Production of toxic vectors

Abstract

The present invention relates generally to the field of medicine. More specifically, it relates to the production of toxic vectors, to the toxic vectors themselves, and to their use for the practice of effective and safe cell therapy.

Description

Technical Field

[0001] This invention generally relates to the pharmaceutical field. More specifically, it relates to the production of toxic vectors, to these toxic vectors themselves, and to their use in the implementation of effective and safe cell therapies. 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 processes of these vectors have been optimized to improve their efficacy and safety in human medicine. Thanks to the significant growth of gene therapy, lentiviral vectors have been involved in numerous clinical trials, despite their limitations and side effects (particularly attributed to their preferential integration into the transcriptional regions of the genome of genetically modified cells).

[0003] Lentiviral vectors possess the ability to transduce proliferating or quiescent cells and integrate transgenes into the genome of their host. Therefore, their use in cell engineering offers a wide range of therapeutic applications. One such application, consisting of the elimination of transduced cells, is a strategy known as “suicide gene therapy,” which is being considered for various medical conditions (anti-tumor therapy, ensuring the safety of transplanted cells, etc.). However, a search of literature databases reveals that few publications report the use of lentiviral vectors for the transfer of cytotoxic genes. This is because these applications face two main technical hurdles: (1) producing lentiviral vectors carrying cytotoxic transgenes, and (2) restricting transgene expression to the targeted cells. Extensive research has been conducted over the years to address these issues, but only partial solutions have been found, particularly in attempts to achieve tissue-specific expression of transgenes. To this end, at least two main strategies are being considered to restrict vector entry or transgene expression to target cells. Lentivirals enter cells through interactions between viral envelope glycoproteins and cell membrane proteins. Therefore, selection from different envelope glycoproteins allows for particle pseudotyping to modulate lentiviral vector tropism by leveraging the diversity of affinity between the viral envelope and cellular receptors. However, existing methods for lentiviral envelope engineering do not allow for sufficient restriction of vector particle tropism to consider targeted gene transfer in vivo solely through this pathway (Li, M., Husic, N., Lin, Y. & Snider, BJJVis.Exp.JoVE e4031 (2012); Ebrahimabadi, S., Shahbazi, M., Akbari, M., Golalipour, M. & Farazmandfar, TJGeneMed.21, e3095 (2019); Gutierrez-Guerrero, A., Costet, F.-L. & Verhoeyen, E.Viruses 12, 1016 (2020)). Therefore, transcriptional restriction is most commonly considered to ensure tissue-specific expression of the transgene. This transcriptional control of transgene expression can be achieved by cloning tissue-specific promoters into expression vectors or by targeting transgene integration under specific active cellular promoters in target cells.

[0004] The main method involves transfecting nucleoprotein complexes (CRISPR / Cas9 and guide RNA) and donor DNA of interest to achieve targeted integration of transgenes into the genome of target cells in vitro.

[0005] For suicide gene transfer, most cytotoxic genes currently used encode proteins whose toxicity depends on the presence of effector molecules (ganciclovir against thymidine kinase in herpes simplex virus, 5-fluorocytosine against cytosine deaminase, a chemical dimerizer against inducible caspase-9, etc.). These enzyme / prodrug systems enable the production of lentiviral vectors carrying these genes without toxicity to producing cells and control the triggering of cell death. However, some of these enzymes (which are of viral or bacterial origin or contain chimeric epitopes) are potentially immunogenic (Berger, C. Blood 107, 2294-2302 (2006)), and prodrugs are not always approved for clinical use.

[0006] Furthermore, the use of transgenes encoding proteins with direct toxicity (diphtheria toxin, enterotoxin, activated caspase, etc.) is being explored, but subsequent gene transfer mainly involves non-lentiviral vectors (Pahle, J. et al. Cancers 13, 4393 (2021); Chang, A., Ling, J., Ye, H., Zhao, H. & Zhuo, X. Bioengineered 12, 6572-6578 (2021); Abbaspour, A., Esmaeilzadeh, A. & Sharaf, A. 3 Biotech 11, 140 (2021)). In fact, the production of lentiviral vectors expressing these transgenes presents technical challenges because they are toxic to the cells producing the vectors, which drastically reduces the titer of the recombinant particles.

[0007] The procedure for producing lentiviral vectors is based on co-transfection in HEK 293T cells of plasmids expressing the following: (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, the transgene is highly expressed during the production process 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)). This is because the plasmids used for the production of recombinant lentiviral vectors have a circular structure and contain at least one strong promoter that controls the transcription of the vector genome that will be capsidated in the recombinant HIV particles. Furthermore, it has recently been shown that plasmids also contain other cryptic promoters capable of initiating transcription on either strand of the plasmid DNA (Muerdter, F. et al. Nat. Methods 15, 141-149 (2018); Lem, NA, Hiraoka, K., Kasahara, N. & Logg, CRN Nucleic Acids Res. 40, 7280-7290 (2012)). Therefore, in the case of packaging cells, basal expression of cytotoxic transgenes significantly reduces the titer of the obtained vector (Maunder, HE et al. Nat. Commun. 8, 14834 (2017)). In some cases, production lines resistant to toxic genes may be used (Lange, MJ, Lyddon, TD & Johnson, MCSci. Rep. 9, 8985 (2019); Li, Y. et al. Cancer Res. 2002 May 1; 62(9): 2576-82), but this is not a universal solution. Summary of the Invention

[0008] Addressing the major challenges of delivering safe and effective in vivo cell therapies, the inventors have constructed an innovative vector genome that, despite expressing suicide or cytotoxic transgenes, still allows for the production of high-titer lentiviral vectors carrying said direct cytotoxic transgenes. Therefore, a first objective of this invention is to make this vector genome and the plasmid containing it available to the pharmaceutical industry for the production of lentiviral vectors. In fact, a second objective of this invention is to provide the use of the vector genome and the plasmid containing it for implementing methods for producing the lentiviral vectors of this invention. Another objective of this invention is to provide the use of said lentiviral vectors for transducing eukaryotic cells, intended for use in safe and effective in vivo cell therapies. Yet another objective of this invention is to provide transduced eukaryotic cells and their use in cell therapies. Detailed Implementation

[0009] In its most general aspect, the subject matter of the present invention relates to a vector genome comprising a lentiviral genome in which an expression cassette containing a promoter located upstream of a toxic or suicide transgene encoding a Noxa protein (wild-type protein or one of its mutants, particularly the S13A mutant) has been introduced.

[0010] The toxic or suicide transgene encoding the Noxa protein is selected from a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1, or whose nucleic acid encodes the Noxa protein, the amino acid sequence of which has at least 80% identity with sequence SEQ ID NO: 2.

[0011] The term "vector genome" refers to a nucleic acid sequence that contains both of the following:

[0012] - Nucleic acid containing the genetic information (=lentiviral genome) required 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 efficient for self-introduction into cells and is non-replicating; and

[0013] - A nucleic acid containing the genetic information of an expression cassette, the expression cassette containing a promoter located upstream of a toxic or suicide transgene, the nucleic acid encoding the toxic or suicide transgene having a nucleic acid sequence at its 3' end that allows transcription of a polyadenylate (polyA) tail, the expression cassette being able to be introduced into the lentiviral genome in a manner that reverses the transcription of the lentiviral genome.

[0014] As described above, the term "lentiviral genome" refers to nucleic acid carrying the genetic information necessary for the production of a lentiviral vector (also known as a (lenti)viral particle) containing the capsidated and enveloped vector genome. This lentiviral vector is also efficient in entering cells, 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 produce a non-integrating provirus (reverse-transcribed vector DNA) in the genome of the transduced cell. Therefore, the vector genome will be in the form of a free organism in the cell nucleus. Typically, after reverse transcription, the structure of the lentiviral genome includes: the cis sequence of the HIV lentiviral genome, consisting of two long terminal repeats (LTRs) flanking the ends of the vector genome, having 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 their LTR are called "self-inactivating" 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 vector RNA genome output, 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 contain an expression cassette located between the two LTRs. As mentioned above, an "expression cassette" refers to a nucleic acid containing an inducible promoter upstream of a virulent or suicide transgene, encoding which may contain a sequence at its 3' end that allows transcription of a polyadenylated tail. This expression cassette is introduced into the lentiviral genome such that its transcription is reversed relative to the transcription of the lentiviral genome.

[0015] The term "promoter" refers to a nucleic acid located upstream of a gene (or transgene) to control the expression of that gene, particularly by regulating its transcription, which can be constitutive or inducible. Subsequent references may include constitutive promoters, inducible promoters, or tissue-specific promoters.

[0016] The term "constitutive promoter" refers to a nucleic acid located upstream of a gene (or transgene) that continuously controls gene (or transgene) expression regardless of its environment, i.e., whether it is in the cellular environment that produces the recombinant vector particles or in other cellular environments with different phenotypes. In this invention, the constitutive promoter may be: SV40 (or pSV40), CMV (or pCMV), UbC (or pUbC), EF1-α core type (or pEF1-α core type), EF1-α long type (or pEF1-α long type), hPGK (or phPGK), CAGG (or pCAGG), RSV (or pRSV), or SFFV (or pSFFV).

[0017] The term "inducible promoter" refers to a nucleic acid located upstream of a gene (or transgene) whose expression control is not constitutive and is only induced upon activation of the promoter in response to an appropriate stimulus. In this invention, the inducible promoter may be: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA or miniCHAC1 YB_TATA 2xAARE YB_TATA 2xAARE TK and miniCHAC1 YB_TATA Activated by inducers of ATF4 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 (especially dexamethasone). 9xHRE YB_TATA The promoter was induced by hypoxia. 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.

[0018] The term "tissue-specific promoter" refers to a nucleic acid located upstream of a gene (or transgene), whose expression control is ineffective in the cellular environment producing recombinant vector particles, but is activated in another cellular environment with a different phenotype to which the tissue-specific promoter is sensitive. In response to this other environment, the tissue-specific promoter will be activated. For example, in one invention, this could be the miR223 promoter, which is specifically induced in myeloid cells, while HEK-293T cells are not myeloid cells.

[0019] The term "toxic or suicide transgene" refers to a nucleic acid located downstream of a promoter (particularly a constitutive, inducible, or tissue-specific promoter) that encodes a toxic, cytotoxic, or suicide protein in genetically modified cells. That is, upon activation of the promoter controlling the expression of the toxic or suicide transgene, the product (i.e., the toxic or suicide protein) produced by transcription and translation of this nucleic acid leads to (cause) cell death in the cells expressing it. In this invention, the latter corresponds to the Noxa protein, the nucleic acid encoding which is selected from a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1, or whose nucleic acid encodes the Noxa protein, the amino acid sequence of which has at least 80% identity with sequence SEQ ID NO: 2. It is a non-secretory toxic or suicide protein, meaning that upon activation of the promoter upstream of the nucleic acid encoding it, its expression is not present in the extracellular medium, and therefore only causes cell death in the cells expressing it.

[0020] Surprisingly, although the Noxa protein is a toxic or suicide gene, meaning its expression induces cell death in cells expressing the protein, its expression in the context of lentiviral vector production of this invention does not interfere with the survival of the latter. Therefore, by implementing an expression cassette that allows Noxa protein expression during the production of recombinant viral vector particles, high-titer lentiviral vectors can be obtained, thereby meeting the medical community's expectation for large-scale supply.

[0021] "Percentage of identity" refers to the percentage determined by a direct comparison of two oligonucleotide sequences (nucleic acid sequences), 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 percentage of identity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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). Furthermore, it should be understood that sequences having at least 80% identity with a reference sequence retain the same properties and functions, or even have improved properties and functions.

[0022] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the toxic or suicide transgene encoding the Noxa protein is selected from a sequence having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 1, or its nucleic acid encodes the Noxa protein, the amino acid sequence of which has at least 90% identity with sequence SEQ ID NO: 2. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the toxic or suicide transgene encoding the Noxa protein has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1, or its nucleic acid encodes the Noxa protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 2.

[0023] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is a constitutive promoter. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is a constitutive promoter specifically selected from the following:

[0024] -SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0025] -CMV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0026] -UbC, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0027] -EF1-α core type, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 6;

[0028] -EF1-α long type, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7;

[0029] -hPGK, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8;

[0030] -CAGG, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 9;

[0031] -RSV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 10; and

[0032] -SFFV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 11.

[0033] In particular, the subject matter of the present invention also relates to vector genomes as described above, wherein the promoter is a constitutive promoter selected from the following: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV, and SFFV, the nucleic acid of which has a sequence having at least 80% identity with the nucleic acids of sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. Advantageously, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is a constitutive promoter selected from the following: SV40, CMV, UbC, EF1-α core type, EF1-α long type, hPGK, CAGG, RSV and SFFV, the nucleic acids of which correspond to the nucleic acids of sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0034] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is an inducible or tissue-specific promoter. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is specifically selected from inducible or tissue-specific promoters of the following:

[0035] -2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 12;

[0036] -2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 13;

[0037] -4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 14;

[0038] -9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 17;

[0039] -GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 16;

[0040] -miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 17; and

[0041] -miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 18.

[0042] In particular, the present invention also relates to a vector genome as described above, wherein the promoter is an inducible or tissue-specific promoter selected from the following: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB_TATA The nucleic acid has a sequence having at least 80% identity with the nucleic acids of sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 18, respectively. Advantageously, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is a constitutive promoter selected from the following: 2xAARE YB_TATA 2xAARE TK 4xSRE YB_TATA 9xHRE YB_TATA GRP78 YB_TATA and miniCHAC1 YB _TATA The nucleic acids correspond to the nucleic acids of sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 18, respectively.

[0043] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the promoter is specifically selected from the following constitutive promoters:

[0044] -SV40, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3;

[0045] -CMV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4;

[0046] -UbC, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5;

[0047] -EF1-α core type, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 6;

[0048] -EF1-α long type, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 7;

[0049] -hPGK, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8;

[0050] -CAGG, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 9;

[0051] -RSV, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 10; and

[0052] -SFFV, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 11.

[0053] Alternatively, the promoter may be specifically selected from the following inducible or tissue-specific promoters:

[0054] -2xAARE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 12;

[0055] -2xAARE TK Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 13;

[0056] -4xSRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 14;

[0057] -9xHRE YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 17;

[0058] -GRP78 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 16;

[0059] -miR223 promoter, whose nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 17; and

[0060] -miniCHAC1 YB_TATA Its nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 18.

[0061] In view of the foregoing, it should be understood that the present invention realizes the vector genome as described above, wherein the expression cassette is selected from sequences having at least 80% identity with sequences SEQ ID NO: 19 to SEQ ID NO: 34. Specifically, the subject matter of the present invention relates to the vector genome as described above, wherein the expression cassette is selected from sequences SEQ ID NO: 19 to SEQ ID NO: 34. In other words, the subject matter of the present invention relates to the vector genome as described above, wherein the expression cassette is a sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, 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, or SEQ ID NO: 34 (see Table 1).

[0062]

[0063] Table 1. List of Expression Boxes

[0064] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the lentiviral genome in which the expression cassette is introduced is a self-inactivating lentivirus (SIN) or a respiratory syncytial virus (RSV).

[0065] According to another embodiment, the subject matter of the present invention relates to a vector genome as described herein, wherein the lentiviral genome incorporating the expression cassette is a self-inactivating lentivirus (SIN). Specifically, the subject matter of the present invention relates to a vector genome as described above, wherein the lentiviral genome incorporating the expression cassette 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: 35. Specifically, the present invention also relates to a vector genome as described above, wherein the lentiviral genome incorporating the expression cassette is a self-inactivating lentivirus (SIN) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 35. It should be noted that the insertion of the expression cassette into the self-inactivating lentivirus (SIN) of sequence SEQ ID NO: 35 may be performed at or at nucleotides 2229 and 2230 of SEQ ID NO: 35. That is, in this case, the expression cassette is located between sequences SEQ ID NO: 36 and SEQ ID NO: 37.

[0066] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the lentiviral genome incorporating the expression cassette is a respiratory syncytial virus (RSV). Specifically, the subject matter of the present invention relates to a vector genome as described above, wherein the lentiviral genome incorporating the expression cassette is a respiratory syncytial virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 38. Specifically, the present invention also relates to a vector genome as described above, wherein the lentiviral genome incorporating the expression cassette is a respiratory syncytial virus (RSV) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 38. It should be noted that the insertion of the expression cassette into the respiratory syncytial virus (RSV) of sequence SEQ ID NO: 38 can be performed at or at nucleotides 1737 and 1738 of SEQ ID NO: 38. That is, in this case, the expression cassette is located between sequences SEQ ID NO: 39 and SEQ ID NO: 40.

[0067] In view of the foregoing, it should be understood that the present invention realizes a vector genome as described above, wherein the vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 41 to SEQ ID NO: 72. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the vector genome is selected from sequences SEQ ID NO: 41 to SEQ ID NO: 72. In other words, the present invention also relates to the vector genome as described above, wherein the vector genome is the sequence 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 or SEQ ID NO: 72 (see Table 2).

[0068]

[0069] Table 2. Vector Genome List

[0070] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the expression cassette further comprises coding or non-coding sequences having post-transcriptional regulatory properties.

[0071] The coding or non-coding sequence having post-transcriptional regulatory properties is located at the 5' end (upstream) of the toxic or suicide transgene encoding the Noxa protein, and is specifically selected from sequences having at least 90% identity with sequences SEQ ID NO: 73 to SEQ ID NO: 99.

[0072] The term "coding or non-coding sequence with post-transcriptional regulatory properties" refers to a nucleic acid sequence that can be either coding (capable of encoding proteins) or non-coding (not encoding proteins) and possesses regulatory properties that function after transcription. That is, after mRNA production, the presence of this nucleic acid sequence inhibits the translation of the mRNA under certain conditions (e.g., normal physiological conditions), and thus inhibits the production of the protein encoded by the mRNA. Under other conditions (e.g., cellular stress), the presence of this nucleic acid sequence in the mRNA no longer has its inhibitory effect on translation. Translation subsequently occurs, and the protein encoded by the mRNA is produced.

[0073] In this invention, the coding or non-coding sequence with post-transcriptional regulatory properties is located in the expression cassette between the transgene of interest and the promoter, with the transgene of interest downstream of the promoter. In other words, the coding or non-coding sequence with post-transcriptional regulatory properties is located at the 5' end (upstream) of the transgene of interest. This arrangement of the expression cassette (5' - promoter - coding or non-coding sequence with post-transcriptional regulatory properties - transcription - transgene of interest - 3') allows for the suppression of translation of unwanted mRNA from the promoter using the coding or non-coding sequence with post-transcriptional regulatory properties, and thus the suppression of the production of the transgene of interest.

[0074] According to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the coding or non-coding sequence having post-transcriptional regulatory properties located at the 5' end (upstream) of the toxic or suicide transgene encoding the Noxa protein is selected from sequences having at least 90% identity with sequences SEQ ID NO: 73 to SEQ ID NO: 99. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the coding or non-coding sequence having post-transcriptional regulatory properties located at the 5' end (upstream) of the toxic or suicide transgene encoding the Noxa protein is selected from sequences SEQ ID NO: 73 to SEQ ID NO: 99. In other words, the present invention also relates to the vector genome as described above, wherein the coding or non-coding sequence having post-transcriptional regulatory properties is the sequence 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: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, 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 or SEQ ID NO: 99 (see Table 3).

[0075]

[0076] Table 3. List of 5'UTR sequences with post-transcriptional regulatory properties

[0077] Advantageously, the subject matter of the present invention relates to a vector genome as described above, wherein the coding or non-coding sequence having post-transcriptional regulatory properties is the 5'UTR sequence of ATF4 of sequence SEQ ID NO: 73.

[0078] In view of the foregoing, it should be understood that the present invention realizes a vector genome as described above, wherein the expression cassette is selected from sequences having at least 80% identity with sequences SEQ ID NO: 100 to SEQ ID NO: 531. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the expression cassette is selected from sequences SEQ ID NO: 100 to SEQ ID NO: 531 (see Table 4).

[0079]

[0080] Table 4. List of expression boxes containing 5' UTR

[0081] In view of the foregoing, it should also be understood that the present invention realizes the vector genome as described above, wherein the vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 532 to SEQ ID NO: 1395. In particular, the subject matter of the present invention relates to the vector genome as described above, wherein the vector genome is selected from sequences SEQ ID NO: 532 to SEQ ID NO: 1395 (see Table 5).

[0082]

[0083] Table 5. List of vector genomes containing 5' UTR

[0084] Interestingly, it should be noted that, in order to optimize the production of the vector genome as described above and increase safety features, the transcriptional direction (5' > 3') of the expression cassette as described above is reversed relative to the transcriptional direction of the lentiviral genome. Furthermore, according to another embodiment, the subject matter of the present invention relates to a vector genome as described above, wherein the transcriptional direction of the expression cassette (5' to 3') is reversed relative to the transcriptional direction of the lentiviral genome.

[0085] Specifically, the expression cassette further includes a nucleic acid sequence that allows transcription of the polyadenylated tail at the 3' end (downstream) of the toxic or suicide transgene encoding the Noxa protein.

[0086] When this embodiment is implemented, the vector genome as described above, particularly at the 3' end of the transgene of interest, contains a polyadenylated nucleotide sequence (e.g., SEQ ID NO: 1396). Indeed, since the transcriptional direction of the expression cassette from 5' to 3' as described above is reversed relative to the transcriptional direction of the lentiviral genome from 5' to 3', adding the polyadenylated nucleotide sequence (e.g., SEQ ID NO: 1396) to the 3' end of the nucleic acid encoding the toxic or suicide transgene promotes mRNA transcription and stability, thereby allowing the translation of the transgene of interest, particularly when the inhibition of translation by the coding or non-coding sequence with post-transcriptional regulatory properties is relieved.

[0087] According to another embodiment, the present invention therefore relates to a vector genome as described above, wherein the expression cassette further comprises a polyadenylated tail having a nucleic acid sequence having at least 80% identity with sequence SEQ ID NO: 1396. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the expression cassette further comprises a polyadenylated tail having sequence SEQ ID NO: 1396.

[0088] In view of the foregoing, it should be understood that the present invention implements a vector genome as described above, wherein the expression cassette further comprises a polyadenylated tail and is selected from sequences having at least 80% identity with sequences SEQ ID NO: 1397 to SEQ ID NO: 1412. In particular, the subject matter of the present invention relates to a vector genome as described above, wherein the expression cassette further comprises a polyadenylated tail and is selected from sequences SEQ ID NO: 1397 to SEQ ID NO: 1412 (see Table 6).

[0089]

[0090] Table 6. List of expression cassettes containing polyadenylate tails

[0091] In view of the foregoing, it should also be understood that the present invention realizes the vector genome as described above, wherein the expression cassette further includes a polyadenylate tail, and wherein the transcription direction from 5' to 3' (5' > 3') is reversed relative to the transcription direction from 5' to 3' of the lentiviral genome.

[0092] The vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 1845 to SEQ ID NO: 1876. In particular, the subject matter of the invention relates to a vector genome as described above, wherein the expression cassette further comprises a polyadenylated tail, and wherein the transcriptional direction from 5' to 3' (5' > 3') is reversed relative to the transcriptional direction from 5' to 3' of the lentiviral genome.

[0093] The vector genome is selected from SEQ ID NO: 1845 to SEQ ID NO: 1876 (see Table 7).

[0094]

[0095] Table 7. List of “reverse” vector genomes containing polyadenylate tails

[0096] In view of the foregoing, it should also be understood that the present invention realizes the vector genome as described above, wherein the expression cassette further comprises a coding or non-coding sequence having post-transcriptional regulatory properties and a polyadenylated tail, and is selected from sequences having at least 80% identity with sequences SEQ ID NO: 1413 to SEQ ID NO: 1844. In particular, the subject matter of the present invention relates to the vector genome as described above, wherein the expression cassette further comprises a coding or non-coding sequence having post-transcriptional regulatory properties and a polyadenylated tail, and is selected from sequences SEQ ID NO: 1413 to SEQ ID NO: 1844 (see Table 8).

[0097]

[0098] Table 8. List of expression cassettes containing 5' UTR and polyadenylate tail

[0099] In view of the foregoing, it should also be understood that the present invention realizes the vector genome as described above, wherein the expression cassette further comprises a coding or non-coding sequence having post-transcriptional regulatory properties and a polyadenylate tail, and wherein the transcription direction from 5' to 3' (5' > 3') is reversed relative to the transcription direction from 5' to 3' of the lentiviral genome.

[0100] The vector genome is selected from sequences having at least 80% identity with sequences SEQ ID NO: 1877 to SEQ ID NO: 2740. In particular, the subject matter of this invention relates to a vector genome as described above, wherein the expression cassette further comprises a coding or non-coding sequence having post-transcriptional regulatory properties and a polyadenylated tail, and wherein the transcriptional direction from 5' to 3' (5' > 3') is reversed relative to the transcriptional direction from 5' to 3' of the lentiviral genome.

[0101] The vector genome is selected from SEQ ID NO: 1877 to SEQ ID NO: 2740 (see Table 9).

[0102]

[0103] Table 9. List of “reverse” vector genomes containing 5' UTR and polyadenylated tail

[0104] According to another aspect, the subject matter of the present invention relates to a method for producing a lentiviral vector comprising a vector genome as described above, said method comprising at least the following steps:

[0105] a. Obtain transfected animal eukaryotic cells by co-transfecting animal eukaryotic cells using the following methods:

[0106] i. A plasmid containing the vector genome as described above and the tool for expressing it;

[0107] ii. Plasmids containing viral envelope proteins and tools for expressing them; and

[0108] iii. Plasmids containing HIV-1 structural proteins and enzymes, as well as tools for expressing them.

[0109] b. Culture the transfected animal eukaryotic cells to produce a lentiviral vector containing the vector genome as described above; and

[0110] c. Harvest and purify the lentiviral vector.

[0111] Such methods are described in the embodiments below and are feasible to practice in light of literature available to those skilled in the art (e.g., Zennou et al., Nat Biotechnol. 2001 May; 19(5): 446-50). Nevertheless, and for illustrative purposes, the following is specified:

[0112] - 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;

[0113] -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: 4595), pHCMV-Mokola, pHCMV-RabiesG, pHCMV-AmphoEnv, pHCMV-LCMV-WE, pHCMV-LCMV-Arm53b and pHCMV-10A1 (Sena-Esteves M et al., Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. Dec 15, 2004; 122(2): 131-9); and

[0114] - The plasmid containing HIV-1 structural proteins and enzymes, as well as tools for expressing it, may be selected from the following plasmids: p8.92 (SEQ ID NO: 4596), p8.92(IN D64V) (SEQ ID NO: 4597), psPAX2 (SEQ ID NO: 4598), pMDLg / pRRE (SEQ ID NO: 4599), and pRSV-Rev (SEQ ID NO: 4600).

[0115] For illustrative purposes and without limitation, the following options are also provided:

[0116] Cells (e.g., HEK 293T) were seeded onto a culture support and allowed to reach 50%–60% confluence after 24 hours of culture. The following day, cells were co-transfected using calcium phosphate precipitation with a plasmid encoding the lentiviral genome and two trans-complementary plasmids encoding the HIV-1 enzyme and structural protein (p8.9) and the vesicular stomatitis virus envelope glycoprotein (pVSVg). Co-transfection was performed at a plasmid ratio of 2:2:1. Transfected cells were incubated with the precipitate for 5 hours, followed by medium replacement. 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 volumes corresponding to a 1000-fold dilution of phosphate-buffered saline (PBS). The obtained suspension was divided into equal portions and stored at -80°C until use.

[0117] According to another embodiment, the subject matter of 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, Freestyle293-F cells (FS293), virus-producing cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8, and PS46.2. Advantageously, the subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells belong to the HEK293T cell line.

[0118] As stated above, although the Noxa protein is a toxic or suicide gene, meaning its expression induces cell death in cells expressing the protein, its expression in the context of producing the lentiviral vector of this invention unexpectedly does not interfere with the latter. That is, it does not cause death in cells producing the lentiviral vector of this invention (e.g., HEK 293T), but it does cause death in genetically modified target cells (e.g., T lymphocytes). With the tools developed by the inventors of this invention, the current gap in gene therapy tools in the medical field is thus completely filled. In this respect, this invention constitutes a true technological breakthrough.

[0119] In this invention, it should be understood that "lentiviral vector" refers to recombinant lentiviral particles obtained using plasmids containing the vector genome as described above and tools for expressing it, which allows for the production of (lentiviral) particles of this invention. This includes 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.

[0120] The term "plasmid" refers to a circular double-stranded DNA molecule that must possess: a bacterial origin of replication and an associated SV40 viral origin of replication, enabling autonomous replication, for example, for amplification in *E. coli*, and, for example, in HEK 293T cells after transfection; and a selection gene that ensures it is not lost during bacterial amplification during cell proliferation. In this invention, the plasmid described in i. further comprises the nucleic acid of the vector genome of the invention and tools for expressing it. That is, it contains the genetic elements necessary for expressing the vector genome of the invention and for producing the lentiviral vector according to the invention. The same applies to the other plasmids in ii. and iii., which respectively allow the expression of viral envelope proteins and structural proteins and enzymes of HIV-1. Advantageously, it should be noted that the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication of the plasmid described in i. is reversed relative to the transcriptional direction of the 5' to 3' of the expression cassette.

[0121] The plasmid containing the vector genome as described above and the tool for expressing it may be a pLV-SIN plasmid or a pLV-RSV plasmid.

[0122] If it is a pLV-SIN plasmid, its nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 2741. Specifically, the pLV-SIN plasmid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2741. It should be noted that the insertion of the vector genome into the pLV-SIN plasmid of sequence SEQ ID NO: 2741 can be performed at nucleotides 4939 and 4940 of the sequence SEQ ID NO: 2741. That is, in this case, the vector genome is located between sequences SEQ ID NO: 2742 and SEQ ID NO: 2743.

[0123] If it is a pLV-RSV plasmid, its nucleic acid has a sequence with at least 80% identity to the nucleic acid of sequence SEQ ID NO: 2744. Specifically, the pLV-RSV plasmid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2744. It should be noted that the insertion of the vector genome into the pLV-RSV plasmid of sequence SEQ ID NO: 2744 can be performed at nucleotides 234 and 235 of SEQ ID NO: 2744. That is, in this case, the vector genome is located between sequences SEQ ID NO: 2745 and SEQ ID NO: 2746.

[0124] In view of the foregoing, it should be understood that, according to another embodiment, the subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein the plasmid i as described above is selected from sequences having 80% identity with sequences SEQ ID NO: 2747 to SEQ ID NO: 2778, SEQ ID NO: 2779 to SEQ ID NO: 3642, SEQ ID NO: 3643 to SEQ ID NO: 3674, and SEQ ID NO: 3675 to SEQ ID NO: 4538. Advantageously, the subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein the plasmid i as described above is selected from the following sequences SEQ ID NO:

[0125] -2747 to 2778 (see Table 10 – “positive” plasmids without 5'UTR);

[0126] -2779 to 3642 (see Table 11 - "positive" plasmids with 5'UTR);

[0127] -3643 to 3674 (see Table 12 – “reverse” plasmids without 5'UTR); and

[0128] -3675 to 4538 (see Table 13 - "reverse" plasmids with 5'UTR).

[0129] It should be noted that the plasmids cited below are themselves part of this invention and constitute one of the purposes of this invention, as are their uses in producing lentiviral vectors containing the vector genome as described above.

[0130]

[0131] Table 10. List of "Forward" Plasmids

[0132]

[0133] Table 11. List of "forward" plasmids +5'UTR

[0134]

[0135] Table 12. List of "Reverse" Plasmids

[0136] Table 13. List of "Reverse" Plasmids +5'UTR

[0137] To facilitate and optimize the production of the lentiviral vector of the present invention, innovative eukaryotic cells expressing specially constructed ribonucleoprotein complexes can also be used to inhibit the expression of toxic or suicide transgenes in the lentiviral vector to be produced. Therefore, according to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells express at least one ribonucleoprotein complex comprising:

[0138] - Fusion proteins containing the following fusions:

[0139] -Inactivated Cas endonuclease (dCas); and

[0140] -KRAB transcriptional repressor domain or methylation domains selected from the following: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L;

[0141] Or a fusion protein containing the following fusions:

[0142] -KRAB transcriptional repressor domain;

[0143] -Inactivated Cas endonuclease (dCas); and

[0144] - Selected from the following methylation domains: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2; and

[0145] -A guide RNA (gRNA) that specifically recognizes the promoter sequence of the inducible promoter.

[0146] 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 is particularly stably expressed.

[0147] According to another embodiment, the subject matter of 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 subject matter of 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).

[0148] The description "animal eukaryotic cells expressing at least one ribonucleoprotein complex" means that the animal eukaryotic cells can express a single ribonucleoprotein complex (i.e., the dCas-KRAB / gRNA interference complex; or one of methylated 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 they can express multiple complexes, especially at least two, namely:

[0149] - Ribonuclear protein interference complex (called CRISPRi) dCas-KRAB / gRNA;

[0150] as well as

[0151] - 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.

[0152] 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 allows targeting of specific sequences via its guide RNA (gRNA). This targeting allows the KRAB domain to block transcription by steric hindrance, and allows the DNMT3A, DNMT3B, DNMT3A-L, or DNMT3B-L domains to block transcription by de novo methylation of the target DNA. Implementing the CRISPRi and / or CRISPRm strategies on the internal sequences of the lentiviral vector during recombinant particle production requires the co-expression of at least one CRISPRi and / or CRISPRm complex and one gRNA in transfected animal eukaryotic cells. Alternatively, the present invention implements a bifunctional CRISPRi / m complex in transfected animal eukaryotic cells, the complex comprising an inactivated or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L or MeCP2 methylation domain, and gRNA.

[0153] The term "gRNA (guide RNA)" refers to RNA that binds to an enzyme or protein complex, allowing the enzyme or protein complex to localize to the complementary RNA or DNA when it pairs with a complementary RNA or DNA sequence.

[0154] The term "dCas-KRAB / gRNA ribonucleoprotein complex" refers to an interference system comprising the binding of a dCas-KRAB fusion protein to guide RNA (gRNA). This guide RNA 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" (pre-spacer adjacent motif) motif, which is unique or repetitive. These two elements can work together, synergistically with the reverse of the expression cassette, and unexpectedly, can produce titers of lentiviral vectors carrying transgenes of interest (particularly virulent or suicide transgenes), a feat never before achieved. In this sense, this approach is truly revolutionary, as it ultimately enables the delivery of the safe and effective cell therapies that patients require.

[0155] The term "ribonucleoprotein complex dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, or dCas-DNMT3B-L / gRNA" refers to a methylation system comprising the combination of a dCas-DNMT3A fusion protein, a 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" (pre-spacer adjacent motif) motif. These two elements can work together, synergistically with the reverse of the expression cassette, and unexpectedly, can produce titers of lentiviral vectors carrying transgenes of interest (particularly virulent or suicide transgenes), which is unprecedented. In this sense, this method is truly revolutionary because it ultimately enables the delivery of the safe and effective cell therapies that patients need.

[0156] The description “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 the binding of a KRAB-dCas-DNMT3A fusion protein, a KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 fusion protein to a guide RNA (gRNA). This 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” (pre-spacer adjacent motif) motif. These three components work together, synergistically with the reverse of the expression cassette, and unexpectedly, are able to produce titers of lentiviral vectors carrying the transgene of interest (particularly toxic or suicide transgenes), a feat never before achieved. In this sense, the method is truly revolutionary, as it ultimately enables the delivery of the safe and effective cell therapies that patients need.

[0157] The term "dCas-KRAB fusion protein" refers to the combination of two amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In this invention, as described above, the two fused components are 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: 4543).

[0158] The term "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 and functional protein. In this invention, as described above, the two fusion components are 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: 4543).

[0159] The terms "KRAB-dCas-DNMT3A fusion protein, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L, or dCas-KRAB-MeCP2" refer to the combination of three amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In this invention, as described above, the three fused components are 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: 4543).

[0160] The term "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, a variety of Cas endonucleases (Cas9, Cas12a, Cas12b, CasX or Cas12e, Cas12f or Cas14, Cas12j or CasΦ) from different hosts have been identified and characterized, or even modified (e.g., to improve their function), which can be inactivated and carried out by the present invention. Therefore, 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:

[0161] - Inactivated Cas9 endonucleases from Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, Neisseria meningitidis, Streptococcus canis, Streptococcus macaae, F. tularensis, Acidaminococcus, Campylobacter jejuni, Streptococcus pneumoniae, and Streptococcus thermophilus;

[0162] - Inactivated Cas12a endonuclease from Lachnospiraceae bacterium and Acidaminococcus sp.

[0163] - Inactivated Cas12b endonuclease from Alicyclobacillus kakegawensis and Bacillus hisashii;

[0164] - Inactivated CasX or Cas12e endonucleases from Deltaproteobacteria and Planctomycetes;

[0165] - Inactivated type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans;

[0166] - Inactivated Cas12j or CasΦ endonucleases from the Biggiephage clade;

[0167] - Inactivated orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from the above-mentioned organisms; and

[0168] - Inactivated mutants or variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas12j or CasΦ derived from the above organisms.

[0169] The term "ortholog" refers to similar Cas endonucleases present in two or more different species that may have become inactive due to evolution, and if not, may be mutated to render them nonfunctional for use in this invention.

[0170] The term "mutant" refers to a Cas endonuclease in which one or more mutations have been introduced, including the deletion, substitution, and / or addition of one or more amino acids. Therefore, the term "inactivated mutant" refers to a Cas protein that has been modified through artificial intervention (e.g., through genetic engineering) to inactivate its DNA-cutting activity.

[0171] The term "inactive variant" refers to Cas endonucleases that have been naturally modified through evolution and do not exhibit DNA cleavage activity.

[0172] According to another embodiment, the subject matter of 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), which 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 subject matter of this 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: 4546, or whose nucleic acid encodes an inactivated Cas9 endonuclease (dCas9) whose amino acid sequence has at least 80% identity with sequence SEQ ID NO: 4547. Specifically, this 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: 4546, or whose nucleic acid encodes an inactivated Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to sequence SEQ ID NO: 4547.

[0173] The term "KRAB transcriptional repressor domain" refers to the KRAB (Kruppel-associated repressor cassette) domain of human zinc finger proteins Kox1 (or zinc finger protein 10) or Zim3, which results in specific inhibition of RNA polymerases II and III. In doing so, the KRAB domain inhibits gene expression present on the DNA to which it is recruited. Specifically, the subject matter of this 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: 4544 or SEQ ID NO: 4552, or whose amino acid sequence has at least 80% identity with the sequence SEQ ID NO: 4545 or SEQ ID NO: 4553. Specifically, this 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: 4544 or SEQ ID NO: 4552, or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4545 or SEQ ID NO: 4553.

[0174] According to another embodiment, the subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein the dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. Specifically, the subject matter of this invention relates to a method for producing lentiviral vectors 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: 4548 or SEQ ID NO: 4554, or its nucleic acid encodes a dCas9-KRAB fusion protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 4549 or SEQ ID NO: 4555. Specifically, this invention also relates to a method for producing lentiviral vectors 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: 4548 or SEQ ID NO: 4554, or its nucleic acid encodes a dCas9-KRAB fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 4549 (from sequence SEQ ID NO: 4548). The plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR encoding NO: 4551 or SEQ ID NO: 4555.

[0175] The description "methylation domain selected from DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2" refers to the functional domain of a DNA methyltransferase that catalyzes covalent modification of DNA by transferring cytosine to 5-methylcytosine via S-adenosyl-1-methionine (SAM). In doing so, the DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 domain inhibits the expression of one or more genes present on the DNA to which it is recruited. In particular, the subject matter of this invention relates to a method for producing lentiviral vectors as described above, wherein the methylation domain:

[0176] -DNMT3A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4556, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 4557;

[0177] -DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4568, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 4569;

[0178] -DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4560, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 4561; or

[0179] -DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4570, or its amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 4571.

[0180] In particular, the present invention also relates to a method for producing lentiviral vectors as described above, wherein the methylation domain:

[0181] -DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 4556, or its amino acid sequence corresponds to sequence SEQ ID NO: 4557;

[0182] -DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 4568, or its amino acid sequence corresponds to sequence SEQ ID NO: 4569;

[0183] -DNMT3A-L is encoded by the nucleic acid of sequence SEQ ID NO: 4560, or its amino acid sequence corresponds to sequence SEQ ID NO: 4561; or

[0184] -DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 4570, or its amino acid sequence corresponds to sequence SEQ ID NO: 4571.

[0185] According to another embodiment, the subject matter of 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: 4572, or its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 4573. In particular, the subject matter of 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: 4572, or its amino acid sequence corresponds to sequence SEQ ID NO: 4573.

[0186] According to another embodiment, the subject matter of the present invention relates to a method for producing lentiviral vectors 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 subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0187] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4562, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, wherein the amino acid sequence of the dCas9-DNMT3A fusion protein is at least 80% identical to the sequence SEQ ID NO: 4563;

[0188] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4574, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B fusion protein is at least 80% identical to the sequence SEQ ID NO: 4575;

[0189] - 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: 4566, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, and the amino acid sequence of the dCas9-DNMT3A-L fusion protein has at least 80% identity to the sequence SEQ ID NO: 4567; or

[0190] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4576, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, and the amino acid sequence of the dCas9-DNMT3B-L fusion protein has at least 80% identity with the sequence SEQ ID NO: 4577.

[0191] In particular, the present invention also relates to a method for producing lentiviral vectors as described above, wherein:

[0192] - The dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4562, or whose nucleic acid encodes the dCas9-DNMT3A fusion protein, and the amino acid sequence of the dCas9-DNMT3A fusion protein corresponds to the sequence SEQ ID NO: 4563;

[0193] - The dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4574, or whose nucleic acid encodes the dCas9-DNMT3B fusion protein, and the amino acid sequence of the dCas9-DNMT3B fusion protein corresponds to the sequence SEQ ID NO: 4575;

[0194] - The dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4566, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, and the amino acid sequence of the dCas9-DNMT3A-L fusion protein corresponds to the sequence SEQ ID NO: 4567; or

[0195] - The dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4576, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, and the amino acid sequence of the dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 4577.

[0196] Interestingly, although the DNMT3L enzyme has no catalytic activity, 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 subject matter of the present invention relates to a method for producing lentiviral vectors as described above, wherein the animal eukaryotic cells further express another ribonucleoprotein complex comprising:

[0197] - Fusion proteins containing the following fusions:

[0198] -Inactivated Cas endonuclease (dCas); and

[0199] - The methylation-promoting domain DNMT3L (dCas-DNMT3L fusion); and

[0200] -A guide RNA (gRNA) that specifically recognizes the promoter sequence of the inducible promoter.

[0201] The other ribonucleoprotein complex dCas-DNMT3L / gRNA is particularly stably expressed.

[0202] According to another embodiment, the subject matter of the 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 subject matter of the invention relates to its use in animal eukaryotic cells as described above, wherein the DNMT3L promethylation domain is located at the C-terminus of the inactivated Cas endonuclease (dCas).

[0203] According to another embodiment, the subject matter of 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: 4558, or its amino acid sequence having at least 80% identity with sequence SEQ ID NO: 4559. In particular, the subject matter of 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: 4558, or its amino acid sequence corresponds to sequence SEQ ID NO: 4559.

[0204] According to another embodiment, the subject matter of the present invention relates to a method for producing a lentiviral vector 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: 4564, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which has at least 80% identity with the sequence SEQ ID NO: 4565. In particular, the subject matter of the present invention relates to a method for producing a lentiviral vector 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: 4564, or the nucleic acid of which encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 4565.

[0205] As described above, the present invention also implements a bifunctional CRISPRi / m complex, the complex comprising:

[0206] - Inactivated or "dead" CRISPR-Cas endonucleases (dCas), KRAB transcriptional repression domains, and fusions with DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, or MeCP2 methylation domains; and

[0207] - A guide RNA (gRNA) that specifically recognizes the promoter sequence of the inducible promoter (i.e., the promoter of the expression cassette of the present invention).

[0208] 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:

[0209] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, whose nucleic acid has a sequence that has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4578 or SEQ ID NO: 4584, 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: 4579 or SEQ ID NO: 4585;

[0210] - 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: 4580 or SEQ ID NO: 4586, 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: 4581 or SEQ ID NO: 4587; or

[0211] - The dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein, whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4582 or SEQ ID NO: 4588, or whose nucleic acid encodes the dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein is at least 80% identical to the sequence SEQ ID NO: 4583 or SEQ ID NO: 4589.

[0212] In particular, the subject matter of this invention relates to a method for producing lentiviral vectors as described above, wherein:

[0213] - The KRAB-dCas-DNMT3A-L fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4578 or SEQ ID NO: 4584, or whose nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein. The amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to the sequence SEQ ID NO: 4579 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-1-P2A-BlastR of sequence SEQ ID NO: 4590) or SEQ ID NO: 4585.

[0214] 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: 4580 or SEQ ID NO: 4586, or its nucleic acid encodes the KRAB-dCas9-DNMT3B-L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 4581 or SEQ ID NO: 4587; or

[0215] - 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: 4582 or SEQ ID NO: 4588, 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: 4583 or SEQ ID NO: 4589.

[0216] Preferably, the subject matter of 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.

[0217] According to another embodiment, the subject matter of 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 having at least 80% identity with, the nucleic acid of sequence SEQ ID NO: 4541 (specifically recognizing the SV40 constitutive promoter (SEQ ID NO: 3)). In particular, the subject matter of 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, sequence SEQ ID NO: 4541.

[0218] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0219] -The fusion protein:

[0220] -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: 4548 or SEQ ID NO: 4554, 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: 4549 or SEQ ID NO: 4555.

[0221] -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: 4562, 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: 4563.

[0222] -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: 4574, 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: 4575.

[0223] -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: 4566, 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: 4567.

[0224] -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: 4576, 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: 4577.

[0225] -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: 4564, 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: 4565.

[0226] -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: 4578 or SEQ ID NO: 4584, 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: 4579 or SEQ ID NO: 4585.

[0227] -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: 4580 or SEQ ID NO: 4586, 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: 4581 or SEQ ID NO: 4587, and / or

[0228] -dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4582 or SEQ ID NO: 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein is at least 80% identical to the sequence SEQ ID NO: 4583 or SEQ ID NO: 4589;

[0229] - The gRNA comprises or is composed of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4541; and

[0230] - 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: 3.

[0231] In particular, the subject matter of this invention relates to a method for producing lentiviral vectors as described above, wherein:

[0232] -The fusion protein:

[0233] -dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4548 or SEQ ID NO: 4554, or whose nucleic acid encodes the dCas9-KRAB fusion protein, wherein the amino acid sequence of the dCas9-KRAB fusion protein corresponds to the sequence SEQ ID NO: 4549 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 4551) or SEQ ID NO: 4555.

[0234] -dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4562, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4563.

[0235] -dCas-DNMT3B is a dCas9-DNMT3B fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4574, or its nucleic acid encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4575.

[0236] -dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4566, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4567.

[0237] -dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4576, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 4577.

[0238] -dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4564, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4565.

[0239] -KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4578 or SEQ ID NO: 4584, or its nucleic acid encodes the KRAB-dCas9-DNMT3A-L fusion protein. The amino acid sequence of the KRAB-dCas9-DNMT3A-L fusion protein corresponds to the sequence SEQ ID NO: 4579 (encoded by plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-1-P2A-BlastR of sequence SEQ ID NO: 4590) or SEQ ID NO: 4585.

[0240] -KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4580 or SEQ ID NO: 4586, or its 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: 4581 or SEQ ID NO: 4587, and / or

[0241] -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: 4582 or SEQ ID NO: 4588, 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 corresponds to the sequence SEQ ID NO: 4583 or SEQ ID NO: 4589;

[0242] - The gRNA comprises or is composed of a nucleic acid corresponding to the sequence SEQ ID NO: 4541; and

[0243] - The constitutive promoter is SV40, and its nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 3.

[0244] According to this embodiment, a lentiviral vector can thus be produced, the lentiviral vector comprising an expression cassette selected from sequences SEQ ID NO: 19, SEQ ID NO: 100 to SEQ ID NO: 126, SEQ ID NO: 1397 and SEQ ID NO: 1412 to SEQ ID NO: 1439, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 532 to SEQ ID NO: 558, SEQ ID NO: 964 to SEQ ID NO: 990, SEQ ID NO: 1845, SEQ ID NO: 1861, SEQ ID NO: 1877 to SEQ ID NO: 1903 and SEQ ID NO: 2309 to SEQ ID NO: 2335, which may be composed of sequences selected from SEQ ID NO: 2747, SEQ ID NO: 2763, SEQ ID NO: 2779 to SEQ ID NO: 2805, SEQ ID NO: 3211 to SEQ ID NO: 3237, SEQ ID NO: 19, SEQ ID NO: 100 to SEQ ID NO: 126, SEQ ID NO: 1397 and SEQ ID NO: 1412 to SEQ ID NO: 1439, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 2747, SEQ ID NO: 2763, SEQ ID NO: 2779 to SEQ ID NO: 2805, SEQ ID NO: 3211 to SEQ ID NO: 3237, SEQ ID NO: 19, SEQ ID NO: 100 to SEQ ID NO: 126, SEQ ID NO: 1397 and SEQ ID NO: 1412 to Plasmid encoding NO: 3643, SEQ ID NO: 3659, SEQ ID NO: 3675 to SEQ ID NO: 3701 and SEQ ID NO: 4107 to SEQ ID NO: 4133.

[0245] According to another embodiment, the subject matter of 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 having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4540 (specifically recognizing the inducible promoter 2xAARE). YB_TATA(SEQ ID NO: 12)). In particular, the subject matter of 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 or consisting of the nucleic acid of sequence SEQ ID NO: 4540.

[0246] According to another embodiment, the present invention relates to a method for producing lentiviral vectors as described above, wherein:

[0247] -The fusion protein:

[0248] -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: 4548 or SEQ ID NO: 4554, 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: 4549 or SEQ ID NO: 4555.

[0249] -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: 4562, 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: 4563.

[0250] -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: 4574, 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: 4575.

[0251] -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: 4566, 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: 4567.

[0252] -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: 4576, 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: 4577.

[0253] -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: 4564, 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: 4565.

[0254] -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: 4578 or SEQ ID NO: 4584, 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: 4579 or SEQ ID NO: 4585.

[0255] -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: 4580 or SEQ ID NO: 4586, 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: 4581 or SEQ ID NO: 4587, and / or

[0256] -dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4582 or SEQ ID NO: 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein, wherein the amino acid sequence of the dCas9-KRAB-MeCP2 / gRNA fusion protein is at least 80% identical to the sequence SEQ ID NO: 4583 or SEQ ID NO: 4589;

[0257] - The gRNA comprises or is composed of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4540; and

[0258] -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: 12.

[0259] In particular, the subject matter of this invention relates to a method for producing lentiviral vectors as described above, wherein:

[0260] -The fusion protein:

[0261] -dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4548 or SEQ ID NO: 4554, or whose nucleic acid encodes the dCas9-KRAB fusion protein, wherein the amino acid sequence of the dCas9-KRAB fusion protein corresponds to the sequence SEQ ID NO: 4549 (encoded by plasmid pLV-EF1a-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 4551) or SEQ ID NO: 4555.

[0262] -dCas-DNMT3A is a dCas9-DNMT3A fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4562, or its nucleic acid encodes the dCas9-DNMT3A fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4563.

[0263] -dCas-DNMT3B is a dCas9-DNMT3B fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4574, or its nucleic acid encodes the dCas9-DNMT3B fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 4575.

[0264] -dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4566, or whose nucleic acid encodes the dCas9-DNMT3A-L fusion protein, the amino acid sequence of which corresponds to sequence SEQ ID NO: 4567.

[0265] -dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein, whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4576, or whose nucleic acid encodes the dCas9-DNMT3B-L fusion protein, wherein the amino acid sequence of the dCas9-DNMT3B-L fusion protein corresponds to the sequence SEQ ID NO: 4577.

[0266] -dCas-DNMT3L is a dCas9-DNMT3L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4564, or its nucleic acid encodes the dCas9-DNMT3L fusion protein, the amino acid sequence of which corresponds to the sequence SEQ ID NO: 4565.

[0267] -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: 4578 or SEQ ID NO: 4584, 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 the sequence SEQ ID NO: 4579 (from sequence SEQ ID NQ: 4). 5 The plasmid pLV-EF1a-KRAB1-dCas9-DNMT3a-1-P2A-BlastR (encoded by 90%) or SEQ ID NO: 4585,

[0268] -KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein, the nucleic acid of which corresponds to the nucleic acid of sequence SEQ ID NO: 4580 or SEQ ID NO: 4586, or its 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: 4581 or SEQ ID NO: 4587, and / or

[0269] -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: 4582 or SEQ ID NO: 4588, 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 corresponds to the sequence SEQ ID NO: 4583 or SEQ ID NO: 4589;

[0270] - The gRNA comprises or is composed of a nucleic acid corresponding to the sequence SEQ ID NO: 4540; and

[0271] -The inducible promoter is 2xAARE YB_TATA Its nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 12.

[0272] According to this embodiment, a lentiviral vector can thus be produced, the lentiviral vector comprising an expression cassette selected from sequences SEQ ID NO: 28, SEQ ID NO: 343 to SEQ ID NO: 369, SEQ ID NO: 1406 and SEQ ID NO: 1656 to SEQ ID NO: 1682, and / or the vector genome of the lentiviral vector may be selected from sequences SEQ ID NO: 50, SEQ ID NO: 66, SEQ ID NO: 775 to SEQ ID NO: 801, SEQ ID NO: 1207 to SEQ ID NO: 1233, SEQ ID NO: 1854, SEQ ID NO: 1870, SEQ ID NO: 2120 to SEQ ID NO: 2146 and SEQ ID NO: 2552 to SEQ ID NO: 2578, which may be composed of sequences selected from SEQ ID NO: 2756, SEQ ID NO: 2772, SEQ ID NO: 3022 to SEQ ID NO: 3048, SEQ ID NO: 3454 to SEQ ID NO: 3480, ... The plasmids are encoded as follows: ID NO: 3652, SEQ ID NO: 3668, SEQ ID NO: 3918 to SEQ ID NO: 3944 and SEQ ID NO: 4350 to SEQ ID NO: 4376.

[0273] Since a method for producing lentiviral vectors has been described, it should be understood that another aspect of the subject matter of this invention relates to lentiviral vectors comprising the vector genome described above. According to this same aspect, the invention also relates to lentiviral vectors that can be obtained by the method for producing lentiviral vectors described above.

[0274] It should also be understood that another aspect of the subject matter of this invention, and the lentiviral agents described above, are used in vitro for transducing animal eukaryotic cells, particularly the animal eukaryotic cells selected from:

[0275] - Cancer cells, especially those that form solid tumors or are involved in blood cancers;

[0276] - Immune cells, especially those selected from: T lymphocytes and NK cells;

[0277] - Myofibroblasts, especially myofibroblasts involved in the fibrosis process;

[0278] - Astrocytes, especially those activated during brain or spinal cord trauma, form glial scars that prevent regeneration; and

[0279] - Induced pluripotent stem cells (iPS).

[0280] According to another embodiment, the subject matter of the present invention relates to the in vitro use of a viral vector as described above, wherein the animal eukaryotic cells are different from pluripotent or totipotent stem cells, or stem cells isolated from the tissues of an organism.

[0281] The term "cancer cell" refers to abnormal cells that have acquired the specific characteristics that cause cancer (or malignancy), a disease characterized by uncontrolled and abnormally significant cell proliferation (tumor) within the normal (healthy) tissues of an organism, threatening the survival of the normal (healthy) tissues. Among these tumors are solid tumors and liquid tumors.

[0282] Therefore, according to another embodiment, the present invention relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are cancer cells, particularly cancer cells forming solid tumors or involved in hematologic malignancies. In particular, the present invention also relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are cancer cells forming solid tumors. In particular, the present invention also relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are cancer cells involved in hematologic malignancies.

[0283] The term "immune cell" refers to cells involved in the functioning of the immune system. Numerous examples exist, such as lymphocytes (e.g., T, B, NK, and NKT), phagocytes (e.g., macrophages and dendritic cells), and granulocytes (e.g., neutrophils, eosinophils, and basophils), as well as their precursor cells. These cells can be autologous, syngeneic, allogeneic, or xenogeneic. Finally, these cells can be genetically modified. It should be noted that this invention specifically relates to T lymphocytes and NK cells. For "T lymphocytes," this invention specifically refers to naive, effector, or memory T lymphocytes possessing helper, cytotoxic, or regulatory properties. For "NK cells," this invention specifically refers to primary, cancerous, or immortalized NK cells.

[0284] Therefore, according to another embodiment, the present invention relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are specifically selected from immune cells of the following types: T lymphocytes and NK cells. In particular, the present invention also relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are T lymphocytes or NK cells.

[0285] The term "myofibroblast" refers to cells characterized by the production, proliferation, migration, and contractile properties of matrix proteins, which are activated during repair and healing. These myofibroblasts are found in certain organs (such as the liver, lungs, heart, or kidneys) during chronic diseases. The process by which myofibroblasts replace tissue is called fibrosis.

[0286] Therefore, according to another embodiment, the present invention relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are myofibroblasts. In particular, the present invention also relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are myofibroblasts involved in the fibrosis process.

[0287] The term "astrocytocyte" refers to glial cells in the central nervous system. They are typically star-shaped and perform a variety of important functions, focusing on supporting and protecting neurons.

[0288] Therefore, according to another embodiment, the present invention relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are astrocytes, particularly astrocytes that are activated during brain or spinal cord injury and form glial scars that prevent regeneration. In particular, the present invention also relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are astrocytes that are activated during brain or spinal cord injury and form glial scars that prevent regeneration.

[0289] The term "stem cell" refers to cells characterized by their undifferentiated state and their ability to differentiate and generate or regenerate tissues such as hematopoietic tissue, muscle, brain (neurons), retina, liver (hepatocytes), pancreatic β cells, kidney, cartilage, bone, or skin. These cells, obtained from adult organisms or induced from differentiated cells (iPS), can be autologous, syngeneic, allogeneic, or xenogeneic.

[0290] Therefore, according to another embodiment, the present invention relates to the in vitro use of the lentiviral vector as described above, wherein the animal eukaryotic cells are induced pluripotent stem cells (iPS).

[0291] According to another aspect of the invention, the subject matter of the invention relates to animal eukaryotic cells transduced via a lentiviral vector as described above, said transduced animal eukaryotic cells being particularly selected from:

[0292] - Cancer cells, especially those that form solid tumors or are involved in blood cancers;

[0293] - Immune cells, especially those selected from: T lymphocytes and NK cells;

[0294] - Myofibroblasts, especially myofibroblasts involved in the fibrosis process;

[0295] - Astrocytes, especially those activated during brain or spinal cord trauma, form glial scars that prevent regeneration; and

[0296] - Induced pluripotent stem cells (iPS).

[0297] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the transduced animal eukaryotic cells are different from pluripotent or totipotent stem cells, or stem cells isolated from organismal tissues.

[0298] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are cancer cells, particularly cancer cells that form solid tumors or are involved in hematologic malignancies. In particular, the present invention also relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are cancer cells that form solid tumors. In particular, the present invention also relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are cancer cells involved in hematologic malignancies.

[0299] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are specifically selected from immune cells of the following types: T lymphocytes and NK cells. In particular, the present invention also relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are T lymphocytes or NK cells.

[0300] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are myofibroblasts. In particular, the present invention also relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are myofibroblasts involved in the fibrosis process.

[0301] According to another embodiment, the subject matter of the invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are astrocytes, particularly astrocytes that are activated during brain or spinal cord injury and form glial scars that prevent regeneration. In particular, the invention also relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are astrocytes that are activated during brain or spinal cord injury and form glial scars that prevent regeneration.

[0302] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above, wherein the animal eukaryotic cells are induced pluripotent stem cells (iPS cells).

[0303] According to another aspect of the invention, the subject matter of the invention relates to transduced animal eukaryotic cells as described above for use in cell therapy, particularly for treating diseases selected from cancer, leukemia, and fibrosis.

[0304] According to another embodiment, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above for the purposes described above, for the treatment of diseases selected from: cancer, leukemia, and fibrosis. In particular, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above for the purposes described above, for the treatment of cancer. In particular, the subject matter of the present invention relates to transfected / transduced animal eukaryotic cells as described above for the purposes described above, for the treatment of leukemia.

[0305] The term "cell therapy" refers to methods known as "biological therapy" that aim to treat an organ or organism by introducing therapeutic cells to replace, replenish, or kill defective cells.

[0306] Alternatively, and according to the same aspect, the subject matter of the invention relates to methods for treating diseases selected from cancer, leukemia, and fibrosis, methods comprising administering to a patient in need transfected / transduced animal eukaryotic cells according to the invention (or a pharmaceutical composition comprising at least one transfected / transduced animal eukaryotic cell according to the invention and a pharmaceutically acceptable excipient).

[0307] The tools of this invention enable effective and safe cell therapies. In particular, these therapies can utilize a technique called a safety switch, which, under the influence of the correct stimulation (and only the correct stimulation), allows activation of the inducible promoter of the expression cassette and / or deactivation of translational repression, thereby enabling the expression of toxic or suicide transgenes. In this context, for example, the following uses in immunocellular therapy:

[0308] - Modified and armed to target and destroy cancer cells; and

[0309] -According to this invention, toxic or suicide-inducing genetically modified organisms are transduced.

[0310] When administered to patients in need, it will exert its targeting and destruction effect on cancer cells. Only when these immune cells become dysfunctional can they be destroyed using the safety system provided by this invention (control of transcriptional induction and / or control of translational repression) to avoid adverse reactions.

[0311] In all respects, it should be noted that the various aspects of the invention and its various embodiments are interdependent. Therefore, the latter can often be combined with each other as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This also applies to all definitions provided in this specification, which apply to all aspects of the invention and its embodiments.

[0312] Furthermore, the present invention is illustrated by the following figures and embodiments, but is not limited thereto. Attached Figure Description

[0313] Figure 1 Production of pLV vector expressing Noxa in HEK-293T cells

[0314] pLV-2xAARE vector produced and titrated in HEK-293T cells YB_TATA -GFP-p2a-Luc(GFP)(SEQ IDNO:4591) and pLV-2xAARE YB_TATARecombinant particles of -GFP-p2a-Noxa (Noxa) (SEQ ID NO: 4592). The concentrated supernatant was titrated by measuring the integrated genome in HEK-293T cells transduced with dilutions of these vectors. The graphs show that the titers of the two vectors were comparable. Each point represents independent production. Statistical analysis: t-test.

[0315] Figure 2 Study on the induction of cell death by Noxa expression in NK-92 cells

[0316] (a) 24 hours after treatment with DMSO or artesunate (2 μM), the transducer pLV-2xAARE was effective. YB_TATA -GFP-p2a-Luc(GFP) (SEQ ID NO: 4591) or pLV-2xAARE YB_TATA (b) The mortality rate of NK-92 cells transduced with the GFP-p2a-Noxa(Noxa) (SEQ ID NO: 4592) was measured. (b) The mortality rate of NK-92 cells transduced with the vector pLV-2xAARE was measured 48 hours after treatment with DMSO or artesunate (2 μM). YB_TATA -GFP-p2a-Luc(GFP) (SEQ ID NO: 4591) or pLV-2xAARE YB_TATA The mortality rate of NK-92 cells containing -GFP-p2a-Noxa (Noxa) (SEQ ID NO: 4592) was measured. Each point represents an independent experiment, and cell mortality was measured by cell counting after staining with annexin V and propidium iodide. Statistical analysis: one-way ANOVA.

[0317] Figure 3 Study on the induction of cell death by Noxa expression in human T lymphocytes

[0318] After 24 hours of treatment with DMSO or artesunate (2 μM), the transducer pLV-2xAARE was effective. YB_TATA -GFP-p2a-Luc(GFP) (SEQ ID NO: 4591) or pLV-2xAARE YB_TATA The mortality rate of lymphocytes containing GFP-p2a-Noxa (Noxa) (SEQ ID NO: 4592) was measured. Each point represents the cell mortality rate measured by cell counting after staining with annexin V and propidium iodide from different donors. Statistical analysis: one-way ANOVA.

[0319] Example - Production of Toxic Carriers

[0320] Materials and Methods

[0321] Cloning of lentiviral plasmids

[0322] The lentiviral plasmid used in the experiment was cloned by enzymatic linearization and homology-based recirculation using the NEBuilder HiFi DNA assembly protocol (New England Biolabs) by inserting a synthesized sequence (Twist Bioscience) into a plasmid (pLV(SIN); SEQ ID NO: 2741) carrying a second-generation self-inactivated lentiviral genome.

[0323] Cell culture

[0324] Cells were cultured in a humidified, controlled atmosphere incubator at 37°C and 5% CO2.

[0325] 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 was induced by treatment with tunicamycin (Sigma Aldrich).

[0326] NK-92 cells were a suspension culture cell line derived from NK lymphocytes from patients with non-Hodgkin's lymphoma (ATCC, CRL-2407). These cells were seeded at a density of 200,000 cells / mL and cultured in RPMI+Glutamax medium (ThermoFisher, catalog number: 61870-01), 20% FBS, and 1% penicillin / streptomycin. They were passaged every 3 to 4 days.

[0327] GIBCO OpTmizer was used in serum-free, nutrient-free medium (SFM) supplemented with Glutamax (Thermo Fischer) and 100 U / mL penicillin / streptomycin. TM CTS TM Primary T lymphocytes isolated from the blood of healthy donors were cultured in Thermo Fischer.

[0328] Production of lentiviral vectors

[0329] 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 (p8.92; SEQ ID NO: 4596 or SEQ ID NO: 4597), and the other encoding the envelope glycoprotein of vesicular stomatitis virus (pVSVg; SEQ ID NO: 4595). 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. The lentiviral suspension was treated with DNase I to remove residual plasmids, filtered (membrane porosity 0.2 μm), and then 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 suspension was aliquoted and stored at -80 °C until use.

[0330] Titration

[0331] 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. 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 (TU).

[0332] Transduction

[0333] 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.

[0334] Establishment of the KRAB (CRISPRi) cell line

[0335] HEK 293T cells were transduced with a lentiviral vector (SEQ ID NO: 4551) carrying a constitutive expression cassette.

[0336] - Under the control of the minimal eF-1α promoter (SEQ ID NO: 4542), the dCas9-KRAB fusion protein (SEQ ID NO: 4548) was fused with the blast fungicide resistance gene (BlastR) (SEQ ID NO: 4550) via 2A (SEQ ID NO: 4601); and

[0337] Guide RNA (SEQ ID NO: 4540) that is specific to the target promoter under the control of the U6 promoter (SEQ ID NO: 4539).

[0338] Two days later, the transduced cells were screened by treating with blastomycin (2 μg / mL). Cells were cloned by limiting dilution, and each clone was characterized.

[0339] Establishment of KRAB (CRISPRi) and DNMT (CRISPRm) cell lines

[0340] HEK 293T cells were transduced with a lentiviral vector (SEQ ID NO: 4590) carrying a constitutive expression cassette.

[0341] - Under the control of the minimal cF-1α promoter (SEQ ID NO: 4542), the KRAB-dCas9-DNMT3A-L fusion protein (SEQ ID NO: 4578) was fused to the blast fungicide resistance gene (BlastR) (SEQ ID NO: 4550) via a 2A peptide (SEQ ID NO: 4601); and

[0342] - A guide RNA (SEQ ID NO: 4540) that is specific to the target promoter under the control of the U6 promoter (SEQ ID NO: 4539).

[0343] Two days later, the transduced cells were treated with blastomycin (2 μg / mL) for selection. HEK-CRISPRi / m was cloned using limiting dilutions. SV40 Cells were analyzed, and each clone was characterized.

[0344] Flow cytometry analysis

[0345] At the end of the experiment, the cultured cells were harvested and washed with PBS. They were then incubated for 30 minutes with surface marker antibodies and Live-or-Dye 405 / 452 vitality markers (Biotium), according to the respective manufacturer's recommendations. The cells were washed and subsequently fixed in PBS containing 1% paraformaldehyde at 4°C for 15 minutes. After washing, transgene expression was analyzed using a MACSQuantAnalyser 10 flow cytometer (Miltenyi).

[0346] result

[0347] Two lentiviral vectors, pLV-2xAARE, were produced in HEK-293T cells. YB_TATA -GFP-p2a-nLuc (SEQ ID NO: 4591) and pLV-2xAARE YB_TATA -GFP-p2a-Noxa (SEQ ID NO: 4592). These vectors carry 2xAARE receptors. YB_TATA (SEQ ID NO: 12) controlled dual reporter transgene GFP-p2a-nLuc (SEQ ID NO: 4602) or reporter gene and pro-apoptotic transgene GFP-p2a-Noxa (SEQ ID NO: 4603). Two vectors, pLV-2xAARE, were used in three independent vector production experiments. YB_TATA - GFP-p2a-nLuc (SEQ ID NO: 4591) and pLV-2xAARE YB_TATA Both -GFP-p2a-Noxa (SEQ ID NO: 4592) and recombinant particle titers were measured to be considerable. Figure 1 ).

[0348] Subsequently, human NK-92 natural killer cells (ATCC CRL 2407 and 2408) were transduced at MOI 10 and treated with artesunate or DMSO solvent. After 24 hours of treatment, artesunate induced a significant increase in the transduction of pLV-2xAARE cells. YB-TATA NK-92 cells transduced with GFP-p2a-Noxa (SEQ ID NO: 4592) died, but cells transduced with the control vector pLV-2xAARE... YB_TATA NK-92 cells containing -GFP-p2a-nLuc (SEQ ID NO: 4591) did not exhibit this phenomenon. Figure 2 a). Furthermore, compared to treatment for only 24 hours, pLV-2xAARE transduction was observed after 48 hours of treatment with artesunate. YB_TATANK-92 cells transduced with GFP-p2a-Noxa (SEQ ID NO: 4592) showed doubled cell death, while in these cells treated with DMSO, and in cells transduced with the control vector pLV-2xAARE... YB_TATA The same treatment effect was not measured in NK-92 cells containing -GFP-p2a-nLuc (SEQ ID NO: 4591). Figure 2 b).

[0349] The induction of Noxa expression in human primary T lymphocytes was also investigated. For this purpose, donor T cells were injected with MOI 10 using the vector pLV-2xAARE. YB_TATA - GFP-p2a-nLuc (SEQ ID NO: 4591) or pLV-2xAARE YB_TATA Transduction was performed using GFP-p2a-Noxa (SEQ ID NO: 4592). The control vector pLV-2xAARE was also used in the transduction. YB_TATA In cells containing p-GFP-p2a-nLuc (SEQ ID NO: 4591), no difference in cell death rate was measured after treatment with artesunate or DMSO. In contrast, no difference was measured after treatment with pLV-2xAARE. YB_TATA T lymphocytes transduced with the GFP-p2a-Noxa (SEQ ID NO: 4592) vector showed a doubling of cell death rate under artesunate treatment, but this phenomenon was not observed after DMSO treatment. Figure 3 ).

[0350] Therefore, surprisingly, these results demonstrate that although the induction of Noxa protein expression in HEK-293T cells does not lead to cell death in the production of the lentiviral vector of the present invention, its identical expression in NK-92 cells and T lymphocytes effectively induces the death of the genetically modified cells.

Claims

1. A vector genome comprising a lentiviral genome, wherein an expression cassette containing a promoter located upstream of a toxic or suicide transgene encoding a Noxa protein has been introduced into the lentiviral genome. The toxic or suicide transgene encoding the Noxa protein is selected from a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1, or whose nucleic acid encodes the Noxa protein, the amino acid sequence of which has at least 80% identity with sequence SEQ ID NO:

2.

2. The vector genome according to claim 1, wherein, The promoter is specifically selected from the following constitutive promoters: -SV40, wherein the nucleic acid of SV40 has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 3; -CMV, wherein the nucleic acid of the CMV has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 4; -UbC, wherein the nucleic acid of UbC has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 5; -EF1-α core type, wherein the nucleic acid of the EF1-a core type has a sequence that has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; -EF1-α long type, wherein the nucleic acid of the EF1-α long type has a sequence that has at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7; -hPGK, wherein the nucleic acid of hPGK has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 8; -CAGG, wherein the nucleic acid of the CAGG has a sequence that is at least 80% identical to the nucleic acid of sequence SEQ ID NO: 9; -RSV, wherein the nucleic acid of said RSV has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10; and -SFFV, wherein the nucleic acid of SFFV has a sequence that has at least 80% identity with the nucleic acid of sequence SEQ ID NO:

11. Or one of them, The promoter is specifically selected from the following inducible or tissue-specific promoters: -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: 12; -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: 13; -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: 14; -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: 17; -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: 16; -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: 17; 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:

18.

3. The vector genome according to claim 1 or 2, wherein, The expression cassette further includes coding or non-coding sequences with post-transcriptional regulatory properties. The coding or non-coding sequence having post-transcriptional regulatory properties is located at the 5' end of the toxic or suicide transgene encoding the Noxa protein, and is specifically selected from sequences having at least 90% identity with sequences SEQ ID NO: 73 to SEQ ID NO:

99.

4. The vector genome according to any one of claims 1 to 3, wherein, The transcription direction of the expression cassette from 5' to 3' is reversed relative to the transcription direction of the lentiviral genome from 5' to 3'. In particular, the expression cassette further includes a nucleic acid sequence at the 3' end of the toxic or suicide transgene encoding the Noxa protein that allows transcription of the polyadenylated tail.

5. A method for producing a lentiviral vector comprising the vector genome according to any one of claims 1 to 4, said method comprising at least the following steps: a. Obtain transfected animal eukaryotic cells by co-transfecting eukaryotic cells using the following methods: i. A plasmid comprising a vector genome according to any one of claims 1 to 4 and a tool for expressing it; ii. Plasmids containing viral envelope proteins and tools for expressing them; and iii. Plasmids containing HIV-1 structural proteins and enzymes, as well as tools for expressing them. b. Culturing the transfected animal eukaryotic cells to produce a lentiviral vector comprising a vector genome according to any one of claims 1 to 4; and c. Harvest and purify the lentiviral vector.

6. The method for producing lentiviral vectors according to claim 5, wherein, The animal eukaryotic cells express at least one ribonucleoprotein complex, the ribonucleoprotein complex comprising: - Fusion proteins containing the following fusions: -Inactivated Cas endonuclease (dCas); and -KRAB transcriptional repression domain or methylated domains selected from the following: DNMT3A, DNMT3B, DNMT3A-L, and DNMT3B-L; or fusion proteins containing the following fusions: -KRAB transcriptional repression domain; -Inactivated Cas endonuclease (dCas); and - Selected from the following methylation domains: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L, and MeCP2; and -A guide RNA (gRNA) that specifically recognizes the promoter sequence of the inducible promoter. 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 is particularly stably expressed.

7. A lentiviral vector comprising a vector genome according to any one of claims 1 to 4.

8. The in vitro use of the lentiviral vector according to claim 7 for transducing animal eukaryotic cells. Specifically, the animal eukaryotic cells are selected from: - Cancer cells, especially those that form solid tumors or are involved in blood cancers; - Immune cells, especially those selected from: T lymphocytes and NK cells; - Myofibroblasts, especially myofibroblasts involved in the fibrosis process; - Astrocytes, especially those activated during brain or spinal cord trauma, form glial scars that prevent regeneration; and - Induced pluripotent stem cells (iPS).

9. Animal eukaryotic cells transduced via the lentiviral vector according to claim 7, wherein the transduced animal eukaryotic cells are particularly selected from: - Cancer cells, especially those that form solid tumors or are involved in blood cancers; - Immune cells, especially those selected from: T lymphocytes and NK cells; - Myofibroblasts, especially myofibroblasts involved in the fibrosis process; - Astrocytes, especially those activated during brain or spinal cord trauma, form glial scars that prevent regeneration; and - Induced pluripotent stem cells (iPS).

10. Animal eukaryotic cells transduced according to claim 9 for use in cell therapy, particularly for the treatment of conditions selected from cancer, leukemia and fibrosis.

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