Promoters inducible by pharmacological molecules

Abstract

The present invention relates generally to the field of medicine. More particularly, it relates to the use of pharmacological molecules for inducing the expression of a transgene downstream of novel inducible cellular promoters. The invention also relates to these novel inducible cell promoters and to the entities associated therewith.

Description

[0001] DESCRIPTION

[0002] TITLE: PROMOTERS INDUCIBLE BY PHARMACOLOGICAL MOLECULES

[0003] FIELD OF INVENTION

[0004] The present invention relates generally to the field of medicine. More particularly, it relates to the use of pharmacological molecules to induce the expression of a transgene downstream of novel inducible cell promoters. In this respect, the invention also relates to these novel inducible cell promoters as such and to objects associated with them.

[0005] PREVIOUS ART

[0006] In eukaryotic cells, the signaling pathways triggered in response to nutrient deprivation, metabolic disturbance, or the influx of foreign nucleic acids are called the Integrated Stress Response (ISR). The unfolded protein detection response (UPR) is an endoplasmic reticulum (ER)-induced signaling pathway due to proteostasis. This pathway induces the ISR through the phosphorylation of the PKR-like endoplasmic reticulum kinase (PERK), as well as through ER-specific responses mediated by the transcription factors XBP1 and ATF6. All stimuli leading to an ISR converge on the phosphorylation of serine 51 of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2a).In mammalian cells, the four kinases, General Control Nonderepressible 2 (GCN2), Heme Regulated Inhibitor (HRI), Protein Kinase R (PKR) and PERK, respectively detect an amino acid deficiency, hypoxia, viral infection or protein misfolding and inactivate eIF2a by phosphorylation (Pakos-Zebrucka, K. et al. (2016). The integrated stress response. EMBO Rep. 17, 1374-1395).

[0007] Phosphorylation of eIF2a blocks the translation of capped mRNAs, but promotes the translation of certain mRNAs possessing a 5'UTR with a characteristic structure; this is the case for the transcript of the ATF4 gene. Thus, in RIS conditions, the ATF4 factor is expressed and activates a genetic program, which promotes cell recovery if the stress is transient, but induces expression of the C / EBP homolog protein (CHOP) and apoptosis in the case of prolonged stress (lurlaro, R., and Munoz-Pinedo, C. (2016). Cell death induced by endoplasmic reticulum stress. The FEBS Journal 283, 2640-2652.).

[0008] A synthetic promoter exists containing a tandem of two amino acid-sensitive elements (2xAAREs) and a TATA YB box (Hansen, J. et al. (2014). Transplantation of prokaryotic two-component signaling pathways into mammalian cells. Proc Natl Acad Sci USA 111, 15705-15710), which binds the transcription factor ATF4 and allows the modulation of transgene expression by feeding with a medium or diet lacking an essential amino acid (EAA) (Chaveroux, C. et al. (2016). Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids. Nat Biotechnol 34, 746-751). Because it enables physiological regulation of transgene expression, this promoter is of great interest for gene transfer into various cell types for use in gene therapy protocols.

[0009] However, in lymphocytes, ATF4 can be expressed upon TCR stimulation (Dougé, A. et al., HLA, 2024 Jan;103(l):el5252). Since the 2XAARE promoter is not actionable in all situations in these cells to induce transgene expression at a chosen time, the use of stress processes in lymphocytes for therapeutic purposes is compromised, if not impossible.

[0010] BRIEF OVERVIEW OF THE INVENTION

[0011] Faced with the major challenge of providing safe and effective in vivo cell and / or gene therapy, the inventors have developed novel inducible promoters whose activation is controlled by pharmaceutical molecules independently of the context and / or cellular environment. In this context, a primary objective of the invention is to make these new inducible promoters, along with expression cassettes containing them, available to the medical community upstream of a transgene. Another objective of the invention is to provide lentiviral vectors (or their genomes) for transducing animal eukaryotic cells in order to offer safe and effective cell and / or gene therapy to patients who need it.In so doing, another aim of the invention is to provide a genetically modified animal eukaryotic cell for use in cell and / or gene therapy and with the use of pharmacological molecules to induce, in a controlled manner in this genetically modified animal eukaryotic cell, the expression of the transgene upstream of said new inducible promoters.

[0012] DETAILED DESCRIPTION

[0013] In its most general aspect, the invention relates to an inducible promoter sequence chosen from sequences having at least 90%, in particular 95%, identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2).

[0014] "Inducible promoter sequence" refers to a nucleic acid located upstream (at the 5' end) of a gene (or transgene) that controls its expression, notably by regulating its transcription. In the invention, this promoter, which was isolated by cloning, is described as inducible because the expression of the gene (or transgene) it controls is not constitutive and is only induced after its activation in response to the appropriate stimulus. In the invention, this appropriate stimulus corresponds to the use (administration of) a clinically applicable pharmacological molecule. With this tool, it then becomes possible to implement sophisticated engineering, particularly with T lymphocytes, allowing for the significant improvement and safety of current gene therapy protocols for cancer, especially with CAR-T cells."% identity" refers to the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequences), by determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides in the longer of the two sequences, and multiplying the result by 100. Therefore, "having at least 80% identity" means that the aforementioned percentage of identity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or is 100%.In this regard, it should be noted that this definition applies to all embodiments of the invention, including when it involves a direct comparison of two polypeptide sequences (amino acid sequences). Furthermore, it is understood that sequences having at least 80% (or at least 90%) identity with a reference sequence retain the same properties and functions, or even that these are improved.

[0015] According to another embodiment, the invention relates to the inducible promoter sequence as described above, said inducible promoter sequence being selected from sequences having at least 95% identity with the SEQ ID NOs: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B), and 4 (SESN2). In particular, the invention relates to the inducible promoter sequence as described above, said inducible promoter sequence being selected from the SEQ ID NOs: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B), and 4 (SESN2). Advantageously, the invention relates to the inducible promoter sequence as described above, said inducible promoter sequence being selected from the SEQ ID NOs: 1 (ULBP1) and 2 (HSPA6).

[0016] According to a second aspect, the invention relates to an expression cassette comprising, upstream (at the 5' end) of a transgene, an inducible promoter sequence as described above. "Expression cassette" refers to a nucleic acid comprising an inducible promoter sequence according to the invention, which is located at the 5' end (upstream) of a transgene. It should be noted that this transgene may include at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail (e.g., SEQ ID NO: 25). In the invention, this transgene may be selected from: oncogenes, anti-oncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic or suicide genes.In fact, and according to another embodiment, the invention relates to the expression cassette as described above, in which said transgene is selected from: oncogenes, anti-oncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic or suicide genes. In particular, the invention relates to the expression cassette as described above, in which said transgene is selected from: transcription factors, cytokines, transporters, nucleases, chimeric antigen receptors (CARs), and toxic or suicide genes.

[0017] “Transcription factors” refers to a protein that interacts with DNA and RNA polymerase and is necessary for the initiation or regulation of gene transcription throughout living organisms (prokaryotes or eukaryotes). In the invention, the following may be cited: c-JUN, FOXO1, B ATF, TBET, and NRF2.

[0018] According to another embodiment, the invention relates to the expression cassette as described above, in which said transgene is selected from: c-JUN, FOXO1, BATF, TBET, and NRF2. In particular, the invention relates to the expression cassette as described above, in which said transgene is c-JUN. In particular, the invention also relates to the expression cassette as described above, in which said transgene is c-JUN, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 129, or the nucleic acid of which codes for an amino acid sequence having at least 80% identity with sequence SEQ ID NO: 130.Advantageously, the invention relates to the expression cassette as described above, in which said transgene is c-JUN whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 129 or whose nucleic acid corresponds to the sequence SEQ ID NO: 130.

[0019] "Cytokines" refers to proteins secreted by immune system cells that allow them to communicate with each other and, consequently, these cytokines participate in activating the immune response. In the invention, the following may be cited: IL2, IFNγ, TNFα, IL18, and IL12.

[0020] According to another embodiment, the invention therefore relates to the expression cassette as described above, in which said transgene is chosen from: IL2, IFNy, TNFa, IL18, and IL12.

[0021] “Transporters” refers to transmembrane proteins whose function is to transport metabolites such as glucose (e.g., GLUT1). According to another embodiment, the invention therefore relates to the expression cassette as described above, in which said transgene is GLUT1.

[0022] "Nucleases" here refers generally to CRISPR-associated proteins (Clustered Regularly Interspaced Short Palindromic Repeats-associated proteins). These correspond to the nucleases of the CRISPR-Cas system, which constitutes a prokaryotic adaptive defense mechanism for destroying invading foreign DNA. This functional framework has since been repurposed from its initial function of prokaryotic immunity and is now applied in biotechnology for the targeting, processing, modification, destruction, and programmed repair of genes. To date, several Cas proteins (Cas9, Casl2a, Casl2b, CasX or Cas12e, Casl2f or Cas14, Casl2j or Cas) from different hosts have been identified and characterized, and even modified (e.g., by improving their function), which can be implemented by the invention for mammalian genome engineering.In general, the Cas proteins mentioned above have decreasing sizes of 1,500 amino acids (AA) for Cas9, 1,000 AA for Casl2a and less than 1,000 AA for CasX, Casl2f and Casl2j.

[0023] According to another embodiment, the invention therefore relates to the expression cassette as described above, in which said transgene is chosen from: ■ the nucleases Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas®;

[0024] ■ the Cas9, Casl2a, Casl2b, CasX or Casl2e orthologs, CRISPR type V, Cas 12j or Cas®; and

[0025] ■ the functional mutants or variants Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas®.

[0026] The term "orthologs" refers to similar Cas proteins found in two or more different species. The term "mutants" refers to a Cas protein into which one or more mutations have been introduced, including the deletion, substitution, and / or addition of one or more amino acids. These mutations can, in particular, increase nuclease activity and / or improve the fidelity of target DNA recognition. The term "functional mutants" refers to Cas proteins modified by humans (e.g., through genetic engineering) to, for example, increase Cas activity. The term "functional variants" refers to Cas proteins modified naturally through evolution, which exhibit, for example, increased activity.

[0027] Advantageously, the invention utilizes Cas9 nucleases such as those of S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae, and S. thermophilus, as well as mutants thereof. According to another embodiment, the invention therefore relates to the expression cassette as described above, in which said transgene is selected from:

[0028] ■ Cas9 nucleases from S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S.

[0029] canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus; And

[0030] ■ Cas9 orthologs and Cas9 mutants or functional variants derived from these organisms.

[0031] In particular, the invention relates to the expression cassette as described above, in which said transgene of interest is saCas9, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 131, or whose nucleic acid codes for a saCas9 protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 132. In particular, the invention also relates to the expression cassette as described above, in which said transgene is saCas9, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 131, or whose nucleic acid codes for a saCas9 protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 132.

[0032] "Chimeric antigen receptors (CARs)" refers to a laboratory-created protein that recognizes and targets proteins present on the surface of cells in the solid tumor being treated. For example, the following CARs can be listed: CAR anti-A-folate receptor, CAR anti-Carbonic anhydrase IX, CAR anti-CD171, CAR anti-CD 19, CAR anti-CD20, CAR anti-CD276, CAR anti-CD319, CAR anti-CEA, CAR anti-cMet, CAR anti-EGFR, CAR anti-EGFRIII, CAR anti-EGFRn, CAR anti-FAP, CAR anti-GD2, CAR anti-GPL 00, CAR anti-GPC3, CAR anti-HER2, CAR anti-IL13Ra2, CAR anti-MAGE, CAR anti-MART-1, CAR anti-MSLN, CAR anti-Mucl, CAR anti-Mucl6, CAR anti-NKG2D, CAR anti-PSMA, CAR anti-TRP-1, CAR anti-TRP2, CAR anti-VEGFR, CAR anti-HLA-G, CAR anti-CLDN18.2, Anti-EPCAM CAR, anti-FAP CAR, CAR anti-RORl, CAR anti-ROR2, CAR anti-PD1, and CAR anti-PSCA (Marie-Thérèse Rubio et al. Biology, concepts and principles of CAR-T cells. Bulletin du Cancer, 2018, 105 (Suppl. 2), pp.S135-S146 ; Belovezhets, T. et al. Comparative Pre-Clinical Analysis of CD20-Specific CAR T Cells Encompassing 1F5-, Leul6-, and 2F2-Based Antigen-Recognition Moieties. Int. J. Mol. Sci. 2023, 24, 3698.).

[0033] According to another embodiment, the invention relates to the expression cassette as described above, in which said transgene is a CAR selected from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD 171 CAR, an anti-CD 19 CAR, an anti-CD20 CAR, an anti-CD276 CAR, an anti-CD319 CAR, an anti-CEA CAR, an anti-cMet CAR, an anti-EGFR CAR, an anti-EGFRIII CAR, an anti-EGFRn CAR, an anti-FAP CAR, an anti-GD2 CAR, an anti-gpl O0 CAR, an anti-GPC3 CAR, an anti-HER2 CAR, an anti-IL13Ra2 CAR, an anti--MAGE CAR, an anti--MART-1 CAR, an anti-MSLN CAR, an anti-Mucl CAR, an anti-Mucl 6 CAR, an anti-NKG2D CAR, a CAR anti-PSMA, an anti-TRP-1 CAR, an anti-TRP2 CAR, an anti-VEGFR CAR, an anti-HLA-G CAR, an anti-CLDN18.2 CAR, an anti-EPCAM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PD1 CAR and an anti-PSCA CAR.

[0034] "Toxic or suicide transgene" refers to a nucleic acid that codes for a toxic, cytotoxic, or suicide protein. Furthermore, after activation of said inducible promoter sequence of the invention using a pharmacological molecule, the expression of said toxic or suicide transgene causes (leads to) cell death in the genetically modified cell expressing it. In the invention, this toxic or suicide transgene can be selected from among the following coding genes: inducible caspase-9 (iC9), Bax (S184del), Noxa (wild-type protein or one of its mutants, in particular the S13A mutant), Gasdermin B (N-ter), constitutively active caspase-3 (V266E), an influenza virus M2 ion channel (H37A), diphtheria toxin (DTA) subunit A and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes), and an enterotoxin (in particular that of C. perfringens). These are non-secreted, toxic, or suicide proteins.This means that the product of the expression (transcription followed by translation) of the nucleic acid encoding them, after activation of the inducible promoter sequence of the invention downstream of it, is not found in the extracellular environment. Therefore, only cell death of the genetically modified cell expressing them occurs.

[0035] According to another embodiment, the invention relates to the expression cassette as described above, in which said transgene, a toxic or suicide transgene, is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active Caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) subunit A and an attenuated mutant aDTA(G128D), a streptolysin O (in particular that of S. pyogenes), and an enterotoxin (in particular that of C. perfringens). According to another embodiment, the invention relates to the expression cassette as described above, in which said toxic or suicide transgene is selected from:

[0036] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5 or whose nucleic acid codes for an inducible Caspase-9 (iC9) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 6;

[0037] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7 or whose nucleic acid codes for a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 8;

[0038] ■ an M2 (H37A) ion channel of influenza virus whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 9 or whose nucleic acid codes an M2 (H37A) ion channel of influenza virus whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 10;

[0039] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 11 or whose nucleic acid codes for a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 12;

[0040] ■ a diphtheria toxin (DTA) subunit A whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 13 or whose nucleic acid codes for a diphtheria toxin (DTA) subunit A whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 14, and an attenuated aDTA (G128D) mutant whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 15 or whose nucleic acid codes for an attenuated aDTA (G128D) mutant whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 16;

[0041] ■ a Gasdermine B (N-ter) protein whose nucleic acid has a sequence with at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17 or whose nucleic acid codes for a Gasdermine B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 18; ■ a Noxa protein whose nucleic acid has a sequence with at least 80% identity with the nucleic acid of sequence SEQ ID NO: 19 or whose nucleic acid codes for a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 20;

[0042] ■ a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 21 or whose nucleic acid codes for a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 22; and

[0043] ■ an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 23 or whose nucleic acid codes for an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 24.

[0044] According to another embodiment, the invention relates to the expression cassette as described above, in which said toxic or suicide transgene is selected from:

[0045] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid corresponds to the sequence SEQ ID NO: 5 or whose nucleic acid codes for an inducible Caspase-9 (iC9) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 6;

[0046] ■ a Bax protein (S 184 del) whose nucleic acid corresponds to the sequence SEQ ID NO: 7 or whose nucleic acid codes for a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 8;

[0047] ■ an M2 (H37A) ion channel of the influenza virus whose nucleic acid corresponds to the sequence SEQ ID NO: 9 or whose nucleic acid codes an M2 (H37A) ion channel of the influenza virus whose amino acid sequence corresponds to the sequence SEQ ID NO: 10;

[0048] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid corresponds to the sequence SEQ ID NO: 11 or whose nucleic acid codes for a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 12;

[0049] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid corresponds to the sequence SEQ ID NO: 13 or whose nucleic acid codes for a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 14, and an attenuated aDTA (G128D) mutant whose nucleic acid corresponds to the sequence SEQ ID NO: 15 or whose nucleic acid codes for an attenuated aDTA (G128D) mutant whose amino acid sequence corresponds to the sequence SEQ ID NO: 16;

[0050] ■ a Gasdermine B (N-ter) protein whose nucleic acid corresponds to the sequence SEQ ID NO: 17 or whose nucleic acid codes for a Gasdermine B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 18;

[0051] ■ a Noxa protein whose nucleic acid corresponds to the sequence SEQ ID NO: 19 or whose nucleic acid codes for a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 20;

[0052] ■ a streptolysin O whose nucleic acid corresponds to the sequence SEQ ID NO:

[0053] 21 or whose nucleic acid codes for a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 22; and

[0054] ■ an enterotoxin whose nucleic acid corresponds to the sequence SEQ ID NO: 23 or whose nucleic acid codes for an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 24.

[0055] In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is an inducible Caspase-9 (iC9) protein whose nucleic acid corresponds to the sequence SEQ ID NO: 5 or whose nucleic acid codes for an inducible Caspase-9 (iC9) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 6. In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a Bax (S 184 del) protein whose nucleic acid corresponds to the sequence SEQ ID NO: 7 or whose nucleic acid codes for a Bax (S 184 del) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 8.In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is an M2 (H37A) ion channel of influenza virus whose nucleic acid corresponds to the sequence SEQ ID NO: 9 or whose nucleic acid encodes an M2 (H37A) ion channel of influenza virus whose amino acid sequence corresponds to the sequence SEQ ID NO: 10. In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a constitutively active caspase-3 protein (V266E) whose nucleic acid corresponds to the sequence SEQ ID NO: 11 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 12.In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a diphtheria toxin (DTA) A subunit whose nucleic acid corresponds to the sequence SEQ ID NO: 13 or whose nucleic acid codes for a diphtheria toxin (DTA) A subunit whose amino acid sequence corresponds to the sequence SEQ ID NO: 14, and an attenuated aDTA (G128D) mutant whose nucleic acid corresponds to the sequence SEQ ID NO: 15 or whose nucleic acid codes for an attenuated aDTA (G128D) mutant whose amino acid sequence corresponds to the sequence SEQ ID NO: 16.In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a Gasdermin B (N-ter) protein whose nucleic acid corresponds to the sequence SEQ ID NO: 17 or whose nucleic acid codes for a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 18. In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a Noxa protein whose nucleic acid corresponds to the sequence SEQ ID NO: 19 or whose nucleic acid codes for a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 20.In particular, the invention relates to the expression cassette as described above, wherein said toxic or suicide transgene is a streptolysin O whose nucleic acid corresponds to the sequence SEQ ID NO: 21 or whose nucleic acid codes for a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 22. In particular, the invention also relates to the expression cassette as described above, wherein said toxic or suicide transgene is an enterotoxin whose nucleic acid corresponds to the sequence SEQ ID NO: 23 or whose nucleic acid codes for an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 24.

[0056] According to another embodiment, the invention relates to the expression cassette as described above, said expression cassette being selected from among those having at least 80% identity with the SEQ ID NOs sequences: 26 to 65. In particular, the invention relates to the expression cassette as described above, said expression cassette being selected from the SEQ ID NOs sequences: 26 to 65. In other words, the invention relates to the expression cassette as described above, said expression cassette being the SEQ ID NO sequence: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 (see Table 1).

[0057]

[0058] Table 1. List of expression cassettes

[0059] iC9: Inducible Caspase-9; Bax: Bax (S184 del); M2: M2 ion channel (H37A); Casp3: Constitutively active caspase-3 (V266E); DTA: Diphtheria toxin A subunit; aDTA: Attenuated mutant aDTA (G128D); GasdB: Gasdermin B (N-ter); Noxa: Noxa protein; StrepO: Streptolysin O; ET: Enterotoxin.

[0060] In view of the foregoing, the invention also relates to the expression cassette as described above, in which said transgene is:

[0061] ■ a transcription factor chosen from: c-JUN, FOXO1, BATF, TBET and NRF2;

[0062] ■ a cytokine chosen from: IL2, IFNy, TNFa, IL18, and IL12;

[0063] ■ the GLUT 1 transporter;

[0064] ■ a nuclease chosen from: Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas®, their orthologs and their functional mutants or variants;

[0065] ■ un CAR choisi parmi : CAR anti-A-folate receptor, CAR anti-Carbonic anhydrase IX, CAR anti-CD 171, CAR anti-CD 19, CAR anti-CD20, CAR anti- CD276, CAR anti-CD319, CAR anti-CEA, CAR anti-cMet, CAR anti-EGFR, CAR anti-EGFRIII, CAR anti-EGFRn, CAR anti-FAP, CAR anti-GD2, CAR anti-gplOO, CAR anti-GPC3, CAR anti-HER2, CAR anti-IL13Ra2, CAR anti- MAGE, CAR anti-MART-1, CAR anti-MSLN, CAR anti-Mucl, CAR anti- Mucl6, CAR anti-NKG2D, CAR anti-PSMA, CAR anti-TRP-1, CAR anti- TRP2, CAR anti-VEGFR, CAR anti-HLA-G, CAR anti-CLDN18.2, CAR anti- EPCAM, CAR anti-FAP, CAR anti-RORl, CAR anti-ROR2, CAR anti-PDl, et CAR anti-PSCA ; ou

[0066] ■ A toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) subunit A and an attenuated aDTA mutant (G128D), a streptolysin O (notably that of S. pyogenes) and an enterotoxin (notably that of C. perfringens).

[0067] According to another embodiment, the invention relates to the expression cassette as described above, said expression cassette further comprising, between said inducible promoter sequence and said transgene (in particular said toxic or suicide transgene), a coding or non-coding sequence having post-transcriptional regulatory properties. The expression "coding or non-coding sequence having post-transcriptional regulatory properties" refers to a nucleic acid sequence that may be either coding (having the capacity to code for proteins) or non-coding (not coding for proteins), and that possesses regulatory properties acting after the transcription process.That is to say, after the production of an mRNA, the presence of said nucleic acid sequence within it inhibits, under certain conditions (for example, normal physiological conditions), the translation of said mRNA and therefore the production of the protein that the mRNA encodes. Under other conditions (for example, conditions of cellular stress), the presence of said nucleic acid sequence within the mRNA no longer has its inhibitory role on translation. Translation then proceeds, and the protein encoded by said mRNA is produced. Among the coding or non-coding sequences having regulatory properties at the post-transcriptional level, the invention particularly utilizes nucleic acid sequences, which are advantageously chosen from among sequences having at least 90% identity with the SEQ ID NOs sequences: 66 to 92.

[0068] According to another embodiment, the invention relates to the expression cassette as described above, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level being chosen from sequences having at least 90% identity with the SEQ ID NOs sequences: 66 to 92. In particular, the invention relates to the expression cassette as described above, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level being chosen from the SEQ ID NOs sequences: 66 to 92. In other words, the invention relates to the expression cassette as described above, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level being the SEQ ID NOs sequences: 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 or 92 (Table 2).

[0069]

[0070] Table 2. List of 5'UTR sequences with post-transcriptional regulatory properties

[0071] Advantageously, the invention relates to the expression cassette as described above, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level being the 5'UTR sequence of ATF4 with sequence SEQ ID NO: 66.

[0072] As stated above, one of the aims of the invention is to provide a genetically modified animal eukaryotic cell for use in cell and / or gene therapy. To achieve this and manufacture said genetically modified animal eukaryotic cell, lentiviral vector technology can be implemented. Therefore, a third aspect of the invention relates to a vector genome comprising a lentiviral genome into which an expression cassette as described above has been introduced.

[0073] "Vector genome" refers to a sequence of nucleic acids comprising both:

[0074] ■ the nucleic acid comprising the genetic information (= lentiviral genome) necessary for the production of a lentiviral vector also called a (lenti-)viral particle, said lentiviral vector or said (lenti-)viral particle comprising said encapsulated and enveloped vector genome, which is efficient at entering the cell and is non-replicative; and

[0075] ■ the nucleic acid comprising the genetic information of an expression cassette according to the invention, i.e. comprising an inducible promoter sequence upstream of a transgene.

[0076] "Lentiviral genome" refers, as mentioned, to the nucleic acid carrying the genetic information necessary for the production of a lentiviral vector, also called a (lenti-)viral particle, the lentiviral vector, or the (lenti-)viral particle comprising the encapsulated and enveloped vector genome. This lentiviral vector is efficient at entering the cell, is non-replicative, and leads to the integration (targeted or untargeted) of the vector genome into the genome of an infected cell. In some cases, the lentiviral particle will contain a mutant integrase (D64V), resulting in a non-integrative provirus (post-reverse transcription vector DNA) into the genome of the transduced cell. Consequently, the genome of this vector will be in episome form within the cell nucleus.Classically, the structure of this lentiviral genome after reverse transcription comprises: The cis sequences of the HIV lentiviral genome, namely two Long Terminal Repeats (LTRs) flanking the ends of the vector genome with the U3, R, and U5 regions, but where the U3 region is mutant (AU3), lacking its enhancer sequence and therefore without promoter activity. Lentiviral vectors with AU3 in the LTRs are called "self-inactivating" since they cannot be transcribed by wild-type HIV. The 5' LTR of the genome is followed by a "psi" sequence for the encapsidation of the vector's RNA genome, a Rev Responsive Element (RRE) sequence for the export of the vector's RNA genome, the Central Polypurine Tract (cppt) and Central Termination Sequence (cts) sequences for the formation of the central DNA triplex during reverse transcription, and a 3' LTR. In addition to the cis sequences of the virus, these vectors contain an expression cassette, placed between the two LTRs.

[0077] According to another embodiment, the invention relates to the vector genome as described above, in which said lentiviral genome into which said expression cassette as described above has been introduced is a Self-inactivating Lentivirus (SIN) or a Respiratory syncytial virus (RSV).

[0078] According to another embodiment, the invention relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN). In particular, the invention relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 93. In particular, the invention also relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 93.Note that the insertion of said expression cassette into said Self-inactivating Lentivirus (SIN) of sequence SEQ ID NO: 93 can be or is carried out at the level of nucleotides 2229 and 2230 of this SEQ ID NO: 93. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 94 and 95.

[0079] According to another embodiment, the invention relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Respiratory Syncytial Virus (RSV). In particular, the invention relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Respiratory Syncytial Virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 96. In particular, the invention also relates to the vector genome as described above, wherein said lentiviral genome into which said expression cassette has been introduced is a Respiratory Syncytial Virus (RSV) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 96.Note that the insertion of said expression cassette into said Respiratory syncytial virus (RSV) of sequence SEQ ID NO: 96 can be or is carried out at the level of nucleotides 1 737 and 1 738 of this SEQ ID NO: 96. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 97 and 98.

[0080] According to another embodiment, the invention relates to the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is:

[0081] ■ a Self-inactivating Lentivirus (SIN) whose nucleic acid sequence has at least 80% identity with the nucleic acid sequence SEQ ID NO: 93; or

[0082] ■ a Respiratory syncytial virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 96;

[0083] and in which said expression cassette (specifically chosen from the SEQ ID NOs sequences: 26 to 65) comprises:

[0084] ■ an inducible promoter chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0085] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (wild-type or one of its mutants, including mutant S 13 A), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) A subunit and an attenuated aDTA mutant (G128D), streptolysin O (including that of S. pyogenes) and an enterotoxin (including that of C. perfringens).

[0086] In view of the foregoing, it is understood that the invention implements in particular a vector genome as described above, which is an artificial construct made from a first building block corresponding to said lentiviral genome, into which has been introduced an expression cassette comprising at least 2 other building blocks, namely:

[0087] - an inducible promoter sequence according to the invention (brick 2);

[0088] - optionally a coding or non-coding sequence having regulatory properties at the post-transcriptional level (block 3 - abbreviated 5'UTR);

[0089] - a transgene (in particular a toxic or suicide transgene - brick 4); and

[0090] - optionally a nucleic acid sequence allowing transcription of a polyA tail (e.g. SEQ ID NO: 25).

[0091] The invention therefore makes available, for example, to the medical profession, at least 4 building blocks from which to reconstitute a vector genome according to the invention, from among all possible combinations (Table 3).

[0092]

[0093]

[0094]

[0095] Table 3. Building blocks of the invention

[0096] The name of the vector genome of the invention then corresponds to the concatenation of the names of each building block / sequence. For example, a vector genome of the invention is LV G2(SIN)-ULBPl-iC9 or LV G2(SIN)-ULBPl-iC9-pA or LV G2(SIN)-ULBP1-5'UTR ATF4-iC9 or LV G2(SIN)-LTLBP1-5'UTR ATF4-iC9-pA depending on whether the optional building blocks / sequences are present or not. In detail, in this example, this vector genome is constructed on the basis of a G2(SIN) lentiviral genome whose sequence has at least 80% identity with the sequence SEQ ID NO: 93,

[0097] in which has been introduced, between nucleotides 2229 and 2230, an expression cassette ULBPl-iC9 or ULBPl-iC9-pA or ULBPl-5'UTR ATF4-iC9-pA, which comprises from the 5' end to the 3' end:

[0098] ■ an inducible ULBP1 promoter having at least 90% identity with the SEQ ID NO sequence: 1; ■ optionally a coding or non-coding sequence having regulatory properties at the L-transcriptional level having at least 90% identity with the SEQ ID NO sequence: 66 (ATF4 5'UTR);

[0099] ■ a toxic or suicide transgene, which encodes an inducible Caspase-9 (iC9) protein whose nucleic acid sequence has at least 80% identity with the nucleic acid sequence SEQ ID NO: 5 or whose nucleic acid encodes an inducible Caspase-9 (iC9) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 6; and

[0100] ■ Optionally a nucleic acid sequence allowing transcription of a polyA tail (e.g. SEQ ID NO: 25).

[0101] This is also the case for all the vector genomes described above.

[0102] Advantageously, the 5' to 3' orientation of the transcription of the expression cassette of the invention is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome. In this case, all the provided sequences relating to said expression cassette of the invention, of which only the directional (5' > 3') sequences are provided, are found on the complementary strand of said vector genome as described above.

[0103] According to another aspect, the invention relates to a plasmid comprising a vector genome as described above and the means of expressing it.

[0104] "Plasmid" refers to a circular, double-stranded DNA molecule that necessarily possesses a bacterial origin of replication and, optionally, an origin of SV40 virus, so that it can replicate autonomously, e.g., in E. coli for its amplification and, e.g., in HEK 293T cells after transfection, and a selection gene to prevent its loss during bacterial amplification over successive cell divisions. In the invention, this further comprises the inducible promoter sequence as described above, the nucleic acid of said expression cassette of the invention or the nucleic acid of said vector genome of the invention, and the means for expressing it.

[0105] "Means of expressing it" refers to the existence in the plasmid of the invention of the genetic elements necessary for the expression of the vector genome of the invention and the production of a viral vector (in particular lentiviral) according to the invention.

[0106] According to another embodiment, the invention relates to the plasmid as described above, said plasmid being a pLV-SIN plasmid or a pLV-RSV plasmid.

[0107] According to another embodiment, the invention relates to the plasmid as described above, said plasmid being a pLV-SIN plasmid. In particular, the invention relates to the plasmid as described above, said plasmid being a pLV-SIN plasmid whose nucleic acid sequence has at least 80% identity with the nucleic acid sequence SEQ ID NO: 99. In particular, the invention also relates to the plasmid as described above, said plasmid being a pLV-SIN plasmid whose nucleic acid sequence corresponds to the nucleic acid sequence SEQ ID NO: 99. It should be noted that the insertion of said vector genome into said pLV-SIN plasmid of sequence SEQ ID NO: 99 can be, or is, carried out at nucleotides 4939 and 4940 of this SEQ ID NO: 99. That is to say, said vector genome is located in this case between sequences SEQ ID NOs: 100 and 101.

[0108] According to another embodiment, the invention relates to the plasmid as described above, said plasmid being a pLV-RSV plasmid. In particular, the invention relates to the plasmid as described above, said plasmid being a pLV-RSV plasmid whose nucleic acid sequence has at least 80% identity with the nucleic acid sequence SEQ ID NO: 102. In particular, the invention also relates to the plasmid as described above, said plasmid being a pLV-RSV plasmid whose nucleic acid sequence corresponds to the nucleic acid sequence SEQ ID NO: 102. It should be noted that the insertion of said vector genome into said pLV-RSV plasmid of sequence SEQ ID NO: 102 can be, or is, carried out at nucleotides 234 and 235 of this SEQ ID NO: 102. That is to say, said vector genome is located in this case between sequences SEQ ID NOs: 103 and 104.

[0109] According to another embodiment, the invention relates to the plasmid as described above, in which the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed with respect to the 5' to 3' orientation of the transcription of said expression cassette as described above.

[0110] According to another embodiment, the invention relates to the plasmid as described above, in which:

[0111] ■ said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 99 and 102;

[0112] ■ said lentiviral genome introduced into said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 93 and 96;

[0113] and in which said expression cassette (specifically chosen from the SEQ ID NOs sequences: 26 to 65) comprises:

[0114] ■ an inducible promoter chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0115] ■ a transgene chosen from: oncogenes, anti-oncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic or suicide genes.

[0116] According to another embodiment, the invention relates to the plasmid as described above, in which:

[0117] ■ said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 99 and 102;

[0118] ■ said lentiviral genome introduced into said plasmid is chosen from sequences having at least 80% identity with SEQ ID NOs sequences 93 and 96; and in which said expression cassette (in particular chosen from SEQ ID NOs sequences 26 to 65) comprises:

[0119] ■ an inducible promoter chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0120] ■ a transgene chosen from: transcription factors, cytokines, transporters, nucleases, chimeric antigen receptors (CARs) and toxic or suicide genes.

[0121] According to another embodiment, the invention relates to the plasmid as described above, in which:

[0122] ■ said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 99 and 102;

[0123] ■ said lentiviral genome introduced into said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 93 and 96;

[0124] and in which said expression cassette (specifically chosen from the SEQ ID NOs sequences: 26 to 65) comprises:

[0125] ■ an inducible promoter chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0126] ■ a transgene chosen from:

[0127] - a transcription factor chosen from: c-JUN, FOXO1, BATF, TBET and NRF2;

[0128] - a cytokine chosen from: IL2, IFNy, TNFa, IL18, and IL12;

[0129] - the GLUT 1 transporter;

[0130] - a nuclease chosen from: Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas®, their orthologs and their functional mutants or variants;

[0131] - un CAR choisi parmi : CAR anti-A-folate receptor, CAR anti-Carbonic anhydrase IX, CAR anti-CD171, CAR anti-CD 19, CAR anti-CD20, CAR anti- CD276, CAR anti-CD319, CAR anti-CEA, CAR anti-cMet, CAR anti-EGFR, CAR anti-EGFRIII, CAR anti-EGFRn, CAR anti-FAP, CAR anti-GD2, CAR anti-gpl 00, CAR anti-GPC3, CAR anti-HER2, CAR anti-IL13Ra2, CAR anti- MAGE, CAR anti-MART-1, CAR anti-MSLN, CAR anti-Mucl, CAR anti- Muclô, CAR anti-NKG2D, CAR anti-PSMA, CAR anti-TRP-1, CAR anti- TRP2, CAR anti-VEGFR, CAR anti-HLA-G, CAR anti-CLDN18.2, CAR anti- EPCAM, CAR anti-FAP, CAR anti-RORl, CAR anti-ROR2, CAR anti-PDl, et CAR anti-PSCA ; et

[0132] - A toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active Caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) subunit A and an attenuated aDTA mutant (G128D), a streptolysin O (particularly that of S. pyogenes), and an enterotoxin (particularly that of C. perfringens). According to another embodiment, the invention relates to the plasmid as described above, in which:

[0133] ■ said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 99 and 102;

[0134] ■ said lentiviral genome introduced into said plasmid is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 93 and 96;

[0135] and in which said expression cassette (specifically chosen from the SEQ ID NOs sequences: 26 to 65) comprises:

[0136] ■ an inducible promoter chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0137] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (wild-type or one of its mutants, including mutant S 13 A), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) subunit A and an attenuated aDTA mutant (G128D), streptolysin O (including that of S. pyogenes) and an enterotoxin (including that of C. perfringens).

[0138] In view of the foregoing, it is understood that the invention implements in particular a plasmid as described above comprising a vector genome as described above, which is an artificial construct made from a first building block corresponding to said lentiviral genome, into which has been introduced an expression cassette comprising at least 2 other building blocks, namely:

[0139] - an inducible promoter sequence according to the invention (brick 2);

[0140] - optionally a coding or non-coding sequence having regulatory properties at the post-transcriptional level (block 3 - abbreviated 5'UTR);

[0141] - a transgene (in particular a toxic or suicide transgene - brick 4); and

[0142] - optionally a nucleic acid sequence allowing transcription of a polyA tail (e.g. SEQ ID NO: 25).

[0143] The invention therefore makes available, for example, to the medical profession, at least 4 building blocks from which to reconstitute a plasmid according to the invention, from among all the possible combinations (Table 3).

[0144] The name of the plasmid of the invention, whose backbone is chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 99 (empty pLV G2 (SIN)) and 102 (empty pLV G3 (RSV)), then corresponds to the concatenation of the names of each building block, with the prefix "p" added to "LV" to indicate that it is a plasmid. For example, a plasmid of the invention is pLV G2(SIN)-ULBPl-iC9 or pLV G2(SIN)-ULBPl-iC9-pA or pLV G2(SIN)-ULBP1-5'UTR ATF4-iC9 or pLV G2(SIN)-ULBP1-5'UTR ATF4-iC9-pA depending on whether the optional building blocks / sequences are present or not.In detail, when discussing this example, this plasmid has as its backbone a sequence having at least 80% identity with the SEQ ID NOs sequence: 99 into which has been introduced a vector genome constructed on the basis of a G2(SIN) lentiviral genome whose sequence has at least 80% identity with the SEQ ID NO: 93 sequence, into which has been introduced, between nucleotides 2229 and 2230, an expression cassette ULBPl-iC9 or ULBPl-iC9-pA or ULBPl-5'UTRATF4-iC9 or ULBPl-5'UTR ATF4-iC9-pA, which includes from the 5' end to the 3' end:.

[0145] ■ an inducible ULBP1 promoter having at least 90% identity with the SEQ ID NO sequence: 1;

[0146] ■ optionally a coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level having at least 90% identity with the sequence SEQ ID NO: 66 (5'UTR of ATF4);

[0147] ■ a toxic or suicide transgene, which encodes an inducible Caspase-9 (iC9) protein whose nucleic acid sequence has at least 80% identity with the nucleic acid sequence SEQ ID NO: 5 or whose nucleic acid encodes an inducible Caspase-9 (iC9) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 6; and

[0148] ■ Optionally a nucleic acid sequence allowing transcription of a polyA tail (e.g. SEQ ID NO: 25).

[0149] This is also the case for all the plasmids described above.

[0150] In another aspect, the invention relates to the use of a plasmid as described above to produce a viral vector (e.g., a lentiviral vector, or a vector derived from an adenovirus or adenovirus-associated virus (AAV)) comprising a vector genome as described above. "Viral vector" means a recombinant viral particle (particularly a lentiviral) obtained using the plasmid of the invention, which enables the production of the (lenti-)viral particle of the invention. This particle comprises said vector genome encapsulated and / or enveloped. It is efficient at entering the cell, it is non-replicative, and it leads either to the integration (targeted or untargeted) of said vector genome into the genome of an infected cell, or to the presence of a non-integrated episome in the cellular genome.

[0151] In another aspect, the invention relates to a method for producing viral vectors comprising a vector genome as described above. In particular, it relates to producing lentiviral vectors. Furthermore, the invention also relates to a method for producing lentiviral vectors comprising a vector genome as described above, said method comprising at least the following steps:

[0152] a. Co-transfection of an animal eukaryotic cell (e.g., HEK 293 T) with:

[0153] ■ a plasmid as described above;

[0154] ■ a plasmid comprising an envelope protein and the means to express it; and

[0155] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means to express it,

[0156] to obtain a transfected animal eukaryotic cell; b. to culture said transfected animal eukaryotic cell to allow the production of lentiviral vectors comprising a vector genome as described above; and

[0157] c. of harvesting and purification of said lentiviral vectors.

[0158] Such a method is illustrated in the examples below and is accessible to those skilled in the art, given the available literature (e.g., Zennou et al. Nat Biotechnol. 2001 May; 19(5):446-50). However, for illustrative purposes, it is specified that:

[0159] ■ said animal eukaryotic cell may be a cell belonging to a cell line chosen from: HEK 293T, HEK 293, CHO, GS-CHO, Free style 293-F cells (FS293), Viral Production Cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8 and PS46.2;

[0160] ■ Said plasmid, comprising a viral envelope protein and the means to express it, may be chosen from the following plasmids: pHCMV-VSV or pVSVg (SEQ ID NO: 105), pHCMV-Mokola, pHCMV-RabiesG, pHCMV-AmphoEnv, pHCMV-LCMV-WE, pHCMV-LCMV-Arm53b and pHCMV-10Al (Sena-Esteves M and aL. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. 2004 Dec 15;122(2):131-9); and

[0161] ■ said plasmid comprising the structural proteins and enzymes of HIV-1, and the means of expressing it may be chosen from the plasmids: p8.92 (SEQ ID NO: 106), p8.92 (IN D64V) (SEQ ID NO: 107), psPAX2 (SEQ ID NO: 108), pMDLg / pRRE (SEQ ID NO: 109) and pRSV-Rev (SEQ ID NO: 110).

[0162] For illustrative purposes and without limitation, the following protocol is also provided: Cells (e.g., HEK 293 T) were seeded onto suitable cell culture media to achieve 50–60% confluence after 24 hours of culture. The following day, the cells were co-transfected, using the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome and two transcomplementing plasmids encoding HIV-1 enzymes and structural proteins (p8.9) and the envelope glycoprotein of vesicular stomatitis virus (pVSVg). Co-transfection was performed at a plasmid ratio of 2:2:1. The transfected cells were cultured for 5 hours in contact with the precipitate, and then the culture medium was renewed. The supernatant containing the lentiviral particles was harvested 48 hours later.The lentiviral suspension was treated with DNase I to remove persistent plasmids, filtered (membrane porosity 0.2 µm), and then ultracentrifuged at 60,000 g for 90 minutes at 4°C. The pellets were resuspended in a volume of phosphate-buffered saline (PBS) at a concentration 1000-fold. The resulting suspensions were aliquoted and stored at -80°C until use.

[0163] According to another embodiment, the invention relates to the method of producing lentiviral vectors as described above, wherein said animal eukaryotic cell belongs to a cell line selected from: HEK 293T, HEK 293, CHO, GS-CHO, Free style 293-F cells (FS293), Viral Production Cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8 and PS46.2. Advantageously, the invention relates to the method of producing lentiviral vectors as described above, wherein said animal eukaryotic cell belongs to the HEK 293 T cell line. Since a method of producing a viral vector (in particular a lentiviral one) is described, it is understood that another aspect of the invention relates to a viral vector comprising a vector genome as described above, in particular said viral vector being a lentiviral vector, or a vector derived from adenovirus or adenovirus-associated virus (AAV).According to this same aspect, the invention also relates to a viral vector (in particular lentiviral) that can be obtained by the method of producing viral vectors (in particular lentivirals) as described above.

[0164] According to another embodiment, the invention relates to the viral vector as described above, said viral vector being a lentiviral vector.

[0165] It is also understood that another aspect of the invention relates to the use:

[0166] - of an inducible promoter sequence as described above;

[0167] - of an expression cassette as described above;

[0168] - of a plasmid as described above; or

[0169] - of a viral vector as described above,

[0170] to transfect / transduce an animal eukaryotic cell.

[0171] According to another embodiment, the invention relates to the use as described above, said animal eukaryotic cell being chosen from:

[0172] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in blood cancer;

[0173] ■ an immune cell specifically chosen from: the T lymphocyte and the NK cell;

[0174] ■ a myofibroblast, in particular a myofibroblast involved in a fibrotic process;

[0175] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord injury and forming a glial scar that prevents regeneration; and

[0176] ■ an induced pluripotent stem cell (iPSC).

[0177] "Cancer cell" refers to an abnormal cell that has acquired specific characteristics leading to cancer (or a malignant tumor), which is a disease characterized by uncontrolled and abnormally rapid cell proliferation (tumor) within a normal (healthy) tissue of the body, such that the survival of the latter is threatened. Among these tumors are solid tumors and liquid tumors. According to another embodiment, the invention relates to the use described above, in which said animal eukaryotic cell is a cancer cell, in particular a cancer cell forming a solid tumor or involved in blood cancer. Specifically, the invention also relates to the use described above, in which said animal eukaryotic cell is a cancer cell forming a solid tumor.In particular, the invention also relates to the use described above, in which said animal eukaryotic cell is a cancer cell involved in blood cancer. "Immune cell" refers to a cell involved in the functioning of the immune system. Numerous examples exist, such as lymphocytes (e.g., T, B, NK, and NKT cells), phagocytes (e.g., macrophages and dendritic cells), and granulocytes (e.g., neutrophils, eosinophils, and basophils), as well as the precursor cells of these cells. These cells may be of autologous, syngeneic, allogeneic, or xenogeneic origin. Finally, these cells may be genetically modified. It should be noted that the invention specifically utilizes a T lymphocyte and an NK cell. The term "T lymphocyte" in the invention refers in particular to a naïve, effector, or memory T lymphocyte, with auxiliary, cytotoxic, or regulatory properties.The term "NK cell" in the invention refers in particular to a primary, cancerous, or immortalized NK cell. According to another embodiment, the invention relates to the use described above, in which said animal eukaryotic cell is an immune cell, specifically selected from among: a T lymphocyte and an NK cell. In particular, the invention also relates to the use described above, in which said animal eukaryotic cell is a T lymphocyte or an NK cell.

[0178] "Myofibroblast" refers to a cell characterized by the production of matrix proteins, proliferation, migration, and contraction, activated in repair and healing processes. These myofibroblasts are found in certain organs such as the liver, lung, heart, or kidney in chronic diseases. The process of tissue replacement by myofibroblasts is called fibrosis. According to another embodiment, the invention relates to the use as described above, in which said animal eukaryotic cell is a myofibroblast. In particular, the invention also relates to the use as described above, in which said animal eukaryotic cell is a myofibroblast involved in a fibrosis process.

[0179] "Astrocyte" refers to a glial cell of the central nervous system. Generally star-shaped, it performs a variety of important functions, primarily supporting and protecting neurons. According to another embodiment, the invention relates to the use described above, wherein said animal eukaryotic cell is an astrocyte, in particular an astrocyte activated during a brain or spinal cord injury and forming a glial scar that prevents regeneration. Specifically, the invention also relates to the use described above, wherein said animal eukaryotic cell is an astrocyte activated during a brain or spinal cord injury that forms a glial scar that prevents regeneration.

[0180] “Stem cell” refers to a cell characterized by its undifferentiated state and its ability to differentiate and generate or regenerate tissue, such as hematopoietic tissue, muscle, brain (neurons), retina, liver (hepatocytes), pancreatic beta cells, kidney, cartilage, bone, or skin. These cells, taken from an adult organism or induced from differentiated cells (iPS), can be of autologous, syngeneic, allogeneic, or xenogeneic origin. According to another embodiment, the invention therefore relates to the use as described above, in which said animal eukaryotic cell is an induced stem cell (iPS).According to another aspect of the invention, it relates to an animal eukaryotic cell (transduced) comprising an inducible promoter sequence as described above or an expression cassette as described above, said animal eukaryotic cell (transduced) being in particular chosen from:.

[0181] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in blood cancer;

[0182] ■ an immune cell specifically chosen from: the T lymphocyte and the NK cell;

[0183] ■ a myofibroblast, in particular a myofibroblast involved in a fibrotic process;

[0184] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord injury and forming a glial scar that prevents regeneration; and

[0185] ■ an induced pluripotent stem cell (iPSC),

[0186] said inducible promoter sequence as described above or said expression cassette being in particular stably inserted into the genome of said (transduced) animal eukaryotic cell.

[0187] According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell, in particular a cancer cell forming a solid tumor or involved in blood cancer. In particular, the invention also relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell forming a solid tumor. In particular, the invention also relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell involved in blood cancer.

[0188] According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is an immune cell, in particular selected from: the T lymphocyte and the NK cell. In particular, the invention also relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a T lymphocyte or an NK cell. According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast. In particular, the invention also relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast involved in a fibrotic process.

[0189] According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is an astrocyte, in particular an astrocyte activated during brain or spinal cord injury and forming a glial scar preventing regeneration. In particular, the invention also relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is an astrocyte activated during brain or spinal cord injury and forming a glial scar preventing regeneration.

[0190] According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above, wherein said animal eukaryotic cell is an induced pluripotent stem cell (iPS).

[0191] According to another aspect of the invention, it relates to the (transduced) animal eukaryotic cell as described above for use in cell and / or gene therapy, in particular for treating a pathology selected from: cancer, leukemia, and fibrosis. "Cell therapy" refers to a method known as "biotherapy," which aims to treat an organ or organism by providing therapeutic cells to complement, replace, supplement, or kill failing cells.

[0192] “Gene therapy” also refers to a method called “biotherapy” which aims to modify a tissue, organ or organism by transferring nucleic acids into the cells of that tissue, organ or organism.

[0193] According to another embodiment, the invention relates to the (transduced) animal eukaryotic cell as described above for its use as described above to treat a pathology selected from: cancer, leukemia, and fibrosis. In particular, the invention relates to the (transduced) animal eukaryotic cell as described above for its use as described above to treat cancer. In particular, the invention relates to the (transduced) animal eukaryotic cell as described above for its use as described above to treat leukemia.

[0194] Alternatively and according to this same aspect, the invention relates to a method of treating a pathology chosen from: cancer, leukemia and fibrosis, said method comprising the administration of an animal eukaryotic cell (transduced) as described above (or of a pharmaceutical composition comprising at least one animal eukaryotic cell (transduced) according to the invention and a pharmaceutically acceptable excipient) to a patient who needs it.

[0195] With the aid of the tools of the invention, it enables the implementation of effective and safe cell and / or gene therapies. These therapies exploit the so-called safety switch technology, which, under the action of the correct stimulus (and only the correct stimulus), allows the activation of the inducible promoter sequence as described above, present in the expression cassette, thus enabling the expression of the transgene of interest, in particular a toxic or suicide transgene. In this case, and by way of example, the therapeutic use of an immune cell:

[0196] ■ modified and armed to target and destroy cancer cells; and

[0197] ■ transduced according to the invention with a toxic or suicide transgene, will, after administration to a patient in need, play its role of targeting and destroying cancer cells. However, if this same immune cell becomes dysfunctional, it will then be possible to destroy it to avoid adverse effects, thanks to the safety systems provided by the invention (control of transcription induction and control of translation inhibition).

[0198] In the invention, the appropriate stimulus mentioned above, which allows for the control of the expression of the transgene of the invention and ensures the safety of the cell and / or gene therapy offered to patients who need it, corresponds to the use (administration of) a clinically applicable pharmacological molecule. Another aspect of the invention therefore relates to the use of a chemical inducer to induce the activation of an inducible promoter sequence as described above or to induce the expression of a transgene contained within an expression cassette as described above, said chemical inducer being:

[0199] ■ of artesunate (Artz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2);

[0200] ■ of asparaginase (Asp) when said inducible promoter sequence or expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0201] ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).

[0202] Alternatively, the invention relates to a method for inducing the activation of an inducible promoter sequence as described above or for inducing the expression of a transgene contained in an expression cassette as described above using a chemical inducer, said chemical inducer being:

[0203] ■ of artesunate (Artz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2);

[0204] ■ of asparaginase (Asp) when said inducible promoter sequence or expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0205] ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence or expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 2 (HSPA6), 3 (HSPA1B), or 4 (SESN2). Whether using a chemical inducer as described above or the induction method as described above, implementation of the invention involves administering said chemical inducer to the patient (in need) who has received a transduced animal eukaryotic cell as described above. This inducer, in particular chosen from artesunate, asparaginase, and bortezomib, may therefore be in the form of a pharmaceutical composition, in particular an injectable pharmaceutical composition.In so doing, the invention also relates to a chemical inducer (or a pharmaceutical composition comprising said chemical inducer) for use in inducing the activation of an inducible promoter sequence as described above or in inducing the expression of a transgene included in an expression cassette as described above, said chemical inducer being:.

[0206] ■ of artesunate (Artz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2);

[0207] ■ of asparaginase (Asp) when said inducible promoter sequence or expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0208] ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).

[0209] According to another embodiment, the invention relates to a chemical inducer for use in cell and / or gene therapy to induce the activation of an inducible promoter sequence chosen from sequences having at least 90% identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2),

[0210] said chemical inducer being:

[0211] ■ of artesunate (Artz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2); ■ of asparaginase (Asp) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0212] ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).

[0213] Alternatively and according to this same aspect, the invention relates to a method for inducing the activation of an inducible promoter sequence chosen from among the sequences having at least 90% identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2), said method comprising administering to a patient in need a chemical inducer (or a pharmaceutical composition comprising a chemical inducer and a pharmaceutically acceptable excipient), said chemical inducer being chosen from: ■ artesunate (Artz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2); ■ of asparaginase (Asp) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0214] ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2),

[0215] in which the said patient who needs it has previously received treatment including the administration of:

[0216] - of an inducible promoter sequence as described above;

[0217] - of an expression cassette as described above;

[0218] - of a plasmid as described above;

[0219] - of a viral vector as described above; or

[0220] - of a eukaryotic cell as described above.

[0221] According to another embodiment, the invention relates to a chemical inducer for use in cell and / or gene therapy as described above, said inducible promoter sequence being chosen from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2).

[0222] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being artesunate (Artz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2).In particular, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being artesunate (Artz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of sequence SEQ ID NO: 3 (HSPA1B) or 4 (SESN2).

[0223] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being asparaginase (Asp) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NO: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2).In particular, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being 1' asparaginase (Asp) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of sequence SEQ ID NO: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2).

[0224] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being bortezomib (Bz or Brtz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with the SEQ ID NOs sequence: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).In particular, the invention relates to the use of a chemical inducer as described above, or of the induction method as described above, or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being bortezomib (Bz or Brtz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of SEQ ID NO: 2 (HSPA6), 3 (HSPA1B), or 4 (SESN2). According to another embodiment, the invention relates to the use of a chemical inducer as described above, or of the induction method as described above, or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being selected from:

[0225] ■ of artesunate (Artz);

[0226] ■ of asparaginase (Asp); and

[0227] ■ bortezomib (Bz or Brtz),

[0228] when said inducible promoter sequence or expression cassette comprises an inducible promoter sequence having at least 90%, in particular 95%, identity with SEQ ID NOs sequences 3 (HSPA1B) or 4 (SESN2). In particular, the invention relates to the use of a chemical inducer as described above or the induction process as described above, or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being selected from:

[0229] ■ of artesunate (Artz);

[0230] ■ of asparaginase (Asp); and

[0231] ■ bortezomib (Bz or Brtz),

[0232] when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of sequence SEQ ID NO: 3 (HSPA1B) or 4 (SESN2).

[0233] Advantageously, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said chemical inducer being asparaginase (Asp) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of sequence SEQ ID NO: 1 (ULBP1).

[0234] Advantageously, the invention also relates to the use of a chemical inducer as described above or of the induction process as described above, said chemical inducer being bortezomib (Bz or Brtz) when said inducible promoter sequence or said expression cassette comprises an inducible promoter sequence of sequence SEQ ID NO: 2 (HSPA6).

[0235] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said inducible promoter sequence being in an expression cassette, said expression cassette comprising downstream of said inducible promoter sequence as described above a transgene.

[0236] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said transgene being selected from: oncogenes, anti-oncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies and toxic or suicide genes.

[0237] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said transgene being a toxic or suicide transgene selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin (DTA) subunit A and an attenuated mutant aDTA(G128D), a streptolysin O and an enterotoxin.

[0238] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said expression cassette being chosen from sequences having at least 80% identity with the SEQ ID NOs sequences: 26 to 65.

[0239] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above, said inducible promoter sequence or said expression cassette comprising downstream of said inducible promoter sequence a transgene being in an animal eukaryotic cell,

[0240] said animal eukaryotic cell being in particular chosen from:

[0241] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in blood cancer;

[0242] ■ an immune cell specifically chosen from: the T lymphocyte and the NK cell; ■ a myofibroblast, specifically a myofibroblast involved in a fibrotic process;

[0243] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord injury and forming a glial scar that prevents regeneration; and

[0244] ■ an induced pluripotent stem cell (iPSC),

[0245] said inducible promoter sequence or said expression cassette being in particular stably inserted into the genome of said animal eukaryotic cell.

[0246] According to another embodiment, the invention relates to the use of a chemical inducer as described above or of the induction process as described above or of a chemical inducer for its use in cell and / or gene therapy as described above to treat a pathology chosen from: cancer, leukemia and fibrosis.

[0247] According to another aspect of the invention, it relates to a kit comprising:

[0248] ■ a nucleic acid comprising an inducible promoter sequence as described above or an expression cassette as described above, or an animal eukaryotic cell (transduced) as described above; and ■ at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz).

[0249] According to this same aspect, the invention also relates to a kit comprising:

[0250] ■ a plasmid as described above or a viral vector as described above;

[0251] And

[0252] ■ at least one chemical inducer chosen from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz).

[0253] According to this same aspect, the invention relates in particular to a kit comprising:

[0254] ■ a nucleic acid comprising an inducible promoter sequence selected from sequences having at least 90% identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and

[0255] ■ at least one chemical inducer chosen from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz),

[0256] said chemical inducer being at least:

[0257] - of artesunate (Artz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2);

[0258] - of asparaginase (Asp) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and

[0259] - bortezomib (Bz or Brtz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2). According to another embodiment, the invention relates to the above kit comprising a nucleic acid including an inducible promoter sequence as described above and at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp) and bortezomib (Bz or Brtz).

[0260] According to another embodiment, the invention relates to the above kit comprising a nucleic acid including an expression cassette as described above and at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz).

[0261] According to another embodiment, the invention relates to the above kit comprising an animal eukaryotic cell (transduced) as described above and at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp), and bortezomib (Bz or Brtz). According to another embodiment, the invention relates to the above kit comprising a plasmid as described above and at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp), and bortezomib (Bz or Brtz).

[0262] According to another embodiment, the invention relates to the above kit comprising a viral vector as described above and at least one chemical inducer selected from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz).

[0263] According to another embodiment, the invention relates to the above kit, in which said nucleic acid is an expression cassette comprising downstream of said inducible promoter sequence a transgene.

[0264] Interestingly, it should be noted that the invention can be described as a kit-of-parts. That is to say, the objects that constitute it can be used in combination or separately in space and / or time.

[0265] In all respects, it should be noted that the various aspects of the invention, as well as its various embodiments, are interdependent. They can therefore be combined as many times as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This also applies to all the definitions provided in this description, which apply to all aspects of the invention and its embodiments.

[0266] Furthermore, the present invention is illustrated, but not limited to, by the following figures and examples. LIST OF FIGURES

[0267] Figure 1. CD8+ transduced with lentiviral vectors carrying GFP under the control of promoters inducible by Artz, Asp or Brtz.

[0268] A) Percentage of CD8+ T lymphocytes transduced with the pLV-ULBPl-GFP-p2a-Luc vector (SEQ ID NO: 119) (ULBP1 promoter) expressing GFP after treatment with PB S (Medium), DMSO (Artz and Brtz vehicle), Brtz, or Asp. B) Mean fluorescence intensity (MFI) per cell after treatment of cells transduced with the pLV-ULBPl-GFP-p2a-Luc vector (SEQ ID NO: 119). C) Percentage of CD8+ T lymphocytes transduced with the pLV-HSPA6-GFP-p2a-Luc vector (SEQ ID NO: 120) expressing GFP after treatment. D) MFI per cell after treatment of cells transduced with the pLV-HSPA6-GFP-p2a-Luc vector (SEQ ID NO: 120). E) Percentage of CD8+ T lymphocytes transduced with the pLV-HSPAlB-GFP-p2a-Luc vector (SEQ ID NO: 121) expressing GFP after treatment. F) MFI per cell after treatment of cells transduced with the pLV-HSPAlB-GFP-p2a-Luc vector (SEQ ID NO: 121).G) Percentage of CD8+ T lymphocytes transduced with the pLV-SESN2-GFP-p2a-Luc vector (SEQ ID NO: 122) expressing GFP after treatment. H) MFI per cell after treatment of cells transduced with the pLV-SESN2-GFP-p2a-Luc vector (SEQ ID NO: 122).

[0269] Figure 2. CD8+ lymphocytes transduced with lentiviral vectors carrying a proapoptotic suicide gene under the control of promoters inducible by Asp or Bz.

[0270] A) Percentage of CD8+ T lymphocytes transduced with the vector pLV-UBC-TagBFP_HSPA6-Noxa (SEQ ID NO: 125) (promoter Hspaô) expressing the death markers Annexin V and propidium iodide (IP) after treatment with PB S (Medium), Artz, Bz, or Asp. B) Percentage of CD8+ T lymphocytes transduced with the vector pLV-UBC-TagBFP_ULBPl-Casp3 (SEQ ID NO: 123) (promoter ULBP1) expressing the death markers Annexin V and IP after treatment with PBS (Medium), Artz, Bz, or Asp. In these experiments, labeled CD3 cells were quantified by flow cytometry 24 h after addition of the inducer to the culture medium. Each dot represents a donor. EXAMPLES

[0271] Pharmaceutical-induced promoters for regulating transgene expression in T lymphocytes: Materials and methods

[0272] Cloning of lentiviral plasmids

[0273] The lentiviral plasmids used in the experiments were cloned by enzymatic linearization and homology recircularization using the NEBuilder HiFi DNA Assembly protocol (New England Biolabs) by inserting synthesized sequences (Twist Bioscience) into a plasmid carrying the second-generation self-inactivating lentiviral genome (pLV(SIN); SEQ ID NO: 99).

[0274] Cell culture

[0275] The cells were cultured in a humidified and controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and 100 U / mL of penicillin / streptomycin. Primary T lymphocytes isolated from the blood of healthy donors were cultured in serum-free, nutrient-free (SFM) GIBCO OpTmizer™ CTS™ medium (Thermo Fisher Scientific) supplemented with Glutamax (Thermo Fisher Scientific) and 100 U / mL of penicillin / streptomycin. Stress induction was achieved by treatment with Artesunate (Artz; 5 pM) (A3731 Merck-Sigma), F Asparaginase (Asp; 1 U / mL) (A3809 Merck-Sigma), Bortezomib (Bz or Brtz; 10 nM) (50431140001, Merck-Sigma).

[0276] Production of lentiviral vectors

[0277] Non-replicating lentiviral particles were obtained using the production protocol described by Zennou et al. (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293 T cells were seeded onto suitable cell culture media to achieve 50–60% confluence after 24 hours of culture. The following day, the cells were co-transfected, by calcium phosphate precipitation, with the lentiviral genome-encoding plasmid (pLV) and two transcomplementary plasmids encoding HIV-1 enzymes and structural proteins (p8.92; SEQ ID NO: 106), and the vesicular stomatitis virus envelope glycoprotein (pVSVg; SEQ ID NO: 105). Co-transfection was performed at a plasmid ratio of 2:2:1. The transfected cells were cultured for 5 hours in contact with the precipitate, and then the culture medium was renewed. The supernatant containing the lentiviral particles was collected 48 hours later.The lentiviral suspension was treated with DNase I to remove persistent plasmids, filtered (membrane porosity 0.2 µm), and then ultracentrifuged at 60,000 g for 90 minutes at 4°C. The pellets were resuspended in a volume of phosphate-buffered saline (PB S) at a concentration 1000-fold. The resulting suspensions were aliquoted and stored at -80°C until use. The lentiviral particle concentration was determined by quantification of the lentiviral capsid protein p24. For this purpose, an aliquot of the lentiviral suspension was thawed, lysed, and diluted according to the manufacturer's protocol for the HIV-1 p24 Antigen ELISA 2.0 titration kit (Zeptometrix ref. 0801008). The concentration of p24 obtained was expressed in pg / pL, the equivalence of which is 1 pg of p24 per 10. 2 transduction units (TU).

[0278] The expression cassettes produced were:

[0279] - ULBPl-GFP-p2a-Luc (SEQ ID NO : 111) ;

[0280] - HSPA6-GFP-p2a-Luc (SEQ ID NO : 112) ;

[0281] - HSPAlB-GFP-p2a-Luc (SEQ ID NO : 113) ;

[0282] - SESN2-GFP-p2a-Luc (SEQ ID NO : 114) ;

[0283] - UBC-TagBFP_ULBP 1 -Casp3 (SEQ ID NO : 115) ;

[0284] - UBC-TagBFP_ULBPl-GFP (SEQ ID NO : 116) ;

[0285] - UBC-TagBFP_HSPA6-Noxa (SEQ ID NO : 117) ; et

[0286] - UBC-TagBFP_HSPA6-GFP (SEQ ID NO : 118),

[0287] et les vecteurs lentiviraux produits ont été :

[0288] - pLV-ULBP 1 -GFP-p2a-Luc (SEQ ID NO : 119) ;

[0289] - pLV-HSPA6-GFP-p2a-Luc (SEQ ID NO : 120) ;

[0290] - pLV-HSPAlB-GFP-p2a-Luc (SEQ ID NO : 121) ;

[0291] - pLV-SESN2-GFP-p2a-Luc (SEQ ID NO : 122) ;

[0292] - pLV-UBC-TagBFP_ULBPl-Casp3 (SEQ ID NO : 123) ;

[0293] - pLV-UBC-TagBFP_ULBPl-GFP (SEQ ID NO: 124);

[0294] - pLV-UBC-TagBFP_HSPA6-Noxa (SEQ ID NO: 125); And

[0295] - pLV-UBC-TagBFP_HSPA6-GFP (SEQ ID NO: 126).

[0296] Transduction

[0297] To integrate a transgene by transduction of a lentiviral vector, the quantity of particles required to achieve the desired infection multiplicity (vector:target ratio) was diluted in culture medium. This lentiviral suspension was then introduced into the target cells. The transduced cells were cultured for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression could be observed or measured as early as the following day by microscopy, flow cytometry, or quantitative PCR.

[0298] Flow cytometry

[0299] For the analysis of cultured cells, they were harvested and rinsed with PBS. They were then incubated for 30 minutes with surface-labeling antibodies and the Live-or-Dye 405 / 452 viability marker (Biotium) according to the respective manufacturers' recommendations. The cells were rinsed and then fixed in PBS with 1% paraformaldehyde for 15 minutes at 4°C. After rinsing, transgene expression was analyzed using a MAC SQuant Analyser 10 flow cytometer (Miltenyi).

[0300] Statistics

[0301] Statistical analyses were performed using GraphPad® Prism 10. Details regarding sample size and graph descriptions can be found in the figure captions for each analysis. To assess statistical variances between two groups, an unpaired Student's t-test or a Mann-Whitney U test was used. Statistical differences between three or more groups were analyzed using analysis of variance (ANOVA) with appropriate multiple comparison tests. In the graphs, significance is indicated by p-values ​​less than 0.05.

[0302] RESULTS

[0303] Cloning of promoters into lentiviral vectors and expression of a reporter gene into lymphocytes

[0304] After developing the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2), these were cloned into lentiviral vectors upstream of a GFP-p2a-Luc transgene, in order to quantify their respective induction rates.

[0305] Recombinant lentiviral vector particles (lVPs) carrying these promoters were obtained and used to transduce donor CD8+ T lymphocytes. After 48 hours, the cells were treated with Artz, Asp, Brtz, or DMSO as a control for the Artz and Brtz vehicle, or with PBS as a control for Asp. Under these conditions, the ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters were observed to produce virtually no background noise in the lentiviral context and to have a high induction rate of 14-fold and 11-fold, respectively, exclusively after treatment with Asp for the former and Brtz for the latter (Fig. 1A-D). Unlike the ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters, which were not expressed under basal conditions in activated CD8+ T lymphocytes, the HSPA1B gene promoter (SEQ ID NO: 3) allowed relatively high basal expression (Fig. 1E and F).Thus, the pLV-HSPAlB-GFP-p2a-Luc vector (SEQ ID NO: 121), which was expressed in the absence of induction, exhibited a 2-fold induction rate after treatment with Brtz (Fig. 1E-F). Similarly, the SESN2 gene promoter (SEQ ID NO: 4) cloned into a lentiviral vector favored high basal expression and a 2- to 3-fold induction rate with Artz, Brtz, and Asp (Fig. 1G-H).

[0306] These promoters, cloned into lentiviral vectors, have therefore enabled transcriptional regulation inducible by pharmacological molecules.

[0307] Use of ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters for the regulated expression of a suicide gene in lymphocytes

[0308] The usefulness of the ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters was tested for the regulation of a cytotoxic transgene within a lentiviral vector. To this end, two lentiviral vectors expressing the Caspase 3 (V266E) (SEQ ID NO: 11 / 12) or Noxa (S12A) (SEQ ID NO: 19 / 20) transgenes, whose mutations promote constitutively pro-apoptotic activity, were generated. These two transgenes were respectively cloned under the control of the ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters to obtain the double-copy vectors, pLV-UBC-TagBFP_ULBPl-Casp3 (SEQ ID NO: 123) and pLV-UBC-TagBFP_HSPA6-Noxa (SEQ ID NO: 125); as control vectors, the reporter transgene eGFP was cloned in place of the cytotoxic genes, Noxa or Casp3 (pLV-UBC-TagBFP_HSPA6-GFP (SEQ ID NO: 126) and pLV-UBC-TagBFP_ULBPl-GFP (SEQ ID NO: 124)).In these vectors, the TagBFP gene (SEQ ID NO: 127) encodes a blue fluorescent protein and allows for the identification of transduced cells, whereas the Noxa (SEQ ID NO: 19 / 20), Casp3 (SEQ ID NO: 11 / 12), and GFP (SEQ ID NO: 128) genes are only detected under induced conditions. Recombinant lentiviral particles were obtained and used to transduce purified CD3 lymphocytes from donors. Forty-eight hours after transduction, the cells were treated with PBS (Milieu), Artz, Bz, or Asp, and then twenty-four hours later, the cells were labeled to detect Annexin V and Propidium iodide (PI), two markers of apoptosis and cell death, respectively. Under these conditions, it was observed that more than 95% of cells transduced with the pLV-UBC-TagBFP_HSPA6-Noxa vector (SEQ ID NO: 125) died by apoptosis only if they were treated with Bz, but not with the other drugs (Fig. 2A).In contrast, cells transduced with the pLV-UBC-TagBFP_ULBPl-Casp3 vector (SEQ ID NO: 123) died only following Asp treatment (Fig. 2B). Cells transduced with the control vectors, on the other hand, were unaffected by any treatment conditions.

[0309] These results demonstrated that the ULBP1 (SEQ ID NO: 1) and HSPA6 (SEQ ID NO: 2) promoters enabled the regulated expression of pro-apoptotic genes by two distinct drugs in activated lymphocytes.

Claims

DEMANDS 1. Chemical inducer for use in cell and / or gene therapy to induce the activation of an inducible promoter sequence chosen from sequences having at least 90% identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2), said chemical inducer being: ■ of artesunate (Artz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2); ■ of asparaginase (Asp) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and ■ of bortezomib (Bz or Brtz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).

2. Chemical inducer for use in cell and / or gene therapy according to claim 1, said inducible promoter sequence being selected from the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2).

3. Chemical inducer for use in cell and / or gene therapy according to claim 1 or 2, said inducible promoter sequence being in an expression cassette, said expression cassette comprising downstream of said inducible promoter sequence a transgene.

4. Chemical inducer for use in cell and / or gene therapy according to claim 3, said transgene being selected from: oncogenes, anti-oncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies and toxic or suicide genes.

5. Chemical inducer for use in cell and / or gene therapy according to claim 3 or 4, said transgene being a toxic or suicide transgene selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a toxin A subunit diphtheria (DTA) and an attenuated mutant aDTA (G128D), a streptolysin O and an enterotoxin.

6. Chemical inducer for use in cell and / or gene therapy according to any one of claims 3 to 5, said expression cassette being selected from sequences having at least 80% identity with SEQ ID NOs sequences: 26 to 65.

7. Chemical inducer for use in cell and / or gene therapy according to any one of claims 1 to 6, said inducible promoter sequence or expression cassette comprising downstream of said inducible promoter sequence a transgene being in an animal eukaryotic cell, said animal eukaryotic cell being in particular chosen from: ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in blood cancer; ■ an immune cell specifically chosen from: the T lymphocyte and the NK cell; ■ a myofibroblast, in particular a myofibroblast involved in a fibrotic process; ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord injury and forming a glial scar that prevents regeneration; and ■ an induced pluripotent stem cell (iPSC), said inducible promoter sequence or said expression cassette being in particular stably inserted into the genome of said animal eukaryotic cell.

8. Chemical inducer for use in cell and / or gene therapy according to any one of claims 1 to 7 for treating a pathology selected from: cancer, leukemia and fibrosis.

9. Kit includes: ■ a nucleic acid comprising an inducible promoter sequence selected from sequences having at least 90% identity with the SEQ ID NOs sequences: 1 (ULBP1), 2 (HSPA6), 3 (HSPA1B) and 4 (SESN2); and ■ at least one chemical inducer chosen from: artesunate (Artz), asparaginase (Asp) and Bortezomib (Bz or Brtz), said at least one chemical inductor being at least: - of artesunate (Artz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NOs sequence: 3 (HSPA1B) or 4 (SESN2); - of asparaginase (Asp) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 1 (ULBP1), 3 (HSPA1B) or 4 (SESN2); and - bortezomib (Bz or Brtz) when said inducible promoter sequence has at least 90% identity with the SEQ ID NO sequence: 2 (HSPA6), 3 (HSPA1B) or 4 (SESN2).

10. Kit according to claim 9, wherein said nucleic acid is an expression cassette comprising downstream of said inducible promoter sequence a transgene.

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