promoters induced by pharmacological molecules

Abstract

The present invention relates generally to the field of medicine. More particularly, it concerns the use of pharmacological molecules in the induction of downstream transgene expression from novel inducible cellular promoters.

Description

Technical Field

[0001] This invention generally relates to the pharmaceutical field. More specifically, it relates to eukaryotic cells expressing ribonucleoprotein complexes and their use in the production of lentiviral vectors. The invention also relates to methods for producing these toxic vectors. Furthermore, the invention relates to the use of pharmacological molecules in inducing downstream transgenic expression of novel inducible cell promoters. Background Technology

[0002] For over 25 years, lentiviral vectors based on the HIV-1 genome have been produced and used for efficient gene transfer into mammalian cells both in vitro and in vivo. The genomes and production methods of these vectors have been optimized to improve their efficacy and safety in human medicine. With the significant advancements in gene therapy, lentiviral vectors have been involved in numerous clinical trials, despite their limitations and side effects, particularly due to their tendency to preferentially integrate into the transcriptional regions of the genome of genetically modified cells.

[0003] Lentiviral vectors possess the ability to transduce proliferating or resting cells and integrate transgenes into their host genome. Therefore, their applications in cell engineering offer a wide range of therapeutic uses. One such use is to eliminate transduced cells, a strategy known as "suicide gene therapy," which is being considered for various medical scenarios (anti-tumor therapy, safeguarding transplanted cells, etc.). However, a search of literature databases reveals very few publications reporting the use of lentiviral vectors to transfer cytotoxic genes. This is because these applications face a significant technical hurdle: the production of lentiviral vectors carrying cytotoxic transgenes.

[0004] There are technical challenges in producing lentiviral vectors that express these cytotoxic transgenes because they are toxic to the cells in which the vectors are produced, thus significantly reducing the titer of the recombinant particles.

[0005] The production process of lentiviral vectors is based on the co-transfection of plasmids expressing the following substances in HEK 293T cells: (1) a lentiviral genome carrying the transgene, (2) envelope proteins, and (3) HIV-1 structural proteins and enzymes (gag, pol, and rev) necessary for the production of non-replicating recombinant lentiviral particles. In lentiviral vectors, the transgene is typically cloned under the control of an internal promoter. Therefore, during production, the transgene is expressed at high levels despite modifications to the lentiviral sequence 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, plasmids used to produce recombinant lentiviral vectors have a circular structure and contain at least one strong promoter that controls the transcription of the vector genome packaged into the recombinant HIV particle. Furthermore, recent studies have shown that plasmids also contain other cryptic promoters that can initiate transcription on either strand of the plasmid DNA (Muerdter, F. et al. Nat. Methods 15, 141–149 (2018); Lemp, NA, Hiraoka, K., Kasahara, N. & Logg, CR NucleicAcids Res. 40, 7280–7290 (2012)). Therefore, in the case of packaging cells, basal expression of cytotoxic transgenes significantly reduces the titer of the resulting vector (Maunder, HE et al. Nat. Commun. 8, 14834 (2017)). In some cases, production cell lines resistant to toxic genes can be used (Lange, MJ, Lyddon, TD & Johnson, MC Sci. Rep. 9, 8985 (2019); Li, Y. et al. CancerRes. 2002 May 1;62(9):2576-82), but this is not a universal solution. Summary of the Invention

[0006] Facing the significant challenge of readily producing lentiviral vectors carrying cytotoxic (or suicide) transgenes in effective quantities, the inventors have developed an innovative eukaryotic cell that expresses a ribonucleoprotein complex specifically designed to suppress the expression of cytotoxic transgenes in the production of lentiviral vectors. Therefore, a primary objective of this invention is to provide such eukaryotic cells for the pharmaceutical field and to propose their use in the production of lentiviral vectors carrying cytotoxic transgenes. Through this use, another objective of the invention is to propose a universal method for producing lentiviral vectors that allows for the acquisition of high titers of lentiviral vectors carrying direct cytotoxic transgenes. Detailed Implementation

[0007] In its most general aspect, the present invention relates to the use of animal eukaryotic cells in the production of lentiviral vectors, said animal eukaryotic cells expressing at least one ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0008] - Fusion proteins containing the following fusions:

[0009] - Inactivated Cas endonuclease (dCas); and

[0010] - KRAB transcriptional repressor domain (dCas-KRAB fusion) or methylated domains selected from the following: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion).

[0011] Alternatively, fusion proteins containing the following fusions:

[0012] - KRAB transcriptional repression domain;

[0013] - Inactivated Cas endonuclease (dCas); and

[0014] - Selected from the following methylation domains: 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 (dCas-KRAB-MeCP2 fusion); and

[0015] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0016] The 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, especially those stably expressed,

[0017] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0018] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0019] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0020] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0021] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the C-terminus of the inactivated Cas endonuclease (dCas); or

[0022] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the N-terminus of the inactivated Cas endonuclease (dCas); or

[0023] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0024] Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0025] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas); and

[0026] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0027] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is selected from: inducible promoters, tissue-specific promoters, and constitutive promoters. Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is an inducible promoter or a tissue-specific promoter.

[0028] "Animal eukaryotic cells expressing at least one ribonucleoprotein complex" means that the animal eukaryotic cells of the present invention can express a single ribonucleoprotein complex (dCas-KRAB / gRNA interference complex; or one of the methylated complexes dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA; or one of the bifunctional complexes KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, or dCas-KRAB-MeCP2 / gRNA), or can express multiple, particularly at least two, namely:

[0029] - Riboprotein interference complex (called CRISPRi) dCas-KRAB / gRNA; and

[0030] - Selected from the following ribonucleoprotein methylation complexes (referred to as CRISPRm): dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, and dCas-DNMT3B-L / gRNA.

[0031] Therefore, the CRISPR interference (CRISPRi) and CRISPR targeted methylation (CRISPRm) strategies according to the present invention are based on the fusion of an inactivated or “dead” CRISPR-Cas endonuclease (dCas) with a KRAB (Krüppel-associated box) transcriptional repression domain or a DNMT3A, DNMT3B, DNMT3A-L, or DNMT3B-L (DNA methyltransferase) methylation domain. dCas, with the aid of its guide RNA (gRNA), allows targeting of specific sequences. This targeting enables the KRAB domain to spatially block transcription, and the DNMT3A, DNMT3B, DNMT3A-L, or DNMT3B-L domains to block transcription through de novo methylation of the targeted DNA. During recombinant particle production, implementing the CRISPRi and / or CRISPRm strategies on the internal sequences of the lentiviral vector requires the co-expression of at least one CRISPRi and / or CRISPRm complex and one gRNA in the transfected animal eukaryotic cells according to the present invention. Alternatively, the present invention implements a bifunctional CRISPRi / m complex comprising an inactivated or “dead” CRISPR-Cas endonuclease (dCas) in transfected animal eukaryotic cells according to the present invention, a KRAB transcriptional repression domain, and a fusion of a DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L or MeCP2 methylation domain, and gRNA.

[0032] "gRNA (guide RNA)" refers to RNA that binds to an enzyme or protein complex. When this RNA pairs with a complementary RNA or DNA sequence, the enzyme or protein complex itself is located on the complementary RNA or DNA.

[0033] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, said animal eukaryotic cells expressing a ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0034] - Fusion proteins containing the following fusions:

[0035] - Inactivated Cas endonuclease (dCas); and

[0036] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0037] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0038] The dCas-KRAB / gRNA ribonucleoprotein complex, especially the stably expressed,

[0039] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0040] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0041] According to another embodiment, the present invention also relates to the use of animal eukaryotic cells as described above, said animal eukaryotic cells expressing a ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0042] - Fusion proteins containing the following fusions:

[0043] - Inactivated Cas endonuclease (dCas); and

[0044] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion); and

[0045] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0046] The ribonucleoprotein complexes dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA, especially those stably expressed, are mentioned.

[0047] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0048] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0049] Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, said animal eukaryotic cells expressing at least first and second ribonucleoprotein complexes, the first ribonucleoprotein complex comprising:

[0050] - Fusion proteins containing the following fusions:

[0051] - Inactivated Cas endonuclease (dCas); and

[0052] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0053] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0054] Furthermore, the second ribonucleoprotein complex comprises:

[0055] - Fusion proteins containing the following fusions:

[0056] - Inactivated Cas endonuclease (dCas); and

[0057] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion); and

[0058] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0059] The first and second ribonucleoprotein complexes are particularly stably expressed.

[0060] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0061] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0062] Advantageously, the present invention also relates to the use of animal eukaryotic cells as described above, said animal eukaryotic cells expressing a bifunctional ribonucleoprotein complex, said bifunctional ribonucleoprotein complex comprising:

[0063] - Fusion proteins containing the following fusions:

[0064] - KRAB transcriptional repression domain;

[0065] - Inactivated Cas endonuclease (dCas); and

[0066] - Selected from the following methylation domains: 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 (dCas-KRAB-MeCP2 fusion); and

[0067] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0068] The bifunctional ribonucleoprotein complexes KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, or dCas-KRAB-MeCP2 / gRNA, especially those stably expressed, are particularly important.

[0069] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0070] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0071] The “dCas-KRAB / gRNA ribonucleoprotein complex” refers to an interference system comprising a combination of a dCas-KRAB fusion protein and guide RNA (gRNA). This guide RNA specifically recognizes a 17-24 nucleotide sequence in the promoter sequence of the promoter present in the genome of the lentiviral vector to be produced, followed by a single or repeated “PAM” motif (Protospacer Adjacent Motif) sequence. These two elements work together, in reverse synergy with the expression cassette, unexpectedly enabling the production of lentiviral vector titers carrying toxic or suicide transgenes—something never before achieved. In this sense, the invention is truly revolutionary because it ultimately makes it possible to provide patients with the safe and effective cell therapies they need.

[0072] "dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA ribonucleoprotein complex" refers to a methylation system comprising a combination of a dCas-DNMT3A fusion protein, dCas-DNMT3B, dCas-DNMT3A-L, or dCas-DNMT3B-L fusion protein and a guide RNA (gRNA) that specifically recognizes a 17- to 24-base sequence in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced, followed by a single or repeated "PAM" motif (prototype spacer adjacent motif). These two elements work together, in reverse synergy with the expression cassette, unexpectedly enabling the production of lentiviral vector titers carrying toxic or suicide transgenes, something never before achieved. In this sense, the invention is truly revolutionary because it ultimately makes it possible to provide patients with the safe and effective cell therapies they need.

[0073] "KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA" refers to a bifunctional interference and methylation system comprising a combination of KRAB-dCas-DNMT3A fusion protein, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 with guide RNA (gRNA). The guide RNA specifically recognizes a 17- to 24-base sequence in the promoter sequence of the promoter present in the genome of the lentiviral vector to be produced, followed by a single or repeated "PAM" motif (prototype spacer adjacent motif). These three components work together, in a reverse synergistic manner with the expression cassette, to unexpectedly achieve the production of lentiviral vector titers carrying toxic or suicide transgenes—something never before accomplished. In this sense, the invention is truly revolutionary because it ultimately enables the provision of safe and effective cell therapies to patients.

[0074] Advantageously, the present invention utilizes interference and / or methylation systems by combining them to obtain tools for producing lentiviral vectors carrying highly toxic or suicide transgenes.

[0075] "dCas-KRAB fusion protein" refers to the combination of two amino acid sequences from different proteins or fragments thereof to create an artificial functional protein. In this invention, the two fusion components are, as described above, an inactivated Cas endonuclease (dCas) and a KRAB transcriptional repression domain, and a nuclear localization signal (NLS) peptide is added to ensure its nuclear localization (e.g., encoded by the nucleic acid of sequence SEQ ID NO: 324).

[0076] "dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L, or dCas-DNMT3B-L fusion protein" refers to the combination of two amino acid sequences from different proteins or fragments thereof to create an artificial functional protein. In this invention, the two fusion components are, as described above, an inactivated Cas endonuclease (dCas) and DNMT3A, DNMT3B, DNMT3A-L, or DNMT3B-L methylation domains. A nuclear localization signal (NLS) peptide is also added to ensure its nuclear localization (e.g., encoded by the nucleic acid of sequence SEQ ID NO: 324).

[0077] "KRAB-dCas-DNMT3A fusion protein, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L, or dCas-KRAB-MeCP2" refers to the combination of three amino acid sequences from different proteins or fragments thereof to create an artificial functional protein. In this invention, the three fusion components are, as described above, a KRAB transcriptional repressor domain, an inactivated Cas endonuclease (dCas), and DNMT3A, DNMT3B, DNMT3A-L, or DNMT3B-L methylation domains. A nuclear localization signal (NLS) peptide is also added to ensure its nuclear localization (e.g., encoded by the nucleic acid of sequence SEQ ID NO: 324).

[0078] "Inactivated Cas endonuclease (dCas)" or the equivalent term "dead" Cas endonuclease (dCas) refers to an endonuclease whose single-stranded or double-stranded DNA cleavage activity is inactive. To date, several Cas endonucleases from different hosts (Cas9, Cas12a, Cas12b, CasX or Cas12e, Cas12f or Cas14, Cas12j or CasΦ) have been identified and characterized, and even modified (e.g., to improve their function), which can be implemented by the present invention after inactivation. Therefore, according to another embodiment, the present invention relates to the use in animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease (dCas) is selected from:

[0079] - Inactivated Cas9 endonucleases from *Streptococcus pyogenes*, *Staphylococcus aureus*, *Corynebacterium diphtheriae*, *Neisseria meningitidis*, *Streptococcus canis*, *Streptococcus macacae*, *F. tularensis*, *Acidaminococcus*, *Campylobacter jejuni*, *Streptococcus pneumoniae*, and *Streptococcus thermophilus*;

[0080] - Inactivated Cas12a endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp.;

[0081] - Inactivated Cas12b endonuclease from Alicyclobacillus kakegawensis and Bacillus hisashii;

[0082] - Inactivated CasX or Cas12e endonucleases from Deltaproteobacteria and Planctomycetes;

[0083] - Inactivated type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans;

[0084] - Inactivated Cas12j or CasΦ endonucleases from the Biggiephage clade;

[0085] - Inactivated orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms; and

[0086] - Inactivated mutants or variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms.

[0087] "Orthologs" refer to similar Cas endonucleases found in two or more different species. They may have become inactive due to evolution, or, if not, can be mutated to render them nonfunctional for use in the purposes of this invention.

[0088] A “mutant” refers to a Cas endonuclease in which one or more mutations (including the deletion, substitution, and / or addition of one or more amino acids) have been introduced. Therefore, the term “inactivating mutant” refers to a Cas protein that has been artificially modified (e.g., through genetic engineering) to inactivate its DNA-cutting activity.

[0089] "Inactive variants" refer to Cas endonucleases that have been naturally modified through evolution and do not exhibit DNA cleavage activity.

[0090] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9). This relates to ribonucleoprotein complexes 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 present invention relates to the use of animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated 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 inactivated Cas9 endonuclease (dCas9) whose amino acid sequence has at least 80% identity with sequence SEQ ID NO: 323. In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322, or whose nucleic acid encodes an inactivated Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to sequence SEQ ID NO: 323.

[0091] The “KRAB transcriptional repression domain” refers to the KRAB (Kruppel-associated repressor cassette) domain of human zinc finger proteins Kox1 (or zinc finger protein 10) or Zim3, which causes specific inhibition of RNA polymerases II and III. In this way, the KRAB domain inhibits the expression of genes present on the DNA to which it is recruited. Specifically, the present invention relates to the use in animal eukaryotic cells as described above, wherein the 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 SEQ ID NO: 751, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 321 or SEQ ID NO: 752. Specifically, the present invention also relates to the use in animal eukaryotic cells as described above, wherein the KRAB transcriptional repression domain is encoded by a nucleic acid of sequence SEQ ID NO: 320 or SEQ ID NO: 751, or its amino acid sequence corresponding to the sequence SEQ ID NO: 321 or SEQ ID NO: 752.

[0092] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. Specifically, the present invention relates to the use of animal eukaryotic cells as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein, the nucleic acid sequence of which has at least 80% identity with the nucleic acid of sequence SEQ ID NO:325 or SEQ ID NO:753, or the nucleic acid of which encodes a dCas9-KRAB fusion protein, wherein the amino acid sequence of the dCas9-KRAB fusion protein has at least 80% identity with the sequence SEQ ID NO:326 or SEQ ID NO:754. In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the 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 SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0093] "The methylation domains selected from the following: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2" refer to the functional domains of DNA methyltransferases that catalyze covalent modification of DNA by transferring cytosine to 5-methylcytosine via S-adenosyl-1-methionine (SAM). In this way, the DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 domains inhibit the expression of genes present on the DNA to which they are recruited. Advantageously, the present invention implements the DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L domains. Therefore, particularly, the present invention relates to the use of animal eukaryotic cells as described above, wherein the methylation domains:

[0094] - DNMT3A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 354;

[0095] - DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 366;

[0096] - DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 358; or

[0097] - DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 368.

[0098] In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the methylated domain:

[0099] - DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence corresponds to sequence SEQ ID NO: 354;

[0100] - DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence corresponds to sequence SEQ ID NO: 366;

[0101] - DNMT3A-L is encoded by the nucleic acid sequence SEQ ID NO: 357, or its amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0102] - DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence corresponds to sequence SEQ ID NO: 368.

[0103] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the 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 its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 370. In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the MeCP2 methylation domain is encoded by a nucleic acid of sequence SEQ ID NO: 369, or its amino acid sequence corresponding to sequence SEQ ID NO: 370.

[0104] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L, or dCasDNMT3B-L fusion proteins are respectively dCas9-DNMT3A, dCas9-DNMT3B, dCas9-DNMT3A-L, or dCas9-DNMT3B-L fusion proteins. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0105] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 360;

[0106] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 372;

[0107] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 364; or

[0108] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 373, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 374.

[0109] In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein:

[0110] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360;

[0111] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372;

[0112] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 363, or the nucleic acid of which encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 364; or

[0113] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372.

[0114] Interestingly, although the DNMT3L enzyme is catalytically inactive, it stimulates the activity of DNMT3A and DNMT3B, thereby silencing the methylation promoter. Therefore, the DNMT3A-L and DNMT3B-L methylation domains are formed by the binding of DNMT3A or DNMT3B to DNMT3L, respectively. Nevertheless, the addition of the dCas-DNMT3L / gRNA ribonucleoprotein complex to the animal eukaryotic cells of the present invention is conceivable. According to another embodiment, the present invention relates to the use of the animal eukaryotic cells described above, wherein the animal eukaryotic cells also express another ribonucleoprotein complex comprising:

[0115] - Fusion proteins containing the following fusions:

[0116] - Inactivated Cas endonuclease (dCas); and

[0117] - DNMT3L methylation-promoting domain (dCas-DNMT3L fusion); and

[0118] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0119] The other ribonucleoprotein complex dCas-DNMT3L / gRNA is particularly stably expressed.

[0120] Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, said animal eukaryotic cells expressing at least first and second ribonucleoprotein complexes, the first ribonucleoprotein complex comprising:

[0121] - Fusion proteins containing the following fusions:

[0122] - Inactivated Cas endonuclease (dCas); and

[0123] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion) and DNMT3B; and

[0124] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0125] Furthermore, the second ribonucleoprotein complex comprises:

[0126] - Fusion proteins containing the following fusions:

[0127] - Inactivated Cas endonuclease (dCas); and

[0128] - DNMT3L methylation-promoting domain (dCas-DNMT3L fusion); and

[0129] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0130] The first and second ribonucleoprotein complexes are particularly stably expressed.

[0131] The lentiviral vector contains a vector genome, which in turn contains a lentiviral genome, in which an expression cassette containing the promoter (the promoter sequence of the promoter) has been introduced upstream of a toxic or suicide transgene.

[0132] The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome. In particular, the invention also relates to the use of animal eukaryotic cells as described above, which further express another ribonucleoprotein complex comprising:

[0133] - Fusion proteins containing the following fusions:

[0134] - Inactivated Cas endonuclease (dCas); and

[0135] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0136] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0137] The dCas-KRAB / gRNA ribonucleoprotein complex is particularly stably expressed.

[0138] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0139] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355, or its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 356. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid of sequence SEQ ID NO: 355, or its amino acid sequence corresponding to sequence SEQ ID NO: 356.

[0140] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein corresponds to the sequence SEQ ID NO: 362.

[0141] As described above, the present invention also implements a bifunctional CRISPRi / m complex comprising:

[0142] - Inactivated or "dead" CRISPR-Cas endonucleases (dCas), KRAB transcriptional repression domains, and fusion with DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 methylation domains; and

[0143] - Guide RNA (gRNA) specifically recognizes the promoter sequence of the promoter (the promoter of the expression cassette).

[0144] Specifically, the bifunctional complex (advantageously expressed in a stable manner) is selected from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, and dCas-KRAB-MeCP2 / gRNA, and advantageously from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA, and dCas9-KRAB-MeCP2 / gRNA. The invention also relates to the use in animal eukaryotic cells as described above, wherein:

[0145] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 376 or SEQ ID NO: 382;

[0146] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0147] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0148] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0149] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382;

[0150] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0151] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0152] "% identity" refers to the percentage determined by a direct comparison of two oligonucleotide sequences (nucleic acid sequences), which is achieved by determining the number of identical nucleotides between the two sequences, dividing that number by the number of nucleotides in the longer of the two sequences, and multiplying the result by 100. Therefore, "having at least 80% identity" means that the aforementioned identity percentage is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In this regard, it should be noted that this definition applies to all embodiments of the invention, including when it involves a direct comparison of two polypeptide sequences (amino acid sequences).

[0153] "The ribonucleoprotein complexes 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, especially those stably expressed," refers to the ribonucleoprotein complex dCas-KRAB / gRNA. The coding sequences of RAB / 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, and the tools for expressing them, can be integrated into the animal eukaryotic cell genome in a targeted or non-targeted manner as described above. In this way, their expression is no longer transient and plasmid-secured, but permanent and genome-secured. Therefore, in particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the ribonucleoprotein complexes 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 are stably expressed by random integration or targeted integration into the genome of the animal eukaryotic cells.Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein the ribonucleoprotein complexes 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 are stably expressed by targeted integration into the genome of the animal eukaryotic cells.

[0154] Animal eukaryotic cells used for producing lentiviral vectors may be selected from various cell lines known to those skilled in the art. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein said animal eukaryotic cells belong to a cell line selected from: HEK 293T, HEK 293, CHO, GS-CHO, Free-style 293-F cells (FS293), virus-producing cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8, and PS46.2. Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein said animal eukaryotic cells belong to the HEK 293T cell line.

[0155] "Lentiviral vector" refers to recombinant lentiviral particles obtained using the tools provided in this invention. This comprises the capsidated and / or enveloped vector genome. It is efficient in entering cells, is non-replicating, and causes the vector genome to integrate (targeted or untargeted) into the genome of the infected cell, or to exist as a free form that does not integrate into the cell genome.

[0156] "Vector genome" refers to a nucleic acid sequence that contains both of the following:

[0157] - Nucleic acid containing the genetic information (=lentiviral genome) necessary for the production of a lentiviral vector (also called a (lenti)viral particle), said lentiviral vector or said (lentiviral particle) containing said capsidated and enveloped vector genome, which is capable of efficiently introducing itself into cells and is non-replicating; and

[0158] - A nucleic acid containing the genetic information of an expression cassette, the expression cassette comprising a promoter selected from inducible promoters, tissue-specific promoters, and constitutive promoters, located upstream of a toxic or suicide transgene, the nucleic acid encoding the toxic or suicide transgene possibly containing a nucleic acid sequence at its 3' end that allows transcription of a polyadenylate (polyA) tail, the expression cassette being introduced into the lentiviral genome such that its transcription is reversed relative to the transcription of the lentiviral genome.

[0159] "Lentiviral genome," as described above, refers to the nucleic acid carrying the genetic information necessary for the production of a lentiviral vector (also known as a (lenti)viral particle), which contains the capsidated and enveloped vector genome. This lentiviral vector is also capable of efficient cell entry, is non-replicating, and causes the vector genome to integrate (targeted or untargeted) into the genome of the infected cell. In some cases, the lentiviral particle will contain a mutant integrase (D64V), which will result in the formation of a non-integrating provirus (reverse-transcribed vector DNA) in the genome of the transduced cell. Therefore, the vector genome will exist in the cell nucleus in free form. Typically, the reverse-transcribed structure of the lentiviral genome includes: the cis sequence of the HIV lentiviral genome, namely two long terminal repeats (LTRs) flanking the ends of the vector genome, with U3, R, and U5 regions, but where the U3 region is mutated (ΔU3), lacking its enhancer sequence and therefore having no promoter activity. Lentiviral vectors with ΔU3 in the LTR are called "self-inactivated" because they cannot be transcribed by wild-type HIV. Following the 5' LTR of the genome are the "psi" sequence for capsidation of the vector RNA genome, the Rev response element (RRE) sequence for output of the vector RNA genome, the central polypurine region (cppt) and central termination sequence (cts) for forming the central DNA triplet during reverse transcription, and the 3' LTR. In addition to the viral cis sequence, these vectors also contain an expression cassette located between the two LTRs. As described above, an "expression cassette" refers to a nucleic acid upstream of a virulent or suicide transgene containing a promoter selected from inducible promoters, tissue-specific promoters, and constitutive promoters. The nucleic acid encoding the virulent or suicide transgene may contain a polyA-tailed nucleic acid sequence at its 3' end that allows transcription. This expression cassette has been introduced into the lentiviral genome such that its transcription is reversed relative to the transcription of the lentiviral genome.

[0160] "The 5' to 3' transcription direction of the expression cassette is reversed relative to the 5' to 3' transcription direction of the lentiviral genome" means that, for example... Figure 3As shown in (pLVi), the transcription of the expression cassette is reversed relative to the transcription of the lentiviral genome. It should be noted that this reverse-assembled construct (selected from inducible promoters, tissue-specific promoters, and constitutive promoters, plus a toxic or suicide transgene) combined with the specific combination of the present invention with animal eukaryotic cells expressing the dCas-KRAB / gRNA ribonucleoprotein complex specific to the selected promoter is a novel and efficient tool that unexpectedly allows for the production of lentiviral vectors encoding toxic or suicide transgenes at previously unattainable titers.

[0161] "Inducible promoters" refer to nucleic acids located upstream of a gene (or transgene) that control its expression (particularly through regulation of its transcription). In this invention, such promoters are described as inducible because the expression of the gene (or transgene) they control is not constitutive but is induced only upon activation in response to a proper stimulus. In this invention, these are advantageously 2xAARE. YB_TATA (SEQ ID NO: 1), 2xAARE TK (SEQ ID NO: 2), 4xSRE YB_TATA (SEQ ID NO:3), 9xHRE YB_TATA (SEQ ID NO: 4), GRP78 YB_TATA (SEQ ID NO: 5) or miniCHAC1 YB_TATA (SEQ ID NO: 7). 2xAARE YB_TATA 2xAARE TK and miniCHAC1 YB_TATA The promoter is activated by inducers that induce ATF4 transcription factor expression. GRP78 YB_TATA The promoter is activated by the inducer of the transcription factor ATF6. 4xSRE YB_TATA The promoter is induced by steroid hormones, particularly dexamethasone. 9xHRE YB_TATA The promoter is hypoxia-induced. Interestingly, the inducible promoter mentioned above depends on the expression of cellular transcription factors that are hardly expressed or not expressed at all under vector production conditions in HEK 293T cells.

[0162] A “tissue-specific promoter” is a nucleic acid located upstream of a gene (or transgene) that controls its expression (particularly by regulating its transcription). In this invention, such a promoter is described as tissue-specific because the expression of the gene (or transgene) it controls is ineffective in the cellular environment producing the recombinant vector particles, but is activated in another cellular environment with a different phenotype that is sensitive to this tissue-specific promoter. In response to this other environment, the tissue-specific promoter will be activated. For example, in this invention, this could be the miR223 promoter (SEQ ID NO: 6), which is specifically induced in myeloid cells, while HEK-293T cells are not myeloid cells.

[0163] A “constitutive promoter” is a nucleic acid located upstream of a gene (or transgene) that controls its expression (particularly by regulating its transcription). In this invention, such a promoter is described as constitutive because the expression of the gene (or transgene) it controls is constant regardless of its environment, i.e., whether it is in the cellular environment that produces recombinant vector particles or in another cellular environment with a different phenotype. In this invention, the latter is advantageously SV40 (or pSV40; SEQ ID NO: 343), CMV (or pCMV; SEQ ID NO: 344), UbC (or pUbC; SEQ ID NO: 345), EF1-α core type (or pEF1-α core type; SEQ ID NO: 346), EF1-α long type (or pEF1-α long type; 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).

[0164] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is an inducible promoter selected from the following: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ;or

[0165] The promoter mentioned is the tissue-specific promoter miR223; or

[0166] The promoters mentioned therein are selected from the following compositional promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV and SFFV.

[0167] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is selected from the following inducible or tissue-specific promoters:

[0168] - 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1;

[0169] - 2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2;

[0170] - 4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0171] - 9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0172] - GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0173] - miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 6; and

[0174] - miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7.

[0175] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is selected from the following inducible or tissue-specific promoters:

[0176] - 2xAARE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0177] - 2xAARE TK Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0178] - 4xSRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0179] - 9xHRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0180] - GRP78 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 5;

[0181] - miR223 promoter, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0182] - miniCHAC1 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0183] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is a constitutive promoter selected from the following:

[0184] - SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 343;

[0185] - CMV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 344;

[0186] - UbC, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 345;

[0187] - EF1-α core type, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 346;

[0188] - EF1-α long type, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 347;

[0189] - hPGK, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 348;

[0190] - CAGG, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 349;

[0191] - RSV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 350; and

[0192] - SFFV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 351.

[0193] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the promoter is a constitutive promoter selected from the following:

[0194] - SV40, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0195] - CMV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0196] - UbC, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0197] - EF1-α core type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 346;

[0198] - EF1-α long type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 347;

[0199] - hPGK, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348;

[0200] - CAGG, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 349;

[0201] - RSV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0202] - SFFV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0203] "Toxic or suicide transgenes" refer to nucleic acids located downstream of the inducible or tissue-specific promoter and encoding toxic, cytotoxic, or suicide proteins in genetically modified cells. In other words, once the inducible or tissue-specific promoter controlling the expression of the toxic or suicide transgene is activated, the product of transcription and translation of this nucleic acid (i.e., the toxic or suicide protein) only causes (leads to) cell death in cells expressing that protein in response to appropriate stimuli or cellular conditions. In this invention, the toxic or suicide transgenes may be selected from genes encoding the following: inducible Caspase-9 (iC9), Bax (S184 del), Noxa (wild-type protein or one of its mutants, particularly the S13A mutant), Gasdermin B (N-ter), constitutively active caspase-3 protein (V266E), the M2 ion channel of influenza virus (H37A), diphtheria toxin subunit A (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (particularly streptococcal hemolysin O of Streptococcus pyogenes), and enterotoxins (particularly enterotoxins of Clostridium perfringens). These are non-secreting toxic or suicide proteins, meaning that their expression is not detected in the extracellular environment after activation of the inducible or tissue-specific promoter upstream of the nucleic acid encoding them, thus leading only to cell death in cells expressing them.

[0204] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the toxic or suicide transgene is selected from: inducible caspase-9 protein (iC9), Bax protein (S184 del), Noxa protein, Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 ion channel (H37A), diphtheria toxin subunit A (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens). In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the toxic or suicide transgene is selected from: inducible Caspase-9 protein (iC9), Bax protein (S184 del), Noxa protein, Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), the M2 ion channel of influenza virus (H37A), diphtheria toxin subunit A (DTA), and attenuated aDTA mutant (G128D).

[0205] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the toxic or suicide transgenic cells are selected from:

[0206] - An inducible Caspase-9 protein (iC9) whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes an inducible Caspase-9 protein (iC9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 9;

[0207] - Bax(S184 del) protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 10, or the nucleic acid of which encodes Bax(S184 del) protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 11;

[0208] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes an M2 (H37A) ion channel of an influenza virus, wherein the amino acid sequence of the M2 (H37A) ion channel of the influenza virus has at least 80% identity with the sequence SEQ ID NO: 13;

[0209] - A constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence that is at least 80% identical 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 is at least 80% identical to the sequence SEQ ID NO: 15;

[0210] - Diphtheria toxin subunit A (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 diphtheria toxin subunit A (DTA), the amino acid sequence of said diphtheria toxin subunit A (DTA) having at least 80% identity with 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 aDTA mutant (G128D), the amino acid sequence of said aDTA mutant (G128D) having at least 80% identity with sequence SEQ ID NO: 19.

[0211] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 21;

[0212] - Noxa protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 23;

[0213] - Streptococcal hemolysin 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 streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O has at least 80% identity with the sequence SEQ ID NO: 756; and

[0214] - Enterotoxin whose nucleic acid has a sequence that is at least 80% identical to 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.

[0215] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the toxic or suicide transgenic cells are selected from:

[0216] - Inducible Caspase-9 protein (iC9), whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes inducible Caspase-9 (iC9), the amino acid sequence of said inducible Caspase-9 (iC9) corresponding to sequence SEQ ID NO: 9;

[0217] - Bax(S184 del) protein, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10, or whose nucleic acid encodes Bax(S184 del) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 11;

[0218] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes the M2 (H37A) ion channel of an influenza virus, the amino acid sequence of the M2 (H37A) ion channel of the influenza virus corresponding to the sequence SEQ ID NO: 13;

[0219] - 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), the amino acid sequence of which corresponds to the sequence SEQ ID NO: 15;

[0220] - Diphtheria toxin subunit A (DTA), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 16, or whose nucleic acid encodes diphtheria toxin subunit A (DTA), the amino acid sequence of which corresponds to sequence SEQ ID NO: 17; and an attenuated mutant aDTA (G128D), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 18, or whose nucleic acid encodes an attenuated aDTA (G128D) mutant, the amino acid sequence of which corresponds to sequence SEQ ID NO: 19;

[0221] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 21;

[0222] - Noxa protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 23;

[0223] - Streptococcal hemolysin O, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 755, or whose nucleic acid encodes streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O corresponds to the sequence SEQ ID NO: 756; and

[0224] - Enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 757, or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0225] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the nucleic acid encoding the toxic or suicide transgene contains a polyA sequence (e.g., SEQ ID NO: 24) at its 3' end. Given that the 5' to 3' orientation of transcription of the expression cassette is reversed relative to the 5' to 3' orientation of transcription of the lentiviral genome, the addition of this polyA sequence (e.g., SEQ ID NO: 24) to the 3' end of the nucleic acid encoding the toxic or suicide transgene after induction of the inducible promoter (and only after induction) promotes the transcription and stability of the mRNA, which allows for the translation of the toxic or suicide transgene.

[0226] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0227] - The promoter is:

[0228] - Selected from the following inductive promoters: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ;

[0229] - The tissue-specific promoter of miR223; or

[0230] - Selected from the following constitutive promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV, and SFFV.

[0231] and

[0232] - The toxic or suicide transgenes are selected from: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein (wild-type protein or one of its mutants, especially the S13A mutant), Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0233] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the expression cassette is selected from sequences having at least 80% identity with sequences SEQ ID NO: 25 to SEQ ID NO: 80 and SEQ ID NO: 391 to SEQ ID NO: 462. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the expression cassette is selected from sequences SEQ ID NO: 25 to SEQ ID NO: 80 and SEQ ID NO: 391 to SEQ ID NO: 462. In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the expression cassette is the sequence SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO: 53. SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66. SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO: 391、SEQ ID NO: 392、SEQ ID NO: 393、SEQ ID NO: 394、SEQ ID NO:395、SEQ ID NO: 396、SEQ ID NO: 397、SEQ ID NO: 398、SEQ ID NO: 399、SEQ ID NO:400、SEQ ID NO: 401、SEQ ID NO: 402、SEQ ID NO: 403、SEQ ID NO: 404、SEQ ID NO:405、SEQ ID NO: 406、SEQ ID NO: 407、SEQ ID NO: 408、SEQ ID NO: 409、SEQ ID NO:410、SEQ ID NO: 411、SEQ ID NO: 412、SEQ ID NO: 413、SEQ ID NO: 414、SEQ ID NO:415、SEQ ID NO: 416、SEQ ID NO: 417、SEQ ID NO: 418、SEQ ID NO: 419、SEQ ID NO:420、SEQ ID NO: 421、SEQ ID NO: 422、SEQ ID NO: 423、SEQ ID NO: 424、SEQ ID NO:425、SEQ ID NO: 426、SEQ ID NO: 427、SEQ ID NO: 428、SEQ ID NO: 429、SEQ ID NO:430、SEQ ID NO: 431、SEQ ID NO: 432、SEQ ID NO: 433、SEQ ID NO: 434、SEQ ID NO:435、SEQ ID NO: 436、SEQ ID NO: 437、SEQ ID NO: 438、SEQ ID NO: 439、SEQ ID NO:440、SEQ ID NO: 441、SEQ ID NO: 442、SEQ ID NO: 443、SEQ ID NO: 444、SEQ ID NO:445、SEQ ID NO: 446、SEQ ID NO: 447、SEQ ID NO: 448、SEQ ID NO: 449、SEQ ID NO:450、SEQ ID NO: 451、SEQ ID NO: 452、SEQ ID NO: 453、SEQ ID NO: 454、SEQ ID NO:455、SEQ ID NO: 456、SEQ IDSEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461 or SEQ ID NO: 462 (see Table 1).

[0234]

[0235] Table 1. List of Expression Boxes

[0236] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is a self-inactivating lentivirus (SIN) or a respiratory syncytial virus (RSV).

[0237] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is a self-inactivating lentivirus (SIN). Specifically, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the 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. Specifically, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the 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 the expression cassette into the self-inactivating lentivirus (SIN) of sequence SEQ ID NO: 81 can be performed at or at nucleotides 2,229 and 2,230 of SEQ ID NO: 81. That is, in this case, the expression cassette is located between sequences SEQ ID NO: 82 and SEQ ID NO: 83.

[0238] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is respiratory syncytial virus (RSV). Specifically, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is 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. Specifically, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is 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 the expression cassette into the respiratory syncytial virus (RSV) of sequence SEQ ID NO: 84 can be performed at or at nucleotides 1,737 and 1,738 of SEQ ID NO: 84. That is, in this case, the expression cassette is located between sequences SEQ ID NO: 85 and SEQ ID NO: 86.

[0239] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the lentiviral genome into which the expression cassette has been introduced is:

[0240] - Self-inactivated lentivirus (SIN) whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 81; or

[0241] - Respiratory syncytial virus (RSV) whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 84;

[0242] And wherein, the expression box includes:

[0243] - Selected from the following inductive promoters: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ;

[0244] Or the tissue-specific promoter of miR223;

[0245] Alternatively, it can be selected from the following constitutive promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV, and SFFV.

[0246] as well as

[0247] - Selected from the following toxic or suicide transgenic proteins: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein (wild-type protein or one of its mutants, especially the S13A mutant), Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0248] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 87 to SEQ ID NO: 198 and SEQ ID NO: 463 to SEQ ID NO: 606. In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein the vector genome is selected from sequences SEQ ID NO: 87 to SEQ ID NO: 198 and SEQ ID NO: 463 to SEQ ID NO: 606. In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the vector genome is the sequence SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113. SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO:138、SEQ ID NO: 139、SEQ ID NO: 140、SEQ ID NO: 141、SEQ ID NO: 142、SEQ IDNO: 143、SEQ ID NO: 144、SEQ ID NO: 145、SEQ ID NO: 146、SEQ ID NO: 147、SEQ IDNO: 148、SEQ ID NO: 149、SEQ ID NO: 150、SEQ ID NO: 151、SEQ ID NO: 152、SEQ IDNO: 153、SEQ ID NO: 154、SEQ ID NO: 155、SEQ ID NO: 156、SEQ ID NO: 157、SEQ IDNO: 158、SEQ ID NO: 159、SEQ ID NO: 160、SEQ ID NO: 161、SEQ ID NO: 162、SEQ IDNO: 163、SEQ ID NO: 164、SEQ ID NO: 165、SEQ ID NO: 166、SEQ ID NO: 167、SEQ IDNO: 168、SEQ ID NO: 169、SEQ ID NO: 170、SEQ ID NO: 171、SEQ ID NO: 172、SEQ IDNO: 173、SEQ ID NO: 174、SEQ ID NO: 175、SEQ ID NO: 176、SEQ ID NO: 177、SEQ IDNO: 178、SEQ ID NO: 179、SEQ ID NO: 180、SEQ ID NO: 181、SEQ ID NO: 182、SEQ IDNO: 183、SEQ ID NO: 184、SEQ ID NO: 185、SEQ ID NO: 186、SEQ ID NO: 187、SEQ IDNO: 188、SEQ ID NO: 189、SEQ ID NO: 190、SEQ ID NO: 191、SEQ ID NO: 192、SEQ IDNO: 193、SEQ ID NO: 194、SEQ ID NO: 195、SEQ ID NO: 196、SEQ ID NO: 197、SEQ IDNO: 198、SEQ ID NO: 463、SEQ ID NO: 464、SEQ ID NO: 465、SEQ ID NO: 466、SEQ IDNO: 467、SEQ ID NO: 468、SEQ ID NO: 469、SEQID NO: 470、SEQ ID NO: 471、SEQ IDNO: 472、SEQ ID NO: 473、SEQ ID NO: 474、SEQ ID NO: 475、SEQ ID NO: 476、SEQ IDNO: 477、SEQ ID NO: 478、SEQ ID NO: 479、SEQ ID NO: 480、SEQ ID NO: 481、SEQ IDNO: 482、SEQ ID NO: 483、SEQ ID NO: 484、SEQ ID NO: 485、SEQ ID NO: 486、SEQ IDNO: 487、SEQ ID NO: 488、SEQ ID NO: 489、SEQ ID NO: 490、SEQ ID NO: 491、SEQ IDNO: 492、SEQ ID NO: 493、SEQ ID NO: 494、SEQ ID NO: 495、SEQ ID NO: 496、SEQ IDNO: 497、SEQ ID NO: 498、SEQ ID NO: 499、SEQ ID NO: 500、SEQ ID NO: 501、SEQ IDNO: 502、SEQ ID NO: 503、SEQ ID NO: 504、SEQ ID NO: 505、SEQ ID NO: 506、SEQ IDNO: 507、SEQ ID NO: 508、SEQ ID NO: 509、SEQ ID NO: 510、SEQ ID NO: 511、SEQ IDNO: 512、SEQ ID NO: 513、SEQ ID NO: 514、SEQ ID NO: 515、SEQ ID NO: 516、SEQ IDNO: 517、SEQ ID NO: 518、SEQ ID NO: 519、SEQ ID NO: 520、SEQ ID NO: 521、SEQ IDNO: 522、SEQ ID NO: 523、SEQ ID NO: 524、SEQ ID NO: 525、SEQ ID NO: 526、SEQ IDNO: 527、SEQ ID NO: 528、SEQ ID NO: 529、SEQ ID NO: 530、SEQ ID NO: 531、SEQ IDNO: 532、SEQ ID NO: 533、SEQ ID NO: 534、SEQ ID NO: 535、SEQ ID NO: 536、SEQ IDNO:537、SEQ ID NO: 538、SEQ ID NO: 539、SEQ ID NO: 540、SEQ ID NO: 541、SEQ IDNO: 542、SEQ ID NO: 543、SEQ ID NO: 544、SEQ ID NO: 545、SEQ ID NO: 546、SEQ IDNO: 547、SEQ ID NO: 548、SEQ ID NO: 549、SEQ ID NO: 550、SEQ ID NO: 551、SEQ IDNO: 552、SEQ ID NO: 553、SEQ ID NO: 554、SEQ ID NO: 555、SEQ ID NO: 556、SEQ IDNO: 557、SEQ ID NO: 558、SEQ ID NO: 559、SEQ ID NO: 560、SEQ ID NO: 561、SEQ IDNO: 562、SEQ ID NO: 563、SEQ ID NO: 564、SEQ ID NO: 565、SEQ ID NO: 566、SEQ IDNO: 567、SEQ ID NO: 568、SEQ ID NO: 569、SEQ ID NO: 570、SEQ ID NO: 571、SEQ IDNO: 572、SEQ ID NO: 573、SEQ ID NO: 574、SEQ ID NO: 575、SEQ ID NO: 576、SEQ IDNO: 577、SEQ ID NO: 578、SEQ ID NO: 579、SEQ ID NO: 580、SEQ ID NO: 581、SEQ IDNO: 582、SEQ ID NO: 583、SEQ ID NO: 584、SEQ ID NO: 585、SEQ ID NO: 586、SEQ IDNO: 587、SEQ ID NO: 588、SEQ ID NO: 589、SEQ ID NO: 590、SEQ ID NO: 591、SEQ IDNO: 592、SEQ ID NO: 593、SEQ ID NO: 594、SEQ ID NO: 595、SEQ ID NO: 596、SEQ IDNO: 597、SEQ ID NO: 598、SEQ ID NO: 599、SEQ ID NO: 600、SEQ ID NO: 601、SEQ IDNO: 602、SEQ ID NO: 603、SEQ ID NO: 604、SEQID NO: 605 or SEQ ID NO: 606 (see Table 2).

[0249]

[0250] Table 2. List of vector genomes

[0251] It should also be noted that, since the transcription direction of the expression cassettes described above is reversed relative to the vector genome described above, all the sequences provided that are associated with the expression cassettes described above are located on the complementary strand of the vector genome described above. This paper only provides the meaningful sequences (5'→3') of the vector genome.

[0252] In this invention, the animal eukaryotic cells used to produce the lentiviral vector express:

[0253] - dCas-KRAB / gRNA interferes with the ribonucleoprotein complex; and / or

[0254] - Selected from the following methylated ribonucleoprotein complexes: dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, and dCas-DNMT3B-L / gRNA, and optionally dCas-DNMT3L / gRNA methylated ribonucleoprotein complex; and / or

[0255] - Selected from the following bifunctional ribonucleoprotein complexes for interference and methylation: KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, and dCas-KRAB-MeCP2 / gRNA.

[0256] The dCas-KRAB component, as defined above, inhibits the expression of genes present on the recruited DNA through steric hindrance (without DNA cleavage), and in this case, inhibits the expression of toxic or suicide transgenes. The dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L, and dCas-DNMT3B-L components, as defined above, methylate the DNA of genes present on the recruited DNA (without DNA cleavage) to inhibit their expression, and in this case, allow for the inhibition of the expression of said toxic or suicide transgenes. On the other hand, the KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L, or dCas-KRAB-MeCP2 component inhibits the expression of genes present on the recruited DNA through steric hindrance (without DNA cleavage) and methylates the DNA of genes present on the recruited DNA (without DNA cleavage) to inhibit their expression. For this purpose, the ribonucleoprotein interference complex and / or the ribonucleoprotein methylation complex and / or the bifunctional ribonucleoprotein interference and methylation complex are guided by a gRNA component that specifically recognizes the DNA sequence in the sequence of the inducible promoter, tissue-specific promoter or constitutive promoter of the expression cassette.

[0257] Therefore, according to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids having at least 80% identity with a complementary sequence of a target sequence of 17 to 24 bases present on the promoter (the promoter sequence of the promoter) controlling the expression of the toxic or suicide transgene.

[0258] The target sequence of the gRNA is determined using tools available to those skilled in the art. In short, computer tools such as CRISPOR (Concordet, J.-P., Haeussler, M., 2018. CRISPOR: intuitive guide selection for CRISPR / Cas9 genome editing experiments and screens. NucleicAcids Res. 46, W242–W245) or CRISPICK (Kim et al., 2018. Deep learning improves sprediction of CRISPR–Cpf1 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 the complementarity of the candidate gRNA sequence to the target and the lack of complementarity with the host cell genome. Therefore, for each inducible, tissue-specific, or constitutive promoter, several gRNAs hybridizing at one or more sites on the promoter are proposed, and then one is selected based on its optimal ability to suppress transgene expression through that promoter. Thus, it is necessary, in addition to computer analysis, to functionally screen the individual candidate gRNA sequences to determine which can implement the most efficient and safest interference and / or methylation strategy according to the invention at the promoter of interest. For this purpose, cells (e.g., HEK-293T cells) are genetically modified to express at least one ribonucleoprotein complex according to the invention and each candidate gRNA. These same cells are then transduced (lentiviral transduction) using an expression cassette containing an inducible, tissue-specific, or constitutive promoter according to the invention upstream of a reporter protein (e.g., FLuc or Luc), the promoter being the one targeted by the candidate gRNA. From the candidate gRNAs, the one that can most effectively target and reduce basal reporter gene expression is selected and used to establish an animal eukaryotic cell clonal line according to the invention, wherein the optimal clone (i.e., the clone with optimal reporter gene expression suppression) is selected and amplified as a tool for producing lentiviral vectors carrying cytotoxic transgenes according to the invention.

[0259] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the gRNA has been identified using CRISPR or CRISPICK tools, and its targeting activity has been determined particularly in vitro.

[0260] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, the following nucleic acids: [the sequence SEQ ID NO: 318 (which specifically recognizes the inducible promoter 2xAARE)]. YB_TATA The nucleic acid of (SEQ ID NO: 318) has at least 80% identity. In particular, the present invention also relates to the use in animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, a nucleic acid corresponding to the sequence SEQ ID NO: 318.

[0261] Advantageously, the present invention also relates to the use of animal eukaryotic cells as described above, wherein:

[0262] - The fusion protein:

[0263] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0264] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0265] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0266] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0267] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0268] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0269] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0270] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0271] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0272] - The gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0273] - The inducible promoter is 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1.

[0274] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0275] - The fusion protein:

[0276] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0277] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0278] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0279] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0280] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0281] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0282] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0283] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0284] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0285] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 318; and

[0286] - The inducible promoter is 2xAARE YB_TATA Its nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0287] Therefore, according to this embodiment, a lentiviral vector can be produced, the lentiviral vector comprising an expression cassette selected from sequences SEQ ID NO: 25 to SEQ ID NO: 32, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 87 to SEQ ID NO: 94 and SEQ ID NO: 143 to SEQ ID NO: 150.

[0288] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the SV40 constitutive promoter (SEQ ID NO: 343)). In particular, the present invention also relates to the use of animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0289] Advantageously, the present invention also relates to the use of animal eukaryotic cells as described above, wherein:

[0290] - The fusion protein:

[0291] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0292] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0293] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0294] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0295] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0296] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0297] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0298] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0299] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0300] - The gRNA comprises, or is composed of, the following nucleic acids: nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and

[0301] - The constitutive promoter is SV40, whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 343.

[0302] In particular, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0303] - The fusion protein:

[0304] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0305] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0306] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0307] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0308] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0309] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0310] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0311] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0312] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0313] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 352; and

[0314] - The constitutive promoter is SV40, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0315] According to this embodiment, a lentiviral vector can thus be produced, the lentiviral vector comprising an expression cassette selected from sequences SEQ ID NO: 391 to SEQ ID NO: 398, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 463 to SEQ ID NO: 470 and SEQ ID NO: 353 to SEQ ID NO: 542.

[0316] According to a second aspect of the invention, the present invention relates to a method for producing a lentiviral vector comprising a vector genome, the vector genome comprising a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) located upstream of a toxic or suicide transgene has been introduced into the lentiviral genome, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0317] The method includes at least the following steps:

[0318] a) Co-transfecting animal eukaryotic cells expressing at least one ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the ribonucleoprotein complex comprises:

[0319] - Fusion proteins containing the following fusions:

[0320] - Inactivated Cas endonuclease (dCas); and

[0321] - KRAB transcriptional repressor domain (dCas-KRAB fusion) or methylated domains selected from the following: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion).

[0322] Or a fusion protein containing the following fusions:

[0323] - KRAB transcriptional repression domain;

[0324] - Inactivated Cas endonuclease (dCas); and

[0325] - Selected from the following methylation domains: 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 (dCas-KRAB-MeCP2 fusion); and

[0326] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (the promoter sequence of the promoter).

[0327] The 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, especially those stably expressed,

[0328] The co-transfection was performed using the following method:

[0329] - Contains the vector genome and a plasmid for expressing it;

[0330] - Contains the envelope protein and plasmids used to express it; and

[0331] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0332] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0333] c) Harvest and purify the lentiviral vector.

[0334] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the KRAB transcriptional repression domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein the KRAB transcriptional repression domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0335] According to another embodiment, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0336] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the C-terminus of the inactivated Cas endonuclease (dCas); or

[0337] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the N-terminus of the inactivated Cas endonuclease (dCas); or

[0338] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0339] Advantageously, the present invention relates to the use of animal eukaryotic cells as described above, wherein:

[0340] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas); and

[0341] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0342] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is selected from: inducible promoters, tissue-specific promoters, and constitutive promoters. Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is an inducible promoter or a tissue-specific promoter.

[0343] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, the lentiviral vector comprising a vector genome containing a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) has been introduced upstream of a toxic or suicide transgene, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0344] The method includes at least the following steps:

[0345] a) Co-transfection of animal eukaryotic cells expressing a ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the ribonucleoprotein complex comprises:

[0346] - Fusion proteins containing the following fusions:

[0347] - Inactivated Cas endonuclease (dCas); and

[0348] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0349] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0350] The dCas-KRAB / gRNA ribonucleoprotein complex, especially the stably expressed,

[0351] The co-transfection was performed using the following method:

[0352] - Contains the vector genome and a plasmid for expressing it;

[0353] - Contains the envelope protein and plasmids used to express it; and

[0354] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0355] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0356] c) Harvest and purify the lentiviral vector.

[0357] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, the lentiviral vector comprising a vector genome containing a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) located upstream of a toxic or suicide transgene has been introduced into the lentiviral genome, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0358] The method includes at least the following steps:

[0359] a) Co-transfection of animal eukaryotic cells expressing a ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the ribonucleoprotein complex comprises:

[0360] - Fusion proteins containing the following fusions:

[0361] - Inactivated Cas endonuclease (dCas); and

[0362] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion); and

[0363] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (the promoter sequence of the promoter).

[0364] The ribonucleoprotein complexes dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA, especially those stably expressed, are mentioned.

[0365] The co-transfection was performed using the following method:

[0366] - Contains the vector genome and a plasmid for expressing it;

[0367] - Contains the envelope protein and plasmids used to express it; and

[0368] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0369] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0370] c) Harvest and purify the lentiviral vector.

[0371] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, the lentiviral vector comprising a vector genome containing a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) located upstream of a toxic or suicide transgene has been introduced into the lentiviral genome, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0372] The method includes at least the following steps:

[0373] a) Co-transfecting animal eukaryotic cells expressing at least a first and a second ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the first ribonucleoprotein complex comprises:

[0374] - Fusion proteins containing the following fusions:

[0375] - Inactivated Cas endonuclease (dCas); and

[0376] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0377] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0378] And the second ribonucleoprotein complex comprises:

[0379] - Fusion proteins containing the following fusions:

[0380] - Inactivated Cas endonuclease (dCas); and

[0381] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion), DNMT3B (dCas-DNMT3B fusion), DNMT3A-L (dCas-DNMT3A-L fusion), and DNMT3B-L (dCas-DNMT3B-L fusion); and

[0382] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0383] The first and second ribonucleoprotein complexes are particularly stably expressed.

[0384] The co-transfection was performed using the following method:

[0385] - Contains the vector genome and a plasmid for expressing it;

[0386] - Contains the envelope protein and plasmids used to express it; and

[0387] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0388] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0389] c) Harvest and purify the lentiviral vector.

[0390] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, the lentiviral vector comprising a vector genome containing a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) located upstream of a toxic or suicide transgene has been introduced into the lentiviral genome, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0391] The method includes at least the following steps:

[0392] a) Co-transfection of animal eukaryotic cells expressing a bifunctional ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the bifunctional ribonucleoprotein complex comprises:

[0393] - Fusion proteins containing the following fusions:

[0394] - KRAB transcriptional repression domain;

[0395] - Inactivated Cas endonuclease (dCas); and

[0396] - Selected from the following methylation domains: 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 (dCas-KRAB-MeCP2 fusion); and

[0397] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0398] The bifunctional ribonucleoprotein complexes KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, or dCas-KRAB-MeCP2 / gRNA, especially those stably expressed, are particularly important.

[0399] The co-transfection was performed using the following method:

[0400] - Contains the vector genome and a plasmid for expressing it;

[0401] - Contains the envelope protein and plasmids used to express it; and

[0402] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0403] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0404] c) Harvest and purify the lentiviral vector.

[0405] This method is illustrated in the following embodiments and is accessible to those skilled in the art, given the availability of literature (e.g., Zennou et al., Nat Biotechnol. 2001 May;19(5):446-50). However, for illustrative purposes, it is specifically described below:

[0406] - The animal eukaryotic cells may be cells belonging to the following cell lines: HEK 293T, HEK 293, CHO, GS-CHO, Free style 293-F cells (FS293), virus-producing cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8 and PS46.2;

[0407] - The plasmid containing the viral envelope protein and the tool for expressing it may be selected from the following plasmids: pHCMV-VSV (SEQ ID NO: 337), pHCMV-Mokola, pHCMV-RabiesG, pHCMV-AmphoEnv, pHCMV-LCMV-WE, pHCMV-LCMV-Arm53b, and pHCMV-10A1 (Sena-Esteves M et al., Optimised large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. 15 December 2004;122(2):131-9); and

[0408] - The plasmid containing HIV-1 structural proteins and enzymes and tools for expressing them may be selected from the following 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).

[0409] For illustrative purposes and without limitation, the following options are also provided:

[0410] Cells expressing at least one ribonucleoprotein complex according to the invention (e.g., HEK 293T) were seeded on a support suitable for cell culture, seeded in a manner that resulted in 50% to 60% confluence after 24 hours of culture. The next day, the cells were co-transfected using calcium phosphate precipitation with a plasmid encoding the lentiviral genome and two trans-complementary plasmids encoding the enzyme and structural protein of HIV-1 (p8.9) and the envelope glycoprotein of vesicular stomatitis virus (pVSVg). Co-transfection was performed at a plasmid ratio of 2:2:1. The transfected cells were incubated with the precipitate for 5 hours, followed by a change of the culture medium. After 48 hours, the supernatant containing lentiviral particles was collected. The lentiviral suspension was treated with DNase I to remove any remaining plasmids, filtered (membrane porosity 0.2 µm), and then ultracentrifuged at 60,000 g for 90 minutes at 4°C. The particle pellet was resuspended in a volume corresponding to 1000-fold concentration of phosphate-buffered saline (PBS). The obtained suspension was divided into equal portions and stored at -80°C until use.

[0411] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the inactivated Cas endonuclease (dCas) is selected from:

[0412] - Inactivated Cas9 endonucleases from *Streptococcus pyogenes*, *Staphylococcus aureus*, *Corynebacterium diphtheriae*, *Neisseria meningitidis*, *Streptococcus canis*, *Streptococcus macacae*, *F. tularensis*, *Acidaminococcus*, *Campylobacter jejuni*, *Streptococcus pneumoniae*, and *Streptococcus thermophilus*;

[0413] - Inactivated Cas12a endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp.;

[0414] - Inactivated Cas12b endonuclease from Alicyclobacillus kakegawensis and Bacillus hisashii;

[0415] - Inactivated CasX or Cas12e endonucleases from Deltaproteobacteria and Planctomycetes;

[0416] - Inactivated type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans;

[0417] - Inactivated Cas12j or CasΦ endonucleases from the Biggiephage clade;

[0418] - Inactivated orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms; and

[0419] - Inactivated mutants or variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms.

[0420] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9). This involves ribonucleoprotein complexes 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. Specifically, the present invention relates to a method for producing lentiviral vectors as described above, wherein the inactivated Cas endonuclease is an inactivated 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 inactivated Cas9 endonuclease (dCas9) whose amino acid sequence has at least 80% identity with sequence SEQ ID NO: 323. Specifically, the present invention also relates to a method for producing lentiviral vectors as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322, or whose nucleic acid encodes an inactivated Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to sequence SEQ ID NO: 323.

[0421] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the KRAB transcriptional repressor domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 320 or SEQ ID NO: 751, or the amino acid sequence thereof having at least 80% identity with the sequence SEQ ID NO: 321 or SEQ ID NO: 752. In particular, the present invention also relates to a method for producing lentiviral vectors as described above, wherein the KRAB transcriptional repressor domain is encoded by a nucleic acid of sequence SEQ ID NO: 320 or SEQ ID NO: 751, or the amino acid sequence thereof corresponds to the sequence SEQ ID NO: 321 or SEQ ID NO: 752.

[0422] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. Specifically, the present invention relates to a method for producing a lentiviral vector as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or the nucleic acid thereon encodes a dCas9-KRAB fusion protein, wherein the amino acid sequence of the dCas9-KRAB fusion protein has at least 80% identity with the sequence SEQ ID NO: 326 or SEQ ID NO: 754. In particular, the present invention also relates to a method for producing a lentiviral vector as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or the nucleic acid of which encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0423] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the methylation domain is:

[0424] - DNMT3A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 354;

[0425] - DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 366;

[0426] - DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 358; or

[0427] - DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 368.

[0428] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the methylation domain is:

[0429] - DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence corresponds to sequence SEQ ID NO: 354;

[0430] - DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence corresponds to sequence SEQ ID NO: 366;

[0431] - DNMT3A-L is encoded by the nucleic acid sequence SEQ ID NO: 357, or its amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0432] - DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence corresponds to sequence SEQ ID NO: 368.

[0433] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the 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 its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 370. In particular, the present invention relates to a method for producing a lentiviral vector as described above, wherein the MeCP2 methylation domain is encoded by a nucleic acid of sequence SEQ ID NO: 369, or its amino acid sequence corresponding to sequence SEQ ID NO: 370.

[0434] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L, or dCasDNMT3B-L fusion proteins are respectively dCas9-DNMT3A, dCas9-DNMT3B, dCas9-DNMT3A-L, or dCas9-DNMT3B-L fusion proteins. In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0435] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 360;

[0436] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 372;

[0437] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 364; or

[0438] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 373, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 374.

[0439] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0440] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360;

[0441] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372;

[0442] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 363, or the nucleic acid of which encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 364; or

[0443] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372.

[0444] Interestingly, although the DNMT3L enzyme is catalytically inactive, it stimulates the activity of DNMT3A and DNMT3B, thereby promoting promoter silencing through methylation. Therefore, the DNMT3A-L and DNMT3B-L methylation domains are formed by the binding of DNMT3A or DNMT3B to DNMT3L, respectively. Nevertheless, the addition of the dCas-DNMT3L / gRNA ribonucleoprotein complex to the animal eukaryotic cells of the present invention is conceivable. According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells also express another ribonucleoprotein complex comprising:

[0445] - Fusion proteins containing the following fusions:

[0446] - Inactivated Cas endonuclease (dCas); and

[0447] - The methylation-promoting domain DNMT3L (dCas-DNMT3L fusion); and

[0448] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0449] The other ribonucleoprotein complex dCas-DNMT3L / gRNA is particularly stably expressed.

[0450] Advantageously, the present invention relates to a method for producing a lentiviral vector as described above, the lentiviral vector comprising a vector genome containing a lentiviral genome, wherein an expression cassette containing a promoter (promoter sequence) located upstream of a toxic or suicide transgene has been introduced into the lentiviral genome, the 5' to 3' transcription direction of the expression cassette being reversed relative to the 5' to 3' transcription direction of the lentiviral genome.

[0451] The method includes at least the following steps:

[0452] a) Co-transfecting animal eukaryotic cells expressing at least a first and a second ribonucleoprotein complex to obtain transfected animal eukaryotic cells, wherein the first ribonucleoprotein complex comprises:

[0453] - Fusion proteins containing the following fusions:

[0454] - Inactivated Cas endonuclease (dCas); and

[0455] - Selected from the following methylation domains: DNMT3A (dCas-DNMT3A fusion) and DNMT3B; and

[0456] - Guide RNA (gRNA) specifically recognizes the promoter sequence of a promoter.

[0457] And the second ribonucleoprotein complex comprises:

[0458] - Fusion proteins containing the following fusions:

[0459] - Inactivated Cas endonuclease (dCas); and

[0460] - DNMT3L methylation-promoting domain (dCas-DNMT3L fusion); and

[0461] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0462] The first and second ribonucleoprotein complexes are particularly stably expressed.

[0463] The co-transfection was performed using the following method:

[0464] - Contains the vector genome and a plasmid for expressing it;

[0465] - Contains the envelope protein and plasmids used to express it; and

[0466] - Contains HIV-1 structural proteins and enzymes, and plasmids for expressing them;

[0467] b) Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the genome of the vector; and

[0468] c) Harvesting and purifying the lentiviral vector. In particular, the invention also relates to a method for producing a lentiviral vector as described above, wherein the animal eukaryotic cells also express another ribonucleoprotein complex comprising:

[0469] - Fusion proteins containing the following fusions:

[0470] - Inactivated Cas endonuclease (dCas); and

[0471] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0472] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0473] The dCas-KRAB / gRNA ribonucleoprotein complex is particularly stably expressed.

[0474] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the DNMT3L promethylation domain is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein the DNMT3L promethylation domain is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0475] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 356. In particular, the present invention relates to a method for producing a lentiviral vector as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid of sequence SEQ ID NO: 355, or its amino acid sequence corresponding to the sequence SEQ ID NO: 356.

[0476] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362. In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein corresponds to the sequence SEQ ID NO: 362.

[0477] As described above, the present invention also implements a bifunctional CRISPRi / m complex comprising:

[0478] - Inactivated or "dead" CRISPR-Cas endonucleases (dCas), KRAB transcriptional repression domains, and fusion with DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 methylation domains; and

[0479] - Guide RNA (gRNA) specifically recognizes the promoter sequence of the promoter (the promoter of the expression cassette).

[0480] Specifically, the bifunctional complex (advantageously expressed in a stable manner) is selected from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, and dCas-KRAB-MeCP2 / gRNA, and advantageously from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA, and dCas9-KRAB-MeCP2 / gRNA. The invention also relates to a method for producing lentiviral vectors as described above, wherein:

[0481] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 376 or SEQ ID NO: 382;

[0482] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0483] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or whose nucleic acid encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, and the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0484] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0485] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382;

[0486] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0487] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0488] Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein the dCas-KRAB / gRNA ribonucleoprotein complex, 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 by targeted integration into the genome of the animal eukaryotic cells.

[0489] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells belong to a cell line selected from: HEK 293T, HEK 293, CHO, GS-CHO, Free-style 293-F cells (FS293), virus-producing cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8, and PS46.2. Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells belong to the HEK 293T cell line.

[0490] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is an inducible promoter selected from the following: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB _TATA and miniCHAC1 YB_TATA ;or

[0491] The promoter mentioned is a tissue-specific promoter of miR223; or

[0492] The promoters mentioned therein are selected from the following compositional promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV and SFFV.

[0493] In another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is selected from the following inducible or tissue-specific promoters:

[0494] - 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1;

[0495] - 2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2;

[0496] - 4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0497] - 9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0498] - GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0499] - miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 6; and

[0500] - miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7.

[0501] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is selected from the following inducible or tissue-specific promoters:

[0502] - 2xAARE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0503] - 2xAARE TK Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0504] - 4xSRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0505] - 9xHRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0506] - GRP78 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 5;

[0507] - miR223 promoter, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0508] - miniCHAC1 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0509] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is a constitutive promoter selected from the following:

[0510] - SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 343;

[0511] - CMV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 344;

[0512] - UbC, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 345;

[0513] - EF1-α core type, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 346;

[0514] - EF1-α long type, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 347;

[0515] - hPGK, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 348;

[0516] - CAGG, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 349;

[0517] - RSV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 350; and

[0518] - SFFV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 351.

[0519] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the promoter is a constitutive promoter selected from the following:

[0520] - SV40, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0521] - CMV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0522] - UbC, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0523] - EF1-α core type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 346;

[0524] - EF1-α long type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 347;

[0525] - hPGK, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348;

[0526] - CAGG, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 349;

[0527] - RSV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0528] - SFFV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0529] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the toxic or suicide transgene is selected from:

[0530] - An inducible Caspase-9 protein (iC9) whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes an inducible Caspase-9 protein (iC9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 9;

[0531] - Bax(S184 del) protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 10, or the nucleic acid of which encodes Bax(S184 del) protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 11;

[0532] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes an M2 (H37A) ion channel of an influenza virus, wherein the amino acid sequence of the M2 (H37A) ion channel of the influenza virus has at least 80% identity with the sequence SEQ ID NO: 13;

[0533] - A constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence that is at least 80% identical 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 is at least 80% identical to the sequence SEQ ID NO: 15;

[0534] - Diphtheria toxin subunit A (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 diphtheria toxin subunit A (DTA), the amino acid sequence of said diphtheria toxin subunit A (DTA) having at least 80% identity with 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 aDTA mutant (G128D), the amino acid sequence of said aDTA mutant (G128D) having at least 80% identity with sequence SEQ ID NO: 19.

[0535] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 21;

[0536] - Noxa protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 23;

[0537] - Streptococcal hemolysin 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 streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O has at least 80% identity with the sequence SEQ ID NO: 756; and

[0538] - Enterotoxin whose nucleic acid has a sequence that is at least 80% identical to 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.

[0539] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the toxic or suicide transgene is selected from:

[0540] - Inducible Caspase-9 protein (iC9), whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes inducible Caspase-9 (iC9), the amino acid sequence of said inducible Caspase-9 (iC9) corresponding to sequence SEQ ID NO: 9;

[0541] - Bax(S184 del) protein, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10, or whose nucleic acid encodes Bax(S184 del) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 11;

[0542] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes the M2 (H37A) ion channel of an influenza virus, the amino acid sequence of the M2 (H37A) ion channel of the influenza virus corresponding to the sequence SEQ ID NO: 13;

[0543] - 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), the amino acid sequence of which corresponds to the sequence SEQ ID NO: 15;

[0544] - Diphtheria toxin subunit A (DTA), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 16, or whose nucleic acid encodes diphtheria toxin subunit A (DTA), the amino acid sequence of which corresponds to sequence SEQ ID NO: 17; and an attenuated mutant aDTA (G128D), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 18, or whose nucleic acid encodes an attenuated aDTA (G128D) mutant, the amino acid sequence of which corresponds to sequence SEQ ID NO: 19;

[0545] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 21;

[0546] - Noxa protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 23;

[0547] - Streptococcal hemolysin O, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 755, or whose nucleic acid encodes streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O corresponds to the sequence SEQ ID NO: 756; and

[0548] - Enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 757, or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0549] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the nucleic acid encoding the toxic or suicide transgene contains a polyA sequence (e.g., SEQ ID NO: 24) at its 3' end.

[0550] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0551] - The promoter is:

[0552] - Selected from the following inductive promoters: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ;

[0553] - The tissue-specific promoter of miR223; or

[0554] - Selected from the following constitutive promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV, and SFFV.

[0555] and

[0556] - The toxic or suicide transgenes are selected from: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein (wild-type protein or one of its mutants, especially the S13A mutant), Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0557] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the expression cassette is selected from sequences having at least 80% identity with sequences SEQ ID NO: 25 to SEQ ID NO: 80 and SEQ ID NO: 391 to SEQ ID NO: 462. In particular, the present invention relates to a method for producing a lentiviral vector as described above, wherein the expression cassette is selected from sequences SEQ ID NO: 25 to SEQ ID NO: 80 and SEQ ID NO: 391 to SEQ ID NO: 462 (see Table 1).

[0558] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 87 to SEQ ID NO: 198 and SEQ ID NO: 463 to SEQ ID NO: 606. In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the vector genome is selected from sequences SEQ ID NO: 87 to SEQ ID NO: 198 and SEQ ID NO: 463 to SEQ ID NO: 606 (see Table 2).

[0559] As described above, the vector genome is carried by a plasmid that allows its expression and thereby the production of the lentiviral vector (or lentiviral particle). Therefore, "a plasmid containing the vector genome and a tool for expressing it" refers to the plasmid, which is a double-stranded circular DNA molecule. This plasmid must possess both a bacterial origin of replication and an SV40 viral origin of replication so that it can replicate autonomously, for example, for amplification in *E. coli*, and replicate post-transfection in, for example, HEK293T cells, and possess a selection gene so that it is not lost during bacterial amplification during cell proliferation. In this invention, it also comprises the nucleic acids of the vector genome of this invention and a tool for expressing it.

[0560] "Tools for expressing it" refers to the genetic elements necessary for expressing the vector genome of the present invention and for producing the lentiviral vector according to the present invention.

[0561] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the plasmid containing the vector genome and a tool for expressing it is a pLV-SIN plasmid or a pLV-RSV plasmid.

[0562] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-SIN plasmid. Specifically, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-SIN plasmid, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 199. Specifically, the present invention also relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-SIN plasmid, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 199. It should be noted that the insertion of the vector genome into the pLV-SIN plasmid of sequence SEQ ID NO: 199 can be performed at or at nucleotides 4,939 and 4,940 of SEQ ID NO: 199. That is, in this case, the vector genome is located between sequences SEQ ID NO: 200 and SEQ ID NO: 201.

[0563] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-RSV plasmid. Specifically, the present invention relates to a method for producing lentiviral vectors as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-RSV plasmid, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 202. Specifically, the present invention also relates to a method for producing lentiviral vectors as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is a pLV-RSV plasmid, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 202. It should be noted that the insertion of the vector genome into the pLV-RSV plasmid of sequence SEQ ID NO: 202 can be performed at or at nucleotides 234 and 235 of SEQ ID NO: 202. That is, in this case, the vector genome is located between sequences SEQ ID NO: 203 and SEQ ID NO: 204.

[0564] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is characterized in that the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed relative to the 5' to 3' transcriptional orientation of the expression cassette.

[0565] Therefore, according to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid having been introduced into the vector genome as described above and the tool for expressing it is:

[0566] - pLV-SIN plasmid, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 199; or

[0567] - pLV-RSV plasmid, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 202;

[0568] The lentiviral genome into which the expression cassette has been introduced is:

[0569] - Self-inactivated lentivirus (SIN) whose nucleic acid has at least 80% sequence identity with the nucleic acid of sequence SEQ ID NO: 81; or

[0570] - Respiratory syncytial virus (RSV) whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 84;

[0571] And wherein, the expression box includes:

[0572] - Selected from the following inductive promoters: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ;

[0573] - Or the tissue-specific promoter of miR223;

[0574] - Alternatively, select from the following constitutive promoters: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV, and SFFV.

[0575] and

[0576] - The toxic or suicide transgenes are selected from: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein (wild-type protein or one of its mutants, especially the S13A mutant), Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0577] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is selected from sequences having at least 80% identity with sequences SEQ ID NO: 205 to SEQ ID NO: 316 and SEQ ID NO: 607 to SEQ ID NO: 750. In particular, the present invention relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it is selected from sequences SEQ ID NO: 205 to SEQ ID NO: 316 and SEQ ID NO: 607 to SEQ ID NO: 750. In particular, the present invention also relates to a method for producing a lentiviral vector as described above, wherein the plasmid comprising the vector genome and a tool for expressing it has a 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, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 70 9, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749 or 750 (see Table 3).

[0578]

[0579] Table 3. Plasmid List

[0580] According to another embodiment, the present invention relates to a method for producing a lentiviral vector as described above, wherein the gRNA comprises, or is composed of, the following nucleic acids: [the sequence SEQ ID NO: 318 (which specifically recognizes 2xAARE)]. YB _TATA The nucleic acid of (SEQ ID NO: 318) has at least 80% identity. In particular, the present invention relates to a method for producing a lentiviral vector as described above, wherein the gRNA comprises, or is composed of, a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 318.

[0581] Advantageously, the present invention also relates to a method for producing lentiviral vectors as described above, wherein:

[0582] - The fusion protein:

[0583] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0584] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0585] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0586] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0587] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0588] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0589] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0590] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0591] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0592] - The gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0593] - The inducible promoter is 2xAARE YB_TATAIts nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1.

[0594] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0595] - The fusion protein:

[0596] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0597] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0598] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0599] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0600] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0601] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0602] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0603] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0604] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0605] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 318; and

[0606] - The inducible promoter is 2xAARE YB_TATA Its nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0607] According to this embodiment, a lentiviral vector can thus be produced comprising an expression cassette selected from sequences SEQ ID NO: 25 to SEQ ID NO: 32, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 87 to SEQ ID NO: 94 and SEQ ID NO: 143 to SEQ ID NO: 150, which may be encoded by plasmids selected from sequences SEQ ID NO: 205 to SEQ ID NO: 212 and SEQ ID NO: 261 to SEQ ID NO: 268.

[0608] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the SV40 constitutive promoter (SEQ ID NO: 343)). In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein the gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0609] Advantageously, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0610] - The fusion protein:

[0611] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0612] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0613] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0614] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0615] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0616] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0617] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0618] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0619] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0620] - The gRNA comprises, or is composed of, the following nucleic acids: nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and

[0621] - The constitutive promoter is SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 343.

[0622] In particular, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0623] - The fusion protein:

[0624] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0625] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0626] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0627] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0628] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0629] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0630] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0631] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0632] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0633] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 352; and

[0634] - The constitutive promoter is SV40, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0635] According to this embodiment, a lentiviral vector can thus be produced comprising an expression cassette selected from sequences SEQ ID NO: 391 to SEQ ID NO: 398, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 463 to SEQ ID NO: 470 and SEQ ID NO: 353 to SEQ ID NO: 542, which may be encoded by plasmids selected from sequences SEQ ID NO: 607 to SEQ ID NO: 614 and SEQ ID NO: 679 to SEQ ID NO: 686.

[0636] According to another aspect, the present invention relates to animal eukaryotic cells expressing at least one ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0637] - Selected from the following fusion proteins:

[0638] - dCas-KRAB, which contains the fusion of an inactivated Cas endonuclease with a KRAB transcriptional repression domain;

[0639] - dCas-DNMT3A, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3A;

[0640] - dCas-DNMT3B, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3B;

[0641] - dCas-DNMT3A-L, which contains the fusion of an inactivated Cas endonuclease with a DNMT3A-L methylation domain;

[0642] - dCas-DNMT3B-L, which contains the fusion of an inactivated Cas endonuclease with a DNMT3B-L methylation domain;

[0643] - KRAB-dCas-DNMT3A-L, which is a fusion of the KRAB transcriptional repression domain, the inactivated Cas endonuclease (dCas), and the DNMT3A-L methylation domain;

[0644] - KRAB-dCas-DNMT3B-L, which comprises a fusion of the KRAB transcriptional repressor domain, an inactivated Cas endonuclease (dCas), and the DNMT3B-L methylation domain; and

[0645] - dCas-KRAB-MeCP2, which comprises a fusion of an inactivated Cas endonuclease (dCas), a KRAB transcriptional repressor domain, and a MeCP2 methylation domain, and

[0646] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of a promoter selected from the following inducible promoters: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB _TATA Alternatively, it may be a tissue-specific promoter of miR223; or a constitutive promoter selected from the following: SV40, CMV, UbC, EF1-α core, EF1-α long, hPGK, CAGG, RSV, and SFFV.

[0647] The ribonucleoprotein complexes 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 are particularly stably expressed.

[0648] Obtaining animal eukaryotic cells according to the invention is within the capabilities of those skilled in the art. However, for illustrative purposes and without limitation, the specific details are as follows: To obtain animal eukaryotic cells (e.g., HEK293T cells) according to the invention, the cells were transduced using a lentiviral vector carrying a constitutive expression cassette, a dCas-KRAB fusion protein fused with an antibiotic resistance gene (e.g., blastcin (BlastR)) via a 2A peptide, and a guide RNA specific to the target promoter. Two days later, the transduced cells were selected by treatment with an antibiotic (e.g., 2 µg / mL). The selected cells were then cloned using a limiting dilution method, and each clone was characterized. Only clones that allowed for the most effective blocking of transgene expression under the promoter control according to the invention were selected and amplified.

[0649] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, which express at least one ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0650] - Selected from the following fusion proteins:

[0651] - dCas-KRAB, which contains the fusion of an inactivated Cas endonuclease with a KRAB transcriptional repression domain;

[0652] - dCas-DNMT3A, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3A;

[0653] - dCas-DNMT3B, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3B;

[0654] - dCas-DNMT3A-L, which comprises the fusion of an inactivated Cas endonuclease with a DNMT3A-L methylation domain; and

[0655] - dCas-DNMT3B-L, which contains the fusion of an inactivated Cas endonuclease with a DNMT3B-L methylation domain;

[0656] - KRAB-dCas-DNMT3A-L, which is a fusion of the KRAB transcriptional repression domain, the inactivated Cas endonuclease (dCas), and the DNMT3A-L methylation domain;

[0657] - KRAB-dCas-DNMT3B-L, which comprises a fusion of the KRAB transcriptional repressor domain, an inactivated Cas endonuclease (dCas), and the DNMT3B-L methylation domain; and

[0658] - dCas-KRAB-MeCP2, which comprises the fusion of an inactivated Cas endonuclease (dCas), a KRAB transcriptional repressor domain, and a MeCP2 methylation domain, and

[0659] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of a promoter, wherein the promoter is:

[0660] - Selected from the following inducible promoters:

[0661] 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1;

[0662] 2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2;

[0663] 4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0664] 9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0665] GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of SEQ ID NO: 5; and

[0666] miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of SEQ ID NO: 7; or

[0667] - The tissue-specific promoter of miR223, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 6; or

[0668] - Selected from the following constitutive promoters:

[0669] SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 343;

[0670] CMV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 344;

[0671] UbC, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 345;

[0672] The EF1-α core has a nucleic acid sequence that has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 346;

[0673] The EF1-α long form has a nucleic acid with at least 80% sequence identity with the nucleic acid of sequence SEQ ID NO: 347;

[0674] hPGK, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 348;

[0675] CAGG, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 349;

[0676] RSV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 350; and

[0677] SFFV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 351,

[0678] The ribonucleoprotein complexes 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 are particularly stably expressed.

[0679] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain or the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0680] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein:

[0681] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the C-terminus of the inactivated Cas endonuclease (dCas); or

[0682] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2 is located at the N-terminus of the inactivated Cas endonuclease (dCas); or

[0683] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0684] Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein:

[0685] - The KRAB transcriptional repression domain is located at the N-terminus of the inactivated Cas endonuclease (dCas), and the methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L is located at the C-terminus of the inactivated Cas endonuclease (dCas); and

[0686] - The KRAB transcriptional repression domain is located at the C-terminus of the inactivated Cas endonuclease (dCas), and the MeCP2 methylation domain is located at the C-terminus of the KRAB transcriptional repression domain.

[0687] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells expressing a ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0688] - dCas-KRAB fusion protein, comprising the fusion of an inactivated Cas endonuclease with a KRAB transcriptional repressor domain; and

[0689] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the present invention.

[0690] The dCas-KRAB / gRNA ribonucleoprotein complex is particularly stably expressed.

[0691] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells expressing a ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0692] - Selected from the following fusion proteins:

[0693] - dCas-DNMT3A, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3A;

[0694] - dCas-DNMT3B, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3B;

[0695] - dCas-DNMT3A-L, which comprises the fusion of an inactivated Cas endonuclease with a DNMT3A-L methylation domain; and

[0696] - dCas-DNMT3B-L, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3B-L, and

[0697] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the present invention.

[0698] The ribonucleoprotein complexes dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA are particularly stably expressed.

[0699] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells comprising at least first and second ribonucleoprotein complexes, the first ribonucleoprotein complex comprising:

[0700] - dCas-KRAB fusion protein, comprising the fusion of an inactivated Cas endonuclease with a KRAB transcriptional repressor domain; and

[0701] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the present invention.

[0702] And the second ribonucleoprotein complex comprises:

[0703] - Selected from the following fusion proteins:

[0704] - dCas-DNMT3A, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3A;

[0705] - dCas-DNMT3B, which contains the fusion of an inactivated Cas endonuclease with the methylation domain of DNMT3B;

[0706] - dCas-DNMT3A-L, which comprises the fusion of an inactivated Cas endonuclease with a DNMT3A-L methylation domain; and

[0707] - dCas-DNMT3B-L, which contains the fusion of an inactivated Cas endonuclease with a DNMT3B-L methylation domain.

[0708] as well as

[0709] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the invention (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0710] The first and second ribonucleoprotein complexes are particularly stably expressed.

[0711] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells expressing a ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0712] - Selected from the following fusion proteins:

[0713] - KRAB-dCas-DNMT3A-L, which is a fusion of the KRAB transcriptional repression domain, the inactivated Cas endonuclease (dCas), and the DNMT3A-L methylation domain;

[0714] - KRAB-dCas-DNMT3B-L, which comprises a fusion of the KRAB transcriptional repressor domain, an inactivated Cas endonuclease (dCas), and the DNMT3B-L methylation domain; and

[0715] - dCas-KRAB-MeCP2, which comprises a fusion of an inactivated Cas endonuclease (dCas), a KRAB transcriptional repressor domain, and a MeCP2 methylation domain, and

[0716] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the present invention.

[0717] The KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, or dCas-KRAB-MeCP2 / gRNA ribonucleoprotein complexes are particularly stably expressed.

[0718] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the guide RNA (gRNA) specifically recognizes promoter sequences selected from the following inducible or tissue-specific promoters:

[0719] - 2xAARE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0720] - 2xAARE TK Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0721] - 4xSRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0722] - 9xHRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0723] - GRP78YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 5;

[0724] - miR223 promoter, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0725] - miniCHAC1 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0726] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the guide RNA (gRNA) specifically recognizes promoter sequences selected from the constitutive promoters:

[0727] - SV40, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 343;

[0728] - CMV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 344;

[0729] - UbC, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 345;

[0730] - EF1-α core type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 346;

[0731] - EF1-α long type, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 347;

[0732] - hPGK, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 348;

[0733] - CAGG, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 349;

[0734] - RSV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 350; and

[0735] - SFFV, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 351.

[0736] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is selected from:

[0737] - Inactivated Cas9 endonucleases from *Streptococcus pyogenes*, *Staphylococcus aureus*, *Corynebacterium diphtheriae*, *Neisseria meningitidis*, *Streptococcus canis*, *Streptococcus macacae*, *F. tularensis*, *Acidaminococcus*, *Campylobacter jejuni*, *Streptococcus pneumoniae*, and *Streptococcus thermophilus*;

[0738] - Inactivated Cas12a endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp.;

[0739] - Inactivated Cas12b endonuclease from Alicyclobacillus kakegawensis and Bacillus hisashii;

[0740] - Inactivated CasX or Cas12e endonucleases from Deltaproteobacteria and Planctomycetes;

[0741] - Inactivated type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans;

[0742] - Inactivated Cas12j or CasΦ endonucleases from the Biggiephage clade;

[0743] - Inactivated orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms; and

[0744] - Inactivated mutants or variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from these organisms.

[0745] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9). This relates to ribonucleoprotein complexes 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. Specifically, the present invention relates to animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated 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 inactivated Cas9 endonuclease (dCas9), the amino acid sequence of which has at least 80% identity with sequence SEQ ID NO: 323. Specifically, the present invention also relates to animal eukaryotic cells as described above, wherein the inactivated Cas endonuclease is an inactivated Cas9 endonuclease (dCas9), whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 322, or whose nucleic acid encodes an inactivated Cas9 endonuclease (dCas9), the amino acid sequence of which corresponds to sequence SEQ ID NO: 323.

[0746] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 320 or SEQ ID NO: 751, or the amino acid sequence thereof having at least 80% identity with the sequence SEQ ID NO: 321 or SEQ ID NO: 752. In particular, the present invention also relates to animal eukaryotic cells as described above, wherein the KRAB transcriptional repressor domain is encoded by a nucleic acid of sequence SEQ ID NO: 320 or SEQ ID NO: 751, or the amino acid sequence thereof corresponds to the sequence SEQ ID NO: 321 or SEQ ID NO: 752.

[0747] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. Specifically, the present invention relates to animal eukaryotic cells as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or the nucleic acid of which encodes a dCas9-KRAB fusion protein, wherein the amino acid sequence of the dCas9-KRAB fusion protein has at least 80% identity with the sequence SEQ ID NO: 326 or SEQ ID NO: 754. In particular, the present invention also relates to animal eukaryotic cells as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or the nucleic acid of which encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0748] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the methylation domain is:

[0749] - DNMT3A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 354;

[0750] - DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 366;

[0751] - DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 357, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 358; or

[0752] - DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 368.

[0753] In particular, the present invention relates to animal eukaryotic cells as described above, wherein the methylated domains are:

[0754] - DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 353, or its amino acid sequence corresponds to sequence SEQ ID NO: 354;

[0755] - DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 365, or its amino acid sequence corresponds to sequence SEQ ID NO: 366;

[0756] - DNMT3A-L is encoded by the nucleic acid sequence SEQ ID NO: 357, or its amino acid sequence corresponds to the sequence SEQ ID NO: 358; or

[0757] - DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 367, or its amino acid sequence corresponds to sequence SEQ ID NO: 368.

[0758] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the 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 its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 370. In particular, the present invention relates to animal eukaryotic cells as described above, wherein the MeCP2 methylation domain is encoded by a nucleic acid of sequence SEQ ID NO: 369, or its amino acid sequence corresponding to sequence SEQ ID NO: 370.

[0759] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L, or dCas-DNMT3B-L fusion proteins are respectively dCas9-DNMT3A, dCas9-DNMT3B, dCas9-DNMT3A-L, or dCas9-DNMT3B-L fusion proteins. In particular, the present invention relates to animal eukaryotic cells as described above, wherein:

[0760] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 360;

[0761] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 372;

[0762] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 364; or

[0763] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 373, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 374.

[0764] In particular, the present invention relates to animal eukaryotic cells as described above, wherein:

[0765] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or the nucleic acid of which encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360;

[0766] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372;

[0767] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 363, or the nucleic acid of which encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 364; or

[0768] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 371, or the nucleic acid of which encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 372.

[0769] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells also expressing another ribonucleoprotein complex, said ribonucleoprotein complex comprising:

[0770] - Fusion proteins containing the following fusions:

[0771] - Inactivated Cas endonuclease (dCas); and

[0772] - DNMT3L methylation-promoting domain (dCas-DNMT3L fusion); and

[0773] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0774] The other ribonucleoprotein complex dCas-DNMT3L / gRNA is particularly stably expressed.

[0775] Advantageously, the present invention relates to animal eukaryotic cells as described above, said animal eukaryotic cells comprising at least first and second ribonucleoprotein complexes, the first ribonucleoprotein complex comprising:

[0776] - Selected from the following fusion proteins:

[0777] - dCas-DNMT3A, which comprises the fusion of an inactivated Cas endonuclease with a DNMT3A methylation domain; and

[0778] - dCas-DNMT3B, which comprises the fusion of an inactivated Cas endonuclease with a DNMT3B methylation domain, and

[0779] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the present invention.

[0780] And the second ribonucleoprotein complex comprises:

[0781] - A fusion protein comprising dCas-DNMT3L, consisting of an inactivated Cas endonuclease fused to a DNMT3L methylation domain, and

[0782] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter according to the invention (i.e., the same promoter targeted by the first ribonucleoprotein complex).

[0783] The first and second ribonucleoprotein complexes are particularly stably expressed. In particular, the invention also relates to animal eukaryotic cells as described above, which also express another ribonucleoprotein complex comprising:

[0784] - Fusion proteins containing the following fusions:

[0785] - Inactivated Cas endonuclease (dCas); and

[0786] - KRAB transcriptional repressor domain (dCas-KRAB fusion); and

[0787] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[0788] The dCas-KRAB / gRNA ribonucleoprotein complex is particularly stably expressed.

[0789] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the DNMT3L pro-methylation domain is located at the N-terminus or C-terminus of the inactivated Cas endonuclease (dCas). Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein the DNMT3L pro-methylation domain is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0790] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 355, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 356. In particular, the present invention relates to animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is encoded by a nucleic acid having a sequence corresponding to SEQ ID NO: 355, or its amino acid sequence corresponding to the sequence SEQ ID NO: 356.

[0791] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362. Specifically, the present invention relates to animal eukaryotic cells as described above, wherein the dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, and the amino acid sequence of the dCas9-DNMT3L fusion protein corresponds to the sequence SEQ ID NO: 362.

[0792] As described above, the present invention also implements a bifunctional CRISPRi / m complex comprising:

[0793] - Inactivated or "dead" CRISPR-Cas endonucleases (dCas), KRAB transcriptional repression domains, and fusion with DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 methylation domains; and

[0794] - Guide RNA (gRNA) specifically recognizes the promoter sequence of the promoter (the promoter of the expression cassette).

[0795] Specifically, the bifunctional complex (advantageously expressed in a stable manner) is selected from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA, and dCas-KRAB-MeCP2 / gRNA, and advantageously from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA, and dCas9-KRAB-MeCP2 / gRNA. The invention also relates to animal eukaryotic cells as described above, wherein:

[0796] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 376 or SEQ ID NO: 382;

[0797] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0798] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0799] In particular, the present invention relates to animal eukaryotic cells as described above, wherein:

[0800] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382;

[0801] - The KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or the nucleic acid of which encodes the KRAB-dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384; or

[0802] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386.

[0803] Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein the ribonucleoprotein complexes 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 are stably expressed by targeted integration into the genome of the animal eukaryotic cells.

[0804] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the animal eukaryotic cells belong to a cell line selected from the following: HEK 293T, HEK 293, CHO, GS-CHO, Free-style 293-F cells (FS293), virus-producing cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8, and PS46.2. Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein the animal eukaryotic cells belong to the HEK 293T cell line.

[0805] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, the following nucleic acids: [the sequence SEQ ID NO: 318 (which specifically recognizes 2xAARE)]. YB_TATA The nucleic acid of the inducible promoter (SEQ ID NO: 1) has at least 80% identity. In particular, the present invention relates to animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid of sequence SEQ ID NO: 318.

[0806] Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein:

[0807] - The fusion protein:

[0808] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0809] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0810] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0811] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0812] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0813] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0814] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0815] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0816] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0817] - The gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 318; and

[0818] - The inducible promoter is 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1.

[0819] In particular, the present invention relates to animal eukaryotic cells as described above, wherein:

[0820] - The fusion protein:

[0821] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0822] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0823] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0824] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0825] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0826] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0827] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0828] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0829] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0830] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 318; and

[0831] - The inducible promoter is 2xAARE YB_TATA Its nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 1.

[0832] According to another embodiment, the present invention relates to animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352 (which specifically recognizes the SV40 constitutive promoter (SEQ ID NO: 343)). In particular, the present invention relates to animal eukaryotic cells as described above, wherein the gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid of sequence SEQ ID NO: 352.

[0833] Advantageously, the present invention relates to animal eukaryotic cells as described above, wherein:

[0834] - The fusion protein:

[0835] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence has at least 80% identity to the sequence SEQ ID NO: 326 or SEQ ID NO: 754.

[0836] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 359, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein has at least 80% identity with the sequence SEQ ID NO: 360.

[0837] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 371, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein has at least 80% identity with the sequence SEQ ID NO: 372.

[0838] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:363, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO:364.

[0839] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO:373, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO:374.

[0840] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 361, or whose nucleic acid encodes the dCas9-DNMT3L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3L fusion protein has at least 80% identity with the sequence SEQ ID NO: 362.

[0841] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 375 or SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 376 or SEQ ID NO: 382.

[0842] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 377 or SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0843] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein has at least 80% identity to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0844] - The gRNA comprises, or is composed of, the following nucleic acids: nucleic acids having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 352; and

[0845] - The constitutive promoter is SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 343.

[0846] In particular, the present invention relates to animal eukaryotic cells as described above, wherein:

[0847] - The fusion protein:

[0848] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 325 or SEQ ID NO: 753, or whose nucleic acid encodes the dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 326 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 328) or SEQ ID NO: 754.

[0849] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 359, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 360.

[0850] - 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 the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 372.

[0851] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO:363, or its nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO:364.

[0852] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid in sequence SEQ ID NO:371, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO:372.

[0853] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 361, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 362.

[0854] - 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 SEQ ID NO: 381, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to sequence SEQ ID NO: 376 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 387) or SEQ ID NO: 382.

[0855] - 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 SEQ ID NO: 383, or whose nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the KRAB-dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 378 or SEQ ID NO: 384, and / or

[0856] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 379 or SEQ ID NO: 385, or the nucleic acid of which encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 380 or SEQ ID NO: 386;

[0857] - The gRNA comprises, or is composed of, nucleic acids corresponding to the nucleic acid sequence SEQ ID NO: 352; and

[0858] - The constitutive promoter is SV40, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 343.

[0859] It should also be noted that the object of the present invention is: a vector genome as described above, which particularly contains an inducible or tissue-specific promoter; a plasmid containing the vector genome as described above; the use of the plasmid as described above in the production of a lentiviral vector containing the vector genome as described above; a lentiviral vector containing the vector genome as described above; the use of the lentiviral vector as described above in the transduction of animal eukaryotic cells; animal eukaryotic cells transduced by the lentiviral vector as described above; and animal eukaryotic cells transduced as described above for use in cell therapy. Regarding this last object, the present invention clearly transcends the scope of enabling the production of lentiviral vectors encoding high titers of toxic (cytotoxic) or suicide transgenes (which has never been achieved before). In fact, it ultimately provides the possibility of implementing effective and safe cell therapies. These therapies utilize a technology known as a safety switch, which, under the action of the correct stimulus (and only the correct stimulus), allows the activation of an inducible or tissue-specific promoter of the expression cassette, thereby allowing the expression of toxic or suicide transgenes, leading to cell death. Additionally, for example, for the use of immune cells in the following treatments…

[0860] - Modified and armed to target and destroy cancer cells; and

[0861] - According to the transduction of the present invention,

[0862] When administered to patients in need, it will target and destroy cancer cells. However, if the immune cells malfunction, they can then be destroyed to avoid adverse effects, thanks to the safety system provided by this invention (i.e., an expression cassette containing an inducible or tissue-specific promoter located upstream of a toxic or suicide transgene).

[0863] Furthermore, as demonstrated in the following examples, the expression of transgenes under the control of the inducible promoter of the present invention can be pharmacologically induced and controlled, said inducible promoter being selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA Especially when it comes to 2xAARE YB_TATA hour.

[0864] Therefore, according to another aspect of the invention, the present invention relates to the use of chemical inducers in the expression of transgenes contained in an inducible promoter activation or inducible expression cassette, wherein the transgene is controlled by the inducible promoter.

[0865] The inducible promoter is selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB _TATA and miniCHAC1 YB_TATA ,and

[0866] The chemical inducers are selected from: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

[0867] Alternatively, the present invention relates to a method for inducing the activation of an inducible promoter or the expression of a transgene contained in an inducible expression cassette, wherein the transgene is controlled by the inducible promoter.

[0868] The inducible promoter is selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB _TATA and miniCHAC1 YB_TATA ,and

[0869] The chemical inducers are selected from: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

[0870] Whether using the chemical inducer described above or the induction method described above, the implementation of an effective and safe cell therapy in the sense of this invention involves administering the chemical inducer to a patient (if in need). Therefore, this inducer, particularly selected from artesunate, asparaginase, and bortezomib, can be a pharmaceutical composition, particularly in the form of an injectable pharmaceutical composition. In doing so, the invention also relates to a chemical inducer (or a pharmaceutical composition containing the chemical inducer) for inducing the activation of an inducible promoter or the expression of a transgene contained in an inducible expression cassette, wherein the transgene is controlled by the inducible promoter.

[0871] The inducible promoter is selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB _TATA and miniCHAC1 YB_TATA ,

[0872] The chemical inducers are selected from: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

[0873] According to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the chemical inducer is artesunate (Art).

[0874] According to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the chemical inducer is asparaginase (Asp).

[0875] According to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the chemical inducer is bortezomib (Bz).

[0876] According to another embodiment, the present invention relates to the use of the chemical inducing agent as described above or the induction method as described above, wherein the inducible promoter is selected from:

[0877] - 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1;

[0878] - 2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2;

[0879] - 4xSRE YB_TATAIts nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0880] - 9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0881] - GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0882] - miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 6; and

[0883] - miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7.

[0884] According to another embodiment, the present invention relates to the use of the chemical inducing agent as described above or the induction method as described above, wherein the inducible promoter is selected from:

[0885] - 2xAARE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1;

[0886] - 2xAARE TK Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2;

[0887] - 4xSRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3;

[0888] - 9xHRE YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4;

[0889] - GRP78 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 5;

[0890] - miR223 promoter, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and

[0891] - miniCHAC1 YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0892] According to another embodiment, the present invention relates to the use of the chemical inducing agent as described above or the induction method as described above, wherein the inducible promoter is 2xAARE. YB_TATA Its nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 1. In particular, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the inducible promoter is 2xAARE. YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1.

[0893] This aspect can also implement expression cassettes. It should be noted that an expression cassette refers to a nucleic acid containing the inducible promoter sequence according to the present invention, located at the 5' end (upstream) of the transgene. It should be noted that the transgene can be selected from: oncogenes, anti-cancer genes, transcription factors, nutritional factors, cytokines, enzymes, hormones, receptors, transport proteins, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic or suicide genes. In fact, and according to another embodiment, the present invention relates to the use of the chemical inducer described above or the induction method described above, wherein the transgene is selected from: oncogenes, anti-cancer genes, transcription factors, nutritional factors, cytokines, enzymes, hormones, receptors, transport proteins, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic or suicide genes. In particular, the present invention relates to the use of the chemical inducer described above or the induction method described above, wherein the transgene is selected from: transcription factors, cytokines, transport proteins, nucleases, chimeric antigen receptors (CARs), and toxic or suicide genes.

[0894] "Transcription factors" are proteins that interact with DNA and RNA polymerases and are essential for the initiation or regulation of gene transcription in all organisms (prokaryotic or eukaryotic). Examples of such factors in this invention include c-JUN, FOXO1, BATF, TBET, and NRF2.

[0895] Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgenic material is selected from: c-JUN, FOXO1, BATF, TBET, and NRF2. In particular, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgenic material is c-JUN. In particular, the present invention also relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgenic material is 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 encodes an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 764. Advantageously, the subject matter of the present invention is the use of the chemical inducer as described above or the induction method as described above, wherein the transgenic material is 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.

[0896] "Cytokines" are proteins secreted by cells of the immune system that enable them to communicate with each other, and thus these cytokines participate in the activation of the immune response. Examples of cytokines in this invention include IL2, IFNγ, TNFα, IL18, and IL12.

[0897] Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is selected from: IL2, IFNγ, TNFα, IL18 and IL12.

[0898] "Transporter protein" refers to a transmembrane protein whose function is to transport metabolites such as glucose (e.g., GLUT1). Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is GLUT1.

[0899] "Nuclease" generally refers to CRISPR-related proteins (clustered regularly spaced short palindromic repeats). It corresponds to the nucleases of the CRISPR-Cas system, a prokaryotic adaptive defense mechanism used to destroy invading foreign DNA. This functional framework has since been diverted from its original prokaryotic immune function and is now used in biotechnology for gene targeting, processing, modification, disruption, and programmed repair. To date, several Cas proteins (Cas9, Cas12a, Cas12b, CasX or Cas12e, Cas12f or Cas14, Cas12j or CasΦ) from different hosts have been identified and characterized, and even modified (e.g., their functions have been improved), all of which can be used by this invention for the engineering of mammalian genomes. Generally, the sizes of the aforementioned Cas proteins decrease sequentially: Cas9 is approximately 1,500 amino acids (AA), Cas12a is approximately 1,000 AA, while CasX, Cas12f, and Cas12j are all less than 1,000 AA.

[0900] Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the genetic material is selected from:

[0901] - Cas9, Cas12a, Cas12b, CasX or Cas12e nucleases, type V CRISPR, Cas12j or CasΦ;

[0902] - Cas9, Cas12a, Cas12b, CasX or Cas12e orthologs, V-type CRISPR, Cas12j or CasΦ; and

[0903] - Mutants or functional variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ.

[0904] "Orthologs" refer to similar Cas proteins found in two or more different species. "Mutants" refer to Cas proteins in which one or more mutations (including deletions, substitutions, and / or additions of one or more amino acids) have been introduced. These mutations can particularly increase nuclease activity and / or increase the fidelity of target DNA recognition. "Functional mutants" refer to Cas proteins that have been artificially modified (e.g., through genetic engineering), for example, to increase Cas activity. "Functional variants" refer to Cas proteins that have been naturally modified through evolution and, for example, exhibit increased activity.

[0905] Advantageously, the present invention uses Cas9 nucleases, such as those from *Streptococcus pyogenes*, *Staphylococcus aureus*, *Corynebacterium diphtheriae*, *Neisseria meningitidis*, *Streptococcus canis*, *Streptococcus simianus*, *Streptococcus tularensis*, *Aminococcus*, *Campylobacter jejuni*, *Streptococcus pneumoniae*, and *Streptococcus thermophilus*, and mutants thereof. Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgenic material is selected from:

[0906] - Cas9 nucleases from *Streptococcus pyogenes*, *Staphylococcus aureus*, *Corynebacterium diphtheriae*, *Neisseria meningitidis*, *Streptococcus canis*, *Streptococcus maculata*, *Streptococcus tularensis*, *Streptococcus aminoacidae*, *Campylobacter jejuni*, *Streptococcus pneumoniae*, and *Streptococcus thermophilus*; and

[0907] - Cas9 orthologs and functional Cas9 mutants or variants derived from these organisms.

[0908] In particular, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is saCas9, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 765, or the nucleic acid thereof encodes the saCas9 protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 766. In particular, the present invention also relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is saCas9, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 765, or the nucleic acid thereof encodes the saCas9 protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 766.

[0909] "Chimeric antigen receptor (CAR)" refers to a protein produced in the laboratory that can recognize and target proteins present on the surface of solid tumor cells to be treated. For example, the following CARs can be listed: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 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-ROR1 CAR, anti-ROR2 CAR, anti-PD1 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 TCells Encompassing 1F5-, Leu16-, and 2F2-Based Antigen-Recognition Moieties. Int. J. Mol. Sci. 2023, 24, 3698.).

[0910] Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is a CAR selected from the following: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 CAR, anti-NKG2D CAR, anti-PSMA CAR, anti-TRP-1 CAR, anti-TRP2 CAR, anti-VEGFR CAR, anti-HLA-G CAR, and anti-CLDN18.2. CAR, anti-EPCAM CAR, anti-FAP CAR, anti-ROR1 CAR, anti-ROR2 CAR, anti-PD1 CAR and anti-PSCACAR.

[0911] "Toxic or suicide transgenes" refer to nucleic acids encoding toxic, cytotoxic, or suicide proteins. Furthermore, upon activation of the inducible promoter sequence of this invention using pharmacological molecules, expression of the toxic or suicide transgene causes (leads to) cell death in genetically modified cells expressing that transgene. In this invention, the toxic or suicide transgene may be selected from genes encoding: inducible Caspase-9 (iC9), Bax (S184 del), Noxa (wild-type protein or one of its mutants, particularly the S13A mutant), Gasdermin B (N-ter), constitutively active caspase-3 protein (V266E), the M2 ion channel of influenza virus (H37A), diphtheria toxin subunit A (DTA) and an attenuated aDTA mutant (G128D), streptococcal hemolysin O (particularly streptococcal hemolysin O of Streptococcus pyogenes), and enterotoxins (particularly enterotoxins of Clostridium perfringens). These are non-secreting toxic or suicide proteins. In other words, once the inducible promoter sequences of this invention downstream are activated, the products of expression (transcription and then translation) of the nucleic acids encoding them are not found in the extracellular environment. Therefore, only genetically modified cells expressing them experience cell death.

[0912] Therefore, according to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is selected from the following toxic or suicide transgenes: inducible Caspase-9 protein (iC9), Bax protein (S184 del), Noxa protein, Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 ion channel (H37A), diphtheria toxin subunit A (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0913] According to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is selected from the following toxic or suicide transgenes:

[0914] - An inducible Caspase-9 protein (iC9) whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes an inducible Caspase-9 protein (iC9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 9;

[0915] - Bax(S184 del) protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 10, or the nucleic acid of which encodes Bax(S184 del) protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 11;

[0916] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes an M2 (H37A) ion channel of an influenza virus, wherein the amino acid sequence of the M2 (H37A) ion channel of the influenza virus has at least 80% identity with the sequence SEQ ID NO: 13;

[0917] - A constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence that is at least 80% identical 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 is at least 80% identical to the sequence SEQ ID NO: 15;

[0918] - Diphtheria toxin subunit A (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 diphtheria toxin subunit A (DTA), the amino acid sequence of said diphtheria toxin subunit A (DTA) having at least 80% identity with 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 aDTA mutant (G128D), the amino acid sequence of said aDTA mutant (G128D) having at least 80% identity with sequence SEQ ID NO: 19.

[0919] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 21;

[0920] - Noxa protein, the nucleic acid of which has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which is at least 80% identical to the sequence SEQ ID NO: 23;

[0921] - Streptococcal hemolysin 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 streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O has at least 80% identity with the sequence SEQ ID NO: 756; and

[0922] - Enterotoxin whose nucleic acid has a sequence that is at least 80% identical to 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.

[0923] According to another embodiment, the present invention relates to the use of the chemical inducer as described above or the induction method as described above, wherein the transgene is selected from the following toxic or suicide transgenes:

[0924] - Inducible Caspase-9 protein (iC9), whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8, or whose nucleic acid encodes inducible Caspase-9 (iC9), the amino acid sequence of said inducible Caspase-9 (iC9) corresponding to sequence SEQ ID NO: 9;

[0925] - Bax(S184 del) protein, whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10, or whose nucleic acid encodes Bax(S184 del) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 11;

[0926] - An M2 (H37A) ion channel of an influenza virus, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12, or the nucleic acid of which encodes the M2 (H37A) ion channel of an influenza virus, the amino acid sequence of the M2 (H37A) ion channel of the influenza virus corresponding to the sequence SEQ ID NO: 13;

[0927] - 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), the amino acid sequence of which corresponds to the sequence SEQ ID NO: 15;

[0928] - Diphtheria toxin subunit A (DTA), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 16, or whose nucleic acid encodes diphtheria toxin subunit A (DTA), the amino acid sequence of which corresponds to sequence SEQ ID NO: 17; and an attenuated mutant aDTA (G128D), whose nucleic acid has a sequence corresponding to the nucleic acid in sequence SEQ ID NO: 18, or whose nucleic acid encodes an attenuated aDTA (G128D) mutant, the amino acid sequence of which corresponds to sequence SEQ ID NO: 19;

[0929] - Gasdermin B (N-ter) protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20, or the nucleic acid of which encodes Gasdermin B (N-ter) protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 21;

[0930] - Noxa protein, the nucleic acid of which has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22, or the nucleic acid of which encodes Noxa protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 23;

[0931] - Streptococcal hemolysin O, whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 755, or whose nucleic acid encodes streptococcal hemolysin O, wherein the amino acid sequence of said streptococcal hemolysin O corresponds to the sequence SEQ ID NO: 756; and

[0932] - Enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid sequence SEQ ID NO: 757, or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 758.

[0933] In view of the foregoing, the present invention also relates to the use of the chemical inducer as described above or the induction method as described above, wherein the genetic modification is:

[0934] - Selected from the following transcription factors: c-JUN, FOXO1, BATF, TBET, and NRF2;

[0935] - Selected from the following cytokines: IL2, IFNγ, TNFα, IL18, and IL12;

[0936] - GLUT1 transporter protein;

[0937] - Selected from the following nucleases: Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ, their orthologs and their functional mutants or variants;

[0938] - Selected from the following CARs: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 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-ROR1 CAR, anti-ROR2 CAR, anti-PD1 CAR, and anti-PSCACAR; or

[0939] - Selected from the following toxic or suicide transgenic proteins: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein, Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0940] According to another aspect of the present invention, the present invention relates to a kit comprising:

[0941] - Nucleic acid comprising the inducible promoter as described above or the transgene contained in the expression cassette as described above, wherein the transgene is controlled by the inducible promoter; and

[0942] - At least one chemical inducer selected from the following: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

[0943] According to another embodiment, the present invention relates to a kit as described above, wherein the chemical inducer is artesunate (Art).

[0944] According to another embodiment, the present invention relates to a kit as described above, wherein the chemical inducer is asparaginase (Asp).

[0945] According to another embodiment, the present invention relates to a kit as described above, wherein the chemical inducer is bortezomib (Bz).

[0946] According to another embodiment, the present invention relates to a kit as described above, wherein the inducible promoter is selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA .

[0947] According to another embodiment, the present invention relates to a kit as described above, wherein the inducible promoter is selected from:

[0948] - 2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1;

[0949] - 2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2;

[0950] - 4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0951] - 9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0952] - GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0953] - miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 6; and

[0954] - miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7.

[0955] According to another embodiment, the present invention relates to a kit as described above, wherein the inducible promoter is 2xAARE. YB_TATAThe nucleic acid has a sequence with at least 80% identity to the nucleic acid of SEQ ID NO: 1. In particular, the present invention relates to a kit as described above, wherein the inducible promoter is 2xAARE. YB_TATA Its nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1.

[0956] According to another embodiment, the present invention relates to a kit as described above, wherein the genetically modified organism is:

[0957] - Selected from the following transcription factors: c-JUN, FOXO1, BATF, TBET, and NRF2;

[0958] - Selected from the following cytokines: IL2, IFNγ, TNFα, IL18, and IL12;

[0959] - GLUT1 transporter protein;

[0960] - Selected from the following nucleases: Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ, their orthologs and their functional mutants or variants;

[0961] - Selected from the following CARs: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 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-ROR1 CAR, anti-ROR2 CAR, anti-PD1 CAR, and anti-PSCACAR; or

[0962] - Selected from the following toxic or suicide transgenic proteins: inducible Caspase-9 protein (iC9), Bax protein (S184del), Noxa protein, Gasdermin B protein (N-ter), constitutively active caspase-3 protein (V266E), influenza virus M2 (H37A) ion channel, diphtheria toxin A subunit (DTA) and attenuated aDTA mutant (G128D), streptococcal hemolysin O (especially streptococcal hemolysin O of Streptococcus pyogenes) and enterotoxin (especially enterotoxin of Clostridium perfringens).

[0963] In all respects, it should be noted that the various aspects and embodiments of the invention are interdependent. The latter can therefore be frequently combined with each other as needed to obtain preferred aspects and / or embodiments of the invention not explicitly described. This also applies to all definitions provided herein, which apply to all aspects and embodiments of the invention.

[0964] Furthermore, the present invention is illustrated by the following figures and embodiments, but is not limited thereto. Attached Figure Description

[0965] Figure 1 Targeting 2xAARE YB_TATA Screening for the most effective gRNA for the promoter (SEQ ID NO: 1).

[0966] The expression of dCas9-KRAB fusion protein (SEQ ID NO: 325 and SEQ ID NO: 326) and targeting 2xAARE YB_TATA Plasmids containing different gRNAs with the promoter sequence (SEQ ID NO: 1) were transfected into a system carrying 2xAARE. YB_TATA HEK-293T cells were transduced with a lentiviral vector containing the FLuc box (SEQ ID NO: 331). Luciferase activity of FLuc was measured 24 hours post-transfection. RLU, relative optical units; n = 4 replicates; Kruskal-Wallis test; ns, P>0.05; *, P≤0.05; **, P≤0.01.

[0967] Figure 2 Expression of dCas9-KRAB fusion protein and gRNA (2xAARE) YB_TATA HEK-CRISPRi 2xAARE Screening of clone populations.

[0968] A) From the expression cassette carrying the dCas9-KRAB fusion protein (SEQ ID NO: 325 and SEQ ID NO: 326) and the previously selected gRNA (2xAARE) YB_TATALimiting dilution clones were created in HEK-293T cells transduced with a lentiviral vector containing dCas9-KRAB (SEQ ID NO: 318). The presence of dCas9-KRAB fusion protein (SEQ ID NO: 325 and SEQ ID NO: 326) and gRNA (2xAARE) in each clone was verified by Western blotting. YB_TATA (SEQ ID NO: 318) expression. B) HEK-293T cells or cells from each expressing dCas9-KRAB fusion protein (SEQ ID NO: 325 and SEQ ID NO: 326) and gRNA (2xAARE) YB _TATA Cells of the clone line (SEQ ID NO: 318) carrying 2xAARE YB_TATA - Lentiviral plasmid transfection with the FLuc box (SEQ ID NO: 331). Luciferase activity derived from FLuc expressed by the plasmid was measured 24 hours later and normalized to the total protein content in the sample. Clonal lines with the most suppressed FLuc transgene expression were selected.

[0969] Figure 3 Carrying 2xAARE YB_TATA (SEQ ID NO: 1) Schematic diagram of promoter-controlled meaningful (pLV; top) and inverse (pLVi; bottom) lentiviral vectors.

[0970] Figure 4 Effects of transcription clamp expression on transgene expression during HEK-293T cell transfection.

[0971] A) Under recombinant particle production conditions, HEK-293T cells (HEK-CRISPRi) 2xAARE ) in CRISPRi 2xAARE The expression of the clamp leads to pLV-2xAARE YB_TATA -Luc (SEQ ID NO: 334) luciferase expression was significantly reduced compared to the same plasmid transfected into HEK-293T cells. B) In HEK-CRISPRi 2xAARE In cells and in the presence of TAT, luciferase expression of the pLVi plasmid (SEQ ID NO: 335) was significantly lower than that of the same plasmid transfected into HEK-293T cells in the presence of TAT. S: significant, I: reversed. Statistical analysis: unpaired t-test, (**) p < 0.01; (***) p < 0.001.

[0972] Figure 5 HEK-CRISPRi 2xAAREExpression of KRAB-dCas9 in cells increases the titer of pLVi carrying the 2xAARE-suicide cassette.

[0973] A) When in HEK-293T-CRISPR 2xAARE When produced in Ci cells, the titer of the 2xAARE-GFP-suicide gene pLVi was significantly higher than that obtained in HEK-293T (WT) cells. Each column represents the mean ± standard deviation of three different experiments. Statistical analysis, two-way ANOVA and Sidak multiple comparison test: (ns) p > 0.05; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001.

[0974] Figure 6 Functionality of pLVi vectors carrying cytotoxic transgenes.

[0975] For by in CRISPRi 2xAARE LVi (2xAARE) produced in HEK cells YB Flow cytometry analysis of human CD3+ lymphocyte mortality induced by the expression of the suicide transgene induced by the transduction and integration of the (eGFP-P2A-GT) vector. Mortality was measured by the percentage of cells double-labeled with annexin V and propidium iodide; n = 3 donors; Art, artesunate; Amt, amantadine; AP1903, a dimerizing agent for iC9. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test or one-way ANOVA and Šídák's multiple comparison test: (ns) p>0.05; (*) p<0.05; (**) p<0.01; (***) p<0.001; (****) p<0.0001.

[0976] Figure 7 GFP expression in genetically modified CD8+ T lymphocytes was activated and treated with the pharmacological molecules asparaginase (Asp, 2 U / ml), bortezomib (Bz, 10 nM), or artesunate (Art, 5 µM).

[0977] Resting CD3+ T cells were transduced with pTRIP-2xAARE-GFP vector, activated with CD3 / CD28 beads, and treated with DMSO, artesunate (Art), asparaginase (Asp), bortezomib (Bz), or tunicamycin (Tm, 4 µg / ml) for 16 h. Analysis was performed by flow cytometry. A) Percentage of cells expressing GFP. B) Mean fluorescence intensity (MFI). Each spot corresponds to one donor. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test, with each condition relative to the resting condition (Rest.) (p>0.05, ns; p≤0.05, *; p≤0.01, **; p≤0.001, ***; p≤0.0001, ****).

[0978] Figure 8 In the presence of stress-inducing drugs, CD8+ T lymphocytes lyse tumor cells.

[0979] A) EST109, melanoma cell line; B) IRNE, melanoma cell line; C) MM1S, multiple myeloma cell line. Each target cell line (T) was co-incubated with increasing numbers of effector CD8+ T cells (E) at ratios of 1:5, 1:25, and 1:50. After 16 hours of culture, the remaining cells in the wells were lysed, and luciferase activity was quantified. Each spot represents the total amount of luciferase measured per well.

[0980] Figure 9 Induction of CAR expression in genetically modified CD3+ lymphocytes.

[0981] Activated CD3+ T lymphocytes were transduced using a 2xAARE-CAR lentiviral vector. Cells were maintained under normal culture conditions (Ctl), or treated with amino acid deficiencies of methionine (Met), tryptophan (Trp), glutamine (Gln), leucine (Leu), arginine (Arg), or lysine (Lys), or with the drugs L-asparaginase (L-Asp) or artesunate (Art). A) Percentage of GFP-expressing T cells as measured by flow cytometry. B) Mean fluorescence intensity (MFI) per cell as measured by flow cytometry. Statistical analysis was performed using a one-way ANOVA (p>0.05, ns; p≤0.05, *; p≤0.01, **; p≤0.001, ***; p≤0.0001, ****).

[0982] Figure 10 Regulation of transcription factor expression in CD3+ lymphocytes.

[0983] CD3+ lymphocytes were transduced using LV-2xAARE-GFP-Tbet or LV-2xAARE-GFP-Foxp3 vectors, and then treated with culture medium, arginase (Arg), asparaginase (Asp), or artesunate (Art) 24 hours after treatment. A) Expression of GFP and T-bet transgenes in modified T cells. B) Expression of IFNγ and TNFα cytokines in T lymphocytes modified with LV-2xAARE-GFP-Tbet vector 24 hours 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. Statistical analysis was performed using one-way ANOVA (p>0.05, ns; p≤0.05, *; p≤0.01, **; p≤0.001, ***; p≤0.0001, ****).

[0984] Example

[0985] Example 1 - Guide RNA (gRNA) Screening

[0986] The CRISPR interference (CRISPRi) and CRISPR targeted methylation (CRISPRm) strategies according to the present invention are based on the fusion of an inactivated or “dead” CRISPR-Cas endonuclease (dCas) with a KRAB (Krüppel-associated box) transcriptional repression domain or a DNMT (DNA methyltransferase) domain. dCas can target specific sequences with the aid of its guide RNA (gRNA).

[0987] For promoter-specific targeting, only the gRNA sequence needs to be adjusted.

[0988] To this end, computational 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–Cpf1 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 the complementarity of the gRNA sequence with the target and the lack of complementarity with the host cell genome. Next, different gRNA sequences must be functionally screened to determine which allows the most efficient CRISPR interference and / or methylation at the promoter in question.

[0989] For example, screening for 2xAARE targets YB_TATA Several RNA sequences of the promoter (SEQ ID NO: 1), namely:

[0990] gRNA 1, sequence SEQ ID NO: 318;

[0991] gRNA 2, sequence number SEQ ID NO: 388;

[0992] gRNA 3, sequence number SEQ ID NO: 389; and

[0993] gRNA 4, sequence SEQ ID NO: 390,

[0994] Its sequence was obtained using CRISPR tools. Therefore, 2xAARE was transduced via lentiviral transduction. YB_TATA-FLuc "target" (SEQ ID NO: 331) was integrated into HEK-293T cells (see Example 2 – Materials and 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 and Methods). The gRNA that most effectively targets CRISPRi to reduce basal FLuc expression was selected ( Figure 1 It is gRNA 1 with the sequence SEQ ID NO: 318.

[0995] Next, establish HEK-CRISPRi 2xAARE Cloning lines were developed (see Example 2 – Materials and Methods). For this purpose, expression cassettes carrying the CRISPRi complex (dCas9-KRAB fusion; SEQ ID NO: 326) and previously selected gRNA (2xAARE) were prepared. YB_TATA (pLVi-2xAARE) YB_TATA The HEK-293T population transduced with the lentiviral vector of (-Luc; SEQ ID NO: 318) was subjected to limiting dilution. Expression of the CRISPRi complex was tested in each clone line by Western blotting. Figure 2 -A).

[0996] Then, the expression of the CRISPRi complex (dCas9-KRAB fusion; SEQ ID NO: 326) and gRNA (2xAARE) was tested. YB_TATA The functionality of each clone of (SEQ ID NO: 318) is described in Example 2 – Materials and Methods. For this purpose, each clone line is equipped with a carrier of 2xAARE. YB_TATA Transfection with a lentiviral plasmid (SEQ ID NO: 335) containing controlled secretory nanoluciferase (nLuc). Clones in which transgene expression was suppressed were selected. Figure 2 -B). It is clone 17.

[0997] Therefore, for any other promoter, a similar gRNA screening step must be performed, followed by the establishment of cloning lines (CRISPRi and / or CRISPRm).

[0998] Example 2 - Production of the Toxicity Carrier (CRISPRi)

[0999] Materials and Methods

[1000] Cloning of lentiviral plasmids

[1001] The lentiviral plasmid used in the experiment was cloned by inserting a synthesized sequence (Twist Bioscience) into a plasmid (pLV(SIN); SEQ ID NO: 199) carrying a second-generation self-inactivated lentiviral genome, and then using the NEBuilder HiFi DNA assembly protocol (New England Biolabs) via enzymatic linearization and homology-based recircularization.

[1002] Cell culture

[1003] Cells were cultured in a humidified, controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (human embryonic kidney, ATTC–CRL-11268) cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL penicillin / streptomycin. Stress induction was achieved by treatment with tunicamycin (4 µg / mL, Sigma-Aldrich). EST109 cells (published under the number 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 (RPMI 1640, Thermo Fischer) medium containing 10% FCS and 100 U / mL penicillin / streptomycin. Primary T lymphocytes isolated from healthy donor blood were cultured in serum-free, nutrient-free medium (SFM) GIBCOOpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL penicillin / streptomycin.

[1004] Production of lentiviral vectors

[1005] Non-replicating lentiviral particles were obtained using a second-generation production protocol described by Zennou et al. in 2001 (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded on cell culture dishes and allowed to reach 50%-60% confluence after 24 hours of culture. The next day, cells were co-transfected using calcium phosphate precipitation with two trans complementary plasmids: one encoding the lentiviral genome (pLV or pLVi), and the other encoding the enzyme and structural protein of HIV-1 (p8.9; SEQ ID NO: 338 or SEQ ID NO: 339), and the other encoding 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 incubated with the precipitate for 5 hours, and then the culture medium was changed. The supernatant containing lentiviral particles was collected after 48 hours. Lentiviral suspensions were treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 µm), and subsequently ultracentrifuged at 60,000 g for 90 min at 4 °C. The particle pellet was resuspended in a volume corresponding to 1000-fold concentration of phosphate-buffered saline (PBS). The resulting suspensions were aliquoted and stored at -80 °C until use.

[1006] titer determination

[1007] The concentration of lentiviral particles was determined by quantifying the lentiviral capsid protein p24. For this purpose, aliquots of the lentiviral suspension were thawed, lysed, and diluted according to the supplier's protocol for the HIV-1 p24 antigen ELISA 2.0 titration kit (Zeptometrix reference number 0801008). The obtained p24 concentration is expressed as pg / µL, representing the concentration per 10^10 μL of lentiviral protein p24. 4 One VSVg pseudotyped lentivirus particle is equivalent to 1 pg of p24 (Dull, T et al. J Virol. 1998 Nov;72(11):8463-71). Tests can improve these values, but on average, 1% of the physical particles obtained are infectious, i.e., 1 pg ≈ 100 transduction units (TUs).

[1008] Transduction

[1009] To integrate the transgene via infection with a lentiviral vector, dilute the amount of particles in the culture medium to obtain the desired multiplicity of infection (vector:target ratio). Contact the lentiviral suspension with the target cells. Culture the infected cells for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression can be observed or measured the following day by microscopy, cell counting, or biochemical analysis.

[1010] Establishment of the KRAB (CRISPRi) cell line

[1011] HEK 293T cells were transduced using a lentiviral vector (SEQ ID NO: 328) carrying the following constitutive expression cassette:

[1012] - Under the control of the minimal eF-1α promoter (SEQ ID NO: 319), the dCas9-KRAB fusion protein (SEQ ID NO: 325) was fused to the blast fungicide resistance gene (BlastR) (SEQ ID NO: 327) via a 2A peptide (SEQ ID NO: 332); and

[1013] - Guide RNA (SEQ ID NO: 318) that is specific to the target promoter under the control of the U6 promoter (SEQ ID NO: 317).

[1014] Two days later, the transduced cells were screened by treatment with blastomycin (2 µg / mL). Cells were cloned by limiting dilutions, and each clone was characterized.

[1015] Flow cytometry analysis

[1016] At the end of the experiment, the cultured cells were collected and washed with PBS. They were then incubated with annexin V (AnV) and propidium iodide (PI) for 30 minutes. The cells were washed again and then fixed with PBS containing 1% paraformaldehyde at 4°C for 15 minutes. After washing, AnV+ / IP+ double labeling was used to measure cell death (the proportion of cells in late apoptosis) using a MACSQuant Analyzer 10 (Miltenyi) flow cytometer.

[1017] Luciferase activity measurement

[1018] The firefly luciferase used in the experiment was an intracellular luciferase. Luciferase activity was measured on cell culture lysates using the Luciferase Assay System kit (Promega) according to the supplier's instructions. Luminescence was measured using a SpectraMax i3x microplate reader (Molecular Devices).

[1019] result

[1020] Effects of transcription clamp on promoters inside the vector

[1021] To block 2xAARE on the plasmid encoding the viral vector during viral particle production. YB_TATATranscription of the transgene upstream of the promoter (SEQ ID NO: 1) yielded a HEK-293T cell line expressing an inactivated Cas9 protein (dCas9) fused with the transcriptional repressor KRAB domain (SEQ ID NO: 326) and 2xAARE. YB_TATA A specific guide RNA (SEQ ID NO: 318) (HEK-CRISPRi) is present at the ATF4 binding site on the promoter. 2xAAR ).

[1022] To compare from 2xAARE YB_TATA The expression level of the transgene (luciferase) in (SEQ ID NO: 1) was determined, and two plasmids were constructed: pLV-2xAARE YB_TATA -Luc (SEQ ID NO: 334) and pLVi-2xAARE YB_TATA -Luc (SEQ ID NO: 335), wherein the 2xAARE-Luc expression cassette (SEQ ID NO: 331) is in a meaningful position (pLV) or an inverted position (pLVi). Figure 3 Then, in the presence of TAT (transcription transactivator), these plasmids were transfected into the HEK-293T cell line or HEK-CRISPRi. 2xAARE In cell lines.

[1023] Ultimately, from 2xAARE YB_TATA The presence of the transcriptional repressor clamp of the promoter (SEQ ID NO: 1) induced a decrease in luciferase expression of the pLV vector by approximately 1.5-fold, while the pLVi vector showed a decrease of more than 10-fold. Figure 4 A and 4B). Therefore, in the presence of TAT, compared with the pLV plasmid, the expression cassette is reversed and CRISPRi 2xAARE The cumulative effect of the clamp reduced transgene expression by more than 20,000 times.

[1024] Increased titer of lentiviral vectors expressing cytotoxic genes

[1025] To produce high-titer lentiviral vectors carrying cytotoxic (or suicide) genes (whose expression can be induced by ATF4), seven reverse lentiviral vectors (pLVi) were obtained and used to produce recombinant particles carrying control reporter transgenes or toxic transgenes. Figure 3 (See figure below). In each vector, the transgene was placed in 2xAARE. YB_TATA(SEQ ID NO: 1) Under the control of the promoter. The control vector (SEQ ID NO: 336) carries the "enhanced green fluorescent protein" reporter transgene (GFP; SEQ ID NO: 329), and six cytotoxic vectors (SEQ ID NO: 205 to SEQ ID NO: 210) carry the eGFP gene associated with the P2A peptide (SEQ ID NO: 332) selected from the following cytotoxic genes:

[1026] - Inducible Caspase-9 (iC9; SEQ ID NO: 8);

[1027] - Bax (S184 del; SEQ ID NO: 10);

[1028] - Influenza virus M2 ion channel (H37A; SEQ ID NO: 12);

[1029] - Constitutively active caspase-3 (V266E; SEQ ID NO: 14); and

[1030] - Diphtheria toxin A subunit (DTA; SEQ ID NO: 16) or attenuated mutant aDTA (G128D; SEQ ID NO: 18).

[1031] The resulting lentiviral vector library contains 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 (SEQID 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).

[1032] In HEK-293T (WT) cell line or HEK-CRISPRi 2xAAREQuantitative analysis of p24 in the culture supernatant after lentiviral particle production in cell lines showed that six vectors carrying the virulence gene were used with HEK-CRISPRi 2xAARE The cell line was produced at a significantly higher titer ( Figure 5 In fact, in HEK-CRISPRi 2xAARE The production of recombinant vector particles in cells increases the titer: approximately 8-fold for Bax, approximately 5-fold for aDTA and Casp3, approximately 4-fold for M2, approximately 3-fold for GFP, and approximately 2-fold for iC9.

[1033] Finally, producing lentiviral particles carrying a toxic (or suicide) transgene located downstream of the promoter using animal eukaryotic cells expressing a ribonucleoprotein complex has proven to be an alternative method for producing high-titer lentiviral vectors, wherein the ribonucleoprotein complex comprises:

[1034] - dCas9-KRAB fusion protein, comprising the fusion of an inactivated Cas9 endonuclease with a KRAB transcriptional repressor domain; and

[1035] - Guide RNA (gRNA) that specifically recognizes the promoter sequence of the promoter.

[1036] The produced reverse lentivirus vector is functional

[1037] Using CRISPRi 2xAARE LVi(2xAARE) produced in HEK cells YB Primary human T cells were transduced with GFP-P2A-GT (MOI = 10). Therefore, the gene:

[1038] - DTA (pLVi-2xAARE YB_TATA -GFP-P2A-DTA; SEQ ID NO: 209),

[1039] - aDTA (pLVi-2xAARE YB_TATA -GFP-P2A-aDTA; SEQ ID NO: 210),

[1040] - Bax (pLVi-2xAARE YB_TATA -GFP-P2A-Bax; SEQ ID NO: 206),

[1041] - Noxa (pLVi-2xAARE YB_TATA -GFP-P2A-Noxa; SEQ ID NO: 212),

[1042] - Casp3 (pLVi-2xAAREYB_TATA -GFP-P2A-Casp3; SEQ ID NO: 208),

[1043] - M2 (pLVi-2xAARE YB_TATA -GFP-P2A-M2; SEQ ID NO: 207) and

[1044] - iC9 (pLVi-2xAARE YB_TATA -GFP-P2A-iC9; SEQ ID NO: 205)

[1045] It was integrated into the genome of cultured primary lymphocytes. In these experiments, cellular stress was induced by treatment with 5 µM artesunate (Art) to activate 2xAARE. YB Art is an antimalarial drug that induces oxidative stress, thereby promoting the expression of ATF4.

[1046] To directly measure the toxicity of the transgene, cells were labeled with annexin V (AnV) and propidium iodide (PI) 24 hours after induction with Art (±5 µM Amt to block the M2 effect, or ±4 µMAP1903 to dimerize iC9). AnV+ / PI+ co-labeling was used to measure mortality (the proportion of cells in late apoptosis) by flow cytometry.

[1047] In these experiments on cells from three donors, Art treatment induced 22.5% LVi (2xAARE) YB -eGFP) transduced cell death, compared to 17.3% in untreated cells (-eGFP). Figure 6 A).

[1048] In the uninduced state, most GTs (Bax, Noxa, Casp3, aDTA, and DTA) caused a baseline mortality rate of 30% (Bax) to 34.8% (aDTA), an increase of 10% compared to the control transgenics. Figure 6 A, white column). However, 24 hours after Art induction, the measured mean mortality rate was significantly higher, particularly for Bax (68.6%), Noxa (68.2%), Casp3 (63.6%), and aDTA (69%). Figure 6 A, shaded column).

[1049] For M2, the mortality rate induced by basal expression (44%) was higher than that of the previous GT. This rate decreased in the presence of Amt (25.3%). Art induction of the transgene significantly increased the mortality rate induced by M2 (68.8%), even in the presence of Amt (48%). Therefore, 2xAAREYB The toxicity of basal expression of the -eGFP-P2A-M2 box can be prevented by Amt treatment, while overexpression of M2 induces cell death. Figure 6 B).

[1050] Finally, 2xAARE YB Baseline expression of the -eGFP-P2A-iC9 cassette induced comparable mean mortality in the absence (35.8%) and presence (43.5%) of AP1903 dimer. This baseline mortality was significantly higher than that obtained in cells transduced with the control vector and treated in the same manner. Art induction of iC9 significantly increased these rates in both the absence (70.1%) and presence (83.6%) of AP1903. Therefore, these data demonstrate the high potency of iC9 in inducing T cell death. Figure 6 C).

[1051] Example 3 - Production of the toxic vector (CRISPRm)

[1052] Materials and Methods

[1053] Cloning of lentiviral plasmids

[1054] The lentiviral plasmid used in the experiment was cloned by inserting a synthesized sequence (Twist Bioscience) into a plasmid (SIN) (pLV; SEQ ID NO: 199) carrying a second-generation self-inactivated lentiviral genome, and then using the NEBuilder HiFi DNA assembly protocol (New England Biolabs) via enzymatic linearization and homology-based recircularization.

[1055] Cell culture

[1056] Cells were cultured in a humidified, controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (human embryonic kidney, ATTC–CRL-11268) cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL penicillin / streptomycin. Stress induction was achieved by treatment with tunicamycin (4 µg / mL, Sigma-Aldrich). EST109 cells (published under the number 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 (RPMI 1640, Thermo Fischer) medium containing 10% FCS and 100 U / mL penicillin / streptomycin. Primary T lymphocytes isolated from healthy donor blood were cultured in serum-free, nutrient-free (SFM) GIBCO OpTmizer™ CTS™ medium (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL penicillin / streptomycin.

[1057] Production of lentiviral vectors

[1058] Non-replicating lentiviral particles were obtained using a second-generation production protocol described by Zennou et al. in 2001 (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded on cell culture medium and allowed to reach 50%-60% confluence after 24 hours of culture. The next day, cells were co-transfected using calcium phosphate precipitation with two trans complementary plasmids: one encoding the lentiviral genome (pLV or pLVi), and the other encoding the enzyme and structural protein of HIV-1 (p8.9; SEQ ID NO: 338 or SEQ ID NO: 339), and the other encoding 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 incubated with the precipitate for 5 hours, followed by a change of culture medium. The supernatant containing lentiviral particles was collected after 48 hours. Lentiviral suspensions were treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 µm), and subsequently ultracentrifuged at 60,000 g for 90 min at 4 °C. The particle pellet was resuspended in a volume corresponding to 1000-fold concentration of phosphate-buffered saline (PBS). The resulting suspensions were aliquoted and stored at -80 °C until use.

[1059] titer determination

[1060] The concentration of lentiviral particles was determined by quantifying the lentiviral capsid protein p24. For this purpose, aliquots of the lentiviral suspension were thawed, lysed, and diluted according to the protocol provided by the supplier of the HIV-1 p24 antigen ELISA 2.0 titration kit (Zeptometrix reference number 0801008). The obtained p24 concentration is expressed as pg / µL and is calculated as 10⁻⁶ pg / µL. 4 One VSVg pseudotyped lentivirus particle is equivalent to 1 pg of p24 (Dull, T et al. J Virol. 1998 Nov;72(11):8463-71). Tests can improve these values, but on average, 1% of the physical particles obtained are infectious, i.e., 1 pg ≈ 100 transduction units (TUs).

[1061] Transduction

[1062] To integrate the transgene via infection with a lentiviral vector, dilute the amount of particles in the culture medium to obtain the desired multiplicity of infection (vector:target ratio). Contact the lentiviral suspension with the target cells. Culture the infected cells for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression can be observed or measured the following day by microscopy, cell counting, or biochemical analysis.

[1063] Establishment of the KRAB (CRISPRi) cell line

[1064] HEK 293T cells were transduced using a lentiviral vector (SEQ ID NO: 328) carrying the following constitutive expression cassette:

[1065] - Under the control of the minimal eF-1α promoter (SEQ ID NO: 319), the dCas9-KRAB fusion protein (SEQ ID NO: 325) was fused to the blast fungicide resistance gene (BlastR) (SEQ ID NO: 327) via a 2A peptide (SEQ ID NO: 332); and

[1066] - A guide RNA (SEQ ID NO: 352) that is specific to the target promoter under the control of the U6 promoter (SEQ ID NO: 317).

[1067] Two days later, transduced cells were selected by treatment with blastomycin (2 µg / mL). The effect of limiting dilution on HEK-CRISPRi cells was also assessed. SV40 Cells are cloned, and each clone is characterized.

[1068] Establishment of KRAB (CRISPRi) and DNMT (CRISPRm) cell lines

[1069] HEK 293T cells were transduced using a lentiviral vector (SEQ ID NO: 387) carrying the following constitutive expression cassette:

[1070] - Under the control of the minimal eF-1α promoter (SEQ ID NO: 319), the KRAB-dCas9-DNMT3A-L fusion protein (SEQ ID NO: 375) was fused to the blast fungicide resistance gene (BlastR) (SEQ ID NO: 327) via a 2A peptide (SEQ ID NO: 332); and

[1071] - A guide RNA (SEQ ID NO: 352) that is specific to the target promoter under the control of the U6 promoter (SEQ ID NO: 317).

[1072] Two days later, transduced cells were selected by treatment with blastomycin (2 µg / mL). The effect of limiting dilution on HEK-CRISPRi / m SV40 Cells are cloned, and each clone is characterized.

[1073] result

[1074] To illustrate the value of the CRISPRm strategy, lentiviral particles were produced from the pLVi (pSV40-eGFP) or pLVi (pSV40-eGFP-P2A-M2) genome plasmid (SEQ ID NO: 609). These plasmids carried, in reverse orientation, a control transgenic eGFP or a virulent transgenic eGFP-P2A-M2 controlled by the early (constitutive) promoter of the SV40 virus (pSV40; SEQ ID NO: 343). Each of these plasmids was co-transfected with plasmid p8.9 (SEQ ID NO: 338 or SEQ ID NO: 339) and pVSVg (SEQ ID NO: 337) into the following cells:

[1075] - HEK-293T;

[1076] - HEK-CRISPRi SV40 (Expressing the dCas9-KRAB complex (SEQ ID NO: 326) and pSV40-specific gRNA (SEQ ID NO: 352)); or

[1077] - HEK-CRISPRi / m SV40(Expresses bifunctional KRAB-dCas9-DNMT3A-L complex (SEQ ID NO:376) and pSV40-specific gRNA (SEQ ID NO:352)).

[1078] In HEK-293T cells, pLV(SV40-eGFP) was significantly more effective than pLVi(SV40-eGFP) in producing LV particles. Furthermore, the titer decreased even further when the transgene was toxic to packaging cells, as observed in the production process using pLVi(SV40-eGFP-P2A-M2) (SEQ ID NO: 609), where the titer was significantly lower than that of pLVi(SV40-eGFP).

[1079] However, when the pLVi(SV40-eGFP-P2A-M2) (SEQ ID NO: 609) vector was used in HEK-CRISPRi SV40 During cell production, the number of lentiviral particles (estimated by measuring p24 concentration in the supernatant) was higher than that obtained in HEK-293T. These results demonstrate the effectiveness of the interference strategy in eliminating the transcription of reverse transgenes, thereby obtaining ≥10 9 Recombinant particle / mL lentiviral particle titer.

[1080] Finally, in HEK-CRISPRi / m SV40 Production in cells even allows for the acquisition of higher yields than in HEK-293T and HEK-CRISPRi (SV40) Higher vector titers are obtained in cells. The presence of the bifunctional CRISPRi / m complex during LV particle production thus allows for obtaining optimal titers of lentiviral vectors carrying constitutively promoter-controlled virulence transgenes.

[1081] To confirm the functionality of lentiviral particles obtained from different HEK lines, their toxicity was tested in an in vitro solid tumor model: 3D (spheroid) culture of EST109 human melanoma cells.

[1082] Each sphere was inoculated from 5 × 10⁶ cells in a 96-well circular plate (ultra-low adhesion plate) without any culture treatment. 3 EST109 cells were formed. Simultaneously with seeding, different cell lines (HEK-293T, HEK-CRISPRi) were used. SV40 and HEK-CRISPRi / m SV40EST109 cells were transduced (MOI = 50) with either the control vector LVi (SV40-eGFP) or the virulent vector LVi (SV40-eGFP-P2A-M2) (SEQ ID NO: 609) produced in the assay. The evolution of fluorescence (eGFP), spheroid size, and cell death marker (7-AAD) was measured using video microscopy (Incucyte S3 system, Sartorius).

[1083] When the transgene is nonviral (eGFP), continuous growth in spheroid size was observed regardless of the production cell line used. However, when the lentiviral vector expressed the M2 ion channel, no growth of transduced spheroids was observed, and all cells were labeled with 7-AAD.

[1084] Therefore, the CRISPRi strategy, especially the combined CRISPRi / m strategy, enables the production of high-titer constitutive virulence lentiviral vectors that lead to transduced cell death.

[1085] Example 4 - Pharmacological Induction

[1086] Materials and Methods

[1087] Cloning of lentiviral plasmids

[1088] The lentiviral plasmid used in the experiment was cloned by inserting a synthesized sequence (Twist Bioscience) into a plasmid (pLV(SIN); SEQ ID NO: 199) carrying a second-generation self-inactivated lentiviral genome, and then using the NEBuilder HiFi DNA assembly protocol (New England Biolabs) via enzymatic linearization and homology-based recircularization.

[1089] Cell culture

[1090] Cells were cultured in a humidified, controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (human embryonic kidney, ATTC–CRL-11268) cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL penicillin / streptomycin. Primary T lymphocytes isolated from healthy donor blood were cultured in serum-free, nutrient-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL penicillin / streptomycin. Stress induction was achieved by treatment with artesunate (Art; 5 µM) (A3731 Merck-Sigma), asparaginase (Asp; 1 U / mL, A3809 Merck-Sigma), bortezomib (Bz or Brtz; 10 nM) (50431140001, Merck-Sigma), and arginase (Arg; 5 U / mL, Merck-Sigma).

[1091] Production of lentiviral vectors

[1092] Non-replicating lentiviral particles were obtained using a production protocol described by Zennou et al. in 2001 (Nat Biotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded on cell culture medium and allowed to reach 50%-60% confluence after 24 hours of culture. The next day, cells were co-transfected using calcium phosphate precipitation with two trans complementary plasmids: one encoding the lentiviral genome (pLV) and the other encoding the enzyme and structural protein of HIV-1 (p8.92; SEQ ID NO: 338), and the other encoding 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 incubated with the precipitate for 5 hours, and then the culture medium was changed. The supernatant containing lentiviral particles was collected after 48 hours. Lentiviral suspensions were treated with DNAse I to remove retained plasmids, filtered (membrane porosity 0.2 µm), and subsequently ultracentrifuged at 60,000 g for 90 min at 4 °C. The particle pellet was resuspended in a volume corresponding to a 1000-fold dilution of phosphate-buffered saline (PBS). The resulting suspensions were aliquoted and stored at -80 °C until use. The concentration of lentiviral particles was determined by quantifying the lentiviral capsid protein p24. For this purpose, aliquots of the lentiviral suspension were thawed, lysed, and diluted according to the supplier's protocol for the HIV-1 p24 antigen ELISA 2.0 titration kit (Zeptometrix reference number 0801008). The obtained p24 concentrations are expressed in pg / µL, where 1 pg p24 is equivalent to 10-1 2 One transduction unit (TU).

[1093] Transduction

[1094] To integrate the transgene via infection with a lentiviral vector, dilute the amount of particles in the culture medium to obtain the desired multiplicity of infection (vector:target ratio). Contact the lentiviral suspension with the target cells. Culture the infected cells for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression can be observed or measured the following day by microscopy, cell counting, or quantitative PCR.

[1095] Flow cytometry

[1096] For analysis of cells in culture, cells were collected and washed with PBS. They were then incubated for 30 minutes with surface marker antibodies and Live-or-Dye 405 / 452 viability markers (Biotium), according to the manufacturer's recommendations. Cells were washed again and then fixed for 15 minutes at 4°C with PBS containing 1% paraformaldehyde. After washing, transgene expression was analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi).

[1097] statistics

[1098] Statistical analyses were performed using GraphPad® Prism 10. Detailed information regarding sample size and graph descriptions can be found in the accompanying text for each analysis. To assess statistical variance between two groups, an unpaired Student t-test or the Mann-Whitney U test was used. Statistical differences between three or more groups were analyzed using analysis of variance (ANOVA) and appropriate multiple comparison tests. In the graphs, significance is expressed as a p-value less than 0.05.

[1099] result

[1100] 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, tunicamycin (Tm) was used as a positive control for endoplasmic reticulum stress induction, and DMSO was used 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 hours, the CD3 / CD28 beads were removed, and the cells were cultured for 5 days in a medium containing IL2, IL7, and IL15. After 5 days under these resting conditions, the cells were reactivated with CD3 / CD28 beads and treated with DMSO, Asp, Art, or Bz, or placed in leucine-deficient medium (Leu-) for 16 hours.

[1101] Four molecules, Art, Asp, Bz, and Tm, were observed to significantly increase the percentage of CD3+ GFP+ T cells, with Tm being the most effective inducer (5-fold induction), followed by Asp and Art (4-fold), and Bz (3-fold). Figure 7 A). Compared with control cells, the mean fluorescence per cell was also significantly increased by Tm (11-fold) and Asp (6-fold). Figure 7 B). These experiments thus demonstrate that the three molecules Asp, Bz, and Art effectively induce 2xAARE receptors in activated CD3+ lymphocytes. YB_TATA Increased expression of GFP tran...

Claims

1. Use of a chemical inducer in the expression of a transgene contained in an inducible promoter activation or inducible expression cassette, wherein the transgene is controlled by the inducible promoter. The inducible promoter is selected from: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA ,and The chemical inducers are selected from: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

2. The use of the chemical inducing agent according to claim 1, wherein the inducible promoter is selected from: - 2xAARE YB_TATA The 2xAARE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1; - 2xAARE TK The 2xAARE TK The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2; - 4xSRE YB_TATA The 4xSRE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3; - 9xHRE YB_TATA The 9xHRE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4; - GRP78 YB_TATA The GRP78 YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5; - miR223 promoter, wherein the nucleic acid of the miR223 promoter has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and - miniCHAC1 YB_TATA The miniCHAC1 YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO:

7.

3. The use of the chemical inducing agent according to claim 1 or 2, wherein, The inducible promoter is 2xAARE YB _TATA The 2xAARE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO:

1.

4. The use of the chemical inducer according to any one of claims 1 to 3, wherein the transgene is selected from: oncogenes, tumor suppressor genes, transcription factors, nutritional factors, cytokines, enzymes, hormones, receptors, transport proteins, ligands, human proteins, heterologous proteins, chimeric proteins, nucleases, immunogenic proteins, chimeric antigen receptors (CARs), antibodies, and toxic genes or suicide genes.

5. The use of the chemical inducing agent according to claim 4, wherein, The genetically modified organism is: - Selected from the following transcription factors: c-JUN, FOXO1, BATF, TBET, and NRF2; - Selected from the following cytokines: IL2, IFNγ, TNFα, IL18, and IL12; - GLUT1 transporter protein; - Selected from the following nucleases: Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ, their orthologs and their functional mutants or variants; - Selected from the following CARs: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 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-ROR1 CAR, anti-ROR2 CAR, anti-PD1 CAR, and anti-PSCA CAR; or - Selected from the following toxic or suicide transgenic proteins: inducible Caspase-9 protein (iC9), Bax protein (S184 del), Noxa protein, Gasdermin B (N-ter) protein, constitutively active caspase-3 protein (V266E), influenza virus M2 ion channel (H37A), diphtheria toxin A subunit (DTA) and its attenuated aDTA mutant (G128D), streptococcal hemolysin O and enterotoxin.

6. A reagent kit, said reagent kit comprising: - A nucleic acid comprising an inducible promoter according to any one of claims 1 to 3, or a transgene contained in an expression cassette according to claim 4 or 5, wherein the transgene is controlled by the inducible promoter; and - At least one chemical inducer selected from: artesunate (Art), asparaginase (Asp), and bortezomib (Bz).

7. The kit according to claim 6, wherein, The inducible promoter is selected from: - 2xAARE YB_TATA The 2xAARE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 1; - 2xAARE TK The 2xAARE TK The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 2; - 4xSRE YB_TATA The 4xSRE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3; - 9xHRE YB_TATA The 9xHRE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4; - GRP78 YB_TATA The GRP78 YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5; - miR223 promoter, wherein the nucleic acid of the miR223 promoter has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and - miniCHAC1 YB_TATA The miniCHAC1 YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO:

7.

8. The kit according to claim 6 or 7, wherein, The inducible promoter is 2xAARE YB_TATA The 2xAARE YB_TATA The nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO:

1.

9. The kit according to any one of claims 6 to 8, wherein the transgenic material is: - Selected from the following transcription factors: c-JUN, FOXO1, BATF, TBET, and NRF2; - Selected from the following cytokines: IL2, IFNγ, TNFα, IL18, and IL12; - GLUT1 transporter protein; - Selected from the following nucleases: Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ, their orthologs and their functional mutants or variants; - Selected from the following CARs: anti-A-folate receptor CAR, anti-carbonic anhydrase IX CAR, anti-CD171 CAR, anti-CD19 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-gp100 CAR, anti-GPC3 CAR, anti-HER2 CAR, anti-IL13Rα2 CAR, anti-MAGE CAR, anti-MART-1 CAR, anti-MSLN CAR, anti-Muc1 CAR, anti-Muc16 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-ROR1 CAR, anti-ROR2 CAR, anti-PD1 CAR, and anti-PSCA CAR; or - Selected from the following toxic or suicide transgenic proteins: inducible Caspase-9 protein (iC9), Bax protein (S184 del), Noxa protein, Gasdermin B (N-ter) protein, constitutively active caspase-3 protein (V266E), influenza virus M2 ion channel (H37A), diphtheria toxin A subunit (DTA) and its attenuated aDTA mutant (G128D), streptococcal hemolysin O and enterotoxin.

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