeat proteins (DARPins), anticalins, avimers, fynomers, and versabodies; aptamers, and mixtures thereof.

[0098] Alternatively, a protein can be directly attached to the transmembrane domain, and recognition of this protein by a receptor specifically expressed on diseased cells would trigger activation of the circuit.

[0099] Antigen-binding domain of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term antigen-binding fragment of an antibody include Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; Fab' fragment, a monovalent fragment consisting of the VL, VH, CL, CH1 domain and hinge region; F(ab')2 fragment, a bivalent fragment containing two Fab' fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH domain of a single chain of an antibody; single domain antibody (sdAb) fragment consisting of the VH or VL domain (Ward et al., 1989 Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be connected by an artificial peptide linker that allows them to be produced, using recombinant techniques, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). A "two-chain Fv" is a VH:VL heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments may form spontaneously by association of monovalent single-chain Fvs, or may be produced by linking monovalent single-chain Fvs through a peptide linker, such as bivalent single-chain (Fv)2. Such single chain antibodies contain one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Unibodies are another type of antibody fragment that lacks the hinge region of an IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent binding region of IgG4 antibodies.Antigen-binding fragments can be incorporated into single domain antibodies, SMIPs (small modular immunopharmaceuticals), maxibodies, minibodies, intrabodies, diabodies, triabodies, and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabody", "tribody" or "tetrabody" refers to a small antibody fragment with multiple antigen-binding sites (2, 3 or 4), which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites. Antigen-binding fragments may also be incorporated into single chain molecules that contain a pair of tandem Fv fragments (VH-CH1-VH-CH1). Together with a complementary light chain polypeptide, it forms a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and US Pat. No. 5,641,870).

[0100] The term "antibody mimic" is intended to refer to molecules that can mimic the ability of antibodies to bind antigens, but are not limited to natural antibody structures. Examples of such antibody mimics include, but are not limited to, adnectins, affibodies, designed ankyrin repeat proteins (DARPins), anticalins, avimers, and versabodies, all of which utilize binding structures that mimic traditional antibody binding but arise from and function via distinct mechanisms. Antigen-binding fragments of antibodies can be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fa3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Affibodies are well known in the art and refer to affinity proteins based on a 58 amino acid residue protein domain derived from one of the IgG-binding domains of Staphylococcus aureus protein A. DARPins (designed ankyrin repeat proteins) are well known in the art and refer to antibody mimic DRP (designed repeat proteins) technology developed to exploit the binding capacity of non-antibody proteins. Anticalins are well known in the art and refer to another antibody mimic technology, where the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting proteins, called duocalins. Avimers are well known in the art and refer to another antibody mimic technology, where avimers are derived from natural A-domain containing proteins. Versabodies are well known in the art and refer to another antibody mimic technology, where they are small proteins of 3-5 kDa with more than 15% cysteines, which form a scaffold with high disulfide density, replacing the hydrophobic core that typical proteins have. Such antibody mimics may be included within the scaffold. The term "scaffold" refers to a polypeptide platform for the engineering of novel products with tailored functions and characteristics.

[0101] Fibronectin scaffolds are based on type III fibronectin domains (e.g., the tenth module (tenth Fn3 domain) of type III fibronectin). Type III fibronectin domains have seven or eight β-strands distributed between two β-sheets that themselves pack against each other to form the core of the protein, and further contain loops (similar to CDRs) that connect the β-strands to each other and expose them to the solvent. There are at least three such loops at each end of the sandwiched β-sheets, where the edges are the boundaries of the protein perpendicular to the direction of the β-strands (see U.S. Pat. No. 6,818,418). Although these fibronectin-based scaffolds are not immunoglobulins, the overall fold is closely related to that of the variable region of the heavy chain, the functionally smallest antibody fragment that contains all the antigen recognition units in camel and llama IgG. Due to this structure, non-immunoglobulin antibodies mimic antigen-binding properties similar in nature and affinity to those of antibodies. These scaffolds can be used for in vitro loop randomization and shuffling strategies, which resemble the in vivo affinity maturation process of antibodies. These fibronectin-based molecules can be used as scaffolds in which the loop regions of the molecules can be replaced with the CDRs of the invention using standard cloning techniques.

[0102] Ankyrin technology is based on using proteins containing ankyrin-derived repeat modules as scaffolds to carry variable regions that can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel α-helices and a β-turn. The binding of the variable regions is mostly optimized by using ribosome display.

[0103] Avimers are derived from naturally occurring A-domain containing proteins, such as LRP-1. These domains are used in nature for protein-protein interactions, and over 250 proteins in humans are structurally based on "A-domain" monomers (2-10) linked via amino acid linkers. For example, methods described in U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844 can be used to generate avimers capable of binding to target antigens.

[0104] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the backbone of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This backbone domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with many ligand variants (see, for example, U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies and they have a molecular weight of 6 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies.

[0105] Anticalins are products developed by Pieris ProteoLab. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body fluids. The structure of the protein is reminiscent of immunoglobulins, with hypervariable loops on a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain, with 160-180 amino acid residues, only slightly larger than one immunoglobulin domain. The set of four loops, which make up the binding pocket, shows remarkable structural plasticity and tolerates a wide variety of side chains. Thus, the binding site can be reshaped in a proprietary process to recognize defined target molecules with various shapes with high affinity and specificity. Anticalins were generated by mutagenesis of a set of four loops using bilin-binding protein (BBP) from Pieris Brassicae, a lipocalin family protein. An example of a patent application describing anticalins is PCT Publication No. WO199916873.

[0106] Affilin molecules are small non-immunoglobulin proteins designed for specific affinity to proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a scaffold protein of different human origin. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are used, one of which is the gamma crystallin, a human structural intraocular lens protein, and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, show high temperature stability, and are almost resistant to pH changes and denaturants. This high stability is mainly due to the amplified beta sheet structure of the protein. Examples of "ubiquitin-like" proteins are described in WO2004106368.

[0107] Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable, providing long shelf lives. Further information regarding Versabodies can be found in U.S. Patent Application No. 2007 / 0191272, which is incorporated herein by reference in its entirety.

[0108] Aptamers are stable DNA or RNA ligands that bind with high affinity and specificity to target antigens, such as small molecules, peptides, proteins, cells, and tissues. These molecules present many advantages: they have a long shelf life, exhibit low batch-to-batch variability, show low or no immunogenicity, and can freely incorporate chemical modifications for enhanced stability and targeting ability. Thus, they find their application in various fields ranging from therapeutics, drug delivery, diagnostics, and functional genomics to biosensing. Aptamers are generally developed in vitro by a very well-defined iterative procedure known as SELEX (Self-Enhanced Exogenous Leukemia-Related Genetic Algorithm). RNA aptamer technology is described in U.S. Patent Nos. 5,789,157; ​​5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774; and 6,387,620.

[0109] The ligand binding domain is capable of binding to a ligand of interest.

[0110] Upon binding to the ligand-binding domain, the ligand of interest will activate the nucleic acid-based sensor component.

[0111] Depending on the condition or disease to be treated, one of skill in the art will know to target a given ligand of interest and therefore select an appropriate ligand binding domain.

[0112] Ligands of interest may be inorganic, such as ions, etc. The presence of these ligands, especially in abnormal amounts, may reflect dysregulation of the microenvironment and may occur in individuals suffering from pathological conditions or diseases, such as cancer.

[0113] Ligands of interest may also be organic, such as peptides or proteins. Such ligands are often present on the outer walls of target cells and organisms, or in the form of free molecules. They are recognized and targeted by the immune system of an individual. Protein ligands may be present, for example, on tumor cells, on "self" cells in the case of autoimmune diseases, on cells derived from transplanted tissues, on viruses, on bacteria, and on other infectious microorganisms and allergens.

[0114] In certain embodiments, the ligand of interest is an antigen.

[0115] In certain embodiments, the ligand of interest is selected from one or more tumor antigens, one or more autoantigens, one or more alloantigens, one or more viral antigens, one or more bacterial antigens, one or more allergen antigens, or one or more markers of neuropathic disorders.

[0116] More generally, any molecule indicative of homeostatic dysregulation and pathological conditions can be used as a ligand.

[0117] Many of these antigens have been previously identified during the study of particular conditions and diseases and are well known in the art.

[0118] Tumor antigens include those expressed by tumor cells, in the case of tumors or cancers, listed further below herein. It may be a tumor-associated antigen, or the perfect candidate would be a tumor-specific antigen, although this is rare.

[0119] Examples of tumor antigens include prostate-specific antigen (PSA), mesothelin, human epithelial receptor 2, oncogenic viral proteins (derived from human papillomavirus and Epstein-Barr virus), carcinoembryonic antigen (CEA), carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC (mucin)-1, epithelial tumor antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE).

[0120] Viral antigens include those antigens presented by the viruses provided in the list further below herein. Bacterial antigens include those antigens presented by the viruses provided in the list further below herein.

[0121] Autoantigens are antigens within an individual's body that are not normally available to the immune system. In the context of autoimmune diseases, autoantigens are those intracellular proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA, and post-translationally modified antigens against which autoantibodies are directed. Indeed, infectious or physical tissue injury, especially in genetically susceptible individuals, or defects in the removal of apoptotic cells by phagocytosis, can terminate immune tolerance to autoantigens and cause autoimmune diseases.

[0122] In some embodiments, the ligand of interest is factor VIII, such as to prevent or treat hemophilia A. Hemophilia A is a disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is the conventional treatment, but a major problem with this type of treatment is that 20% to 30% of these patients are immunologically intolerant to this human protein and therefore produce anti-FVIII neutralizing antibodies (inhibitors). The goal is to make the patient tolerant to this protein so that their immune system stops recognizing it as foreign and stops degrading it.

[0123] Transmembrane domains of the B cell receptor or T cell receptor As used herein, the term "transmembrane domain of a B cell receptor or a T cell receptor" preferably refers to a domain that is the transmembrane domain of the membrane-anchored immunoglobulin part of the B-cell receptor, i.e. the transmembrane domain of the membrane-anchored immunoglobulin heavy chain, or -refers to the TCR transmembrane domain, which connects the extracellular variable-like region (VR) and constant-like region (CR) and the stalk segment that allows for disulfide bond pairing of the T cell receptor to the short cytoplasmic tail. The transmembrane helices of both the α and β chains are unusual in that they contain positively charged residues (two and one, respectively) along with hydrophobic transmembrane helices.

[0124] These domains are essential for anchoring the sensor components to the cell.

[0125] Signal transduction domains that control cell activation of the B cell receptor or T cell receptor Interaction of the extracellular ligand recognition domain of the sensor component with an antigen of interest will result in activation of the signaling domain of the B cell receptor, T cell receptor, or chimeric immunoreceptor, thus resulting in various signaling pathways within the host cell. The B cell receptor and T cell receptor signaling pathways are essential for proper B cell and T cell development, activation, proliferation, differentiation, and, consequently, humoral immune responses.

[0126] As used herein, "signaling domains that control cellular activation of the B cell receptor" refers to disulfide-linked dimers of immunoglobulin (Ig)-α and immunoglobulin-β / γ subunits, also known as CD79a and CD79b, which are the products of the mb-1(α) and B29(β / γ) genes.

[0127] Binding of antigen to the B cell receptor induces membrane translocation and aggregation of B cell receptor components, leading to phosphorylation of ITAMs (immunoreceptor tyrosine-based activation motifs) in the cytoplasmic tails of CD79a and CD79b. The latter involves LYN, a member of the SRC kinase family. The phosphorylated ITAMs recruit spleen tyrosine kinase (SYK) to the receptor, where it becomes activated by tyrosine phosphorylation and propagates signal activation to downstream signaling proteins. SYK activation plays a key role in B cell receptor signaling, initiating the formation of the B cell receptor signalosome, adaptor proteins such as CD19 and B cell linker (BLNK), and Bruton's tyrosine kinase (BTK), and signaling enzymes such as PLCγ2, PI3K, and Vav. Signals emanating from such signalosomes initiate and regulate downstream signaling cascades, including the RAS / RAF / MEK / ERK pathway, which are important in determining B cell fates, such as proliferation, survival, differentiation, and cell death.

[0128] Thus, in certain embodiments, the signaling domain that controls cellular activation of the B cell receptor comprises CD79a and CD79b.

[0129] In certain embodiments, the signaling domain that controls cellular activation of the B cell receptor comprises a protein domain that binds to immunoglobulins anchored at the cell surface.

[0130] As used herein, "signaling domains that control cellular activation of the T cell receptor" refers to the signaling subunits gamma, delta, epsilon, and zeta of the T cell receptor (CD3 complex) with or without combination with costimulatory domains (such as ICOS (inducible T cell costimulatory molecule), 41BB, CD28, etc.).

[0131] The stimulus that drives T cell activation is a foreign antigen, particularly a peptide, bound to a molecule encoded by the major histocompatibility complex (MHC) presented on the surface of professional antigen-presenting cells (APCs), such as dendritic cells (DCs).

[0132] Thus, in a particular embodiment, the signaling domain that controls the cellular activation of the T cell receptor comprises the CD3 complex, which contains the subunits γ, δ, ε and ζ of the T cell receptor. Binding of the major histocompatibility complex on the T cell leads to phosphorylation of the ITAM motif by Lck. ZAP-70 binds to the ITAM of the phosphorylated ζ chain and becomes phosphorylated and activated. Activated ZAP-70 then phosphorylates LAT and SLP-70, which are bound to GADS. GADS:SLP-76:LAT recruits and activates PLC-γ through its phosphorylation by Itk. PLC-γ will then generate calcium influx and indirect activation of NFAT (nuclear factor of activated T cells) and NF-κB transcription factors.

[0133] The presence of sequence (c) encoding a signalling domain, which controls cellular activation of the B cell receptor or T cell receptor, is optional in the nucleic acid-based sensor component of the invention.

[0134] Indeed, in certain embodiments, cells, particularly B cells or T cells, that will be transformed with the nucleic acid system of the invention will already contain on their surface a signaling domain that controls the cellular activation of the B cell receptor or T cell receptor. In this case, the sensor component of the nucleic acid system of the invention may only comprise (a) a sequence encoding at least one extracellular ligand recognition domain and (b) a sequence encoding the transmembrane domain of the B cell receptor (BCR) or T cell receptor (TCR). When activated by a ligand via the extracellular ligand recognition domain, this sensor component will consequently activate the signaling domain already present on the surface of the B cell or T cell, which will then activate the transducer / effector component. A similar mechanism applies to chimeric receptors.

[0135] In such cases, the sensor components will not form a complete receptor such as a B cell receptor, a T cell receptor, a chimeric immune receptor (TCR), e.g., a CAR-T cell, a CAR-NK cell, a B cell antibody receptor (BAR) or a chimeric autoantibody receptor T (CAAR-T) cell. The membrane-anchored sensor components will restrict the specificity of the target antigen and take some of the elements of such receptors and use other elements, e.g., signaling domains and transcription factors, that are already naturally present on / within the surface of the cell.

[0136] In another embodiment, the nucleic acid-based sensor component of the invention will include (a) a sequence encoding at least one extracellular ligand recognition domain, (b) a sequence encoding a transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR), and (c) a sequence encoding a signaling domain that controls cellular activation of the B cell receptor or the T cell receptor. This embodiment will be particularly applicable when the cellular host does not contain such signaling domains on its surface.

[0137] In a particular embodiment, the nucleic acid based sensor component forms an immunoglobulin, in particular an antigen receptor.

[0138] In certain embodiments, the extracellular ligand recognition domain (a), the transmembrane domain (b) and optionally the signaling domain (c) form a B cell receptor, a T cell receptor, a chimeric immune receptor (CIR), such as a CAR-T cell, a CAR-NK cell, a B cell antibody receptor (BAR) or a chimeric autoantibody receptor T (CAAR-T) cell, in particular a B cell receptor.

[0139] In particular, when the sensor component forms a B cell receptor, the sensor component preferably does not contain (c) a sequence encoding a signaling domain that controls cellular activation of the B cell receptor or T cell receptor.

[0140] In particular, when the sensor component forms a T cell receptor, the sensor component preferably does not contain (c) a sequence encoding a signaling domain that controls cellular activation of the B cell receptor or the T cell receptor.

[0141] In particular, when the sensor component forms a chimeric immune receptor, such as a CAR-T cell, a CAR-NK cell, a B cell antibody receptor, or a CAAR-T cell, the sensor component preferably comprises (c) a sequence encoding a signaling domain that controls cellular activation of the T cell receptor.

[0142] Definitions of B cell receptors, T cell receptors, and chimeric immunoreceptors are provided above.

[0143] Thus, in some embodiments, the nucleic acid-based sensor components disclosed herein encode proteins that include, from their N-terminus to their C-terminus, a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor, a transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR), and at least one extracellular ligand recognition domain.

[0144] In some other embodiments, the nucleic acid-based sensor components disclosed herein encode a protein that consists, from its N-terminus to its C-terminus, of a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor, a transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR), and at least one extracellular ligand recognition domain.

[0145] Transducer / Effector Components pNR4A1 promoter The effector component of the nucleic acid system of the present invention comprises (d) a sequence encoding the pNR4A1 promoter, or a functional fragment thereof.

[0146] The NR4A1 gene, also known as the Nur77 gene, is an immediate early gene whose expression is rapidly upregulated by B cell receptor or T cell receptor signaling in mouse cells and human thymocytes. It has been shown to be specifically induced in human T cells and B cells after T cell receptor and B cell receptor stimulation, respectively.

[0147] Thus, the pNR4A1 promoter is an inducible promoter that is activated upon T cell receptor or B cell receptor activation.

[0148] The pNRA41 sequence is known in the art. The isolated promoter sequence consists of SEQ ID NO:1.

[0149] In a particular embodiment, the transducer / effector component of the nucleic acid system of the invention comprises the pNR4A1 promoter consisting of SEQ ID NO:1.

[0150] In another embodiment, the transducer / effector component of the nucleic acid system of the invention comprises a functional fragment of the pNR4A1 promoter, said functional fragment therefore consisting of a functional fragment of the sequence SEQ ID NO:1.

[0151] As used herein, a "functional fragment" of the pNR4A1 promoter is a fragment that maintains the promoter activity of pNR4A1. Thus, a functional fragment of pNR4A1 can be activated upon activation of the T cell receptor or B cell receptor, just like the complete NR4A1. It can also be recognized by RNA polymerase and associated transcription factors, and therefore can initiate transcription of genes downstream of the fragment.

[0152] In a particular embodiment, the functional fragment of the pNR4A1 promoter has a length of 200 bp to 2210 bp, in particular of 500 bp to 2210 bp.

[0153] Fragments from 200 bp to 2210 bp include fragments of the following lengths: [ka] TIFF2025510588000002.tif235165 TIFF2025510588000003.tif236165 TIFF2025510588000004.tif234165 TIFF2025510588000005.tif125165

[0154] Indeed, the present inventors have discovered that pNR4A1 fragments as small as 200 bp, and especially as small as 500 bp, can be used as efficient promoters.

[0155] In a particular embodiment, the pNR4A1 promoter is selected from a fragment (functional fragment) having a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, and SEQ ID NO:23, in particular from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0156] In certain embodiments, the pNR4A1 promoter is selected from fragments having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, or SEQ ID NO:23, in particular to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 ... SEQ ID NO:5, SEQ ID NO:22, or SEQ ID NO:23, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, or SEQ ID NO:23.

[0157] In a particular embodiment, the pNR4A1 promoter is selected from a fragment consisting of the sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22 or SEQ ID NO:23, in particular the sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0158] In particular, the pNR4A1 promoter is a fragment having the sequence of SEQ ID NO:5.

[0159] In particular, the pNR4A1 promoter is a fragment having at least 90% sequence identity to SEQ ID NO:5, in particular at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5.

[0160] In a particular embodiment, the pNR4A1 promoter is a fragment consisting of SEQ ID NO:5.

[0161] A sequence encoding at least one effector protein of interest. Activation of the pNR4A1 promoter or a functional fragment thereof will result in the transcription of at least one effector protein of interest.

[0162] In the context of the present invention, the term "effector protein" refers to a protein that is involved in the regulation of a biological signaling pathway. Preferably, the effector protein is an immune effector protein or a cell death-inducing effector protein.

[0163] By "immune effector protein" is meant herein a protein that is involved in regulating pathways of the immune system. Examples of immune effector proteins include cytokines.

[0164] By "cell death-inducing effector protein" is meant herein a protein that is involved in a cell death signaling pathway, in particular the apoptosis signaling pathway.

[0165] The effector protein of interest may be formed by a single peptide or protein EfP, or by the combination of two effector protein subunits EfP1 and EfP2, the combination of these two subunits forming the active effector protein EfP. By "combination" herein is meant any interaction linking the two subunits as defined above, such as a disulfide bond.

[0166] The effector proteins of interest according to the present invention will vary depending on the disease to be treated or the physiological condition to be modified.

[0167] Thus, when treating a tumor, the at least one effector molecule may be selected from a pro-inflammatory molecule or a cell death-inducing molecule and mixtures thereof.

[0168] When treating an immune disease, the at least one effector molecule may be selected from a tolerizing molecule or an anti-inflammatory cytokine, and mixtures thereof.

[0169] In the case of avoiding graft rejection, the at least one effector molecule may be selected from tolerizing / tolerogenic molecules and mixtures thereof.

[0170] When treating allergies, the at least one effector molecule may be selected from antihistamine molecules and mixtures thereof.

[0171] When treating an infectious disease, the at least one effector molecule may be selected from antiviral proteins and mixtures thereof.

[0172] When treating a neurological disorder, the at least one effector molecule may be selected from a matrix metalloproteinase inhibitor, a neurotrophic factor such as nerve growth factor (which promotes nerve growth), an antibody against beta amyloid sheets, an interfering RNA, a modified dopamine, and the like.

[0173] In certain embodiments, the at least one effector protein of interest is an immunostimulatory or immunosuppressive protein.

[0174] In particular, the at least one effector protein of interest is selected from proinflammatory cytokines, such as IL-18, gamma-interferon, and tumor necrosis factor; anti-inflammatory cytokines, such as IL-10 and IL-4; costimulatory molecules, such as CD80, CD86, and CD40; and inhibitory molecules, such as Fas ligand and Fas.

[0175] However, any therapeutic molecule that can be genetically encoded may be instrumented as an effector of the circuit.

[0176] In a particular embodiment, (i) the sensor component and (ii) the transducer / effector component of the nucleic acid system of the invention are both under the control of the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0177] In a particular embodiment, said pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof is the only promoter present in the nucleic acid system. Thus, according to a particular embodiment, (i) the sensor component and (ii) the transducer / effector component of the nucleic acid system of the invention are both under the control of the same pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0178] When the (i) sensor component and (ii) transducer / effector component of the nucleic acid system of the invention are both under the control of the same pNR4A1 promoter consisting of SEQ ID NO: 1 or a functional fragment thereof, the nucleic acid system further comprises a separating sequence of an open reading frame. Such a sequence may allow polycistronic expression of the two components (i.e. sensor and transducer / effector). In particular, said separating sequence of an open reading frame is located between the (i) sensor component and the (ii) transducer / effector component.

[0179] Thus, in a particular embodiment, the nucleic acid system further comprises a separating sequence of an (iii) open reading frame located between the (i) sensor component and the (ii) transducer / effector component, wherein both the (i) sensor component and the (ii) transducer / effector component are under the control of the same pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0180] An open reading frame separation sequence is a nucleic acid sequence used to obtain more than one separate protein from an open reading frame under the control of a single promoter. Examples of open reading frame separation sequences are known in the art. Thus, the sensor and effector components can be placed in the same transcription unit using an open reading frame separation sequence between them.

[0181] Examples of isolated sequences of open reading frames include two amino acid self-cleaving peptides. 2A peptides consist of peptides of 18-22 amino acids in length that can induce ribosome skipping during translation of proteins in cells. This allows the generation of two proteins, i.e. (i) a sensor component and (b) a transducer / effector component, by causing the failure of the ribosome to create a peptide bond. These sequences are described, for example, in Liu et al. (Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017)).

[0182] Another separating sequence of open reading frames that can be used to allow co-expression of two proteins is the internal ribosome entry site (IRES) (Maillot et al., Viral internal ribosomal entry sites: four classes for one goal, WIREs RNA 2018, 8:e1458), e.g., an IRES from a picornavirus.

[0183] The separation sequence of an open reading frame may also contain a cleavage sequence recognized by an intracellular protease.

[0184] Another example of an isolating sequence of an open reading frame is a linker sequence of 74 nucleotides (nt) between a first ORF and a second ORF, such as a sensor component and an effector / transducer component, which does not contain any ATG triplets and provides an optimal distance between the stop codon of the first ORF and the start codon of the second ORF to allow reinitiation of translation (Kozak, M. 1987. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol. 7:3438-3445).

[0185] In certain embodiments, the ORF separation sequence may be selected from a two amino acid self-cleaving peptide, such as T2A, an IRES, a cleavage sequence recognized by an intracellular protease, or a linker sequence. In particular, the ORF separation sequence is a two amino acid self-cleaving peptide.

[0186] In a particular embodiment, the ORF isolation sequence is T2A having the nucleic acid sequence of SEQ ID NO:18.

[0187] Therefore, the sensor and effector components can be located within the same transcription unit using the T2A sequence.

[0188] According to a particular embodiment, the present invention provides a method for producing a method for the treatment of a pulmonary arthritis, comprising: (i)(a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) optionally a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor; A sensor component comprising a sequence encoding (ii)(d) the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof; and (e) at least one effector protein of interest. a transducer / effector component comprising a sequence encoding (iii) an open reading frame isolating sequence located between the (i) sensor component and the (ii) transducer / effector component; (wherein (i) the sensor component and (ii) the transducer / effector component of the nucleic acid system are both under the control of the pNR4A1 promoter consisting of SEQ ID NO:1) The present invention relates to a nucleic acid system comprising:

[0189] vector A further subject of the present invention relates to a vector comprising a nucleic acid system as defined above.

[0190] Typically, the nucleic acid system of the present invention is a DNA or RNA molecule, which may be comprised in any suitable vector, such as a linear DNA, a plasmid, a cosmid, an episome, an artificial chromosome, a phage, or a viral vector.

[0191] As used herein, the terms "vector," "cloning vector," and "expression vector" refer to a vehicle that introduces DNA or RNA sequences (e.g., foreign genes) into a cell, thereby transforming it and promoting the expression (e.g., transcription and translation) of the introduced sequences.

[0192] Such vectors may further comprise regulatory sequences, such as promoters, enhancers, terminators, etc., that cause or direct expression of the nucleic acid system upon administration to a subject.

[0193] Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0194] In certain embodiments, the vector further comprises at least one safety sequence that will allow the end user to activate and deactivate the nucleic acid system of the present invention at will. The safety sequence will add another level of regulation to the system, and in particular will avoid ectopic expression of the system in the absence of the antigen of interest. Safety sequences that can be used according to the present invention include inducible promoters, suicide genes, Boolean logic gates, etc.

[0195] In certain embodiments, a vector as described herein further comprises at least one sequence encoding an inducible promoter and an effector protein of interest.

[0196] In a particular embodiment, the vector as described further comprises at least one sequence encoding a suicide gene.

[0197] As used herein, a "suicide gene" is a gene that will send an apoptotic death signal to the cell expressing it, causing its destruction. By including such a gene in the vector according to the present invention, any serious adverse events can be avoided by switching off the nucleic acid system.

[0198] Any expression vector for animal cells can be used as long as it is possible to insert and express a gene encoding a human antibody C region.

[0199] Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4- (Miyaji H et al. 1990), and the like. Other examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral vectors, retroviral vectors, herpes viral vectors, and adeno-associated viral vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO94 / 19478.

[0200] In a particular embodiment, said vector is a viral vector, in particular a retroviral vector.

[0201] Retroviral vectors can be vector plasmids that contain the nucleic acid system that is to be introduced into target cells.Thus, retroviral vectors typically contain a retroviral backbone, which contains the retroviral cis-acting gene sequences required for the vector to infect target cells, and the nucleic acid system described in the present invention.Thus, retroviral backbone typically contains long terminal repeats (LTRs) for the control of transcription and integration, psi sequences required for encapsidation, and primer binding sites (PBS) and polypurine track (PPT) sequences required for reverse transcription of the retroviral genome.

[0202] By "retrovirus" is meant a virus, a member of the Retroviridae family, whose genome consists of an RNA molecule and contains the enzyme reverse transcriptase. Retroviruses are classified as oncogenic viruses, lentiviruses, and spumaviruses. Preferably, the retrovirus is an oncogenic virus, such as Moloney leukemia virus, ALS, Rous sarcoma virus, or Mason-Pfizer monkey virus (MPMV), a lentivirus, such as human immunodeficiency virus (HIV)-1, HIV-2, simian immunodeficiency virus, equine infectious anemia virus, or a spumavirus, such as human foamy virus. The genomes of these retroviruses are readily available in databanks. More preferably, the retrovirus is a lentivirus, in particular HIV-1, HIV-2, or simian immunodeficiency virus.

[0203] As is well known to those skilled in the art, various types of transfer retroviral backbones can be used depending on the type of retroviral packaging system intended, i.e., first, second, third, or fourth generation retroviral packaging system.

[0204] As is well known to those skilled in the art, in first generation retroviral packaging systems, live viral particles are produced from one transfer retroviral vector carrying all retroviral genes, i.e. genes encoding retroviral core proteins, enzymes, and accessory factors, together with a transgene, and from a separate plasmid carrying the envelope gene. The transgene is typically under the control of the wild-type 5'-long terminal repeat (LTR).

[0205] In contrast, as is well known to those skilled in the art, in second generation retroviral packaging systems, five of the nine retroviral genes have been deleted, leaving only the gag / pol and tat / rev regions. The transgene is typically under the control of the wild-type 5'-LTR. The gag / pol / tat / rev regions are typically present on a separate plasmid.

[0206] As known to those skilled in the art, third generation retroviral packaging systems contain only the gag, pol, and rev genes. The gag / pol and rev genes are typically present on two separate plasmids. The transgene is typically under the control of a chimeric 5'-LTR to ensure transcription in the absence of tat. In this chimeric 5'-LTR, the U3 region is typically replaced by a constitutively active promoter / enhancer, such as Rous sarcoma virus or cytomegalovirus.

[0207] Finally, as is well known to those skilled in the art, in fourth generation retroviral packaging systems, the gag and pol genes are further codon optimized and reside in two separate plasmids.

[0208] Preferably, the retroviral backbone contained in a retroviral vector as described herein is a third or fourth generation retroviral backbone.

[0209] In a particularly preferred embodiment, the retroviral backbone contained in the retroviral vector is a self-inactivating retroviral backbone.

[0210] By "self-inactivating retroviral backbone" herein is meant a retroviral construct that has a deletion within the U3 sequence of the 3'-LTR of the construct, which after replication also results in a deletion within the promoter and enhancer of the 5'-LTR and prevents transcription from cell-specific internal promoters, which may activate otherwise silent intracellular oncogenes.

[0211] In a particularly preferred embodiment, the retroviral backbone contained in a retroviral vector as described herein is a third or fourth generation self-inactivating retroviral backbone, more particularly a third or fourth generation self-inactivating lentiviral backbone.

[0212] Thus, in certain embodiments, the retroviral backbone contained in the retroviral vector comprises, in sequence, (11) a modified 5'LTR containing a cytomegalovirus enhancer replaced in the U3 region, (12) psi and gag sequences, (13) a central polypurine tract (cPPT) / DNA flap sequence, (14) a Rev response element sequence (RRE), (15) a woodchuck hepatitis virus posttranscriptional regulatory element sequence (WPRE), and (16) a self-inactivating 3'LTR containing a deletion in the U3 region that renders the integrated proviral 5'LTR transcriptionally inactive.

[0213] By "Rev response element sequence" or "RRE" is meant herein a highly structured RNA segment present within the envelope coding region of unspliced ​​and partially spliced ​​viral mRNAs. In the presence of Rev, retroviral mRNAs containing the RRE can be exported from the nucleus to the cytoplasm for translation and further packaging.

[0214] By "central polypurine tract (cPPT) / DNA flap sequence" is meant herein the initiation site from which DNA synthesis typically begins with the polypurine tract (PPT) during lentiviral reverse transcription. The resulting plus-strand overlap is called the central DNA flap (99 nucleotides), which is known to play a role in enhancing lentiviral proviral nuclear import. In the field of lentivector technology, it is now common knowledge that the introduction of this cis-acting cPPT sequence into a transfer vector plasmid greatly enhances the transduction efficiency of the vector in certain cell types, particularly in hematopoietic stem cells, as described, for example, in Van Maele et al. (2003) J. Virol. 77:4685-4694.

[0215] By "Woodchuck Hepatitis Virus Post-transcriptional Regulatory Sequence" or "WPRE" is meant herein a DNA sequence that, when transcribed, creates a tertiary structure that enhances expression. Thus, the presence of the WPRE, particularly in combination with the cPPT, allows for increased transduction efficiency and expression of the transgene. By WPRE is also meant herein an improved WPRE, such as the improved WPRE described in Zanta-Boussif et al. (2009) Gene Therapy 16:605-619.

[0216] In this particular embodiment, the nucleic acid system according to the invention is preferably located in reverse orientation between sequences (14) and (15).

[0217] Thus, in a particular embodiment, a retroviral vector of the invention comprises, in order: (11) A modified 5'LTR containing a cytomegalovirus enhancer replaced for the U3 region; (12) psi and gag sequences, (13) central polypurine tract (cPPT) / DNA flap sequence, (14) Rev response element sequence (RPE), (15) Woodchuck hepatitis virus posttranscriptional regulatory element sequence (WPRE), and (16) A self-inactivating 3' LTR that contains a deletion in the U3 region, rendering the integrated proviral 5' LTR transcriptionally inactive. (wherein the nucleic acid system is located in reverse orientation between sequences (14) and (15)).

[0218] In a particular embodiment, the vector is a lentiviral vector.

[0219] In a particular embodiment, (i) the sensor component and (ii) the transducer / effector component of the nucleic acid system of the invention are comprised in a single lentiviral vector.

[0220] In this case, the vector may be considered an "all-in-one" vector and may be labeled as such throughout the specification. An example of such a vector is provided in Example 2 below.

[0221] Thus, according to a particular embodiment, the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: (i)(a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) optionally a signaling domain that controls cellular activation of a B cell receptor or a T cell receptor; A sensor component comprising a sequence encoding (ii)(d) the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof; and (e) at least one effector protein of interest. a transducer / effector component comprising a sequence encoding (iii) an open reading frame isolating sequence located between the (i) sensor component and the (ii) transducer / effector component; (wherein (i) the sensor component and (ii) the transducer / effector component of the nucleic acid system are both under the control of the pNR4A1 promoter consisting of SEQ ID NO:1) The present invention relates to a vector comprising a nucleic acid system comprising the nucleic acid system.

[0222] Each of elements (a) through (e) and (iii) may be as described above.

[0223] Thus, at least one effector protein of interest (e) and elements of the sensor component, (a) at least one extracellular ligand recognition domain, (b) the transmembrane domain of the B cell receptor (BCR) or T cell receptor (TCR), and, if present, (c) the signal transduction domain controlling cellular activation of the B cell receptor or T cell receptor, are all under the control of the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0224] In particular, the pNR4A1 promoter or a functional fragment thereof is the only promoter present in the vector, in which case the pNR4A1 promoter or a functional fragment thereof controls both the sensor and the effector, which provides an autoregulatory feedback loop.

[0225] In certain embodiments, at least one effector protein of interest (e) and elements of the sensor component, (a) at least one extracellular ligand recognition domain, (b) the transmembrane domain of the B cell receptor (BCR) or T cell receptor (TCR), and, if present, (c) the signaling domain controlling cellular activation of the B cell receptor or T cell receptor, are all under the control of a single pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0226] In certain embodiments, at least one effector protein of interest (e) and the elements of the sensor component, (a) at least one extracellular ligand recognition domain, (b) the transmembrane domain of the B cell receptor (BCR) or T cell receptor (TCR), and, if present, (c) the signaling domain controlling cellular activation of the B cell receptor or T cell receptor, and the pNR4A1 promoter or functional fragment thereof consisting of SEQ ID NO:1, are all in the same orientation.

[0227] According to a particular embodiment, the vector according to the invention may comprise, in order, the pNR4A1 promoter consisting of SEQ ID NO: 1 or a functional fragment thereof, at least one effector protein of interest, an ORF isolation sequence, in particular a T2A linker of SEQ ID NO: 18, a sensor component, a Woodchuck Hepatitis Virus post-transcriptional regulatory element sequence (WPRE) and a long terminal repeat (LTR).

[0228] In a particular embodiment, the vector according to the invention may comprise, from 5' to 3', the pNR4A1 promoter consisting of SEQ ID NO: 1 or a functional fragment thereof, at least one effector protein of interest, an ORF isolation sequence, in particular a T2A linker of SEQ ID NO: 18, a sensor component, a Woodchuck Hepatitis Virus post-transcriptional regulatory element sequence (WPRE) and a long terminal repeat (LTR).

[0229] According to another embodiment, the sensor component is contained in a first vector and the transducer / effector component is contained in a second vector. Thus, according to a particular aspect, the subject matter of the present invention also relates to a first vector comprising a sensor component; - a second vector containing a transducer / effector component. Also described is a kit comprising:

[0230] The vector according to this embodiment may be as described above.

[0231] In a particular embodiment, the first vector is a retroviral vector, in particular a lentiviral vector. In a particular embodiment, the second vector is a retroviral vector, in particular a lentiviral vector. In a particular embodiment, both the first and second vectors are retroviral vectors, in particular lentiviral vectors.

[0232] In a particular embodiment, in the first vector containing the sensor component, the elements (a) at least one extracellular ligand recognition domain, (b) the transmembrane domain of the B cell receptor (BCR) or T cell receptor (TCR), and, if present, (c) the signaling domain controlling cellular activation of the B cell receptor or T cell receptor are under the control of the pNR4A1 promoter consisting of SEQ ID NO:1 or a functional fragment thereof.

[0233] Use and Instructions Transformed cells A further aspect of the invention relates to a cell transfected, infected or transformed by a nucleic acid, by a vector or by a kit according to the invention.

[0234] The term "transformation" means the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA sequence, or RNA sequence into a cell so that the cell will express the introduced gene or sequence and express a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0235] The nucleic acid system of the present invention can be used to produce one or more effector proteins in a suitable expression system. The term "expression system" refers to a host cell and a competent vector under suitable conditions for the expression of a protein, e.g., encoded by foreign DNA carried in a vector and introduced into the host cell.

[0236] Common expression systems include mammalian cell lines (e.g., Vero cells, Chinese hamster ovary cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. The present invention also relates to a method for producing a recombinant host cell expressing a nucleic acid system according to the present invention, said method comprising the steps of (i) introducing, in vitro or ex vivo, in particular ex vivo, a nucleic acid system, vector or kit as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, in particular ex vivo, the obtained recombinant host cell, and (iii) optionally selecting cells expressing the system.

[0237] In particular, the nucleic acid system according to the invention may also be directly introduced in vivo using a viral vector or a DNA vector.

[0238] Such recombinant cells may be transfected into a patient in need thereof as part of a therapeutic strategy, as presented in the applications further below.

[0239] In a particular embodiment, said cells may be selected from B cells and T cells, which are described further above herein.

[0240] Thus, a particular embodiment of the present invention relates to B cells or T cells transfected with a nucleic acid system, with a vector, with a kit of the invention as described above.

[0241] In a preferred embodiment, the B and T cells transfected according to the invention are those of the patient to be treated (autologous setting). The B and T cells of said individual can be harvested ex vivo, transformed to introduce the nucleic acid system of the invention into their genome and reintroduced into the individual's organism.

[0242] In another embodiment, the B and T cells transfected according to the invention are those of a donor (allogeneic setting).

[0243] Pharmaceutical Compositions A particular subject of the present invention refers to a pharmaceutical composition comprising a nucleic acid system, a vector, a kit or a cell as previously described, and a pharma- ceutically acceptable vehicle.

[0244] "Pharmaceutically" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when appropriately administered to a mammal, especially a human.

[0245] A pharma- ceutically acceptable vehicle or excipient refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0246] In the pharmaceutical compositions of the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the nucleic acid system, vector, kit or cells can be administered to animals and humans in a unit dosage form and in admixture with conventional pharmaceutical supports.

[0247] Suitable unit dosage forms include oral route dosage forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and intranasal dosage forms, and rectal dosage forms. Application of herbal medicines may be performed for specific delivery to the small or large intestine.

[0248] Preferably, the pharmaceutical composition contains a vehicle that is pharma- ceutically acceptable for injectable formulations. These may be, in particular, isotonic, sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dried, in particular lyophilized, compositions that allow the reconstitution of an injectable solution when sterile water or saline is added, as the case may be. Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0249] Applicable As mentioned above, the effectiveness of the nucleic acid system of the present invention can be applied to many pathologies, such as tumors, autoimmune diseases, transplant rejection, allergies, neurological disorders, and infectious diseases.Indeed, the main advantage of this approach is its complete reprogramming ability in terms of recognized signals and output functions, which can be applied to targeted diseases with the same goal of continuous sensing of disease-specific biomarkers and triggering physiological expression of therapeutic molecules in response in vivo.

[0250] The term "tumor" or "cancer" as used herein refers to a condition in which abnormally replicating cells originating from the host are present in a subject in detectable amounts. It includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer or tumor includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" or "tumor" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, cancer may be classified as any of the following histological types, in particular: malignant neoplasms; cell carcinoma; undifferentiated cell carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial cell carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; familial adenomatous polyposis. Adenocarcinoma; solid cell carcinoma; malignant carcinoid tumor; bronchiolar / alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; Follicular adenocarcinoma; Non-encapsulated sclerosing cell carcinoma; Adrenocortical carcinoma; Adenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease of the breast;Pancreatic acinar cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Malignant thymoma;Malignant ovarian stromal tumor;Malignant theca cell tumor;Malignant granulosa cell tumor;Malignant neuroblastoma;Sertoli cell carcinoma;Malignant Leydig cell tumor;Malignant lipid cell tumor;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Malignant glomus tumor ;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stroma sarcoma;Malignant mixed tumor;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Malignant mesenchymoma;Malignant Brenner tumor;Malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian thyroid tumor;choriocarcinoma;malignant mesonephroma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;malignant odontogenic tumor;ameloblastoma;malignant ameloblastoma;ameloblastic fibrosarcoma;malignant pinealoma;chordoma;malignant glioma;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrillary astrocytoma;astroblastoma;glioma;oligodendroglioma;oligodendroglioma;anaplastic neuroectodermal tumor;cerebellar sarcoma;ganglionoblastoma;neuroblastoma The cancer may be, but is not limited to, a tumor; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; lateral granuloma; small lymphocytic lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. The cancer may be a malignant cancer or a non-malignant cancer. Cancers include, but are not limited to, biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasia, leukemia, lymphoma, liver cancer, lung cancer, malignant melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and cell carcinoma and sarcoma. Cancers may be primary or metastatic.

[0251] As used herein, the term "infectious disease" refers to a condition in which an infectious microorganism or pathogen is present in detectable amounts in the blood or in normally sterile tissues or normally sterile compartments of a subject. Infectious microorganisms and pathogens include viruses, mycobacteria, bacteria, fungi, and parasites. The term encompasses both acute and chronic infections, as well as sepsis.

[0252] In some embodiments, the viral infection comprises an infection with one or more viruses selected from the group consisting of Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepeviridae, Leviviridae, Luteoviridae, Mononegavirales, Mosaicviruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae, or Papillomaviridae viruses. Relevant taxonomic RNA virus families include, but are not limited to, Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepeviridae, Leviviridae, Luteoviridae, Mononegavirales, Mosaicviruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnaviruses, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuiviruses, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.In some embodiments, the viral infection is caused by a virus, such as adenovirus, rhinovirus, hepatitis virus, immunodeficiency virus, poliovirus, measles virus, Ebola virus, coxsackievirus, rhinovirus, West Nile virus, smallpox virus, encephalitis virus, yellow fever virus, dengue virus, influenza virus (including human, avian, and porcine), Lassa fever virus, lymphocytic choriomeningitis virus, Junin virus, Machupo virus, Guanarito virus, Hantavirus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever virus, Marburg virus, Japanese encephalitis virus, Kyasanur Forest disease virus, or any of the following: , Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Severe Acute Respiratory Syndrome (SARS) virus, Parainfluenza virus, Respiratory syncytial virus, Punta Toro virus, Tacaribe virus, Pachindae virus, Adenovirus, Dengue virus, Influenza A virus and Influenza B virus (including human, avian, and porcine), Junin virus, Measles virus, Parainfluenza virus, Pichinde virus, Punta Toro virus, Respiratory syncytial virus, Rhinovirus, Rift Valley fever virus, Severe Acute Respiratory Syndrome (SARS) virus, Tacaribe virus, Venezuelan equine encephalitis virus, Includes infection with one or more viruses selected from the group consisting of West Nile and Yellow Fever viruses, Tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan encephalitis virus, Rocio virus, Louping ill virus, Banzi virus, Ilheus virus, Cocobera virus, Kunjin virus, Alfuy virus, Bovine diarrhea virus and Kyasanur Forest disease virus.

[0253] Bacterial infections that may be treated according to the present invention include, but are not limited to, infections caused by: Staphylococcus; Streptococcus (including Streptococcus pyogenes); Enterococcus; Bacillus (including Bacillus anthracis), and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella (including G. vaginalis); Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponema; Campylobacter, Pseudomonas (including Pseudomonas aeruginosa); Legionella; Neisseria (including Neisseria gonorrhoeae and Neisseria meningitidis); Flavobacterium (F. meningosepticum and F. odoraturn). odoraturn); Brucella; Bordetella (including B. pertussis and B. bronchiseptica); Escherichia (including E. coli), Klebsiella; Enterobacter, Serratia (including B. niger and S. liquefaciens); Edwardsiella; Proteus (including P. mirabilis and P. vulgaris); Streptobacillus; Rickettsiae (including R. fickettsfi), Chlamydia (including C. psittaci and C. trachomatis); Mycobacterium (including M. tuberculosis, M. intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasiikansasii, M. intracellulare, and M. lepraernurium); and Nocardia.

[0254] Protozoal infections that may be treated according to the present invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma.

[0255] A complete list of infectious diseases can be found on the website of the National Institute of Infectious Diseases (NCID) of the Centers for Disease Control and Prevention (CDC) at the World Wide Web (www) https: / / www.cdc.gov / DiseasesConditions / , which list is incorporated herein by reference. All of said diseases are candidates for treatment using the nucleic acid system described in the present invention.

[0256] As used herein, the term "autoimmune disease" refers to a condition when the body's natural defense system cannot tell the difference between its own cells and foreign cells, causing the body to mistakenly attack normal cells.

[0257] Autoimmune diseases that may be treated according to the present invention include type 1 diabetes, rheumatoid arthritis (RA), psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, and celiac disease.

[0258] The term "graft rejection" as used herein occurs when transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Graft rejection can be attenuated by using immunosuppressants after transplantation, by determining molecular similarities between donor and recipient. The use of the nucleic acid system described in the present invention allows the reprogramming of the recipient's graft B and T cells, so that they no longer recognize the graft's cells as foreign cells that should be destroyed.

[0259] The term "allergy" or "allergic disease" as used herein refers to a number of conditions caused by hypersensitivity of the immune system to typically harmless substances in the environment. Allergen immunotherapy involves exposing people to larger and greater amounts of allergens in an attempt to change the immune system's response. Similarly, the use of the nucleic acid system of the present invention will allow individuals to overcome their sensitivity to allergens by reprogramming B and T cells to no longer recognize these substances as foreign.

[0260] Examples of types of allergies include allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom, drug allergies, and food allergies.

[0261] The term "neurological disorder" as used herein refers to any disorder of the nervous system. They may be caused by defective genes or problems with the way the nervous system develops, they may be degenerative diseases, in which nerve cells are damaged or die, and they may also be caused by diseases of blood vessels supplying the brain, injuries, seizure disorders, cancer, or infections. The use of the nucleic acid system described in the present invention will allow the reprogramming of B cells and T cells to secrete molecules locally, thereby promoting the recovery of destroyed tissues or restoring correct neuronal signaling, for example.

[0262] Neurological disorders include, but are not limited to, acute spinal cord injury, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, brain tumors, cerebral aneurysms, epilepsy and seizures, Guillain-Barre syndrome, headaches, head injuries, Huntington's disease, hydrocephalus, lumbar disc disease (herniated disc), meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, Parkinson's disease, spina bifida, seizures (strokes), cluster headaches, tension-type headaches, migraines, encephalitis, sepsis, and myasthenia gravis.

[0263] According to a particular aspect, the present invention relates to a method for preventing and / or treating a tumor, an infectious disease, an immune disease, a transplant rejection and / or an allergy, comprising at least one step of administering to an individual in need thereof a nucleic acid system, a vector, a kit, a cell or a pharmaceutical composition as described herein.

[0264] As used herein, an "individual" or "subject" is a mammal, most preferably a human. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Most preferably, the individual or subject is a human. In particular, an "individual in need thereof" is an individual suffering from a tumor, an infection, an immune disorder, a transplant rejection, and / or an allergy.

[0265] The present invention further relates to a nucleic acid system, a vector, a kit, a cell, or a pharmaceutical composition as described herein for use as a medicament.

[0266] Another aspect of the present invention relates to a nucleic acid system, a vector, a kit, a cell, or a pharmaceutical composition as described herein for its use in the prevention and / or treatment of tumors, infectious diseases, immune diseases, transplant rejection, and / or allergies.

[0267] The present invention also relates to the use of a nucleic acid system, a vector, a kit, a cell, or a pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of tumors, infectious diseases, immune diseases, transplant rejection, and / or allergies.

[0268] It is to be understood that the disclosure encompasses all variations, combinations, and permutations of at least one limitation, element, clause, description requirement, etc. from at least one of the enumerated claims that are introduced into another claim that is dependent on the same base claim (or any other claim, as related), unless otherwise specified or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. When elements are presented as a list, such as a Markush group or similar format, it is to be understood that each subgroup of elements is also disclosed, and any element(s) can be removed from the group. In general, when the disclosure, or aspects of the disclosure, are referred to as including certain elements, features, etc., it should be understood that they also encompass embodiments that consist of, or consist essentially of, such elements, features, etc. For purposes of simplicity, such embodiments have not in every case been specifically set forth in so many words herein. It should be understood that any embodiment or aspect of the disclosure may be specifically excluded from the claims, regardless of whether a specific exclusion is set forth herein. Publications and other reference materials referred to herein to explain the background of the disclosure and to provide additional detail regarding its practice are hereby incorporated by reference.

[0269] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0270] Working Example Example 1: Nucleic acid system of the invention in the reprogramming of B and T cells for better control and regulation of the immune response Materials and Methods Construction of plasmids encoding components of the nucleic acid system The NR4A1 promoter was amplified from human genomic DNA and cloned into the EcoRI and BamHI restriction enzyme sites of the pHRSIN vector. Primers of the following sequences shown as SEQ ID NOs: 6-9 and 22-23 were used in combination with a primer having the sequence shown as SEQ ID NO: 10.

[0271] [Table 1]

[0272] A diagram of lentiviral vectors containing the pNR4A1(2204), pNR4A1(1750), pNR4A1(1251), or pNR4A1(734) fragments is included in Figure 1A.

[0273] The ectopic B cell receptor was cloned by inserting the variable regions of a monoclonal antibody (OBI and ADRI sequences) synthesized by GenScript into the published FAM0 construct.

[0274] Destabilized TurboGFP was a kind gift from Dr. Mangeot. They were cloned after the promoter between the BamHI and SbfI restriction enzyme sites to replace GFP in the pNR41(608) and pNR4A1(415) constructs.

[0275] Cell lines and primary cells Jurkat cells (ACC-282), Namalwa cells (Burkitt lymphoma (BL) subtype PNT (ACC-69), Raji cells (ACC-319), and BL-2 cells (ACC-625) were purchased from DSMZ (Braunschweig, Germany). BJAB cells (ACC-757) and Ramos cells (ACC-603) were kind gifts from Pr. Belot and Dr. Gruffat. B cell lines were grown in culture flasks in RPMI-1640 supplemented with 10%-20% heat-inactivated fetal bovine serum and containing 50 μg / ml penicillin and streptomycin at 37°C in a humidified atmosphere of 95% air / 5% carbon dioxide as recommended on the DSMZ website. 293T (human kidney epithelial cells) were grown in Dulbecco's modified Eagle's medium (Gibco, Invitrogen) supplemented in the same way.

[0276] Peripheral blood mononuclear cells (PMBCs) were isolated from human donor peripheral blood by human Ficoll gradient. Human primary B cells were then isolated by positive selection using magnetic beads conjugated to anti-CD19 antibodies (Miltenyi). They were cultured at 1 × 10 in complete RPMI-1640 medium supplemented with 2 mM glutamine, 100 IU / ml penicillin, 100 mg / ml streptomycin, 55 μM B-ME, 1% HEPES. 6 Cross-linked CD40L (2 μg / ml), IL-4 (2 ng / ml), and BAFF (10 ng / ml) were added to the medium.

[0277] Lentivirus titration Infectious titers (TU / ml) of lentivirus (LV) were quantified by addition of serial dilutions of lentivirus to 293T target cells. Ten days after transduction, genomic DNA was extracted from target cells for quantitative PCR analysis of viral genome copy numbers. Quantitative PCR was performed using 5 μl of DNA on a StepOnePlus system with specific primers for detection of integrated lentiviral backbone. Titers were normalized to human actin gene copies. Two control samples were processed in parallel for each run.

[0278] Lentivirus production and transduction of B cells Lentiviruses encoding each of the described constructs were generated by transient transfection of 293T cells via calcium phosphate precipitation. For pseudotyping with VSV-G (vesicular stomatitis virus G protein) and BRL glycoproteins, 2.7 μg and 7 μg of envelope plasmids were transfected together with gagpol packaging plasmid (8.6 μg) and plasmids encoding lentiviral constructs (8.6 μg), respectively. 18 h after transfection, the medium was replaced with Opti-MEM supplemented with 10 mM Hepes and 1% penicillin-streptomycin. Viral supernatants were collected 48 h after transfection and filtered through a 40 μm filter. Low-speed concentration was performed by overnight centrifugation of the viral supernatant at 3,000 g at 4°C.

[0279] For transduction of in vitro B cell lines, cells were cultured in supplemented RPMI and transduced at a multiplicity of infection of 10. For transduction of primary human B cells, protamine sulfate was added to the medium (8 μg / ml).

[0280] Cell stimulation 1.5×10 5 ~2.0×10 5B cells were stimulated with antibodies or immune stimulants for the indicated times in supplemented RPMI-1640 medium in 96-well plates. Stimuli included CpG oligodeoxynucleotides (ODNs) 2006 (InvivoGen), lipopolysaccharide from E. coli (Sigma-Aldrich), anti-human F(ab')2 IgM and F(ab')2 IgG (Southern Biotech), ionomycin (EMD Millipore), and PMA (Cell Signaling Technology) for B cells. After stimulation, cells were washed and resuspended in PBS and then acquired by flow cytometry. For antigen-specific stimulation, 100 antigen-coated beads per B cell were added to the supernatant.

[0281] For stimulation of Jurkat T cells, 2 x 10 6 Cells were plated in 96-well plates and stimulated with 1 μg / mL of CD3 / CD8 antibodies (Invitrogen) or TransAct (Miltenyi Biotec) according to the manufacturer's instructions. For kinetic evaluation, plates were washed 3 times with 200 μl of PBS to remove the stimulatory molecules.

[0282] Preparation of synthetic particulate antigen beads coated with ovalbumin Synthetic particulate antigen (SPAg) was produced as previously described. Briefly, 0.4 μM Flash streptavidin beads were incubated with monobiotinylated ovalbumin (OVA, Sigma), then washed twice with PBS containing 2% bovine serum albumin and filtered through 0.1 μm and 0.65 μm columns (Durapore, Merck Millipore) to remove unbound molecules and bead aggregates, respectively. Bead concentration was assessed using a standard curve by reading Flash Red fluorescence on a Tecan plate reader.

[0283] Internalization assay 1×10 5Cells were plated in 96-well plates and incubated with 100 SPAgs-OVA per B cell for 1, 6, 24, or 48 h. After incubation, cells were washed with PBS 2% SVF and then surface stained with anti-OVA antibody.

[0284] Confocal microscopy analysis B cells loaded with synthetic particulate antigens were cultured overnight. 6 B cells were plated on 17 mm glass coverslips (Zeiss) preincubated with 0.01% poly-L-lysine (Sigma) for 4 h. Cells were permeabilized with 0.1% triton, then incubated with blocking solution (PBS-1% bovine serum albumin) for 30 min at room temperature and stained with eFluor570 anti-B220 (clone RA3-6B2, BD) and anti-LAMP1 (clone H4A3, BD) monoclonal antibodies conjugated to AlexaFluor488 for 1 h at room temperature. After three washes with PBS, cells were stained with Hoechst (1 / 10,000) for 5 min. After three further washes, the coverslips were mounted on slides with mowiol mounting medium. Confocal 3D image stacks were acquired with a Confocal Spectrum LM610 (Zeiss). Images were analyzed with FIJI software.

[0285] Flow cytometry staining and analysis For staining, 2 x 10 5 Cells were resuspended in PBS containing 2% fetal bovine serum and incubated with optimal dilutions of fluorochrome-conjugated antibodies, anti-IgM-APC (Miltenyi), anti-IgG-PE (Miltenyi), anti-CD86-VB (Miltenyi), anti-HLA-DR-APC-Vio7 (Miltenyi), anti-IFNg-APC (Miltenyi) and anti-OVA-FITC (Cell Signaling), at 4°C for 30 min and then washed with supplemented PBS.

[0286] Data was acquired using a FACSCantoII (BD Biosciences) and analyzed using FlowLogic™.

[0287] Preparation of cell extracts and Western blots The following antibodies were used: anti-IgM (LsBio), anti-IgG (LsBio60606, Cliniscience, France), and anti-calnexin (SPA-860, Stressgen Biotechnologies, Canada). 6 Cells were lysed in cell lysis buffer containing 20 mM Tris-HCl (pH 8.0), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 1 mM PMSF (phenylmethylsulfonyl fluoride). Proteins were heated at 95° C. for 3 min in denaturing buffer, separated under reducing conditions by 12% SDS-PAGE, and revealed by Western blot using a horseradish peroxidase revealing kit (IgG, IgM, and calnexin) or an Odyssey instrument (IL-18).

[0288] statistical analysis Statistical significance was assessed using Prism software. Data are expressed as mean ± standard error of the mean, and differences were considered significant (p < 0.05; * );highly significant (p<0.01; ** ) and highly significant (p<0.001; *** ) was determined.

[0289] result Generation of a small B cell receptor-inducible promoter construct It has been shown that Nur77 (NR4A1) protein is specifically induced in B cells after B cell receptor stimulation (Ashouri et al.). Based on this observation, we isolated several fragments of the NR4A1 promoter of decreasing size that terminate at the transcription start site and placed them upstream of the GFP reporter gene in a lentiviral vector (Figure 1B) or upstream of a destabilized TurboGFP gene. In particular, these fragments may contain binding sequences for the NFAT and NF-κB transcription factors involved in the B cell receptor signaling cascade. The fragments are as follows: [Table 2]

[0290] To assess the effect of B cell receptor stimulation on promoter inducibility, a B cell line, BJAB cells, were transduced with lentiviral vectors encoding either an inducible reporter construct or a constitutively active (spleen focus forming virus, SFFV) (control) promoter and then stimulated with the endogenous IgM B cell receptor for 24 h (Gagnepain et al.).

[0291] For all constructs except pNR4A1(169)-GFP, an increase in GFP or destabilized TurboGFP expression was observed.

[0292] For the pNR4A1(2204)-GFP, pNR4A1(1750)-GFP, pNR4A1(1251)-GFP and pNR4A1(734)-GFP constructs, an approximately 2.5-fold increase in GFP expression was observed, within the same range as the positive control containing ionomycin in combination with PMA, which has been shown to activate the B cell receptor signaling cascade without requiring direct binding of the B cell receptor (Figure 1C). The length of the ectopic promoter did not affect the magnitude of induction after B cell receptor stimulation, but played a significant role in the transduction rate, which increased with decreasing promoter size, with the 734 bp construct resulting in >90% transduction (Figure 1D).

[0293] Searching for the minimal sequence of pNR4A1 that maintained promoter induction by the B cell receptor, we found that a fragment truncated to 415 bp on the 5' side showed induction similar to the 734 bp construct.

[0294] Notably, no induction was observed for the 169 bp fragment, suggesting that important regulatory sequences reside between positions -305 and -60.

[0295] Given the similar inducibility of the constructs and the limited packaging capacity of lentiviral vectors, we decided to focus on the smaller promoter construct (734 bp) for subsequent experiments.

[0296] Characterization of the inducible 734-bp NR4A1 promoter The promoter dose response was monitored 24 hours after stimulation with F(ab')2 IgM molecules and peaked at a concentration of 2.5 μg / ml (FIG. 2A).

[0297] Importantly, we showed that the NR4A1 reporter construct was specifically induced after antigen receptor signaling, but not after stimulation with TLR-4 (lipopolysaccharide) and TLR-9 (CpG) in B cells (Figure 2B). All previous experiments were performed in BJAB cells, but promoter inducibility was also evaluated in other B cell lines expressing the IgMB cell receptor. As in BAJB cells, a 2.5-fold promoter induction was observed in BL-2 cells after B cell receptor ligation, but not in other cell lines, where an increase in GFP expression was only detected after incubation with PMA and ionomycin.

[0298] Kinetics of promoter induction, extinction, and reversibility To refine promoter kinetics, the GFP promoter placed behind a constitutive or inducible promoter was replaced by a destabilized TurboGFP, which has a shorter maturation time and a shorter half-life. First, promoter induction kinetics was assessed after 2 to 24 h of continuous stimulation.

[0299] The induction was quite rapid, after 4 h, a 2-fold increase of TurboGFPdes was already observed, which reached 12-fold after 24 h of stimulation, demonstrating the rapid response of the promoter (Figure 3A). Alternatively, the extinction kinetics was also investigated to see if this induction could be reversed if the stimulus was removed. To address this question, cells transduced with TurboGFPdes under the control of a constitutive or inducible promoter were stimulated through their B cell receptor for 1, 4, 8 or 24 h, after which the inducer molecule was removed. Three days after the stimulus was removed, the median value of TurboGFPdes again acquired the basal value of unstimulated cells and remained stable for 10 days, demonstrating the reversibility of the induction (Figure 3B).

[0300] However, we then wanted to know whether this system could be induced multiple times and whether it was still fully reversible after several inductions. Three stimulations were performed on the transduced cells by alternating 8 hours of stimulation (gray) with 80 hours of rest (white). The fold induction, normalized by unstimulated cells, increased slightly from 3 to 5 stimulations. Importantly, the induction was fully reversible even after 3 cycles of stimulation / rest, which is particularly interesting from a therapeutic point of view to treat disease flares in chronic patients (Figure 3C).

[0301] Construction of a vector encoding a membrane-anchored B cell receptor. After validating the B cell receptor-inducible promoter, the inventors then attempted to create a complete synthetic circuit for reprogramming B cells, i.e., the nucleic acid system of the present invention. Towards this goal, a unique sensor (membrane-anchored B cell receptor) was developed to control the induction of the promoter. This membrane-anchored B cell receptor "sensor" was encoded by a lentiviral vector containing the variable regions of a monoclonal antibody directed either against the HbS protein of the Hepatitis B virus (FAM0-ADRI) (Cerino et al.) or against the ovalbumin (OVA) protein (FAM0-OVA) (Dougan et al.), together with a constant IgG / κ human immunoglobulin domain fused to a transmembrane IgG domain. Notably, the intron region allowing the conditional secretion of immunoglobulin upon B cell activation was removed to express only the membrane-anchored immunoglobulin (Fusil et al.). The nucleic acid sequence of the FAM0-OVA construct is provided in SEQ ID NO: 11. In this construct, the variable regions of the monoclonal antibody directed against the ovalbumin (OVA) protein are embodied in bold in the sequence of SEQ ID NO: 11 (from nucleotide 561 to nucleotide 3149).

[0302] To validate the components of this circuit, both in terms of expression and functionality, BJAB cells were transduced with lentiviral vectors encoding the sensor. BJAB cells endogenously express IgM immunoglobulins but are negative for IgG immunoglobulins. After transduction, IgG immunoglobulins were detected by Western blot (Figure 4A) and surface cytometry staining to validate membrane expression of the sensor components. Synthetic particulate antigen beads (SPAG), consisting of 400 nm fluorescent beads coated with ovalbumin molecules, were incubated with the transduced cells to validate antigen-specific recognition by the sensor. After 24 h of incubation with SPAG-OVA beads, only cells transduced with the OVA-specific sensor were positive for SPAG-OVA (approximately 50% bead-positive cells were detected by hemocytometry). The percentage of SPAG-OVA positive cells as well as the average number of beads per positive cell were assessed by immunofluorescence staining and were 50% and 4 for the FAM0-OVA condition, respectively, which is consistent with the hemocytometry analysis (Figure 4B).

[0303] Notably, little nonspecific binding was observed in untransduced cells and in cells transduced with the FAM0-ADRI construct (less than 5% positive cells and only one SPAG-OVA bead). To verify the functionality and signaling of the sensor after antigen-specific recognition, the expression of activation markers in sensor-transduced cells was quantified by hemocytometry after 24 h of incubation with SPAG-OVA beads (Figure 4C). CD86 was upregulated in cells expressing the OVA sensor compared to untransduced cells, suggesting antigen-specific activation.

[0304] Surprisingly, no upregulation of HLA-DR (MHC-H) was observed after antigen binding on the FAM0-OVA sensor. Indeed, after antigen recognition, the B cell receptor-antigen complex naturally underwent endocytosis and intracellular processing, resulting in presentation of the antigen on the MHC-II complex (Figure 4C).

[0305] The percentage of bead-positive cells did not increase over the course of incubation.

[0306] Assembling all ectopic components into a functional synthetic circuit Going one step further, all circuit components, i.e. sensor (membrane-anchored B cell receptor), transducer (734 bp NR4A1 inducible promoter) and effector (here TurboGFPdes) were introduced together into BJAB cells by double transduction with two lentiviral vectors encoding the sensor and TurboGFPdes, respectively, under the control of a B cell receptor inducible promoter or an SFFV constitutive promoter as a control. The double transduced cells were then stimulated through their ectopic sensor with anti-IgG molecules, and a specific upregulation of TurboGFPdes expression was observed after stimulation in cells transduced with the sensor and B cell receptor inducible promoter. Notably, the slight reduction in activation in cells transduced with the inducible promoter together with the sensor correlated with a reduction in IgM expression, compared to cells in the absence of the sensor after stimulation of the B cell receptor with endogenous IgM. Indeed, ectopic expression of novel B cell receptors has already been shown to reduce the expression of endogenous B cell receptors, possibly due to competition in the processes at hand.

[0307] No differences were observed in cells carrying the SFFV promoter after stimulation with IgG (Fig. 5A ).

[0308] We then wanted to verify the antigen-specific induction of the whole nucleic acid system by stimulating the doubly transduced cells with ovalbumin-coated beads, either alone or in combination with the costimulatory molecule CD40L. After 24 hours of stimulation with ovalbumin-coated beads and CD40L, the whole system was specifically antigen-specifically activated (3-fold), since no activation was detected after stimulation with spike RBD (receptor binding site)-coated beads (Figure 5B).

[0309] Inducibility of the promoter in T cells Finally, we tested the 734 bp NR4A1 reporter construct in Jurkat T cells. After 24 h of T cell receptor stimulation with CD3 and CD28 coated beads, we observed a 3-fold increase in GFP expression (Figures 6A and 6B), indicating inducibility of the promoter by the T cell receptor in T cells, suggesting that it should also be activated by the CAR construct.

[0310] conclusion Taken together, the results presented above also demonstrate the efficacy of the nucleic acid system of the present invention in reprogramming B and T cells to better control and regulate immune responses. Upon binding of a target molecule to a dedicated sensor (targeted to a given pathological signal), the transducer pNR4A1 is specifically activated, leading to the expression of effector therapeutic molecules placed under its control.

[0311] Example 2: Development of a self-amplifying all-in-one vector To shift the present invention towards a clinical setting based on human primary B cells, we developed an "all-in-one vector" encoding all components within the same lentiviral vector construct. Indeed, as shown below, the transduction efficiency of human primary B cells is improved by equipping it with an all-in-one lentiviral vector, given that it allows to increase the number of cells co-expressing all circuit components, thus reducing variability.

[0312] Materials and Methods Construction of plasmids encoding synthetic circuit components The NR4A1 promoter fragment was amplified from human genomic DNA and cloned into the EcoRI and BamHI restriction sites of the pHRSIN vector (Demaison, Christophe, Kathryn Parsley, Gaby Brouns, Michaela Scherr, Karin Battmer, Christine Kinnon, Manuel Grez, et Adrian J. Thrasher. « High-Level Transduction and Gene Expression in Hematopoietic Repopulating Cells Using a Human Imunodeficiency Virus Type 1-Based Lentiviral Vector Containing an Internal Spleen Focus Forming Virus Promoter ». Human Gene Therapy 13, no 7 (mai 2002): 803-13) to replace the SFFV promoter originally controlling the GFP transgene. The following primers were used as in Example 1: [Table 3]

[0313] Destabilized TurboGFP was a kind gift from Dr. Mangeot and was cloned after the promoter between the BamHI and SbfI restriction enzyme sites to replace GFP in the modified pHRSIN vector described above.

[0314] The fragment encoding the synthetic circuit transgene for the self-amplifying vector was ordered from GenScript and then inserted into the original pHRSIN vector by restriction cloning.

[0315] Cell lines and primary cells BJAB (ACC-757) cells derived from Burkitt's lymphoma were a kind gift from Pr. Belot. B cell lines were grown in culture flasks in RPMI 1640 medium containing 50 μg / ml penicillin and streptomycin supplemented with 10%-20% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere of 95% air / 5% carbon dioxide as recommended on the DSMZ website. 293T cells (human kidney epithelial) were grown in Dulbecco's modified Eagle's medium (Gibco, Invitrogen) medium supplemented with 10% fetal calf serum.

[0316] Production of lentiviral vectors (LV) and transduction of B cells Lentiviruses were produced by transient transfection of 293T cells via calcium phosphate precipitation. For pseudotyping of lentiviruses with VSV-G glycoprotein, 2.7 μg of envelope plasmid was co-transfected with 8.6 μg of gagpol packaging plasmid (psPAX2, Addgene, plasmid #12260) and the plasmid encoding the lentiviral construct (8.6 μg). 18 h after transfection, the medium was replaced with Opti-MEM (Gibco) supplemented with 10 mM Hepes and 1% penicillin-streptomycin. Viral supernatants were collected 48 h after transfection and filtered through a 40 μm filter. Low-speed concentration of the viral supernatant was performed by centrifugation at 3,000 × g at 4 °C overnight.

[0317] Lentivirus titration Lentivirus was titrated by adding serial dilutions of lentivirus to 293T target cells. Ten days after transduction, genomic DNA was extracted from target cells (Macherey Nagel) for analysis of viral genome copy number by quantitative PCR. Quantitative PCR was performed using 5 μl of DNA in a StepOnePlus system with specific primers for detection of integrated lentivirus: primer F: 5′-TGT GTG CCC GTC TGT TGT GT (SEQ ID NO: 12), primer R: 5′-GAG TCC TGC GTC GAG AGA GC (SEQ ID NO: 13), and probe 5′-CAG TGG CGC CCG AAC AGG GA (SEQ ID NO: 14). Genomic vector copies in each sample were normalized to human actin gene copies as previously described (Fusil, Floriane, Sara Calattini, Fouzia Amirache, Jimmy Mancip, Caroline Costa, Justin B Robbins, Florian Douam, et al. « A Lentiviral Vector Allowing Physiologically Regulated Membrane-Anchored and Secreted Antibody Expression Depending on B-Cell Maturation Status ». Molecular Therapy 23, no 11 (novembre 2015): 1734-47) using specific primers: primer F5′ TCC GTG TGG ATC GGC GGC TCC A (SEQ ID NO: 15): primer R5′-CTG CTT GCT GAT CCA CAT CTG (SEQ ID NO: 16) and probe CCT GGC CTC GCT GTC CAC CTT CCA (SEQ ID NO: 17). Titers were normalized to human actin gene copies. Two control samples were processed in parallel for each run.

[0318] Transduction of B cells For in vitro transduction of B cell lines, cells were cultured in supplemented RPMI-1640 medium and transduced at a multiplicity of infection of 10.

[0319] Cell stimulation 1.5×10 5 ~2.0×10 5 B cells were stimulated with antibodies or immunostimulants in supplemented RPMI-1640 medium in 96-well plates for the indicated times. Stimuli included anti-human F(ab')2 IgM and F(ab')2 IgG (Southern Biotech), ionomycin (EMD Millipore), PMA (Cell Signaling Technology), and cross-linked human CD40L (Miltenyi). After stimulation, cells were washed and resuspended in PBS before being subjected to flow cytometry analysis. For antigen-specific stimulation, 100 antigen-coated beads per B cell were added to the supernatant.

[0320] Preparation of ovalbumin-coated synthetic particulate antigen (SPAG) beads SPAG beads were prepared as previously described (Sicard, Antoine, Alice Koenig, Stephanie Graff-Dubois, Sebastien Dussurgey, Angeline Rouers, Valerie Dubois, Pascal Blanc, et al. ≪ B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform To Generate Antigen-Specific Helper T Cells for Cell Therapy ≫. Nano Letters 16, no 1 (13 Janvier 2016): 297-308).

[0321] Briefly, 0.4 μm Flash Red streptavidin beads (Bangslabs) were incubated with monobiotinylated ovalbumin (OVA, Sigma) or spiked RBD (ligand binding domain) (Miltenyi), then washed twice with PBS containing 2% bovine serum albumin and filtered through 0.1 μm and 0.65 μm columns (Durapore, Merck Millipore) to remove unbound molecules and bead aggregates, respectively. Bead concentrations were assessed using a standard curve by reading Flash Red fluorescence on a Tecan plate reader.

[0322] Flow cytometry staining and analysis For staining, 2 x 10 5 The cells were resuspended in PBS containing 2% fetal bovine serum. The following fluorescent dyes were used: anti-IgM-APC antibody (Miltenyi), anti-IgG-PE antibody (Miltenyi), anti-CD86-VB antibody (Miltenyi), anti-HLA-DR-APC-Vio7 antibody (Miltenyi), and anti-OVA-FITC antibody (Cell Signaling Technology).

[0323] Data was acquired using a FACSCantoII (BD Biosciences) and analyzed using FlowLogic™.

[0324] statistical analysis Statistical significance was assessed using Prism software. Data are expressed as mean ± standard error of the mean, and differences were considered significant (p < 0.05; * );highly significant (p<0.01; ** ) and highly significant (p<0.001; *** ) was determined.

[0325] result We constructed an autonomously amplifying all-in-one vector in which a short inducible NR4A1 promoter drives both the effector and sensor transgenes using a T2A sequence (SEQ ID NO: 18) between the two coding sequences (Figure 7A). Indeed, we predicted a leaky activity of the inducible promoter, which could induce the basal sensor expression required to activate the circuit upon further stimulation. Thus, upon stimulation of a specific sensor, the inducible promoter would be fully activated, resulting in effective expression of both the effector and sensor molecules, which would amplify the composite circuit response, thus creating a positive feed-forward loop. We note that the insertion of the T2A motif after the effector and before the open reading frame of the sensor adds four amino acids fused to the TurboGFPdes marker, which may impair its destabilization and therefore turnover. This may explain the higher basal fluorescence levels in cells transduced with the self-amplifying construct (pNR4A1(734)-TurboGFPdes-T2A-FAM0-OVA) compared to cells expressing only the effector (pNR4A1(734)-TurboGFPdes) (Figure 8).

[0326] After 24 hours of stimulation with anti-IgG antibody molecules, we found that the expression of both TurboGFPdes and the sensor was upregulated 2-fold and 6-fold, respectively, in transduced cells (Figures 7B and 7C). Importantly, we demonstrated antigen-specific induction of this self-amplifying synthetic circuit using OVA-coated beads (Figure 7D). Indeed, the level of induction upon stimulation with OVA-coated beads combined with stimulation with CD40L was approximately 2-fold after 24 hours and reached 4-fold after 48 hours (Figures 7D-7G). Similarly, upregulation of effectors and sensors persisted 48 hours after stimulation with anti-B cell receptor antibodies (Figures 7E and 7F). Effector expression remained stable after 48 h of stimulation through the B cell receptor with IgM but increased after stimulation of the B cell receptor with IgG compared to 24 h (4-fold after 24 h to 5-fold after 48 h), suggesting the onset of self-amplification (Figures 7E-7G).

[0327] However, this induction was reversible upon removal of the sensor stimulation, and the circuitry was terminated within 3 days (FIG. 9).

[0328] conclusion Taken together, we demonstrated that the autoregulated construct enabled specific expression of the effector upon sensor stimulation.

[0329] References [Table 4] TIFF2025510588000010.tif186165

[0330] array SEQ ID NO:1 [ka] TIFF2025510588000012.tif114165 SEQ ID NO:2 [ka] TIFF2025510588000014.tif198165 SEQ ID NO:3 [ka] SEQ ID NO:4 [ka] SEQ ID NO:5 [ka] SEQ ID NO:6 [ka] SEQ ID NO:7 [ka] SEQ ID NO:8 [ka] SEQ ID NO:9 [ka] SEQ ID NO:10 [ka] SEQ ID NO:11 [ka] TIFF2025510588000024.tif236165 TIFF2025510588000025.tif237165 TIFF2025510588000026.tif236165 TIFF2025510588000027.tif236165 TIFF2025510588000028.tif34165 SEQ ID NO:12 [ka] SEQ ID NO:13 [ka] SEQ ID NO:14 [ka] SEQ ID NO:15 [ka] SEQ ID NO:16 [ka] SEQ ID NO:17 [ka] SEQ ID NO:18 [ka] SEQ ID NO:19 [ka] SEQ ID NO:20 [ka] SEQ ID NO:21 [ka] SEQ ID NO:22 [ka] SEQ ID NO:23 [ka]

Claims

1. (i) Sensor components, (a) at least one extracellular ligand recognition domain; (b) the transmembrane domain of a B cell receptor (BCR) or a T cell receptor (TCR); and (c) A signaling domain that, depending on the case, regulates the cellular activation of B cell receptors or T cell receptors. A sensor component including an array that codes for, (ii) Transducer / effector component, (d) the pNR4A1 promoter or a functional fragment thereof consisting of Sequence ID No. 1; and (e) at least one target effector protein The transducer / effector components include an array that codes for and Nucleic acid-based substances, including those mentioned above.

2. The nucleic acid system according to claim 1, wherein the transducer / effector component comprises (d) a functional fragment of the pNR4A1 promoter.

3. The nucleic acid system according to claim 1, wherein the functional fragment of the pNR4A1 promoter has a length of 200 bp to 2210 bp or 500 bp to 2210 bp.

4. The nucleic acid system according to claim 1, wherein the functional fragment of the pNR4A1 promoter has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, and SEQ ID NO:

23.

5. The nucleic acid system according to claim 1, wherein the extracellular ligand recognition domain (a), the transmembrane domain (b), and, if present, the signal transduction domain (c) form a B cell receptor, a T cell receptor, a chimeric immune receptor (CIR), a CAR-NK cell, a B cell antibody receptor (BAR), or a chimeric autoantibody receptor T (CAR-T) cell.

6. The nucleic acid system according to claim 1, wherein the extracellular ligand recognition domain (a) comprises at least one ligand-binding fragment that binds to a target ligand.

7. The nucleic acid system according to claim 1, wherein the antigen-binding domain is selected from the antigen-binding domain of an antibody; from Fd fragments, single-domain antibodies (sdAb), complementarity-determining regions (CDRs), Fv fragments, single-stranded FV (scFV), double-stranded Fvs, and single-stranded (Fv)2; from the antigen-binding domain of an antibody mimetic; and from aptamers and mixtures thereof.

8. The nucleic acid system according to claim 6, wherein the target ligand is selected from one or more tumor antigens, one or more autoantigens, one or more alloantigens, one or more viral antigens, one or more bacterial antigens, one or more allergen antigens, or one or more neurological disorder markers.

9. The nucleic acid system according to claim 1, wherein at least one target effector protein is an immunostimulatory protein or an immunosuppressive protein.

10. The nucleic acid system according to claim 1, wherein at least one target effector protein is selected from pro-inflammatory cytokines; costimulatory molecules; and inhibitory molecules.

11. The nucleic acid system according to claim 1, further comprising (iii) an ORF isolation sequence between the (i) sensor component and the (ii) transducer / effector component, wherein both the (i) sensor component and the (ii) transducer / effector component are under the control of the pNR4A1 promoter or a functional fragment thereof, which is sequence number 1.

12. A vector comprising the nucleic acid system described in claim 1.

13. The vector according to claim 12, which is a retrovirus vector.

14. A first vector comprising the nucleic acid system sensor components described in claim 1, A second vector comprising the nucleic acid-based transducer / effector component described in claim 1, and A kit that includes this.

15. Cells transformed by the nucleic acid system described in claim 1, the vector described in claim 12, or the kit described in claim 14.

16. A pharmaceutical composition comprising a nucleic acid system according to claim 1, a vector according to claim 12, a kit according to claim 14, or cells according to claim 15, and a pharmaceutically acceptable vehicle.

17. A composition for preventing and / or treating tumors, immunodeficiencies, graft rejection, allergies, neurological disorders, and / or infectious diseases, comprising the nucleic acid system according to claim 1, the vector according to claim 12, the kit according to claim 14, the cells according to claim 15, or the pharmaceutical composition according to claim 16.

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