Production of toxic vectors

Abstract

The present invention relates in general to the field of medicine. More particularly, it relates to an eukaryotic cell expressing a ribonucleoprotein complex and to the use thereof for producing toxic vectors. The invention also relates to the method for producing these toxic vectors.

Description

[0001] PRODUCTION OF TOXIC VECTORS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of medicine. More particularly, it relates to a eukaryotic cell expressing a ribonucleoprotein complex and its use for the production of toxic vectors. The method for producing these toxic vectors also belongs to the invention.

[0004] PRIOR ART

[0005] For more than twenty-five years, lentiviral vectors based on the HIV-1 genome have been produced and used for efficient gene transfer into mammalian cells in vitro and in vivo. The genome of these vectors and the production processes have been optimized to improve their efficacy and safety for use in human medicine. Taking advantage of the significant growth in gene therapy, lentiviral vectors are involved in numerous clinical trials despite their limitations and side effects due in particular to their preferential integration into the transcribed regions of the genome of the genetically modified cell.

[0006] Lentiviral vectors have the ability to transduce proliferative or quiescent cells as well as to integrate a transgene into the genome of their host. Their use in cell engineering thus offers possibilities for very varied therapeutic applications. One of these consists of the elimination of the transduced cell, a strategy called "suicide gene therapy", envisaged to address various medical situations (anti-tumor treatment, securing of grafted cells, etc.). However, by consulting bibliographic databases, publications reporting the use of lentiviral vectors for the transfer of a cytotoxic gene are rare. Indeed, these applications are faced with a major technological obstacle: producing lentiviral vectors carrying a cytotoxic transgene.

[0007] Indeed, the production of lentiviral vectors expressing these cytotoxic transgenes poses a technical problem due to their toxicity on the cells where the vectors are produced, which drastically reduces the titers of recombinant particles.

[0008] The procedure for producing lentiviral vectors is based on the co-transfection into HEK 293 T cells of plasmids expressing: (1) the lentiviral genome carrying the transgene, (2) an envelope protein and (3) the structural proteins and HIV-1 enzymes necessary for the production of non-replicating recombinant lentiviral particles (gag, pol and rev). In lentiviral vectors, the transgene is classically cloned under the control of an internal promoter. Also, during the production process, the transgene is highly expressed, despite modifications to the lentiviral sequences and the use of inducible or tissue-specific internal promoters (Pourzadegan, F. et al. Cancer Gene Ther. 23, 7-12 (2016); Brandtner, EM et al. J. Gene Med. 10, 113-122 (2008)).Indeed, the plasmids used for the production of recombinant lentiviral vectors have a circular structure and contain at least one strong promoter, controlling the transcription of the vector genome that will be packaged in the recombinant HIV particles. In addition, it has recently been shown that plasmids also contain other, cryptic promoters, which can be the origin of transcription starts on either of the two DNA strands of the plasmid (Muerdter, F. et al. Nat. Methods 15, 141-149 (2018); Lemp, NA, Hiraoka, K., Kasahara, N. & Logg, CR Nucleic Acids Re s. 40, 7280-7290 (2012)). Thus, in the case of packaging cells, basal expression of a cytotoxic transgene strongly reduces the titer of the vectors obtained (Maunder, HE et al. Nat. Commun. 8, 14834 (2017)). In some cases, the use of a production line resistant to the toxic gene is possible (Lange, MJ, Lyddon, TD & Johnson, MC Sci. Rep.9, 8985 (2019); Li, Y. et al. Cancer Res. 2002 May l;62(9):2576-82), but this is not a universal solution.

[0009] BRIEF OVERVIEW OF THE INVENTION

[0010] Faced with this major challenge of being able to easily produce lentiviral vectors carrying a cytotoxic (or suicide) transgene and in an effective quantity, the inventors have developed an innovative eukaryotic cell which expresses a ribonucleoprotein complex constructed specifically to repress the expression of the cytotoxic transgene of the lentiviral vector to be produced. A first aim of the invention is therefore to provide the medical profession with this eukaryotic cell and to propose its use for producing lentiviral vectors carrying a cytotoxic transgene. Via this use, another aim of the invention is to propose a universal method for producing lentiviral vectors which makes it possible to obtain high titers of lentiviral vectors carrying a directly cytotoxic transgene.

[0011] DETAILED DESCRIPTION

[0012] In its most general aspect, the invention relates to the use of an animal eukaryotic cell to produce a lentiviral vector, said animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising:

[0013] ■ a fusion protein comprising the fusion:

[0014] - an inactive Cas endonuclease (dCas); and

[0015] - a KRAB transcriptional repression domain (dCas-KRAB fusion) or a methylation domain selected from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion), or a fusion protein comprising the fusion:

[0016] - a KRAB transcriptional repression domain;

[0017] - an inactive Cas endonuclease (dCas); and

[0018] <h2 style=";text-align:left;direction:ltr">- from a methylation domain chosen from: DNMT3A (fusion KRAB-dCas-DNMT3A), DNMT3B (fusion KRAB-dCas-DNMT3B), DNMT3A-L (fusion KRAB-dCas-DNMT3A-L), DNMT3B-L (fusion KRAB-dCas-DNMT3B-L), and MeCP2 (fusion dCas-KRAB-MeCP2), etc.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0019] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the orientation 5' to 3' of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0020] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0021] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which:

[0022] ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the C-terminal position of said inactive Cas endonuclease (dCas); or

[0023] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the N-terminal position of said inactive Cas endonuclease (dCas); or

[0024] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0025] Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0026] ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas); and said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0027] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said promoter is chosen from: an inducible promoter, a tissue-specific promoter and a constitutive promoter. Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said promoter is an inducible promoter or a tissue-specific promoter.

[0028] By "animal eukaryotic cell expressing at least one ribonucleoprotein complex", reference is made to the fact that the animal eukaryotic cell of the invention can express a single ribonucleoprotein complex (the interference one dCas-KRAB / gRNA; or one of the methylation ones dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA or dCas-DNMT3B-L / gRNA; or one of the bifunctional ones KRAB-dCas-DNMT3A, gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA) as it can express several and in particular at least two, namely:

[0029] ■ a dCas-KRAB / gRNA interference ribonucleoprotein complex (CRISPRi); and

[0030] ■ a ribonucleoprotein methylation complex (called CRISPRm) chosen from: dCas- DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA and dCas- DNMT3B-L / gRNA.

[0031] The CRISPR interference (CRISPRi) and CRISPR-targeted methylation (CRISPRm) strategies according to the invention are therefore based on the fusion of an inactive or "dead" CRISPR-Cas endonuclease (dCas) and a KRAB (Krüppel-Associated Box) transcriptional repression domain or a DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L methylation domain (DNA methyltransferase). The dCas, thanks to its guide RNA (gRNA), makes it possible to target a specific sequence. This targeting allows the KRAB domain to sterically block transcription and the DNMT3 A, DNMT3B, DNMT3A-L or DNMT3B-L domains to block transcription by de novo methylation of the targeted DNA. The implementation of a CRISPRi and / or CRISPRm strategy on a sequence internal to the lentiviral vector during the production of recombinant particles requires the co-expression of at least one CRISPRi and / or CRISPRm complex and a gRNA in the transfected animal eukaryotic cells according to the invention.Alternatively, the invention implements a bifunctional CRISPRi / m complex comprising the fusion of an inactive or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L or MeCP2 methylation domain, and a gRNA in the transfected animal eukaryotic cells according to the invention. By “gRNA (guide RNA)” is meant an RNA associating with an enzyme or a protein complex, which, when it pairs with a complementary RNA or DNA sequence, allows the enzyme or protein complex to position itself on this complementary RNA or DNA.

[0032] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, said animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0033] ■ a fusion protein comprising the fusion:

[0034] - an inactive Cas endonuclease (dCas); and

[0035] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0036] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0037] According to another embodiment, the invention also relates to the use of an animal eukaryotic cell as described above, said animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0038] ■ a fusion protein comprising the fusion:

[0039] - an inactive Cas endonuclease (dCas); and

[0040] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion) and

[0041] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said ribonucleoprotein complex dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA or dCas-DNMT3B-L / gRNA being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0042] Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, said animal eukaryotic cell expressing at least a first and a second ribonucleoprotein complex, said first ribonucleoprotein complex comprising: ■ a fusion protein comprising the fusion:

[0043] - an inactive Cas endonuclease (dCas); and

[0044] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0045] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, and said second ribonucleic complex comprising:

[0046] ■ a fusion protein comprising the fusion:

[0047] - an inactive Cas endonuclease (dCas); and

[0048] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion) and

[0049] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (i.e. the same as that targeted by said first ribonucleoprotein complex). said first and second ribonucleoprotein complexes being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0050] Advantageously, the invention also relates to the use of an animal eukaryotic cell as described above, said animal eukaryotic cell expressing a bifunctional ribonucleoprotein complex comprising:

[0051] ■ a fusion protein comprising the fusion:

[0052] - a KRAB transcriptional repression domain;

[0053] - an inactive Cas endonuclease (dCas); and

[0054] - a methylation domain chosen from: DNMT3A (KRAB-dCas-DNMT3A fusion), DNMT3B (KRAB-dCas-DNMT3B fusion), DNMT3A-L (KRAB-dCas-DNMT3A-L fusion), DNMT3B-L (KRAB-dCas-DNMT3B-L fusion) and MeCP2 (fusion dCas-KRAB-MeCP2) and

[0055] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said bifunctional ribonucleoprotein complex KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0056] The term "dCas-KRAB / gRNA ribonucleoprotein complex" refers to an interference system comprising the association of a dCas-KRAB fusion protein and a guide RNA (gRNA) specifically recognizing a sequence of 17 to 24 bases followed by a single or repeated "PAM" (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These two elements work together, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce previously unattainable titres of lentiviral vector carrying a toxic or suicide transgene. In this sense, the invention is a true revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0057] By "dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA or dCas-DNMT3B-L / gRNA ribonucleoprotein complex" is meant a methylation system comprising the association of a dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L or dCas-DNMT3B-L fusion protein and a guide RNA (gRNA) specifically recognizing a succession of 17 to 24 bases followed by a single or repeated "PAM" (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These two elements work together, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce lentiviral vector titers carrying a toxic or suicide transgene never before achieved. In this sense, the invention is a real revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0058] By "KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA ribonucleoprotein complex" is meant a bifunctional interference and methylation system comprising the association of a KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 fusion protein and a guide RNA (gRNA) specifically recognizing a succession of 17 to 24 bases followed by a single or repeated "PAM" (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These three elements work together, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce lentiviral vector titers carrying a toxic or suicide transgene never before achieved.In this sense, the invention is a real revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0059] Advantageously, the invention takes advantage of interference and / or methylation systems by combining them so as to obtain a tool for producing a lentiviral vector carrying a high-performance toxic or suicide transgene. By "dCas-KRAB fusion protein" is meant the combination of two amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In the invention, these two fused components are, as mentioned, an inactive Cas endonuclease (dCas) and a KRAB transcriptional repression domain, and a nuclear import signal (NLS) peptide is added to ensure its nuclear localization (e.g. encoded by a nucleic acid of sequence SEQ ID NO: 324).

[0060] By "dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L or dCas-DNMT3B-L fusion protein" is meant the combination of two amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In the invention, these two fused components are, as mentioned, an inactive Cas endonuclease (dCas) and a DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L methylation domain. A nuclear import signal (NLS) peptide is also added to ensure its nuclear localization (e.g. encoded by a nucleic acid of sequence SEQ ID NO: 324).

[0061] By "KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 fusion protein" is meant the combination of three amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In the invention, these three fused components are, as mentioned, a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L methylation domain. A nuclear import signal (NLS) peptide is also added to ensure its nuclear localization (e.g. encoded by a nucleic acid of sequence SEQ ID NO: 324).

[0062] By "inactive Cas endonuclease (dCas)" or the equivalent expression "dead" Cas endonuclease (dCas), reference is made to endonucleases whose activity of cleaving one or two DNA strands does not function. To date, several Cas endonucleases (Cas9, Casl2a, Casl2b, CasX or Cas 12e, Casl2f or Cas 14, Casl2j or Cas®) from different hosts have been identified and characterized, or even modified (e.g. improvement of their function), which can be implemented by the invention after their inactivation. According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said inactive Cas endonuclease (dCas) is chosen from:

[0063] ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, '

[0064] ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, '

[0065] ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, '

[0066] ■ inactive CasX or Casl2e endonucleases from Deltaproteobacteria and Planctomycetes, ' ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans

[0067] ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade

[0068] ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and

[0069] ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

[0070] By "orthologs" is meant similar Cas endonucleases present in two or more different species. These, due to evolution, may be inactive and if this is not the case, it is possible to render them non-functional by mutation to exploit them for the purposes of the invention.

[0071] By "mutants" we mean a Cas endonuclease into which one or more mutations have been introduced, including the deletion, substitution and / or addition of one or more amino acids. By "inactive mutants" we mean Cas proteins modified by human intervention (e.g. by genetic engineering) in order to inactivate their DNA cleavage activity.

[0072] By "inactive variants" we refer to Cas endonucleases naturally modified through evolution, which do not exhibit DNA cleavage activity.

[0073] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9). This is a ribonucleoprotein complex dCas9-KRAB / gRNA, dCas9-DNMT3A / gRNA, dCas9-DNMT3B / gRNA, dCas9-DNMT3A-L / gRNA, dCas9-DNMT3B-L / gRNA, KRAB-dCas9-DNMT3A / gRNA, KRAB-dCas9-DNMT3B / gRNA, KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA or dCas9-KRAB-MeCP2 / gRNA. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid encodes an inactive Cas9 endonuclease (dCas9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 323.In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid codes for an inactive Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 323.

[0074] By "KRAB transcriptional repression domain" is meant the KRAB (Kruppel-associated suppression box) domain of the human zinc finger protein Koxl (or zinc finger protein 10) or Zim3, which causes specific inhibition of RNA polymerases II and III. In doing so, this KRAB domain inhibits the expression of the gene(s) present on the DNA to which it is recruited. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 321 or 752.In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain is encoded by the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence corresponds to the sequence SEQ ID NO: 321 or 752.

[0075] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754.In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF 1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or 754.

[0076] By "methylation domain selected from: DNMT3 A, DNMT3B, DNMT3 AL, DNMT3B-L and MeCP2", reference is made to the functional domain of DNA methyltransferase, which catalyzes the covalent modification of DNA by transfer of S-adenosyl-1-methionine (SAM) by converting cytosine into 5-methylcytosine. In doing so, this DNMT3 A, DNMT3B, DNMT3A-L, DNMT3B-L or MeCP2 domain inhibits the expression of the gene(s) present on the DNA where it is recruited. Advantageously, the invention uses the DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L domains. In particular, the subject of the invention is therefore the use of an animal eukaryotic cell as described above, in which said methylation domain:

[0077] ■ DNMT3 A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 354;

[0078] ■ DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 366;

[0079] ■ DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 358; or ■ DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 368.

[0080] In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said methylation domain:

[0081] ■ DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 354;

[0082] ■ DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 366;

[0083] ■ DNMT3A-L is encoded by the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0084] ■ DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 368.

[0085] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said MeCP2 methylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 370. In particular, the subject of the invention is also the use of an animal eukaryotic cell as described above, in which said MeCP2 methylation domain is encoded by the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 370.

[0086] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said dCas-DNMT3 A, dCas-DNMT3B, dCas-DNMT3A-L or dCasDNMT3B-L fusion protein is respectively a dCas9-DNMT3A, dCas9-DNMT3B, dCas9-DNMT3A-L or dCas9-DNMT3B-L fusion protein. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0087] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360;

[0088] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372;

[0089] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364; or

[0090] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374.

[0091] In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which:

[0092] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360;

[0093] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372;

[0094] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364; or

[0095] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372.

[0096] Interestingly, if the DNMT3L enzyme is catalytically inactive, it stimulates the activity of DNMT3A and DNMT3B and thus promotes the silencing of a promoter by methylation. Hence the DNMT3 AL and DNMT3B-L methylation domains where respectively DNMT3A or DNMT3B have been combined with DNMT3L. This being the case, it is possible to envisage the addition of a dCas-DNMT3L / gRNA ribonucleoprotein complex in the animal eukaryotic cell of the invention. According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, said animal eukaryotic cell further expressing another ribonucleoprotein complex comprising:

[0097] ■ a fusion protein comprising the fusion:

[0098] - an inactive Cas endonuclease (dCas); and

[0099] - a domain promoting DNMT3L methylation (dCas-DNMT3E fusion) ■ and

[0100] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said other dCas-DNMT3L / gRNA ribonucleoprotein complex being in particular stably expressed. Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, said animal eukaryotic cell expressing at least a first and a second ribonucleoprotein complex, said first ribonucleoprotein complex comprising:

[0101] ■ a fusion protein comprising the fusion:

[0102] - an inactive Cas endonuclease (dCas); and

[0103] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion) and DNMT3B; and

[0104] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, and said second ribonucleic complex comprising:

[0105] ■ a fusion protein comprising the fusion:

[0106] - an inactive Cas endonuclease (dCas); and

[0107] - a domain promoting DNMT3L methylation (dCas-DNMT3L fusion) ■ and

[0108] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (i.e. the same as that targeted by said first ribonucleoprotein complex), said first and second ribonucleoprotein complexes being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter (said promoter sequence of said promoter) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome. In particular, the invention also relates to the use of an animal eukaryotic cell as described above, said animal eukaryotic cell further expressing another ribonucleoprotein complex comprising:

[0109] ■ a fusion protein comprising the fusion:

[0110] - an inactive Cas endonuclease (dCas); and

[0111] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0112] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed.

[0113] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0114] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 356. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is encoded by the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 356.

[0115] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a fusion protein dCas9-DNMT3L whose amino acid sequence corresponds to the sequence SEQ ID NO: 362.

[0116] As mentioned above, the invention also implements a bifunctional CRISPRi / m complex comprising:

[0117] ■ the fusion of an inactive or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L or MeCP2 methylation domain; and

[0118] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter (that of the expression cassette).

[0119] In particular, this bifunctional complex (advantageously expressed in a stable manner) is chosen from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA and dCas-KRAB-MeCP2 / gRNA, and is advantageously chosen from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA and dCas9-KRAB-MeCP2 / gRNA. Also, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0120] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382; ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384; or

[0121] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386.

[0122] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0123] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 (encoded by the plasmid pLV-EFla-KRABl-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382;

[0124] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384; or

[0125] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386.

[0126] By "% identity" is meant the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequence), determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides in the longer sequence of the two, and multiplying the result by 100. By "having at least 80% identity" is therefore meant that the aforementioned percentage 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 less than 94%, at least 95%, at least 96%, at least 97%, at least 98%, 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 is a direct comparison of two polypeptide sequences (amino acid sequence).By "said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3L / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed", reference is made to the possibility of integrating, in a targeted or non-targeted manner, into the genome of the animal eukaryotic cell as described the coding sequences of said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / sgRNA, dCas-DNMT3B / sgRNA, dCas-DNMT3L / sgRNA, dCas-DNMT3A-L / sgRNA, dCas-DNMT3B-L / sgRNA, KRAB-dCas-DNMT3A / sgRNA, KRAB-dCas-DNMT3B / sgRNA, KRAB-dCas-DNMT3A-L / sgRNA, KRAB-dCas-DNMT3B-L / sgRNA or dCas-KRAB-MeCP2 / sgRNA and the means to express it. In this way, its expression is no longer transient and ensured by plasmids but permanent and ensured by the genome.In particular, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3L / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA is stably expressed either via random integration or via targeted integration into the genome of said animal eukaryotic cell.Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3L / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA is stably expressed via targeted integration into the genome of said animal eukaryotic cell.

[0127] Concerning said animal eukaryotic cell used to produce a lentiviral vector, the latter can be chosen from different cell lines known to those skilled in the art. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said animal eukaryotic cell belongs 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. Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said animal eukaryotic cell belongs to the HEK 293 T cell line.

[0128] By "lentiviral vector" is meant a recombinant lentiviral particle obtained using the tools provided by the invention. This comprises said encapsidated and / or enveloped vector genome. It is efficient for introduction into the cell, it is non-replicative and it leads either to the integration (targeted or not) of said vector genome into the genome of an infected cell, or to the presence of a non-integrated episome into the cellular genome. By "vector genome" is meant a nucleic acid sequence comprising both:

[0129] ■ 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 encapsidated and enveloped vector genome, which is efficient for introduction into the cell and is non-replicative; and

[0130] ■ the nucleic acid comprising the genetic information of an expression cassette comprising a promoter chosen from: an inducible promoter, a tissue-specific promoter and a constitutive promoter, upstream of a toxic or suicide transgene, the nucleic acid encoding said toxic or suicide transgene being able to comprise at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail, said expression cassette having been introduced into said lentiviral genome such that its transcription is reversed compared to that of said lentiviral genome.

[0131] By "lentiviral genome", we mean, as mentioned above, a nucleic acid carrying the genetic information necessary for the production of a lentiviral vector, also called a (lenti-)viral particle, said lentiviral vector or said (lenti-)viral particle comprising said encapsidated and enveloped vector genome. This lentiviral vector is also efficient for entering the cell, it is non-replicative, and it leads to the integration (targeted or not) of said vector genome into the genome of an infected cell. In certain cases, the lentiviral particle will contain a mutant integrase (D64V), which will result in a non-integrating provirus (post-reverse transcription vector DNA) in the genome of the transduced cell. Consequently, the genome of this vector will be in episome form in the cell nucleus.Classically, the structure of this lentiviral genome after reverse transcription includes: The cis sequences of the HIV lentiviral genome, i.e. two Long Terminal Repeats (LTR) 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 LTR are called "self-inactivating" since they cannot be transcribed by a wild-type HIV virus. The 5' LTR of the genome is followed by a "psi" sequence for the encapsidation of the vector RNA genome, a Rev Responsive element (RRE) sequence for the export of the vector 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.By "expression cassette", as mentioned above, is meant a nucleic acid comprising a promoter chosen from: an inducible promoter, a tissue-specific promoter and a constitutive promoter, upstream of a toxic or suicide transgene, the nucleic acid encoding said toxic or suicide transgene being able to comprise at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail, said expression cassette having been introduced into said lentiviral genome such that its transcription is inverted with respect to that of said lentiviral genome. By "the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome", reference is made, as illustrated in Figure 3 (pLVi), to the fact that the transcription of said expression cassette is inverted with respect to that of said lentiviral genome.It should be noted that the particular association of this construction (promoter chosen from: an inducible promoter, a tissue-specific promoter and a constitutive promoter + toxic or suicide transgene) mounted in reverse with the animal eukaryotic cells of the invention expressing a dCas-KRAB / gRNA ribonucleoprotein complex specific to said chosen promoter is a novel and effective tool which makes it possible to unexpectedly produce lentiviral vectors encoding toxic or suicide transgenes with previously unattained titers.

[0132] By "inducible promoter" is meant a nucleic acid located upstream of a gene (or transgene) and which controls its expression, in particular by regulating its transcription. In the invention, this promoter is described as inducible because the expression of the gene (or transgene) that it controls is not constitutive and is only induced after its activation in response to the right stimulus. In the invention, these are advantageously the 2xAARE YB TATA (SEQ ID NO: 1), the 2xAARE TK (SEQ ID NO: 2), the 4xSRE YB TATA (SEQ ID NO: 3), the 9xHRE YB TATA (SEQ ID NO: 4), GRP78 YB TATA (SEQ ID NO: 5) or miniCHACl YB - TATA (SEQ ID NO: 7). The 2XAARE promoters YB - TATA , 2xAARE TK and miniCHACl YB TATA are activated by an inducer inducing the expression of the transcription factor ATF4. The GRP78 promoter YB TATA is activated by an inducer inducing the transcription factor ATF6. The 4xSRE promoterYB TATA is induced by steroid hormones and more specifically by dexamethasone. The 9xHRE promoter YB TATA is induced by hypoxia. Interestingly, the inducible promoters mentioned above are dependent on the expression of cellular transcription factors that are poorly or not expressed under vector production conditions in HEK 293 T cells.

[0133] By "tissue-specific promoter" is meant a nucleic acid located upstream of a gene (or transgene) and which controls its expression, in particular by regulating its transcription. In the invention, this promoter is described as tissue-specific because the expression of the gene (or transgene) that it controls is not effective in the cellular environment where the recombinant vector particles are produced, but is activated in another cellular environment with a distinct phenotype to which said tissue-specific promoter is sensitive. In response to this other environment, said tissue-specific promoter will be activated. For example, in the invention it may be the miR223 promoter (SEQ ID NO: 6) which is specifically induced in myeloid cells, which is not a HEK-293T cell.

[0134] By "constitutive promoter" is meant a nucleic acid located upstream of a gene (or transgene) and which controls its expression, in particular by regulating its transcription. In the invention, this promoter is described as constitutive because the expression of the gene (or transgene) that it controls is constant regardless of its environment, i.e. whether it is in the cellular environment where the recombinant vector particles are produced or in another cellular environment with a distinct phenotype. In the invention, the latter are advantageously SV40 (or pSV40; SEQ ID NO: 343), CMV (or pCMV; SEQ ID NO: 344), UbC (or pUbC; SEQ ID NO: 345), EFl-a core (or pEFl-a core; SEQ ID NO: 346), EFl-a long (or pEFl-a long; SEQ ID NO: 347), hPGK (or phPGK; SEQ ID NO: 348), CAGG (or pCAGG; SEQ ID NO: 349), RSV (or pRSV; SEQ ID NO: 350) and SFFV (or pSFFV; SEQ ID NO: 351).

[0135] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said promoter is an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB - TATA , the 9XHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA ; or wherein said promoter is the tissue-specific promoter of miR223; or wherein said promoter is a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV.

[0136] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said promoter is an inducible or tissue-specific promoter chosen from:

[0137] ■ the 2XAARE YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1;

[0138] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2;

[0139] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0140] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0141] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0142] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and

[0143] ■ the miniCHAClYB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7.

[0144] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said promoter is an inducible or tissue-specific promoter chosen from:

[0145] ■ the 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0146] ■ the 2XAARE TK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0147] ■ the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0148] ■ the 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4; ■ GRP78 YB TATAwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5;

[0149] ■ the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0150] ■ the miniCHACl YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0151] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said promoter is a constitutive promoter chosen from:

[0152] ■ SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343;

[0153] ■ CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 344;

[0154] ■ UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 345;

[0155] ■ the EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 346;

[0156] ■ the long EFl-a whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 347;

[0157] ■ hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 348;

[0158] ■ CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 349;

[0159] ■ RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 350; and

[0160] ■ SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 351.

[0161] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said promoter is a constitutive promoter chosen from:

[0162] ■ SV40 whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0163] ■ CMV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0164] ■ UbC whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0165] ■ the EFl-a core whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 346;

[0166] ■ the long EFl-a whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 347;

[0167] ■ hPGK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348; ■ CAGG whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 349;

[0168] ■ RSV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0169] ■ SFFV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0170] By "toxic or suicide transgene" is meant a nucleic acid located downstream of said inducible or tissue-specific promoter and which codes for a toxic, cytotoxic or suicide protein in the genetically modified cell. That is to say, after the activation of said inducible or tissue-specific promoter controlling the expression of said toxic or suicide transgene and only in response to the right stimulus or the right cellular environment, the product (i.e. the toxic or suicide protein) resulting from the transcription and translation of this nucleic acid causes (leads to) the cell death of the cell expressing it.In the invention, the latter may be chosen from the genes coding: inducible Caspase-9 (iC9), Bax (S 184 del), Noxa (the wild-type protein or one of its mutants, in particular the S 13 A mutant), Gasdermin B (N-ter), constitutively active caspase-3 (V266E), an M2 ion channel (H37A) of the influenza virus, the A subunit of diphtheria toxin (DTA) 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, i.e. whose expression after activation of said inducible or tissue-specific promoter upstream of the nucleic acid encoding them is not found in the extracellular medium and therefore only results in the cell death of the cell expressing them.

[0171] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which said 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated mutant aDTA (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D).

[0172] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said toxic or suicide transgene is chosen from:

[0173] ■ 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: 8 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: 9;

[0174] ■ 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: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 11;

[0175] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 13;

[0176] ■ 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: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 15;

[0177] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 19;

[0178] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 21;

[0179] ■ a Noxa protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a protein a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 23;

[0180] ■ a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 756; and

[0181] ■ an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 758.

[0182] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said toxic or suicide transgene is chosen from: ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid codes for an inducible Caspase-9 (iC9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 9;

[0183] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 11;

[0184] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence corresponds to the sequence SEQ ID NO: 13;

[0185] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 15;

[0186] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence corresponds to the sequence SEQ ID NO: 19;

[0187] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 21;

[0188] ■ a Noxa protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 23;

[0189] ■ a streptolysin O whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 756; and

[0190] ■ an enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0191] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which the nucleic acid coding for said toxic or suicide transgene comprises at its 3' end a polyA sequence (eg SEQ ID NO: 24). Insofar as the 5' to 3' orientation of the transcription of said expression cassette is inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, the addition of this polyA sequence (eg SEQ ID NO: 24) to the 3' end of the nucleic acid coding for said toxic or suicide transgene promotes, after (and only after) induction of said inducible promoter, the transcription and stability of an mRNA, which authorizes the translation of said toxic or suicide transgene.

[0192] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which:

[0193] ■ the said promoter is:

[0194] - an inducible promoter chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA ;

[0195] - is the tissue-specific promoter of miR223; or

[0196] - is a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, and

[0197] ■ said toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0198] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said expression cassette is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 25 to 80 and 391 to 462. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said expression cassette is chosen from the sequences SEQ ID NOs: 25 to 80 and 391 to 462. In particular, the subject of the invention is also the use of an animal eukaryotic cell as described above, in which said expression cassette is the sequence SEQ ID NO: 25, 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, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 391, 392, 393, 394,

[0199] 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,

[0200] 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,

[0201] 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,

[0202] 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 or 462 (see Table 1).

[0203]

[0204] Table 1. List of expression cassettes

[0205] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) or a Respiratory syncytial virus (RSV).

[0206] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN). In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which 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: 81.In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which 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: 81. It should be noted that the insertion of said expression cassette into said Self-inactivating Lentivirus (SIN) of sequence SEQ ID NO: 81 can be or is carried out at nucleotides 2229 and 2230 of this SEQ ID NO: 81. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 82 and 83.

[0207] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Respiratory syncytial virus (RSV). In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which 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: 84.In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which 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: 84. It should be noted that the insertion of said expression cassette into said Respiratory syncytial virus (RSV) of sequence SEQ ID NO: 84 can be or is carried out at nucleotides 1737 and 1738 of this SEQ ID NO: 84. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 85 and 86.

[0208] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said lentiviral genome into which said expression cassette has been introduced is:

[0209] ■ 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: 81; or

[0210] ■ 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: 84; and in which said expression cassette comprises: ■ an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA ; or a tissue-specific promoter of miR223; or a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, and

[0211] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0212] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 87 to 198 and 463 to 606. In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 87 to 198 and 463 to 606. In particular, the subject of the invention is also the use of an animal eukaryotic cell as described above, in which said vector genome is the sequence SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,

[0213] 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,

[0214] 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

[0215] 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,

[0216] 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,

[0217] 198, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,

[0218] 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,

[0219] 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,

[0220] 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,

[0221] 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,

[0222] 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,

[0223] 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,

[0224] 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605 or 606 (see Table 2).

[0225]

[0226]

[0227]

[0228] Table 2. List of vector genomes

[0229] It should also be noted that due to the inversion of the expression cassette as described above with respect to the orientation of the transcription of the vector genome as described above, all of the sequences provided relating to said expression cassette as described above are found on the complementary strand of said vector genome as described above of which only the sense sequences (5' > 3') are provided.

[0230] In the invention, said animal eukaryotic cell used to produce a lentiviral vector expresses:

[0231] ■ a dCas-KRAB / gRNA interference ribonucleoprotein complex; and / or

[0232] ■ a methylation ribonucleoprotein complex selected from: dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA and dCas-DNMT3B-L / gRNA, and optionally a dCas-DNMT3L / gRNA methylation ribonucleoprotein complex; and / or

[0233] ■ a bifunctional ribonucleoprotein interference and methylation complex chosen from: KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3 AL / gRNA, KRAB-dCas-DNMT3B-L / RNAg and dCas-KRAB-MeCP2 / gRNA.

[0234] The dCas-KRAB component as defined above makes it possible to inhibit the expression of the gene(s) present on the DNA where it is recruited (without cleavage of the DNA) by steric hindrance and in this case, it makes it possible to inhibit the expression of said toxic or suicide transgene. The dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L and dCas-DNMT3B-L component as defined above makes it possible to methylate the DNA of the gene(s) present on the DNA where it is recruited (without cleavage of the DNA) to inhibit its expression and in this case, it makes it possible to inhibit the expression of said toxic or suicide transgene. The KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 component allows, for its part, to inhibit the expression of the gene(s) present on the DNA where it is recruited (without cleavage of the DNA) by steric hindrance and to methylate the DNA of the gene(s) present on the DNA where it is recruited (without cleavage of the DNA) to inhibit its expression.For this, the interference ribonucleoprotein complex and / or the methylation ribonucleoprotein complex and / or the bifunctional interference and methylation ribonucleoprotein complex are guided by the gRNA component which specifically recognizes a DNA sequence in the sequence of said inducible, tissue-specific or constitutive promoter of the expression cassette.

[0235] According to another embodiment, the invention therefore relates to the use of an animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the complementary sequence of a target sequence of 17 to 24 bases present on said promoter (said promoter sequence of said promoter) controlling the expression of said toxic or suicide transgene.

[0236] The target sequence of said gRNA is determined using tools accessible to those skilled in the art. In brief, computational tools such as CRISPOR (Concordet, J. -P., Haeussler, M., 2018. CRISPOR: intuitive guide selection for CRISP R / Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242-W245) or CRISPICK (Kim et al. 2018. Deep learning improves prediction of CRISPR-Cpfl guide RNA activity. Nat. Biotechnol. 36, 239-241; Sanson et al. 2018. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416) test and propose candidate gRNA sequences based on their complementarity with the target and the absence of complementarity with the host cell genome. Thus, for each promoter, inducible, tissue-specific or constitutive, several gRNAs hybridizing to one or more locations of the promoter are proposed and then one is selected based on its optimal capacity to inhibit the expression of a transgene by this promoter.It is therefore necessary to complete the in silico analysis with a functional screening of the different candidate gRNA sequences to determine the one that allows the most effective and safest interference and / or methylation strategy according to the invention to be implemented on the promoter considered. For this, cells, e.g., HEK-293T are genetically modified so as to express at least one ribonucleoprotein complex according to the invention and each of the candidate gRNAs. These same cells are then transduced (lentiviral transduction) with an expression cassette comprising the inducible, tissue-specific or constitutive promoter according to the invention upstream of a reporter protein (e.g., FLuc or Luc), said promoter being the one targeted by the candidate gRNAs.Of the candidate gRNAs, the one allowing the most effective targeting to reduce the basal expression of the reporter gene is selected and is used to establish a clonal line of animal eukaryotic cell according to the invention where the best clone, i.e. the one where the repression of the expression of the reporter gene is the best, is selected and amplified as a tool for producing lentiviral vectors carrying a cytotoxic transgene according to the invention. According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said gRNA has been determined using the CRISPOR or CRISPICK tools and whose targeting activity has in particular been determined in vitro.

[0237] According to another embodiment, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318 (which specifically recognizes the inducible promoter 2XAARE YB TATA (SEQ ID NO: 1)). In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318.

[0238] Advantageously, the invention also relates to the use of an animal eukaryotic cell as described above, in which:

[0239] ■ said fusion protein:

[0240] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754,

[0241] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360,

[0242] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0243] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0244] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0245] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0246] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0247] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0248] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0249] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0250] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1.

[0251] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0252] ■ said fusion protein:

[0253] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or 754,

[0254] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0255] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372 - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0256] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0257] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0258] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0259] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0260] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0261] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318; and

[0262] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0263] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 25 to 32, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 87 to 94 and 143 to 150.

[0264] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the constitutive promoter SV40 (SEQ ID NO: 343)). In particular, the invention also relates to the use of an animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0265] Advantageously, the invention also relates to the use of an animal eukaryotic cell as described above, in which:

[0266] ■ said fusion protein:

[0267] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754,

[0268] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360,

[0269] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0270] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0271] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0272] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0273] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0274] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0275] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0276] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and

[0277] ■ said constitutive promoter is SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343.

[0278] In particular, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0279] ■ said fusion protein:

[0280] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 325) or 754,

[0281] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0282] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372

[0283] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0284] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0285] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0286] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0287] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0288] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0289] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352; and

[0290] ■ said constitutive promoter is SV40 whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0291] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 391 to 398, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 463 to 470 and 353 to 542.

[0292] According to a second aspect of the invention, the invention relates to a method for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising: ■ a fusion protein comprising the fusion:

[0293] - an inactive Cas endonuclease (dCas); and

[0294] - a KRAB transcriptional repression domain (dCas-KRAB fusion) or a methylation domain selected from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion), or a fusion protein comprising the fusion:

[0295] - a KRAB transcriptional repression domain;

[0296] - an inactive Cas endonuclease (dCas); and

[0297] - a methylation domain chosen from: DNMT3A (KRAB-dCas-DNMT3A fusion), DNMT3B (KRAB-dCas-DNMT3B fusion), DNMT3A-L (KRAB-dCas-DNMT3A-L fusion), DNMT3B-L (KRAB-dCas-DNMT3B-L fusion) and MeCP2 (fusion dCas-KRAB-MeCP2) and

[0298] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (said promoter sequence of said promoter), said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B- L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed, with:

[0299] ■ a plasmid comprising said vector genome and the means for expressing it;

[0300] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0301] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.

[0302] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3 A, DNMT3B, DNMT3 AL and DNMT3B-L is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3 A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0303] According to another embodiment, the invention relates to the use of an animal eukaryotic cell as described above, in which: ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain chosen from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the C-terminal position of said inactive Cas endonuclease (dCas); or

[0304] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the N-terminal position of said inactive Cas endonuclease (dCas); or

[0305] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0306] Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which:

[0307] ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas); and

[0308] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0309] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said promoter is chosen from: an inducible promoter, a tissue-specific promoter and a constitutive promoter. Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said promoter is an inducible promoter or a tissue-specific promoter.

[0310] According to another embodiment, the subject of the invention is the method as described above for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0311] ■ a fusion protein comprising the fusion:

[0312] - an inactive Cas endonuclease (dCas); and

[0313] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0314] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed, with:

[0315] ■ a plasmid comprising said vector genome and the means for expressing it;

[0316] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0317] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.

[0318] According to another embodiment, the subject of the invention is the method as described above for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0319] ■ a fusion protein comprising the fusion:

[0320] - an inactive Cas endonuclease (dCas); and

[0321] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion) and

[0322] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (said promoter sequence of said promoter), said ribonucleoprotein complex dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA or dCas-DNMT3B-L / gRNA being in particular stably expressed, with:

[0323] ■ a plasmid comprising said vector genome and the means for expressing it;

[0324] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0325] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.According to another embodiment, the subject of the invention is the method as described above for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfecting an animal eukaryotic cell expressing at least a first and a second ribonucleoprotein complex, said first ribonucleoprotein complex comprising:

[0326] ■ a fusion protein comprising the fusion:

[0327] - an inactive Cas endonuclease (dCas); and

[0328] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0329] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, and said second ribonucleic complex comprising:

[0330] ■ a fusion protein comprising the fusion:

[0331] - an inactive Cas endonuclease (dCas); and

[0332] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion) and

[0333] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (i.e. the same as that targeted by said first ribonucleoprotein complex), said first and second ribonucleoprotein complexes being in particular stably expressed, with:

[0334] ■ a plasmid comprising said vector genome and the means for expressing it;

[0335] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0336] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.

[0337] According to another embodiment, the subject of the invention is the method as described above for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing a bifunctional ribonucleoprotein complex comprising:

[0338] ■ a fusion protein comprising the fusion:

[0339] - a KRAB transcriptional repression domain;

[0340] - an inactive Cas endonuclease (dCas); and

[0341] - a methylation domain chosen from: DNMT3 A (KRAB-dCas-DNMT3A fusion), DNMT3B (KRAB-dCas-DNMT3B fusion), DNMT3A-L (KRAB-dCas-DNMT3A-L fusion), DNMT3B-L (KRAB-dCas-DNMT3B-L fusion) and MeCP2 (dCas-KRAB-MeCP2 fusion) ; And

[0342] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said bifunctional ribonucleoprotein complex KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed, with:

[0343] ■ a plasmid comprising said vector genome and the means for expressing it;

[0344] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0345] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.

[0346] Such a method is illustrated in the examples below and is accessible to those skilled in the art in view of the literature at their disposal (eg Zennou et al. Nat Biotechnol. 2001 May; 19(5):446-50)). Nevertheless, and for the purposes of illustration, it is specified that:

[0347] ■ 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;

[0348] ■ said plasmid comprising a viral envelope protein and the means for expressing it may be chosen from the plasmids: pHCMV-VSV (SEQ ID NO: 337), pHCMV-Mokola, pHCMV-RabiesG, pHCMV-AmphoEnv, pHCMV-LCMV-WE, pHCMV-LCMV-Arm53b and pHCMV-10Al (Sena-Esteves M et al. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. 2004 Dec 15;122(2): 131-9); and ■ said plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it can be chosen from the plasmids: p8.92 (SEQ ID NO: 338), p8.92 (IN D64V) (SEQ ID NO: 339), psPAX2 (SEQ ID NO: 340), pMDLg / pRRE (SEQ ID NO: 341) and pRSV-Rev (SEQ ID NO: 342).

[0349] For illustrative purposes and in a non-limiting manner, the following protocol is also provided: Cells expressing at least one ribonucleoprotein complex according to the invention (eg HEK 293 T) were seeded on supports suitable for cell culture and so as to reach a confluence of 50 to 60% after 24 hours of culture. The following day, the cells were cotransfected, by the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome and two transcomplementing plasmids encoding the enzymes and structural proteins of HIV-1 (p8.9) and the envelope glycoprotein of the vesicular stomatitis virus (pVSVg). The co-transfection was carried out at the plasmid ratio 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 ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0350] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease (dCas) is chosen from:

[0351] ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, '

[0352] ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, '

[0353] ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, '

[0354] ■ inactive CasX or Casl2e endonucleases from Deltaproteobacteria and Planctomycetes, '

[0355] ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans,'

[0356] ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade, -

[0357] ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and

[0358] ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

[0359] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9). This is a ribonucleoprotein complex dCas9-KRAB / gRNA, dCas9-DNMT3A / gRNA, dCas9-DNMT3B / gRNA, dCas9-DNMT3A-L / gRNA, dCas9-DNMT3B-L / gRNA, KRAB-dCas9-DNMT3A / gRNA, KRAB-dCas9-DNMT3B / gRNA, KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA or dCas9-KRAB-MeCP2 / gRNA. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid encodes an inactive Cas9 endonuclease (dCas9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 323.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid codes for an inactive Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 323.

[0360] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 321 or 752. In particular, the subject of the invention is also the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain is encoded by the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence corresponds to the sequence SEQ ID NO: 321 or 752.

[0361] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EFla-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or 754.

[0362] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said methylation domain: ■ DNMT3 A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 354;

[0363] ■ DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 366;

[0364] ■ DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 358; or

[0365] ■ DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 368.

[0366] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said methylation domain:

[0367] ■ DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 354;

[0368] ■ DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 366;

[0369] ■ DNMT3A-L is encoded by the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0370] ■ DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 368.

[0371] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said MeCP2 methylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 370. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said MeCP2 methylation domain is encoded by the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 370.

[0372] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L or dCasDNMT3B-L fusion protein is respectively a dCas9-DNMT3 A, dCas9-DNMT3B, dCas9-DNMT3 AL or dCas9-DNMT3B-L fusion protein. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0373] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360; ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372;

[0374] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364; or

[0375] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374.

[0376] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which

[0377] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360;

[0378] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372;

[0379] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364; or

[0380] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372.

[0381] Interestingly, if the DNMT3L enzyme is catalytically inactive, it stimulates the activity of DNMT3A and DNMT3B and thus promotes the silencing of a promoter by methylation. Hence the DNMT3 AL and DNMT3B-L methylation domains where respectively DNMT3A or DNMT3B have been combined with DNMT3L. This being the case, it is possible to envisage the addition of a dCas-DNMT3L / gRNA ribonucleoprotein complex in the animal eukaryotic cell of the invention. According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell further expresses another ribonucleoprotein complex comprising:

[0382] ■ a fusion protein comprising the fusion of: - an inactive Cas endonuclease (dCas); and

[0383] - a domain promoting DNMT3L methylation (dCas-DNMT3L fusion) ■ and

[0384] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said other dCas-DNMT3L / gRNA ribonucleoprotein complex being in particular stably expressed.

[0385] Advantageously, the subject of the invention is the method as described above for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter (a promoter sequence of a promoter) upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing at least a first and a second ribonucleoprotein complex, said first ribonucleoprotein complex comprising:

[0386] ■ a fusion protein comprising the fusion:

[0387] - an inactive Cas endonuclease (dCas); and

[0388] - a methylation domain chosen from: DNMT3A (dCas-DNMT3A fusion) and DNMT3B; and

[0389] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, and said second ribonucleic complex comprising:

[0390] ■ a fusion protein comprising the fusion:

[0391] - an inactive Cas endonuclease (dCas); and

[0392] - a domain promoting DNMT3L methylation (dCas-DNMT3L fusion) ■ and

[0393] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter (i.e. the same as that targeted by said first ribonucleoprotein complex), said first and second ribonucleoprotein complexes being in particular stably expressed, with:

[0394] ■ a plasmid comprising said vector genome and the means for expressing it;

[0395] ■ a plasmid comprising an envelope protein and the means for expressing it; and

[0396] ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors. In particular, the invention also relates to the method for producing lentiviral vectors as described above, said animal eukaryotic cell further expressing another ribonucleoprotein complex comprising:

[0397] ■ a fusion protein comprising the fusion:

[0398] - an inactive Cas endonuclease (dCas); and

[0399] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0400] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed.

[0401] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said DNMT3L methylation-promoting domain is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said DNMT3L methylation-promoting domain is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0402] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said methylation-promoting domain DNMT3L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 356. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said methylation-promoting domain DNMT3L is encoded by the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 356.

[0403] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a fusion protein dCas9-DNMT3L whose amino acid sequence corresponds to the sequence SEQ ID NO: 362.As mentioned above, the invention also implements a bifunctional CRISPRi / m complex comprising:.

[0404] ■ the fusion of an inactive or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L or MeCP2 methylation domain; and

[0405] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter (that of the expression cassette).

[0406] In particular, this bifunctional complex (advantageously expressed in a stable manner) is chosen from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA and dCas-KRAB-MeCP2 / gRNA, and is advantageously chosen from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA and dCas9-KRAB-MeCP2 / gRNA. Also, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0407] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382;

[0408] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384; or

[0409] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386.

[0410] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0411] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 (encoded by the plasmid pLV-EFla-KRABl-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382;

[0412] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384; or ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386.

[0413] Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said ribonucleoprotein complex dCas-KRAB / sgRNA, dCas-DNMT3A / sgRNA, dCas-DNMT3B / sgRNA, dCas-DNMT3L / sgRNA, dCas-DNMT3A-L / sgRNA, dCas-DNMT3B-L / sgRNA, KRAB-dCas-DNMT3A / sgRNA, KRAB-dCas-DNMT3B / sgRNA, KRAB-dCas-DNMT3A-L / sgRNA, KRAB-dCas-DNMT3B-L / sgRNA or dCas-KRAB-MeCP2 / sgRNA is stably expressed via targeted integration into the genome of said animal eukaryotic cell.

[0414] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell belongs 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. Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell belongs to the HEK 293T cell line.

[0415] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said promoter is an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB - TATA , the 9XHRE YB - TATA , the GRP78 YB - TATAand the miniCHACl YB - TATA ; or wherein said promoter is the tissue-specific promoter of miR223; or wherein said promoter is a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV.

[0416] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said promoter is an inducible or tissue-specific promoter chosen from:

[0417] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1;

[0418] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2;

[0419] ■ the 4XSRE YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0420] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0421] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5; ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and

[0422] ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7.

[0423] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said promoter is an inducible or tissue-specific promoter chosen from:

[0424] ■ the 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0425] ■ the 2XAARE TK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0426] ■ the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0427] ■ the 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0428] ■ the GRP78 YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5;

[0429] ■ the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0430] ■ the miniCHACl YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0431] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said promoter is a constitutive promoter chosen from:

[0432] ■ SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343;

[0433] ■ CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 344;

[0434] ■ UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 345;

[0435] ■ the EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 346;

[0436] ■ the long EFl-a whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 347;

[0437] ■ hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 348;

[0438] ■ CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 349;

[0439] ■ RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 350; and

[0440] ■ SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 351. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said promoter is a constitutive promoter chosen from:

[0441] ■ SV40 whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0442] ■ CMV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0443] ■ UbC whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0444] ■ the EFl-a core whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 346;

[0445] ■ the long EFl-a whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 347;

[0446] ■ hPGK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348;

[0447] ■ CAGG whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 349;

[0448] ■ RSV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0449] ■ SFFV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0450] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said toxic or suicide transgene is chosen from:

[0451] ■ 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: 8 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: 9;

[0452] ■ 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: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 11;

[0453] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 13;

[0454] ■ 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: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 15;

[0455] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 19;

[0456] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 21;

[0457] ■ a Noxa protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a protein a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 23;

[0458] ■ a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 756; and

[0459] ■ an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 758.

[0460] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said toxic or suicide transgene is chosen from:

[0461] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid encodes an inducible Caspase-9 (iC9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 9;

[0462] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 11;

[0463] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence corresponds to the sequence SEQ ID NO: 13;

[0464] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 15;

[0465] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence corresponds to the sequence SEQ ID NO: 19;

[0466] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 21;

[0467] ■ a Noxa protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 23;

[0468] ■ a streptolysin O whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 756; and

[0469] ■ an enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0470] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which the nucleic acid coding for said toxic or suicide transgene comprises at its 3' end a polyA sequence (eg SEQ ID NO: 24).

[0471] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which:

[0472] ■ the said promoter is:

[0473] - an inducible promoter chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA ;

[0474] - is the tissue-specific promoter of miR223; or

[0475] - is a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, and

[0476] ■ said toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens). According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said expression cassette is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 25 to 80 and 391 to 462.In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said expression cassette is chosen from the sequences SEQ ID NOs: 25 to 80 and 391 to 462 (see Table 1).

[0477] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 87 to 198 and 463 to 606. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 87 to 198 and 463 to 606 (see Table 2).

[0478] As mentioned above, said vector genome is carried by a plasmid which allows it to be expressed and therefore to produce said lentiviral vectors (or lentiviral particles). Also, by "a plasmid comprising said vector genome and the means for expressing it", reference is made to this plasmid which is a double-stranded circular DNA molecule. This plasmid necessarily has a bacterial origin of replication and, incidentally, the SV40 virus, so that it can replicate autonomously, e.g., in A. coli for its amplification and, e.g., in the HEK 293 T cell after transfection, and a selection gene so that it is not lost during its bacterial amplification over the course of cell multiplications. In the invention, this further comprises the nucleic acid of said vector genome of the invention and the means for expressing it.

[0479] By "the means of expressing it" is meant the genetic elements necessary for the expression of the vector genome of the invention and the production of a lentiviral vector according to the invention.

[0480] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-SIN plasmid or a pLV-RSV plasmid.

[0481] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-SIN plasmid. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-SIN plasmid whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 199. In particular, the subject of the invention is also the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-SIN plasmid whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 199.It should be noted that the insertion of said vector genome into said plasmid pLV-SIN of sequence SEQ ID NO: 199 can be or is carried out at nucleotides 4939 and 4940 of this SEQ ID NO: 199. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 200 and 201.

[0482] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-RSV plasmid. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-RSV plasmid whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 202. In particular, the subject of the invention is also the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is a pLV-RSV plasmid whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 202.It should be noted that the insertion of said vector genome into said plasmid pLV-RSV of sequence SEQ ID NO: 202 can be or is carried out at nucleotides 234 and 235 of this SEQ ID NO: 202. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 203 and 204.

[0483] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is characterized by the fact that 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.

[0484] According to another embodiment, the invention therefore relates to the method for producing lentiviral vectors as described above, in which said plasmid into which a vector genome as described above and the means for expressing it has been introduced is:

[0485] ■ a plasmid pLV-SIN whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 199; or

[0486] ■ a plasmid pLV-RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 202; said lentiviral genome into which said expression cassette has been introduced being:

[0487] ■ 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: 81; or

[0488] ■ 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: 84; and wherein said expression cassette comprises:

[0489] ■ an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4XSRE YB - TATA , the 9XHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA ; or a tissue-specific promoter of miR223; or a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, and

[0490] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0491] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205 to 316 and 607 to 750. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it is chosen from the sequences SEQ ID NOs: 205 to 316 and 607 to 750.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said plasmid comprising said vector genome and the means for expressing it has the sequence SEQ ID NO: 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,.

[0492] 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,

[0493] 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,

[0494] 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,

[0495] 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,

[0496] 311, 312, 313, 314, 315, 316, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,

[0497] 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,

[0498] 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657,

[0499] 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,

[0500] 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,

[0501] 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714,

[0502] 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733,

[0503] 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749 or 750 (see Table 3).

[0504]

[0505] Table 3. List of plasmids

[0506] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318 (which specifically recognizes the inducible promoter 2XAARE YB TATA (SEQ ID NO: 1)). In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318.

[0507] Advantageously, the invention also relates to the method for producing lentiviral vectors as described above, in which:

[0508] ■ said fusion protein:

[0509] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754, - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360,

[0510] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0511] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0512] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0513] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0514] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0515] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0516] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0517] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0518] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1.

[0519] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0520] ■ said fusion protein:

[0521] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 325) or 754,

[0522] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0523] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0524] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0525] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0526] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0527] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0528] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0529] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0530] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318; and

[0531] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0532] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 25 to 32, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 87 to 94 and 143 to 150, which can be encoded by a plasmid chosen from the sequences SEQ ID NOs: 205 to 212 and 261 to 268.

[0533] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the constitutive SV40 promoter (SEQ ID NO: 343)). In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0534] Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0535] ■ said fusion protein:

[0536] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754,

[0537] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360, - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0538] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0539] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0540] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0541] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0542] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0543] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0544] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and

[0545] ■ said constitutive promoter is SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0546] ■ said fusion protein:

[0547] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 325) or 754,

[0548] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0549] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372

[0550] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0551] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0552] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0553] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0554] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0555] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0556] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352; and

[0557] ■ said constitutive promoter is SV40 whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0558] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 391 to 398, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 463 to 470 and 353 to 542, which can be encoded by a plasmid chosen from the sequences SEQ ID NOs: 607 to 614 and 679 to 686.

[0559] According to another aspect, the invention relates to an animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising:

[0560] ■ a fusion protein chosen from:

[0561] - dCas-KRAB comprising the fusion of an inactive Cas endonuclease and a KRAB transcriptional repression domain;

[0562] - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain;

[0563] - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain;

[0564] - dCas-DNMT3 AL comprising the fusion of an inactive Cas endonuclease and a DNMT3 AL methylation domain;

[0565] - dCas-DNMT3B-L comprising the fusion of an inactive Cas endonuclease and a DNMT3B-L methylation domain;

[0566] - KRAB-dCas-DNMT3A-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3 AL methylation domain;

[0567] - KRAB-dCas-DNMT3B-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3B-L methylation domain; and

[0568] - dCas-KRAB-MeCP2 comprising the fusion of an inactive Cas endonuclease (dCas), a KRAB transcriptional repression domain and a MeCP2 methylation domain, and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said promoter being an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB TATA and the miniCHACl YB TATA ; or the tissue-specific promoter of miR223; or a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed.

[0569] Obtaining such an animal eukaryotic cell according to the invention is accessible to those skilled in the art who have the knowledge thereof. Nevertheless, for the purposes of illustration and in a non-limiting manner, it is specified that to obtain the animal eukaryotic cell according to the invention (e.g. HEK 293 T cell), cells were transduced with a lentiviral vector carrying the constitutive expression cassettes, of the dCas-KRAB fusion protein fused by a 2 A peptide to an antibiotic resistance gene (e.g. blasticidin (BlastR)), and of F guide RNA specific to the targeted promoter. Two days later, the transduced cells were selected by treatment with an antibiotic (e.g. 2 pg / mL). The selected cells were then cloned by limiting dilution and each clone was characterized. Only the clone allowing the most effective blocking of the expression of the transgene under the control of a promoter according to the invention was selected and amplified.

[0570] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above expressing at least one ribonucleoprotein complex comprising:

[0571] ■ a fusion protein chosen from:

[0572] - dCas-KRAB comprising the fusion of an inactive Cas endonuclease and a KRAB transcriptional repression domain;

[0573] - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain;

[0574] - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain;

[0575] - dCas-DNMT3 AL comprising the fusion of an inactive Cas endonuclease and a DNMT3 AL methylation domain; and

[0576] - dCas-DNMT3B-L comprising the fusion of an inactive Cas endonuclease and a DNMT3B-L methylation domain;

[0577] - KRAB-dCas-DNMT3A-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3 AL methylation domain; - KRAB-dCas-DNMT3B-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3B-L methylation domain; and

[0578] - dCas-KRAB-MeCP2 comprising the fusion of an inactive Cas endonuclease (dCas), a KRAB transcriptional repression domain and a MeCP2 methylation domain, and a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said promoter being:

[0579] - an inducible promoter chosen from: 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1; the 2XAARE TKwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2; the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3; the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4; GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5; and miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7, or

[0580] - the tissue-specific promoter of miR223 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6, or

[0581] - a constitutive promoter chosen from: SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343; CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 344; UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 345; EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 346; EFl-a long whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 347; hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 348; CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 349;RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 350; and SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 351, said ribonucleoprotein complex dCas-KRAB / sgRNA, dCas-DNMT3A / sgRNA, dCas-DNMT3B / sgRNA, dCas-DNMT3A-L / sgRNA, dCas-DNMT3B-L / sgRNA, KRAB-dCas-DNMT3A / sgRNA, KRAB-dCas-DNMT3B / sgRNA, KRAB-dCas-DNMT3A-L / sgRNA, KRAB-dCas-DNMT3B-L / sgRNA or dCas-KRAB-MeCP2 / sgRNA being in particular stably expressed.;

[0582] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3 A, DNMT3B, DNMT3 AL and DNMT3B-L is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0583] According to another embodiment, the invention relates to the animal eukaryotic cell as described above, in which:

[0584] ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the C-terminal position of said inactive Cas endonuclease (dCas); or

[0585] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2 is in the N-terminal position of said inactive Cas endonuclease (dCas); or

[0586] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0587] Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0588] ■ said KRAB transcriptional repression domain is in the N-terminal position of said inactive Cas endonuclease (dCas), and said methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas); and

[0589] ■ said KRAB transcriptional repression domain is in the C-terminal position of said inactive Cas endonuclease (dCas), and said MeCP2 methylation domain is in the C-terminal position of said KRAB transcriptional repression domain.

[0590] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell expressing a ribonucleoprotein complex comprising: ■ a dCas-KRAB fusion protein comprising the fusion of an inactive Cas endonuclease and a KRAB transcriptional repression domain; and

[0591] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter according to the invention, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed.

[0592] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0593] ■ a fusion protein chosen from:

[0594] - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain;

[0595] - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain;

[0596] - dCas-DNMT3 AL comprising the fusion of an inactive Cas endonuclease and a DNMT3 AL methylation domain; and

[0597] - dCas-DNMT3B-L comprising the fusion of an inactive Cas endonuclease and a DNMT3B-L methylation domain, and

[0598] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter according to the invention, said ribonucleoprotein complex dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA or dCas-DNMT3B-L / gRNA being in particular stably expressed.

[0599] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell at least one first and one second ribonucleoprotein complex, said first ribonucleoprotein complex comprising:

[0600] ■ a dCas-KRAB fusion protein comprising the fusion of an inactive Cas endonuclease and a KRAB transcriptional repression domain; and

[0601] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter according to the invention, and said second ribonucleic complex comprising:

[0602] ■ a fusion protein chosen from:

[0603] - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain;

[0604] - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain;

[0605] - dCas-DNMT3 AL comprising the fusion of an inactive Cas endonuclease and a DNMT3 AL methylation domain; and

[0606] - dCas-DNMT3B-L comprising the fusion of an inactive Cas endonuclease and a DNMT3B-L methylation domain, and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter according to the invention (i.e. the same as that targeted by said first ribonucleoprotein complex). said first and second ribonucleoprotein complexes being in particular stably expressed.

[0607] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0608] ■ a fusion protein chosen from:

[0609] - KRAB-dCas-DNMT3A-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3 AL methylation domain;

[0610] - KRAB-dCas-DNMT3B-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3B-L methylation domain; and

[0611] - dCas-KRAB-MeCP2 comprising the fusion of an inactive Cas endonuclease (dCas), a KRAB transcriptional repression domain and a MeCP2 methylation domain, and

[0612] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter according to the invention, said ribonucleoprotein complex KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed.

[0613] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said guide RNA (gRNA) specifically recognizes the promoter sequence of an inducible or tissue-specific promoter chosen from:

[0614] ■ the 2XAARE YB TATAwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0615] ■ the 2XAARE TK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0616] ■ the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0617] ■ the 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0618] ■ the GRP78 YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5;

[0619] ■ the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0620] ■ the miniCHACl YB TATAwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7. According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said guide RNA (gRNA) specifically recognizes the promoter sequence of a constitutive promoter chosen from:

[0621] ■ SV40 whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0622] ■ CMV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0623] ■ UbC whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0624] ■ the EFl-a core whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 346;

[0625] ■ the long EFl-a whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 347;

[0626] ■ hPGK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348;

[0627] ■ CAGG whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 349;

[0628] ■ RSV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0629] ■ SFFV whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0630] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said inactive Cas endonuclease is chosen from:

[0631] ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, '

[0632] ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, '

[0633] ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, '

[0634] ■ inactive CasX or Casl2e endonucleases from Deltaproteobacteria and Planctomycetes, '

[0635] ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans,'

[0636] ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade, -

[0637] ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and

[0638] ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

[0639] According to another embodiment, the invention relates to the animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9). This is a ribonucleoprotein complex dCas9-KRAB / gRNA, dCas9-DNMT3A / gRNA, dCas9-DNMT3B / gRNA, dCas9-DNMT3A-L / gRNA, dCas9-DNMT3B-L / gRNA, KRAB-dCas9-DNMT3A / gRNA, KRAB-dCas9-DNMT3B / gRNA, KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA or dCas9-KRAB-MeCP2 / gRNA. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid encodes an inactive Cas9 endonuclease (dCas9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 323.In particular, the invention also relates to the animal eukaryotic cell as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322 or whose nucleic acid codes for an inactive Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 323.

[0640] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 321 or 752. In particular, the subject of the invention is also the animal eukaryotic cell as described above, in which said KRAB transcriptional repression domain is encoded by the nucleic acid of sequence SEQ ID NO: 320 or 751, or whose amino acid sequence corresponds to the sequence SEQ ID NO: 321 or 752.

[0641] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754.In particular, the invention also relates to the animal eukaryotic cell as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 (encoded by the plasmid pLV-EFla-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or 754.

[0642] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said methylation domain:

[0643] ■ DNMT3 A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 354; ■ DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 366;

[0644] ■ DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 358; or

[0645] ■ DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 368.

[0646] In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said methylation domain:

[0647] ■ DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 354;

[0648] ■ DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 366;

[0649] ■ DNMT3A-L is encoded by the nucleic acid of sequence SEQ ID NO: 357 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0650] ■ DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 368.

[0651] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said MeCP2 methylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 370. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said MeCP2 methylation domain is encoded by the nucleic acid of sequence SEQ ID NO: 369 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 370.

[0652] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L or dCasDNMT3B-L fusion protein is respectively a dCas9-DNMT3A, dCas9-DNMT3B, dCas9-DNMT3A-L or dCas9-DNMT3B-L fusion protein. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0653] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360;

[0654] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372;

[0655] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364; or

[0656] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374.

[0657] In particular, the subject of the invention is the animal eukaryotic cell as described above, in which

[0658] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360;

[0659] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372;

[0660] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364; or

[0661] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372.

[0662] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell further expressing another ribonucleoprotein complex comprising:

[0663] ■ a fusion protein comprising the fusion:

[0664] - an inactive Cas endonuclease (dCas); and

[0665] - a domain promoting DNMT3L methylation (dCas-DNMT3E fusion) ■ and

[0666] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said other dCas-DNMT3L / gRNA ribonucleoprotein complex being in particular stably expressed. Advantageously, the subject of the invention is the animal eukaryotic cell as described above, said animal eukaryotic cell at least one first and one second ribonucleoprotein complex, said first ribonucleoprotein complex comprising:

[0667] ■ a fusion protein chosen from:

[0668] - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain; and

[0669] - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain, and

[0670] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter according to the invention, and said second ribonucleic complex comprising:

[0671] ■ a fusion protein comprising dCas-DNMT3L comprising the fusion of an inactive Cas endonuclease and a DNMT3L methylation domain, and

[0672] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter according to the invention (i.e. the same as that targeted by said first ribonucleoprotein complex), said first and second ribonucleoprotein complexes being in particular stably expressed. In particular, the invention also relates to the animal eukaryotic cell as described above, said animal eukaryotic cell further expressing another ribonucleoprotein complex comprising:

[0673] ■ a fusion protein comprising the fusion:

[0674] - an inactive Cas endonuclease (dCas); and

[0675] - a KRAB transcriptional repression domain (dCas-KRAB fusion) ■ and

[0676] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said dCas-KRAB / gRNA ribonucleoprotein complex being in particular stably expressed.

[0677] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0678] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said methylation-promoting domain DNMT3L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 356. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said methylation-promoting domain DNMT3L is encoded by the nucleic acid of sequence SEQ ID NO: 355 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 356.

[0679] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362. In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose sequence of amino acids corresponds to the sequence SEQ ID NO: 362.

[0680] As mentioned above, the invention also implements a bifunctional CRISPRi / m complex comprising:

[0681] ■ the fusion of an inactive or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L or MeCP2 methylation domain; and

[0682] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter (that of the expression cassette).

[0683] In particular, this bifunctional complex (advantageously expressed in a stable manner) is chosen from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA and dCas-KRAB-MeCP2 / gRNA, and is advantageously chosen from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA and dCas9-KRAB-MeCP2 / gRNA. Also, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0684] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382;

[0685] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384; or

[0686] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386.

[0687] In particular, the subject of the invention is the animal eukaryotic cell as described above, in which: ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 (encoded by the plasmid pLV-EFla-KRABl-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382;

[0688] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384; or

[0689] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386.

[0690] Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3L / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA is stably expressed via targeted integration into the genome of said animal eukaryotic cell.

[0691] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said animal eukaryotic cell belongs 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. Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which said animal eukaryotic cell belongs to the HEK 293T cell line.

[0692] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318 (which specifically recognizes the inducible promoter 2XAARE YB TATA(SEQ ID NO: 1)). In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318.

[0693] Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which: ■ said fusion protein:

[0694] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754,

[0695] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360,

[0696] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0697] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0698] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0699] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0700] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0701] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0702] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0703] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0704] ■ said inducible promoter is 2XAAREYB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1.

[0705] In particular, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0706] ■ said fusion protein:

[0707] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 325) or 754,

[0708] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0709] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372

[0710] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0711] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0712] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0713] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0714] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0715] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0716] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318; and

[0717] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0718] According to another embodiment, the subject of the invention is the animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the constitutive promoter SV40 (SEQ ID NO: 343)). In particular, the subject of the invention is the animal eukaryotic cell as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0719] Advantageously, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0720] ■ said fusion protein:

[0721] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 326 or 754,

[0722] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 360,

[0723] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 372,

[0724] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 364,

[0725] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 373 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 374,

[0726] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 362,

[0727] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 376 or 382,

[0728] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 378 or 384, and / or

[0729] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 380 or 386;

[0730] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and ■ said constitutive promoter is SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343.

[0731] In particular, the subject of the invention is the animal eukaryotic cell as described above, in which:

[0732] ■ said fusion protein:

[0733] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 326 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 325) or 754,

[0734] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 359 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 360,

[0735] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372

[0736] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 363 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 364,

[0737] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 371 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 372,

[0738] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 361 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 362,

[0739] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 375 or 381, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 376 encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or 382,

[0740] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 377 or 383, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 378 or 384, and / or

[0741] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 379 or 385, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 380 or 386;

[0742] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 352; and

[0743] ■ said constitutive promoter is SV40 whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0744] It should also be noted that among the objects of the invention are: a vector genome as described above comprising in particular an inducible or tissue-specific promoter, a plasmid comprising said vector genome as described above, the use of said plasmid as described above to produce a lentiviral vector comprising a vector genome as described above, a lentiviral vector comprising a vector genome as described above, the use of said lentiviral vector as described above to transduce an animal eukaryotic cell, an animal eukaryotic cell transduced by a lentiviral vector as described above and an animal eukaryotic cell transduced as described above for its use in cell therapy.With this last object, it appears that the invention goes beyond allowing the production at high titers of lentiviral vectors coding for toxic (cytotoxic) or suicide transgenes, hitherto never achieved. Indeed, it finally offers the possibility of implementing effective and safe cell therapies. These exploit the so-called safety switch technology, which under the action of the right stimulus (and only the right stimulus) allows the activation of the inducible or tissue-specific promoter of said expression cassette, making possible the expression of the toxic or suicide transgene, causing the death of the cell. Also and for example, the therapeutic use of an immune cell:.

[0745] ■ modified and armed to target and destroy cancer cells; and

[0746] ■ transduced according to the invention, will, after its administration to a patient who needs it, play its role of targeting and destroying cancer cells. Only if this same immune cell becomes dysfunctional, it will then be possible to destroy it in order to avoid adverse effects, thanks to the safety system provided by the invention (i.e. expression cassette comprising an inducible or tissue-specific promoter upstream of a toxic or suicide transgene). Furthermore, and as demonstrated by the examples below, it is possible to pharmacologically induce and pharmacologically control the expression of the transgene under the control of the inducible promoter of the invention, which is chosen from: 2XAARE YB - TATA the 2XAARE TK , the 4xSRE YB - TATA , L e 9xHRE YB - TATA , L e GRP78 YB - TATA and the miniCHACl YB - TATAEspecially when it comes to 2xAARE YB - TATA .

[0747] According to another aspect of the invention, the subject of the invention is therefore the use of a chemical inducer to induce the activation of an inducible promoter or to induce the expression of a transgene included in an expression cassette, said transgene being under the control of said inducible promoter, said inducible promoter being chosen from: 2XAARE YB TATA , the 2xAARE TK , the 4XSRE YB I I the 9XHRE YB - TATA , L e GRP78 YB - TATA and the miniCHAC I YB T I \ and said chemical inducer being chosen from: artesunate (Art), asparaginase (Asp) and bortezomib (Bz).

[0748] Alternatively, the subject of the invention is a method for inducing the activation of an inducible promoter or for inducing the expression of a transgene included in an expression cassette, said transgene being under the control of said inducible promoter, said inducible promoter being chosen from: 2XAARE YB TATA , the 2xAARE TK , the 4XSRE YB I I the 9XHRE YB - TATA , L e GRP78 YB - TATA and the miniCHAC I YB T I \ and said chemical inducer being chosen from: artesunate (Art), asparaginase (Asp) and bortezomib (Bz).

[0749] Whether it is the use of a chemical inducer as described above or the induction method as described above, the implementation of an effective and safe cell therapy within the meaning of the invention involves the administration to the patient (who needs it) of said chemical inducer. This, in particular chosen from: artesunate, asparaginase and bortezomib, can therefore be in the form of a pharmaceutical composition, in particular an injectable pharmaceutical composition. In doing so, the invention also relates to a chemical inducer (or a pharmaceutical composition comprising said chemical inducer) for its use in inducing the activation of an inducible promoter or inducing the expression of a transgene included in an expression cassette, said transgene being under the control of said inducible promoter, said inducible promoter being chosen from: 2XAARE YB TATA , the 2xAARE TK , the 4XSRE YB I I the 9XHRE YB -TATA , L e GRP78 YB - TATA and the miniCHAC I YB TATA , said chemical inducer being chosen from: artesunate (Art), asparaginase (Asp) and bortezomib (Bz).

[0750] 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, said chemical inducer being artesunate (Art). 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, said chemical inducer being asparaginase (Asp).

[0751] 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, said chemical inducer being bortezomib (Bz).

[0752] 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, said inducible promoter chosen from:

[0753] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1;

[0754] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2;

[0755] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0756] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0757] ■ the GRP78 YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0758] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and

[0759] ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7.

[0760] 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, said inducible promoter chosen from:

[0761] ■ the 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0762] ■ the 2XAARE TKwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0763] ■ the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0764] ■ the 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0765] ■ the GRP78 YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5;

[0766] ■ the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0767] ■ the miniCHACl YB TATAwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7. According to another embodiment, the subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1. In particular, the subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1.

[0768] This aspect may also implement an expression cassette, which as a reminder designates a nucleic acid comprising an inducible promoter sequence according to the invention, which is located 5' (upstream) of a transgene, it should be noted that this transgene may be chosen from: oncogenes, antioncogenes, transcription factors, trophic factors, cytokines, enzymes, hormones, receptors, transporters, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CAR), antibodies and toxic or suicide genes.In fact and according to another embodiment, the subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from: oncogenes, antioncogenes, 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 subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from: transcription factors, cytokines, transporters, nucleases, chimeric antigen receptors (CARs) and toxic or suicide genes.

[0769] “Transcription factors” refers to a protein that interacts with DNA and RNA polymerase and is necessary for the initiation or regulation of gene transcription in all living organisms (prokaryotes or eukaryotes). Examples of the invention include: c-JUN, FOXO1, B ATF, TBET and NRF2.

[0770] According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from: c-JUN, FOXO1, BATF, TBET and NRF2. In particular, the invention relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being c-JUN. In particular, the invention also relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being c-JUN whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 763 or whose nucleic acid codes for an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 764.Advantageously, the subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said transgene being c-JUN whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 763 or whose nucleic acid corresponds to the sequence SEQ ID NO: 764.

[0771] "Cytokines" refers to proteins secreted by cells of the immune system that allow them to communicate with each other and, consequently, these cytokines participate in the activation of the immune response. In the invention, the following may be cited: IL2, IFNy, TNFa, IL18, and IL12.

[0772] According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from: IL2, IFNy, TNFa, IL18, and IL12.

[0773] “Transporters” refers to transmembrane proteins whose function is to ensure the transport of metabolites such as glucose (eg GLUT1). According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being GLUT1.

[0774] “Nucleases” herein 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 diverted 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 Cas 12e, Casl2f or Cas 14, Casl2j or Cas®) from different hosts have been identified and characterized, or even modified (e.g., improvement of their function), which can be implemented by the invention, for the engineering of mammalian genomes.Generally, the Cas proteins mentioned above have decreasing sizes of 1,500 amino acids (AA) for Cas9, 1,000 AA for Cas 12a and less than 1,000 AA for CasX, Casl2f and Casl2j.

[0775] According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from:

[0776] ■ Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® nucleases;

[0777] ■ Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® orthologs; and

[0778] ■ Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® functional mutants or variants. The term “orthologs” refers to similar Cas proteins present in two or more different species. The term “mutants” refers to a Cas protein in which one or more mutations have been introduced, including the deletion, substitution and / or addition of one or more amino acids. These mutations may, in particular, increase nuclease activity and / or increase the fidelity of target DNA recognition. The term “functional mutants” refers to Cas proteins modified by human intervention (e.g., by genetic engineering), in order, for example, to increase Cas activity. The term “functional variants” refers to Cas proteins modified naturally through evolution, which exhibit, e.g., increased activity.

[0779] Advantageously, the invention uses 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, the mutants thereof. According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being chosen from:

[0780] ■ Cas9 nucleases from S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus; And

[0781] ■ Cas9 orthologs and Cas9 mutants or functional variants derived from these organisms.

[0782] In particular, the subject of the invention is the use of a chemical inducer as described above or of the induction method as described above, said transgene being saCas9 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 765 or whose nucleic acid encodes a saCas9 protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 766. In particular, the subject of the invention is also the use of a chemical inducer as described above or of the induction method as described above, said transgene being saCas9 whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 765 or whose nucleic acid encodes a saCas9 protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 766.

[0783] “Chimeric antigen receptors (CARs)” refers to a laboratory-created protein that can recognize and target proteins 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, CAR anti-FAP, anti-ROR1 CAR, anti-ROR2 CAR, anti-PDI CAR, and anti-PSCA CAR (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-, Leulô- , and 2F2-Based Antigen-Recognition Moieties. Int. J. Mol. Sci. 2023, 24, 3698.).

[0784] According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being a CAR chosen from: an anti-A-folate receptor CAR, an anti-Carbonic anhydrase IX CAR, an anti-CD171 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 antigpl 00 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, a CAR anti-Mucl6, an anti-NKG2D CAR, an anti-PSMA CAR, 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-EPC AM CAR, an anti-FAP CAR, an anti-RORl CAR, an anti-ROR2 CAR, an anti-PDI CAR and an anti-PSCA CAR.

[0785] “Toxic or suicide transgene” refers to a nucleic acid that codes for a toxic, cytotoxic or suicide protein. Also, and 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 of the genetically modified cell that expresses it. In the invention, this toxic or suicide transgene can be chosen from the genes coding: inducible Caspase-9 (iC9), Bax (S 184 del), Noxa (the wild-type protein or one of its mutants, in particular the S 13 A mutant), Gasdermin B (N-ter), constitutively active caspase-3 (V266E), an M2 ion channel (H37A) of the influenza virus, the A subunit of diphtheria toxin (DTA) 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.That is to say that the product of the expression (transcription then translation) of the nucleic acid encoding them, after activation of said inducible promoter sequence of the invention downstream of it, is not found in the extracellular medium. Also, only the cell death of the genetically modified cell expressing them occurs.

[0786] According to another embodiment, the invention therefore relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being a toxic or suicide transgene 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) 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 use of a chemical inducer as described above or of the induction method as described above, said transgene being a toxic or suicide transgene 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated mutant aDTA (G128D), a streptolysin O (in particular that of S. pyogenes and an enterotoxin (in particular that of C. perfringens). above, said transgene being a toxic or suicide transgene chosen from:

[0787] ■ 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: 8 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: 9;

[0788] ■ 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: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 11;

[0789] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 13;

[0790] ■ 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: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 15;

[0791] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 19;

[0792] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 21;

[0793] ■ a Noxa protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a protein a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 23;

[0794] ■ a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 756; and

[0795] ■ an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 758.

[0796] 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, said transgene being a toxic or suicide transgene chosen from:

[0797] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid encodes an inducible Caspase-9 (iC9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 9;

[0798] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 11;

[0799] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence corresponds to the sequence SEQ ID NO: 13;

[0800] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 15;

[0801] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence corresponds to the sequence SEQ ID NO: 19;

[0802] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 21;

[0803] ■ a Noxa protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 23;

[0804] ■ a streptolysin O whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 755 or whose nucleic acid encodes a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 756; and

[0805] ■ an enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 757 or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0806] In view of the above, the invention also relates to the use of a chemical inducer as described above or of the induction method as described above, said transgene being:

[0807] ■ a transcription factor chosen from: c-JUN, F0X01, BATF, TBET and NRF2;

[0808] ■ a cytokine chosen from: IL2, IFNy, TNFa, IL18, and IL12;

[0809] ■ the GLUT 1 transporter;

[0810] ■ a nuclease chosen from: Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas®, their orthologs and their mutants or functional variants;

[0811] ■ 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- 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 ; ou

[0812] ■ 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated mutant aDTA (G128D), a streptolysin O (notably that of S. pyogenes) and an enterotoxin (notably that of C. perfringens).

[0813] According to another aspect of the invention, the subject of the invention is a kit comprising:

[0814] ■ a nucleic acid comprising an inducible promoter as described above or a transgene comprised in an expression cassette as described above, said transgene being under the control of said inducible promoter; and

[0815] ■ at least one chemical inducer chosen from: artesunate (Art), F asparaginase (Asp) and Bortezomib (Bz).

[0816] According to another embodiment, the invention relates to the kit as described above, said chemical inducer being artesunate (Art). According to another embodiment, the invention relates to the kit as described above, said chemical inducer being asparaginase (Asp).

[0817] According to another embodiment, the invention relates to the kit as described above, said chemical inducer being Bortezomib (Bz).

[0818] According to another embodiment, the invention relates to the kit as described above, said inducible promoter being chosen from: 2xAARE YB T I the 2xAARE TK , the 4xSRE YB I I the 9XHRE YB I I the GRP78 YB - TATA e t the miniCHACl YB - TATA

[0819] According to another embodiment, the invention relates to the kit as described above, said inducible promoter being chosen from:

[0820] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1;

[0821] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2;

[0822] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0823] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0824] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0825] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and

[0826] ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7.

[0827] According to another embodiment, the invention relates to the kit as described above, said inducible promoter being 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1. In particular, the subject of the invention is the kit as described above, said inducible promoter being 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1.

[0828] According to another embodiment, the invention relates to the kit as described above, said transgene being:

[0829] ■ a transcription factor chosen from: c-JUN, FOXO1, BATF, TBET and NRF2;

[0830] ■ a cytokine chosen from: IL2, IFNy, TNFa, IL 18, and IL 12;

[0831] ■ the GLUT 1 transporter;

[0832] ■ a nuclease chosen from: Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas®, their orthologs and their mutants or functional variants; ■ a CAR chosen from: 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-Muclô, 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, anti-RORl CAR, anti-ROR2 CAR, anti-PDl CAR, and anti-PSCA CAR; Or

[0833] ■ 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 M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated mutant aDTA (G128D), a streptolysin O (notably that of S. pyogenes) and an enterotoxin (notably that of C. perfringens).

[0834] In any respect, it should be noted that the various aspects of the invention, as well as the various embodiments thereof, are interdependent. The latter can therefore be combined with each other as much as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This is also valid for all the definitions provided in this description, which applies to all aspects of the invention and its embodiments.

[0835] Further, the present invention is illustrated, but not limited to, the following figures and examples.

[0836] LIST OF FIGURES

[0837] Figure 1. Screening for the best performing gRNA to target the 2XAARE promoter YB TATA (SEQ ID NO: 1).

[0838] Plasmids expressing the dCas9-KRAB fusion protein (SEQ ID NOs: 325 and 326) and different gRNAs targeting a 2XAARE promoter sequence YB TATA (SEQ ID NO: 1) were transfected into HEK-293T cells transduced with a lentiviral vector carrying the 2XAARE cassette YB TATA -FLUC (SEQ ID NO: 331). The luciferase activity of FLuc was measured 24 h after transfection. RLU, Relative Light Units; n = 4 replicates; Kruskal-Wallis test; ns, P>0.05; *, P<0.05; **, P<0.01. Figure 2. Screening of HEK-CRISPRi clonal populations 2xAARE expressing the dCas9-KRAB fusion protein and the gRNA(2xAARE YB TATA ).

[0839] A) Cloning by limiting dilution was performed from HEK-293T cells transduced with a lentiviral vector carrying an expression cassette of the dCas9-KRAB fusion protein (SEQ ID NOs: 325 and 326) and the gRNA(2xAARE YB - TATA) previously selected (SEQ ID NO: 318). The expression of the dCas9-KRAB fusion protein (SEQ ID NOs: 325 and 326) and the gRNA(2xAARE YB - TATA ) (SEQ ID NO: 318) was verified in each clone by immunoblotting. B) HEK-293T cells or each clonal line expressing the dCas9-KRAB fusion protein (SEQ ID NOs: 325 and 326) and gRNA(2xAARE YB TATA ) (SEQ ID NO: 318) were transfected with a lentiviral plasmid carrying the 2XAARE cassette YB TATA -FLUC (SEQ ID NO: 331). Luciferase activity from plasmid-expressed FLuc, normalized to the total amount of protein in the sample, was measured 24 hours later. The clonal line in which FLuc transgene expression was most repressed was selected.

[0840] Figure 3. Schematics of forward (pLV; top panel) and reverse (pLVi; bottom panel) lentiviral vectors carrying a transgene under the control of the AARE 2 promoter YB - TATA(SEQ ID NO: 1).

[0841] Figure 4. Effect of transcriptional clamp expression on transgene expression upon transfection into HEK-293T cells.

[0842] A) Under the conditions of production of recombinant particles, the expression of the CRISPRi clamp 2xAARE in HEK-293T cell (HEK-CRISPRi 2xAARE ) allowed a significant decrease in luciferase expression by a plasmid pLV-2xAARE YB TATA -Luc (SEQ ID NO: 334) compared to the same plasmid transfected into HEK-293T cells. B) A significant decrease in luciferase expression by a plasmid pLVi (SEQ ID NO: 335), was observed in HEK-CRISPRi cells 2xAARE and in the presence of TAT compared to the same plasmid transfected into HEK-293T cells and in the presence of TAT. S: sense, I: reversed. Statistics, Unpaired T-test, (**) p<0.01; (***) p0.001).

[0843] Figure 5. KRAB-dCas9 expression in HEK-CRISPRi cells 2xAAREincreases the titers of pLVi carrying a 2xAARE-suicide gene cassette.

[0844] A) The titers of pLVi 2xAARE-GFP-suicide gene were significantly increased when produced in HEK-293T-CRISPR cells 2XAARE (Ci) compared to the titers obtained in HEK-293T cells (WT). Each bar represents the mean ± standard deviation of 3 different experiments. Statistics, Two-way ANOVA and Sidak multiple comparison test: (ns) p>0.05; (*) p<0.05; (**) p<0.01; (***) p0.001; (****) p0.001.

[0845] Figure 6. Functionality of pLVi vectors carrying a cytotoxic transgene.

[0846] Cytometric analysis of human CD3+ lymphocyte mortality induced by expression of suicide transgenes integrated by transduction with LVi(2xAARE) vectors YB - eGFP-P2A-GT) produced in HEK CRISPRi cells 2xAARE. The mortality rate was measured by the percentage of cells co-labeled with F Annexin-V and Propidium Iodide; n = 3 donors; Art, artesunate; Amt, amantadine; AP1903, dimerizer for iC9. Statistics, Two-way ANOVA and Tukey multiple comparison test or One-way ANOVA and Sidâk multiple comparison test: (ns) p>0.05; (*) p<0.05; (**) p<0.01; (***) p<0.001; (****) p0.001.

[0847] Figure 7. GFP expression in genetically modified CD8+ T lymphocytes, activated and treated with the pharmacological molecules, asparaginase (Asp, 2U / ml), bortezomib (Bz, 10 nM) or artesunate (Art, 5 pM).

[0848] Resting CD3+ T cells transduced with the pTRIP-2xAARE-GFP vector, activated with CD3 / CD28 beads and treated with DMSO, artesunate (Art), asparaginase (Asp), bortezomib (Bz) or tunicamycin (Tm, 4pg / ml) for 16h, and analyzed by flow cytometry. A) Percentage of cells expressing GFP. B) Mean fluorescence intensity (MFI). Each point corresponds to one donor. Statistics, one-way ANOVA and Dunnett's multiple comparison test for each condition compared to the resting condition (Rest.) (p >0.05, ns; p<0.05, *; p <0.01, **; p <0.001, ***; p <0.0001, ****).

[0849] Figure 8. Allogeneic lysis of tumor cells by CD8+ T lymphocytes in the presence of stress-inducing drugs.

[0850] A) EST 109, melanoma cell line, B) IRNE, melanoma cell line, C) MM1 S, multiple myeloma cell line. Each target cell line (T) was incubated with increasing numbers of effector CD8+T cells (E), in a ratio of 1:5, 1:25 and 1:50. After 16 h of culture, the remaining cells were lysed in the well and luciferase activity was quantified. Each point represents the total amount of luciferase measured per well.

[0851] Figure 9. Induction of CAR expression in genetically modified CD3+ lymphocytes.

[0852] Activated CD3+ T cells were transduced with a 2xAARE-CAR lentiviral vector. Cells were maintained either under normal culture conditions (Ctl), or treated with a deficiency of the amino acids methionine (Met), or tryptophan (Trp), or glutamine (Gin), or leucine (Leu), or arginine (Arg), or lysine (Lys), or treated with the drugs L-asparaginase (L-Asp) or artesunate (Art). A) Percentage of T cells expressing GFP measured by flow cytometry. B) Mean fluorescence intensity (MFI) per cell, measured by flow cytometry. Statistics, One-way ANOVA (p >0.05, ns; p<0.05, *; p <0.01, **; p <0.001, ***; p <0.0001, ****)

[0853] Figure 10. Regulated expression of transcription factors in CD3+ lymphocytes.

[0854] CD3+ lymphocytes were transduced with an LV-2xAARE-GFP-Tbet or LV-2xAARE-GFP-Foxp3 vector and then treated with medium, arginase (Arg), asparaginase (Asp) or artesunate (Art) 24h after treatment. A) Expression of GFP and T-bet transgenes in modified T cells. B) Expression of IFNγ and TNFα cytokines by T cells modified with the LV-2xAARE-GFP-Tbet vector, 24h after treatment. C) Expression of GFP and Foxp3 transgenes in modified T cells. D) Expression of regulatory T cell differentiation markers in genetically modified CD4+ cells. Statistics, One-way ANOVA (p >0.05, ns; p<0.05, *; p <0.01, **; p <0.001, ***; p <0.0001,

[0855] ****\

[0856] EXAMPLES

[0857] EXAMPLE I - GUIDE RNA (gRNA) SCREENING

[0858] The CRISPR interference (CRISPRi) and CRISPR-targeted methylation (CRISPRm) strategies of the invention are based on the fusion of an inactive or "dead" CRISPR-Cas endonuclease (dCas) and a KRAB (Krüppel-Associated Box) transcriptional repression domain or DNMT (DNA methyltransferase) methylation domain. dCas, thanks to its guide RNA (gRNA), makes it possible to target a specific sequence.

[0859] For specific targeting of a promoter, only the gRNA sequence needs to be adapted.

[0860] For this, computer analysis tools such as CRISPOR (Concordet, J. -P., Haeussler, M., 2018. CRISPOR: intuitive guide selection for CRISPR / Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242-W245) or CRISPICK (Kim et al. 2018. Deep learning improves prediction of CRISPR-Cpfl guide RNA activity. Nat. Biotechnol. 36, 239-241; Sanson et al. 2018. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416) test and propose candidate gRNA sequences according to their complementarity with the target and the absence of complementarity with the host cell genome. Then, it is necessary to functionally screen the different gRNA sequences to determine which one allows the most efficient CRISPR interference and / or methylation on the promoter considered.

[0861] For example, several gRNA sequences targeting the 2XAARE promoter were screened YB - TATA(SEQ ID NO: 1) namely:

[0862] ■ gDNA 1 of sequence SEQ ID NO: 318;

[0863] ■ gDNA 2 of sequence SEQ ID NO: 388;

[0864] ■ gDNA 3 of sequence SEQ ID NO: 389; and

[0865] ■ gDNA 4 of sequence SEQ ID NO: 390, the sequences of which were obtained using the CRISPOR tool. For this, a 2XAARE cassette YB TATA -FLUC “target” (SEQ ID NO: 331) was integrated into HEK-293T cells by lentiviral transduction (see Example 2 - Materials & Methods). These genetically modified cells were transfected with a plasmid co-expressing the CRISPRi complex (dCas9-KRAB fusion; SEQ ID NO: 326) and each gRNA (see Example 2 - Materials & Methods). The gRNA allowing the most efficient CRISPRi targeting to reduce the basal expression of FLuc was selected (Fig. 1). This is gRNA 1 with sequence SEQ ID NO: 318.

[0866] Then, a clonal line of HEK-CRISPRi 2xAAREwas established (see Example 2 - Materials & Methods). For this, a limiting dilution was carried out on a population of HEK-293T transduced with a lentiviral vector carrying an expression cassette of the CRISPRi complex (dCas9-KRAB fusion; SEQ ID NO: 326) and the gRNA (2xAARE YB - TATA ) (pLVi- 2xAARE YB TATA -Luc; SEQ ID NO: 318) previously selected. Expression of the CRISPRi complex was tested in each clonal line by immunoblotting (Fig. 2-A).

[0867] The functionality of each clone expressing the CRISPRi complex (dCas9-KRAB fusion; SEQ ID NO: 326) and the gRNA(2xAARE YB - TATA ) (SEQ ID NO: 318) was then tested (c Example 2 - Materials & Methods). For this, each clonal line was transfected with a lentiviral plasmid carrying the secreted nanoLuciferase (nLuc), under the control of 2XAARE YB TATA(SEQ ID NO: 335). The clone in which transgene expression was most repressed was selected (Fig. 2-B). This is clone 17.

[0868] Thus, for any other promoter, a similar procedure of gRNA screening and then establishment of a clonal line (CRISPRi and / or CRISPRm) must be carried out.

[0869] EXAMPLE 2 - PRODUCTION OF TOXIC VECTORS (CRISPRÎ)

[0870] MATERIALS & METHODS

[0871] Cloning of lentiviral plasmids

[0872] 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 (SIN) lentiviral genome (pLV; SEQ ID NO: 199).

[0873] Cell culture

[0874] Cells were cultured in a humidified and controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) were cultured in High Glucose Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL of Penicillin / Streptomycin. Stress induction was performed by treatment with tunicamycin (4 pg / mL, Sigma Aldrich). EST109 cells (published as UKRV-Mel-2 [ECACC 13012436]; Artuc M et al. Biochem Biophys Res Commun. 213(2):699-705) were cultured under the same conditions but in Roswell Park Memorial Institute medium (RPMI 1640, Thermo Fischer) containing 10% FCS and 100 U / mL Penicillin / Streptomycin.Primary T lymphocytes isolated from the blood of healthy donors were cultured in serum-free, feeder-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL Penicillin / Streptomycin.

[0875] Production of lentiviral vectors

[0876] Non-replicating lentiviral particles were obtained using the second-generation production protocol described by Zennou et al. in 2001 (NatBiotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded onto suitable cell culture media and cultured to reach 50-60% confluence after 24 hours of culture. The following day, cells were co-transfected, by the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome (pLV or pLVi) and two transcomplementing plasmids encoding HIV-1 enzymes and structural proteins (p8.9; SEQ ID NO: 338 or 339) and the vesicular stomatitis virus envelope glycoprotein (pVSVg; SEQ ID NO: 337). 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 ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0877] Titration

[0878] The concentration of lentiviral particles was determined by quantification of the lentiviral capsid protein p24. For this, an aliquot of lentiviral suspension was thawed, lysed and diluted according to the supplier protocol of the HIV-1 p24 Antigen ELISA 2.0 titration kit (Zeptometrix ref. 0801008). The concentration of p24 obtained was expressed in pg / pL, the equivalent of which is 1 pg of p24 for 104 VSVg-pseudotyped physical lentiviral particles (Dull, T et al. J Virol. 1998 Nov;72(l 1 ):8463-71 ). Tests can specify these values ​​but, on average, 1% of the physical particles obtained are infectious, i.e. 1 pg ~ 100 transduction units (TU).

[0879] Transduction

[0880] To integrate a transgene by infection of a lentiviral vector, the quantity of particles necessary to obtain the desired multiplicity of infection (vector:target ratio) was diluted in culture medium. This lentiviral suspension was brought into contact with the target cells. The infected cells were cultured for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression could be observed or measured the following day by microscopy, cytometry, or biochemical analysis.

[0881] Establishment of a KRAB line (CRISPRi)

[0882] HEK 293T cells were transduced with a lentiviral vector (SEQ ID NO: 328) carrying the constitutive expression cassettes:

[0883] ■ the dCas9-KRAB fusion protein (SEQ ID NO: 325) fused by a 2A peptide (SEQ ID NO: 332) to the blasticidin resistance gene (BlastR) (SEQ ID NO: 327) under the control of the eF-1a minimal promoter (SEQ ID NO: 319); and

[0884] ■ of guide RNA specific to the targeted promoter (SEQ ID NO: 318) under the control of the U6 promoter (SEQ ID NO: 317).

[0885] Two days later, transduced cells were selected by treatment with blasticidin (2 μg / mL). Cells were cloned by limiting dilution and each clone was characterized. Flow cytometry analysis

[0886] At the end of the experiment, the cultured cells were harvested and rinsed with PB S. They were then incubated for 30 minutes with F Annexin-V (AnV) and propidium iodide (PI). The cells were rinsed and then fixed in PBS with 1% paraformaldehyde for 15 minutes at 4°C. After rinsing, the AnV+ / PI+ co-labeling allows the mortality rate (proportion of cells in the late phase of apoptosis) to be measured using the MACSQuant Analyzer 10 cytometer (Miltenyi).

[0887] Luciferase activity measurement

[0888] The firely-Luciferase used in the experiments presented is an intracellular luciferase. Luciferase activity was measured on cell culture lysate using the Luciferase Assay System kit (Promega) according to the supplier's instructions. Luminescence was measured using a SpectraMax i3x plate reader (Molecular Devices).

[0889] RESULTS

[0890] Effect of a transcriptional clamp on the internal promoter of the vector.

[0891] In order to block the transcription of the transgene upstream of the 2XAARE promoter YB - TATA (SEQ ID NO: 1) on the plasmid encoding the vector during the production of viral particles, a HEK-293T cell line expressing an inactivated Cas9 protein (dCas9) fused to the KRAB transcription inhibition domain (SEQ ID NO: 326), and a guide RNA (SEQ ID NO: 318) specific for the ATF4 binding site on the 2XAARE promoter YB TATA (HEK- CRISPRi 2xAARE ) was obtained.

[0892] In order to compare the expression rate of the transgene (here luciferase) from the 2XAARE promoter YB - TATA (SEQ ID NO: 1), two plasmids were constructed: pLV-2xAARE YB - TATA - Luc (SEQ ID NO: 334) and pLVi-2xAARE YB TATA-Luc (SEQ ID NO: 335) in which the 2xAARE-Luc expression cassette (SEQ ID NO: 331) is either in the forward position (pLV) or in the reverse position (pLVi) (Fig. 3). These plasmids were then transfected either into a HEK-293T cell line or into the HEK-CRISPRi cell line 2xAARE , in the presence of TAT (transcription transactivation factor).

[0893] Finally, the presence of the transcription inhibitory clamp from the 2XAARE promoter YB TATA (SEQ ID NO: 1) induced a decrease in luciferase expression of approximately 1.5-fold with the pLV vector and more than 10x with pLVi (Fig. 4 A and B). Thus, the cumulative effect of the inversion of the expression cassette and the CRISPRi clamp 2xAARE reduced transgene expression by more than 20,000x compared to a pLV plasmid in the presence of TAT.

[0894] Increased titers of lentiviral vectors expressing a cytotoxic gene.

[0895] In order to produce high titers of lentiviral vectors carrying cytotoxic (or suicide) genes, whose expression is inducible by ATF4, seven inverted lentiviral vectors (pLVi) were obtained and used to produce recombinant particles carrying a control reporter transgene, or a toxic transgene (Fig. 3, bottom panel). In each vector, the transgene was placed under the control of the 2XAARE promoter YB - TATA (SEQ ID NO: 1). The control vector (SEQ ID NO: 336) carries the reporter transgene “enhanced Green Fluorescent Protein” (GFP; SEQ ID NO: 329), and the six toxic vectors (SEQ ID NOs: 205 to 210) respectively carry the eGFP gene associated by a P2A peptide (SEQ ID NO: 332) with the cytotoxic genes chosen from:

[0896] ■ inducible Caspase-9 (iC9; SEQ ID NO: 8);

[0897] ■ Bax (S 184 del; SEQ ID NO: 10);

[0898] ■ the M2 ion channel (H37A; SEQ ID NO: 12) of the influenza virus;

[0899] ■ constitutively active caspase-3 (V266E; SEQ ID NO: 14); And

[0900] ■ the diphtheria toxin A subunit (DTA; SEQ ID NO: 16) or an attenuated aDTA mutant (G128D; SEQ ID NO: 18).

[0901] The lentiviral vector library thus produced includes the following seven reverse lentiviral vectors: pLVi-2xAARE YB TATA -GFP (SEQ ID NO: 336), pLVi-2xAARE YB TATA -GFP-P2A- iC9 (SEQ ID NO: 205), pLVi-2xAARE YB TATA -GFP-P2A-Bax (SEQ ID NO: 206), pLVi- 2xAARE YB TATA -GFP-P2A-Casp3 (SEQ ID NO: 208), pLVi-2xAARE YB TATA -GFP-P2A-M2 (SEQ ID NO: 207), pLVi-2xAARE YB TATA -GFP-P2A-aDTA (SEQ ID NO: 210), and pLVi- 2XAARE YB TATA -GFP-P2A-DTA (SEQ ID NO: 209).

[0902] After production of lentiviral particles in a HEK-293T (WT) cell line or in the HEK-CRISPRi cell line 2xAARE, it was observed by quantification of p24 in the culture supernatant that the six vectors carrying a toxic gene were produced at significantly higher titers with the HEK-CRISPRi line 2xAARE (Fig. 5). Indeed, the production of recombinant vector particles in HEK-CRISPRi cells 2xAARE increased titers by approximately 8x for Bax, 5x for aDTA and Casp3, 4x for M2, 3x for GFP, and 2x for iC9.

[0903] Finally, the production of lentiviral particles carrying a toxic (or suicide) transgene downstream of a promoter with an animal eukaryotic cell expressing a ribonucleoprotein complex comprising:

[0904] ■ a dCas9-KRAB fusion protein comprising the fusion of an inactive Cas9 endonuclease and a KRAB transcriptional repression domain; and

[0905] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, has proven to be a method of choice for producing high titres of lentiviral vectors.

[0906] The produced reverse lentiviral vectors are functional.

[0907] Human primary T lymphocytes were transduced (MOI = 10) with LVi(2xAARE YB - GFP-P2A-GT) produced in HEK CRISPRi cells 2xAARE . Thus, the genes:

[0908] ■ of the DTA (pLVi-2xAARE YB TATA -GFP-P2A-DTA; SEQ ID NO: 209), ■ of 1' aDT A (pLVi-2xAARE YB TATA -GFP-P2 A-aDT A; SEQ ID NO: 210),

[0909] ■ of Bax (pLVi-2xAARE YB TATA -GFP-P2A-Bax; SEQ ID NO: 206),

[0910] ■ of Noxa (pLVi-2xAARE YB TATA -GFP-P2A-Noxa; SEQ ID NO: 212),

[0911] ■ of Casp3 (pLVi-2xAARE YB - TATA -GFP-P2A-Casp3; SEQ ID NO: 208),

[0912] ■ of M2 (pLVi-2xAARE YB TATA-GFP-P2A-M2; SEQ ID NO: 207) and

[0913] ■ of iC9 (pLVi-2xAARE YB TATA -GFP-P2A-iC9; SEQ ID NO: 205) were integrated into the genome of primary lymphocytes in culture. In these experiments, cellular stress to activate the 2xAARE YB was induced by 5pM artesunate (Art) treatment. Art is an antimalarial drug that causes oxidative stress that induces ATF4 expression.

[0914] To directly measure the toxicity of the transgenes, 24 hours after their induction with F Art (± 5pM Amt to block the effect of M2 or ± 4pM AP1903 to dimerize iC9), the cells were labeled with Annexin-V (AnV) and propidium iodide (PI). Detected by cytometry, the AnV+ / PI+ co-labeling allowed the measurement of the mortality rate (proportion of cells in the late phase of apoptosis).

[0915] In these experiments, carried out on cells from 3 donors, Art treatment induced the death of 22.5% of cells transduced with a LVi(2xAARE YB -eGFP), compared to 17.3% in the absence of treatment (Fig. 6A).

[0916] In the absence of induction, most GTs (Bax, Noxa, Casp3, aDTA, and DTA) induced basal mortality of 30% (Bax) to 34.8% (aDTA), a 10% increase compared to the control transgene (Fig. 6A, white bars). However, after 24 h of Art induction, the mean mortality rate measured was significantly higher, particularly with Bax (68.6%), Noxa (68.2%), Casp3 (63.6%), and aDTA (69%) (Fig. 6A, hatched bars).

[0917] Regarding M2, its basal expression induced a mortality rate (44%) higher than previous GTs. This rate was reduced in the presence of Amt (25.3%). Induction of the transgene by Art significantly increased the mortality rate caused by M2 (68.8%) even in the presence of Amt (48%). Thus, the toxicity of basal expression of the 2xAARE cassette YB - eGFP-P2A-M2 could be prevented by F Amt treatment and overexpression of M2 induced cell death (Fig. 6B).

[0918] Finally, the basal expression of the 2xAARE cassette YB-eGFP-P2A-iC9 caused comparable mean mortality rates in the absence (35.8%) or presence (43.5%) of the dimerizer AP 1903. This basal mortality was significantly higher than that obtained in cells transduced with the control vector and treated identically. Induction of FiC9 by Art significantly increased these rates in the absence (70.1%) and presence (83.6%) of AP1903. These data therefore demonstrated a very good efficacy of FiC9 in inducing T cell death (Fig. 6C). EXAMPLE 3 - PRODUCTION OF TOXIC VECTORS (CRisPRm)

[0919] MATERIALS & METHODS

[0920] Cloning of lentiviral plasmids

[0921] 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 (SIN) lentiviral genome (pLV; SEQ ID NO: 199).

[0922] Cell culture

[0923] Cells were cultured in a humidified and controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) were cultured in High Glucose Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL of Penicillin / Streptomycin. Stress induction was achieved by treatment with tunicamycin (4 pg / mL, Sigma Aldrich). EST 109 cells (published as UKRV-Mel-2 [ECACC 13012436]; Artuc M et al. Biochem Biophys Res Commun. 213(2):699-705) were cultured under the same conditions but in Roswell Park Memorial Institute medium (RPMI 1640, Thermo Fischer) containing 10% FCS and 100 U / mL Penicillin / Streptomycin.Primary T lymphocytes isolated from the blood of healthy donors are cultured in serum-free, feeder-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL Penicillin / Streptomycin.

[0924] Production of lentiviral vectors

[0925] Non-replicating lentiviral particles are obtained using the second-generation production protocol described by Zennou et al. in 2001 (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293T cells are seeded on supports suitable for cell culture and grown to 50-60% confluence after 24 hours of culture. The next day, the cells are co-transfected, by the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome (pLV or pLVi) and two transcomplementing plasmids encoding the HIV-1 enzymes and structural proteins (p8.9; SEQ ID NO: 338 or 339) and the envelope glycoprotein of the vesicular stomatitis virus (pVSVg; SEQ ID NO: 337). Co-transfection is carried out at a plasmid ratio of 2:2:1. The transfected cells are cultured for 5 hours in contact with the precipitate and then the culture medium is renewed.The supernatant containing the lentiviral particles is harvested 48 hours later. The lentiviral suspension is treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 μm) and then ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets are taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1000. The suspensions obtained are aliquoted and stored at -80°C until use.

[0926] Titration The concentration of lentiviral particles is determined by quantification of the lentiviral capsid protein p24. For this, an aliquot of lentiviral suspension is thawed, lysed and diluted according to the supplier protocol of the HIV-1 p24 Antigen ELISA 2.0 titration kit (Zeptometrix ref. 0801008). The concentration of p24 obtained is expressed in pg / pL, the equivalent of which is 1 pg of p24 for 104 VSVg-pseudotyped physical lentiviral particles (Dull, T et al. J Virol. 1998 Nov;72(l 1 ):8463-71 ). Tests can specify these values ​​but, on average, 1% of the physical particles obtained are infectious, i.e. 1 pg ~ 100 transduction units (TU).

[0927] Transduction

[0928] To integrate a transgene by infection of a lentiviral vector, the quantity of particles necessary to obtain the desired multiplicity of infection (vector:target ratio) is diluted in culture medium. This lentiviral suspension is brought into contact with the target cells. The infected cells are cultured for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression can be observed or measured the next day by microscopy, cytometry, or biochemical analysis.

[0929] Establishment of a KRAB line (CRISPRi)

[0930] HEK 293T cells are transduced with a lentiviral vector (SEQ ID NO: 328) carrying the constitutive expression cassettes:

[0931] ■ the dCas9-KRAB fusion protein (SEQ ID NO: 325) fused by a 2A peptide (SEQ ID NO: 332) to the blasticidin resistance gene (BlastR) (SEQ ID NO: 327) under the control of the eF-1a minimal promoter (SEQ ID NO: 319); and

[0932] ■ of guide RNA specific to the targeted promoter (SEQ ID NO: 352) under the control of the U6 promoter (SEQ ID NO: 317).

[0933] Two days later, transduced cells are selected by treatment with blasticidin (2 pg / mL). HEK-CRISPRi cells SV40 are cloned by limiting dilution and each clone is characterized.

[0934] Establishment of a KRAB (CRISPRi) and DNMT (CRISRPm) line

[0935] HEK 293 T cells are transduced with a lentiviral vector (SEQ ID NO: 387) carrying the constitutive expression cassettes:

[0936] ■ the KRAB-dCas9-DNMT3A-L fusion protein (SEQ ID NO: 375) fused by a 2A peptide (SEQ ID NO: 332) to the blasticidin resistance gene (BlastR) (SEQ ID NO: 327) under the control of the eF-la minimal promoter (SEQ ID NO: 319); and

[0937] ■ guide RNA specific to the targeted promoter (SEQ ID NO: 352) under the control of the U6 promoter (SEQ ID NO: 317).

[0938] Two days later, transduced cells were selected by treatment with blasticidin (2 pg / mL). HEK-CRISPRi / m cells SV40 are cloned by limiting dilution and each clone is characterized.

[0939] RESULTS To illustrate the interest of the CRISPRm strategy, lentiviral particles were produced from the genome plasmids pLVi(pSV40-eGFP) or pLVi(pSV40-eGFP-P2A-M2) (SEQ ID NO: 609) which carry, in reverse orientation, the control transgene eGFP or the toxic transgene eGFP-P2A-M2, under the control of the early (constitutive) promoter of the SV40 virus (pSV40; SEQ ID NO: 343). Each of these plasmids was co-transfected, with the plasmids p8.9 (SEQ ID NO: 338 or 339) and pVSVg (SEQ ID NO: 337), into cells:

[0940] ■ HEK-293T;

[0941] ■ HEK-CRISPRi SV40 (expressing a dCas9-KRAB complex (SEQ ID NO: 326) and a pSV40-specific gRNA (SEQ ID NO: 352)); or

[0942] ■ HEK-CRISPRi / m SV40 (expressing a bifunctional KRAB-dCas9-DNMT3 A-L complex (SEQ ID NO: 376) and a pSV40-specific gRNA (SEQ ID NO: 352)).

[0943] In HEK-293T cells, LV particle production is significantly more efficient with pLV(SV40-eGFP) than with pLVi(SV40-eGFP). In addition, the titer is further reduced when the transgene is toxic to packaging cells, as observed during production with pLVi(SV40-eGFP-P2A-M2) (SEQ ID NO: 609), the titer of which is significantly lower than for pLVi(SV40-eGFP).

[0944] However, when the production of the vector pLVi(SV40-eGFP-P2A-M2) (SEQ ID NO: 609) is carried out in HEK-CRISPRi cells SV40 the quantity of lentiviral particles (estimated by measuring the p24 concentration in the supernatants) is higher than that obtained in HEK-293T. These results demonstrate the effectiveness of the interference strategy to abolish transcription from the inverted transgene and therefore obtain lentiviral particle titers > 10 9 recombinant particles / mL.

[0945] Finally, production in HEK-CRISPRi / m cells SV40 allows the obtaining of even higher vector titers than the titers obtained in HEK-293T and HEK-CRISPRi SV40 . During the production of LV particles, the presence of the bifunctional CRISPRi / m complex therefore makes it possible to obtain the best titres of lentiviral vectors carrying a toxic transgene controlled by a constitutive promoter.

[0946] To confirm the functionality of the lentiviral particles obtained in the different HEK lines, their toxicity is tested on an in vitro model of solid tumor: the 3D culture (spheroid) of human melanoma cells EST 109.

[0947] Each spheroid is formed from 5.10 3EST 109 cells seeded in 96-well round-bottom plates and untreated for culture (ultra low adherence plates). Simultaneously with seeding, EST109 cells are transduced (MOI = 50) with the control vector LVi(SV40-eGFP) or the toxic vector LVi(SV40-eGFP-P2A-M2) (SEQ ID NO: 609), produced in the different lines (HEK-293T, HEK-CRISPRi SV40 and HEK-CRISPRi / m SV40 ). The evolution of fluorescence (eGFP), spheroid size and cell death labeling (7-AAD) is measured by videomicroscopy (Incucyte S3 system, Sartorius). I ll

[0948] When the transgene is non-toxic (eGFP), continuous growth in spheroid size is observed, regardless of the production line used. In contrast, when the M2 ion channel is expressed by the lentiviral vector, growth of transduced spheroids is zero and all cells are labeled with 7-AAD.

[0949] Thus, the CRISPRi strategy and in particular the CRISPRi / m combinatorial strategy make it possible to obtain high titres of constitutively toxic lentiviral vectors which can cause the death of the transduced cells.

[0950] EXAMPLE 4 - PHARMACOLOGICAL INDUCTION

[0951] MATERIALS & METHODS

[0952] Cloning of lentiviral plasmids

[0953] 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: 199).

[0954] Cell culture

[0955] Cells were cultured in a humidified and controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) were cultured in High Glucose Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL Penicillin / Streptomycin. Primary T lymphocytes isolated from the blood of healthy donors were cultured in serum-free and feed-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL Penicillin / Streptomycin. Stress induction was performed 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), F Arginase (Arg, 5U / mL A3233, Merck-Sigma).

[0956] Production of lentiviral vectors

[0957] Non-replicating lentiviral particles were obtained using the production protocol described by Zennou et al. (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded onto suitable cell culture media and grown to 50-60% confluence after 24 hours of culture. The next day, cells were cotransfected, using the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome (pLV) and two transcomplementing plasmids encoding HIV-1 enzymes and structural proteins (p8.92; SEQ ID NO: 338), and the envelope glycoprotein of vesicular stomatitis virus (pVSVg; SEQ ID NO: 337). Co-transfection was performed at a plasmid ratio of 2:2:1. Transfected cells were cultured for 5 hours in contact with the precipitate, 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 ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1,000. The suspensions obtained were aliquoted and stored at -80°C until use. The concentration of lentiviral particles was determined by quantification of the lentiviral capsid protein p24. For this, an aliquot of lentiviral suspension was thawed, lysed and diluted according to the supplier protocol of the HIV-1 p24 Antigen ELISA 2.0 titration kit (Zeptometrix ref. 0801008). The concentration of p24 obtained was expressed in pg / pL, the equivalent of which is 1 pg of p24 for 10. 2 transduction units (TU).

[0958] Transduction

[0959] To integrate a transgene by transduction of a lentiviral vector, the quantity of particles necessary to obtain the desired multiplicity of infection (vector:target ratio) was diluted in culture medium. This lentiviral suspension was placed in contact with 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 the next day by microscopy, cytometry or quantitative PCR.

[0960] Flow cytometry

[0961] For analysis of cultured cells, they were harvested and rinsed with PB S. They were then incubated for 30 minutes with surface labeling antibodies and the viability marker Live-or-Dye 405 / 452 (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 MACSQuant Analyzer 10 cytometer (Miltenyi).

[0962] Statistics

[0963] Statistical analyses were performed using GraphPad® Prism 10. Details regarding sample size and graph descriptions are provided in the figure legends corresponding to 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.

[0964] RESULTS

[0965] The induction of GFP transgene expression in CD3+ lymphocytes transduced with the lentiviral vector pTRIP 2xAARE-GFP (SEQ ID NO: 759) by Asparaginase (Asp), Bortezomib (Bz) or Artesunate (Art) was tested. In this experiment, the drug Tunicamycin (Tm) was used as a positive control for the induction of reticulum stress, and DMSO as a negative control. CD3+ T cells from four donors were activated with CD3 / CD28 beads and transduced with the lentiviral vector pTRIP 2xAARE-GFP (SEQ ID NO: 759) at an MOI of 10. After 48 h, the CD3 / CD28 beads were removed and the cells were maintained in culture with IL2, IL7 and IL15 for 5 days. After 5 days in these resting conditions, cells were reactivated with CD3 / CD28 beads and treated with DMSO, Asp, Art, Bz, or placed in leucine-deficient medium (Leu-), for 16 h.

[0966] It was observed that the four molecules Art, Asp, Bz or Tm, significantly increased the percentage of CD3+ GFP+ T cells; Tm being the most efficient inducer (5x induction), followed by Asp and Art (4x), and Bz (3x) (Fig. 7A). The average fluorescence per cell was also significantly increased by Tm (1 Ix) and Asp (6x) compared to control cells (Fig. 7B). These experiments thus demonstrated that the three molecules, Asp, Bz and Art, effectively induced an increase in the expression of the GFP transgene placed under the control of the 2XAARE promoter YB - TATA (SEQ ID NO: 1) in activated CD3+ lymphocytes.

[0967] In parallel, it was assessed whether the cytotoxic activity of CD3+ T cells was affected in the presence of these drugs (Tm, Asp, Bz and Art). For this purpose, CD3+ T cells were cultured with human tumor cell lines, MM1S (multiple myeloma), IRNE (UKRV-Mel-14a, melanoma) or EST-109 (UKRV-Mel-2, melanoma), and their killing by allogeneic cytotoxicity was measured. As these cell lines constitutively express luciferase (Luc), the lysis of allogeneic cells was inversely proportional to the remaining Luc activity in each well after 16 h of co-culture with CD3+ T cells. The measured allogeneic cytotoxicity appeared less effective with EST 109 than with IRNE and MM1S cells, and it was shown that the drugs (Asp, Bz and Art) did not affect the lysis of target cells, regardless of the treatment or the lymphocyte / target cell ratio (Fig. 8).This experiment thus demonstrated that the drugs Tm, Art, Asp or Bz do not affect the cytotoxic activity of CD8+ T lymphocytes.

[0968] To measure whether the drugs Asp and Art can induce the expression of a chimeric antigen receptor (CAR) in these cells, CD3+ lymphocytes were transduced with a lentiviral vector carrying an anti-CD19 CAR under the control of the 2xAARE promoter. YB TATA (SEQ ID NO: 1): LV-2xAARE-CAR (SEQ ID NO: 760). These cells were treated with either Art, Asp, or different amino acid deficiencies: methionine (Met), tryptophan (Trp), glutamine (Gin), leucine (Leu), arginine (Arg), or lysine (Lys). It was observed that all amino acid deficiencies induced CAR expression, and that Asp and Art induced even greater CAR expression than amino acid deficiencies 24 hours after treatment (Fig. 9A and B).

[0969] Furthermore, to assess the possibility of modifying the phenotype or functionality of CD3+ T lymphocytes, these cells were transduced with a lentiviral vector carrying a transgene encoding the transcription factors T-bet or Foxp3, which when induced in the context of an immune response, promote an effector or regulatory phenotype, respectively. In activated CD3+ cells transduced with the LV-2xAARE-GFP-p2a-Tbet vector (SEQ ID NO: 761), it was observed that treatments with arginase, asparaginase or artesunate all led to the induction of T-bet expression and that each of these three drugs was able to induce in these genetically modified T cells the expression of the cytokines IFNγ and TNFα, characteristics of effector cells (Fig. 10A and B).In activated CD3+ cells transduced with the LV-2xAARE-GFP-p2a-Foxp3 vector (SEQ ID NO: 762), it was observed that treatments with arginase, F asparaginase or artesunate all led to the induction of greater Foxp3 expression than in uninduced cells. In addition, it was observed that genetically modified CD3+ cells induced with each of these three drugs displayed a characteristic phenotype of regulatory T cells, characterized by the absence of CD127 expression (CD127') and a strong expression of CD25 (CD25. hi ) and Foxp3 (Foxp3 hi ) (Fig. 10C and D).

[0970] Ultimately, all of these data demonstrated the possibility of inducing the expression of a transgene whatever its nature (CAR, transcription factor, etc.) by the pharmacological induction of the 2XAARE promoter. YB TATA (SEQ ID NO: 1).

Claims

CLAIMS 1. Use of an animal eukaryotic cell for producing a lentiviral vector, said animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising: ■ a fusion protein comprising the fusion: - an inactive Cas endonuclease (dCas); and - a KRAB transcriptional repression domain or a methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L, or a fusion protein comprising the fusion: - a KRAB transcriptional repression domain; - an inactive Cas endonuclease (dCas); and - a methylation domain chosen from: DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L and MeCP2; and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA, dCas-DNMT3B- L / gRNA, KRAB-dCas-DNMT3 A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed, said lentiviral vector comprising a vector genome comprising a lentiviral genome into which an expression cassette comprising said promoter has been introduced upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

2. Use of an animal eukaryotic cell according to claim 1, said inactive Cas9 endonuclease being chosen from: ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, ' ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, ' ■ inactive Casl2b endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, ' ■ inactive CasX or Caslle endonucleases of Deltaproteobacteria and Planctomycetes ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

3. Use of an animal eukaryotic cell according to claim 1 or 2, wherein said animal eukaryotic cell belongs 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.

4. Use of an animal eukaryotic cell according to any one of claims 1 to 3, wherein said promoter is an inducible promoter chosen from: 2XAARE YB - TATA , the 2XAARE TK , the 4xSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA; or wherein said promoter is the tissue-specific promoter of miR223; or wherein said promoter is a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV.

5. Use of an animal eukaryotic cell according to any one of claims 1 to 4, wherein said 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 A subunit (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O and an enterotoxin.

6. Method for producing lentiviral vectors comprising a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter upstream of a toxic or suicide transgene, the 5' to 3' orientation of the transcription of said expression cassette being reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said method comprising at least the steps: a) co-transfection of an animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising: ■ a fusion protein comprising the fusion: - an inactive Cas endonuclease (dCas); and - a KRAB transcriptional repression domain (dCas-KRAB fusion) or a methylation domain selected from: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion) and DNMT3B-L (dCas-DNMT3B-L fusion), or a fusion protein comprising the fusion: - a KRAB transcriptional repression domain; - an inactive Cas endonuclease (dCas); and - a methylation domain chosen from: DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L and MeCP2; and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said promoter, said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas- DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA, dCas- DNMT3B-L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas- DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B- L / RNAg or dCas-KRAB-MeCP2 / RNAg being in particular stably expressed, with: ■ a plasmid comprising said vector genome and the means for expressing it; ■ a plasmid comprising an envelope protein and the means for expressing it; and ■ a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b) culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising said vector genome; and c) harvesting and purifying said lentiviral vectors.

7. Method for producing lentiviral vectors according to claim 6, said inactive Cas9 endonuclease being chosen from: ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, ' ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, ' ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, ' ■ inactive CasX or Caslle endonucleases of Deltaproteobacteria and Planctomycetes ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

8. Animal eukaryotic cell expressing at least one ribonucleoprotein complex comprising: ■ a fusion protein chosen from: - dCas-KRAB comprising the fusion of an inactive Cas endonuclease and a KRAB transcriptional repression domain; - dCas-DNMT3 A comprising the fusion of an inactive Cas endonuclease and a DNMT3 A methylation domain; - dCas-DNMT3B comprising the fusion of an inactive Cas endonuclease and a DNMT3B methylation domain; - dCas-DNMT3 AL comprising the fusion of an inactive Cas endonuclease and a DNMT3 AL methylation domain; and - dCas-DNMT3B-L comprising the fusion of an inactive Cas endonuclease and a DNMT3B-L methylation domain; - KRAB-dCas-DNMT3A-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3 AL methylation domain; - KRAB-dCas-DNMT3B-L comprising the fusion of a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3B-L methylation domain; and - dCas-KRAB-MeCP2 comprising the fusion of an inactive Cas endonuclease (dCas), a KRAB transcriptional repression domain and a MeCP2 methylation domain, and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of a promoter, said promoter being an inducible promoter chosen from: 2XAARE YB - TATA , the 2XAARE TK , the 4xSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB TATA and the miniCHACl YB TATA ; or the tissue-specific promoter of miR223; or a constitutive promoter selected from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV, said ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas- DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular stably expressed.

9. Animal eukaryotic cell according to claim 8, said inactive Cas9 endonuclease being chosen from: ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, ' ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, ' ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, ' ■ inactive CasX or Casl2e endonucleases from Deltaproteobacteria and Planctomycetes, ' ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans,' ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade, - ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms.

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