Production of toxic vectors

Abstract

The present invention relates in general to the field of medicine. More particularly, it relates to the production of toxic vectors, to these toxic vectors as such and to the use of same for the implementation of effective and safe cellular therapies.

Description

[0001] PRODUCTION OF TOXIC VECTORS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of medicine. More particularly, it relates to the production of toxic vectors, these toxic vectors as such and their use for the implementation of effective and safe cell therapies.

[0004] PRIOR ART

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

[0006] Lentiviral vectors have the ability to transduce proliferative or quiescent cells as well as to integrate a transgene into the genome of their host. Their use in cell engineering thus offers possibilities for very varied therapeutic applications. One of these consists of the elimination of the transduced cell, a strategy called "suicide gene therapy", envisaged to address various medical situations (anti-tumor treatment, securing of grafted cells, etc.). However, by consulting bibliographic databases, publications reporting the use of lentiviral vectors for the transfer of a cytotoxic gene are rare. Indeed, these applications are faced with two major technological obstacles: (1) producing lentiviral vectors carrying a cytotoxic transgene and (2) limiting the expression of the transgene to the targeted cells.To address these issues, much work has been undertaken over the past several years, and only partial solutions have been found, particularly in attempts to achieve tissue-specific expression of the transgene. To this end, at least two major strategies are being considered to restrict either vector entry or transgene expression to target cells only. Lentiviruses enter cells through interaction between a viral envelope glycoprotein and a cell membrane protein. Thus, the choice among different envelope glycoproteins allows for particle pseudotyping to exploit the diversity of affinities between viral envelopes and cellular receptors to modulate the tropism of lentiviral vectors.However, current methods of lentiviral envelope engineering do not allow sufficient restriction of vector particle tropism to envisage targeted gene transfer, in vivo, by this route alone (Li, M., Husic, N., Lin, Y. & Snider, BJJ Vis. Exp. JoVE e4031 (2012); Ebrahimabadi, S., Shahbazi, M., Akbari, M., Golalipour, M. & Farazmandfar, TJ Gene Med. 21, e3095 (2019); Gutierrez-Guerrero, A., Cosset, F.-L. & Verhoeyen, E. Viruses 12, 1016 (2020)). Therefore, transcriptional restriction is most often considered to ensure tissue-specific expression of the transgene. This transcriptional control of transgene expression can be achieved by cloning a tissue-specific promoter into an expression vector or by targeting transgene integration under a cellular promoter specifically active in target cells.

[0007] Targeted integration of the transgene into the genome of the target cell is mainly developed ex vivo through the transfection of nucleoprotein complexes (CRISPR / Cas9 and guide RNA) and a donor DNA of interest.

[0008] For suicide gene transfer, the majority of cytotoxic genes used today encode a protein whose toxicity is conditioned by the presence of an effector molecule (Ganciclovir for the thymidine kinase of the Herpes Simplex Virus, 5-fluorocytosine for cytosine deaminase, chemical dimerizer for inducible Caspase-9, etc.). These enzyme / pro-drug systems make it possible to produce lentiviral vectors carrying these genes without toxicity for the producer cells, and to control the triggering of cell death. However, some of these enzymes, of viral or bacterial origin or containing chimeric epitopes, are potentially immunogenic (Berger, C. Blood 107, 2294-2302 (2006)), and the pro-drug is not always approved for clinical use.

[0009] Furthermore, the use of transgenes encoding a protein with direct toxicity is also explored (diphtheria toxin, enterotoxins, activated caspase, etc.) but transgenesis then 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. & Sharafi, A. 3 Biotech 11, 140 (2021)). Indeed, the production of lentiviral vectors expressing these transgenes poses a technical problem due to their toxicity on the cells where the vectors are produced, which drastically reduces the titers of recombinant particles.

[0010] The procedure for producing lentiviral vectors is based on the co-transfection into HEK 293 T cells of plasmids expressing: (1) the lentiviral genome carrying the transgene, (2) an envelope protein and (3) the structural proteins and HIV-1 enzymes necessary for the production of non-replicating recombinant lentiviral particles (gag, pol and rev). In lentiviral vectors, the transgene is classically cloned under the control of an internal promoter. Also, during the production process, the transgene is highly expressed, despite modifications to the lentiviral sequences and the use of inducible or tissue-specific internal promoters (Pourzadegan, F. et al. Cancer Gene Ther. 23, 7-12 (2016); Brandtner, EM et al. J. Gene Med. 10, 113-122 (2008)).Indeed, the plasmids used for the production of recombinant lentiviral vectors have a circular structure and contain at least one strong promoter, controlling the transcription of the vector genome that will be packaged in the recombinant HIV particles. In addition, it has recently been shown that plasmids also contain other, cryptic promoters, which can be the origin of transcription starts on either of the two DNA strands of the plasmid (Muerdter, F. et al. Nat. Methods 15, 141-149 (2018); Lemp, NA, Hiraoka, K., Kasahara, N. & Logg, CR Nucleic Acids Re s. 40, 7280-7290 (2012)). Thus, in the case of packaging cells, basal expression of a cytotoxic transgene strongly reduces the titer of the vectors obtained (Maunder, HE et al. Nat. Commun. 8, 14834 (2017)). In some cases, the use of a production line resistant to the toxic gene is possible (Lange, MJ, Lyddon, TD & Johnson, MC Sci. Rep.9, 8985 (2019); Li, Y. et al. Cancer Res. 2002 May l;62(9):2576-82), but this is not a universal solution.

[0011] BRIEF OVERVIEW OF THE INVENTION

[0012] Faced with this major challenge of being able to promise safe and effective in vivo cell therapy, the inventors have constructed an innovative vector genome making it possible, despite the expression of a suicide or toxic transgene, to produce high titers of lentiviral vectors carrying said directly cytotoxic transgene. Thus, a first aim of the invention is to make this vector genome and the plasmid comprising it available to the medical profession to produce a lentiviral vector. De facto, a second aim of the invention corresponds to proposing the use of the vector genome of the invention and the plasmid comprising it to implement a method for producing the lentiviral vector of the invention. Another aim of the invention is to make available the use of said lentiviral vector to transduce a eukaryotic cell, which is intended to be used in safe and effective in vivo cell therapy.Also, another aim of the invention is to propose a transduced eukaryotic cell and its use in cell therapy.

[0013] DETAILED DESCRIPTION

[0014] In its most general aspect, the subject of the invention is a vector genome comprising a lentiviral genome into which has been introduced an expression cassette comprising a promoter upstream of a toxic or suicide transgene encoding a Noxa protein (the wild-type protein or one of its mutants, in particular the S13A mutant, said toxic or suicide transgene encoding a Noxa protein being chosen from the sequences having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1 or whose nucleic acid encodes a protein, a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 2.

[0015] By “vector genome” is meant a nucleic acid sequence comprising both:

[0016] ■ the nucleic acid comprising the genetic information (= lentiviral genome) necessary for the production of a lentiviral vector also called a (lenti-)viral particle, said lentiviral vector or said (lenti-)viral particle comprising said encapsidated and enveloped vector genome, which is efficient for introduction into the cell and is non-replicative; and

[0017] ■ the nucleic acid comprising the genetic information of an expression cassette comprising a promoter upstream of a toxic or suicide transgene, the nucleic acid encoding said toxic or suicide transgene being able to comprise at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail, said expression cassette being able to be introduced into said lentiviral genome such that its transcription is reversed compared to that of said lentiviral genome.

[0018] By "lentiviral genome", as mentioned above, is meant a nucleic acid carrying the genetic information necessary for the production of a lentiviral vector also called a (lenti-)viral particle, said lentiviral vector or said (lenti-)viral particle comprising said encapsidated and enveloped vector genome. This lentiviral vector is also efficient for introduction into the cell, it is non-replicative, and it leads to the integration (targeted or not) of said vector genome into the genome of an infected cell. In certain cases, the lentiviral particle will contain a mutant integrase (D64V) which will result in a non-integrating provirus (post reverse transcription vector DNA) in the genome of the transduced cell. Consequently, the genome of this vector will be in the form of an episome in the cell nucleus.Classically, the structure of this lentiviral genome after reverse transcription includes: The cis sequences of the HIV lentiviral genome, i.e. two Long Terminal Repeats (LTR) flanking the ends of the vector genome with the U3, R and U5 regions but where the U3 region is mutant (AU3), devoid of its enhancer sequence and therefore without promoter activity. Lentiviral vectors with AU3 in the LTR are called "self-inactivating" since they cannot be transcribed by a wild-type HIV virus. The 5' LTR of the genome is followed by a "psi" sequence for the encapsidation of the vector RNA genome, a Rev Responsive element (RRE) sequence for the export of the vector RNA genome, the Central Polypurine Tract "cppt" and Central Termination Sequence "cts" sequences, for the formation of the central DNA triplex during reverse transcription and a 3' LTR. In addition to the cis sequences of the virus, these vectors contain an expression cassette, placed between the two LTRs.By "expression cassette", as mentioned above, is meant a nucleic acid comprising an inducible promoter upstream of a toxic or suicide transgene, the nucleic acid encoding said toxic or suicide transgene possibly comprising at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail, said expression cassette having been introduced into said lentiviral genome such that its transcription is reversed compared to that of said lentiviral genome.

[0019] The term "promoter" refers to a nucleic acid located upstream of a gene (or transgene) and which controls its expression, in particular by regulating its transcription, which can be constitutive or induced. Reference is then made to a constitutive promoter, or to an inducible or tissue-specific promoter.

[0020] By "constitutive promoter" is meant a nucleic acid located upstream of a gene (or transgene) whose expression it controls is constant regardless of its environment, i.e. whether it is in the cellular environment where the recombinant vector particles are produced or in another cellular environment with a distinct phenotype. In the invention, these may be: SV40 (or pSV40), CMV (or pCMV), UbC (or pUbC), EFl-a core (or pEFl-a core), EFl-a long (or pEFl-a long), hPGK (or phPGK), CAGG (or pCAGG), RSV (or pRSV) or SFFV (or pSFFV).

[0021] By "inducible promoter" is meant a nucleic acid located upstream of a gene (or transgene) whose expression it controls is not constitutive and is only induced after its activation in response to the right stimulus. In the invention, these can be: the 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB- TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA or the miniCHACl YB - TATA The promoters of 2XAARE YB - TATA , the 2xAARE TK , and miniCHACl YB TATA are activated by an inducer inducing the expression of the transcription factor ATF4. The GRP78 promoter YB TATA is activated by an inducer inducing the transcription factor ATF6. The 4xSRE promoter YB TATA is induced by steroid hormones and more specifically by dexamethasone. The 9xHRE promoter YB TATA is induced by hypoxia. Interestingly, the inducible promoters mentioned above are dependent on the expression of cellular transcription factors that are poorly or not expressed under vector production conditions in HEK 293T cells.

[0022] By "tissue-specific promoter" is meant a nucleic acid located upstream of a gene (or transgene) whose expression that it controls is not effective in the cellular environment where the recombinant vector particles are produced, but is activated in another cellular environment with a distinct phenotype to which said tissue-specific promoter is sensitive. In response to this other environment, said tissue-specific promoter will be activated. For example, in the invention it may be the miR223 promoter which is specifically induced in myeloid cells, which is not a HEK-293T cell.

[0023] By "toxic or suicide transgene" is meant a nucleic acid located downstream of said promoter, in particular constitutive, inducible or tissue-specific, and which codes for a toxic, cytotoxic or suicide protein in the genetically modified cell. That is to say that after the activation of said promoter controlling the expression of said toxic or suicide transgene, the product (i.e. the toxic or suicide protein) resulting from the transcription and translation of this nucleic acid causes (leads to) the cell death of the cell expressing it. In the invention, the latter corresponds to the Noxa protein whose nucleic acid encoding it is chosen from the sequences having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1 or whose nucleic acid encodes a protein, a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 2. This is a non-secreted toxic or suicide protein, iewhose expression after activation of said promoter upstream of the nucleic acid encoding it is not found in the extracellular medium and therefore only results in the cell death of the cell expressing them.

[0024] Surprisingly, while the Noxa protein is a toxic or suicide transgene, i.e. its expression induces the death of the cell that expresses it, its expression in the context of production of the lentiviral vector of the invention does not interfere with the survival of the latter. High titers of lentiviral vectors can therefore be obtained by implementing an expression cassette allowing the expression of the Noxa protein during the production of recombinant viral vector particles and thus satisfying the medical profession's expectation of having them available in abundance.

[0025] By "% identity" is meant the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequence), determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides in the longer sequence of the two, and multiplying the result by 100. By "having at least 80% identity" is therefore meant that the aforementioned percentage identity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least less than 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%.In this regard, it should be noted that this definition applies to all embodiments of the invention, including when it involves a direct comparison of two polypeptide sequences (amino acid sequence). Furthermore, it is understood that sequences having at least 80% identity with a reference sequence retain the same properties and functions, or even these are improved.

[0026] According to another embodiment, the subject of the invention is the vector genome as described above, said toxic or suicide transgene encoding a Noxa protein being chosen from the sequences having at least 90% identity with the nucleic acid of sequence SEQ ID NO: 1 or whose nucleic acid encodes a Noxa protein whose amino acid sequence is at least 90% identical to the sequence SEQ ID NO: 2. In particular, the subject of the invention is the vector genome as described above, said toxic or suicide transgene encoding a Noxa protein having a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1 or whose nucleic acid encodes a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 2.

[0027] According to another embodiment, the subject of the invention is the vector genome as described above, in which said promoter is a constitutive promoter. In particular, the subject of the invention is the vector genome as described above, in which said promoter is a constitutive promoter chosen in particular from:

[0028] ■ SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0029] ■ CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0030] ■ UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0031] ■ the EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6;

[0032] ■ the long EFl-a whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7;

[0033] ■ hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 8;

[0034] ■ CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 9;

[0035] ■ RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10; and ■ SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 11.

[0036] In particular, the subject of the invention is also the vector genome as described above, in which said promoter is a constitutive promoter chosen from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV whose nucleic acid respectively has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10 and 11. Advantageously, the subject of the invention is the vector genome as described above, in which said promoter is a constitutive promoter chosen from: SV40, CMV, UbC, EFl-a core, EFl-a long, hPGK, CAGG, RSV and SFFV whose nucleic acid corresponds respectively to the nucleic acid of sequence SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10 and 11.

[0037] According to another embodiment, the subject of the invention is the vector genome as described above, in which said promoter is an inducible or tissue-specific promoter. In particular, the subject of the invention is the vector genome as described above, in which said promoter is an inducible or tissue-specific promoter, in particular chosen from:

[0038] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12;

[0039] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 13;

[0040] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 14;

[0041] ■ the 9XHRE YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17;

[0042] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16;

[0043] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17; and

[0044] ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18.

[0045] In particular, the invention also relates to the vector genome as described above, in which said promoter is an inducible or tissue-specific promoter chosen from: 2XAARE YB - TATA , the 2XAARE TK , the 4xSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB- TATA and the miniCHACl YB TATA whose nucleic acid respectively has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12, 13, 14, 15, 16, 18 and 18. Advantageously, the subject of the invention is the vector genome as described above, in which said promoter is a constitutive promoter chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA and the miniCHACl YB - TATA whose nucleic acid corresponds respectively to the nucleic acid of sequence SEQ ID NO: 12, 13, 14, 15, 16, 18 and 18.

[0046] According to another embodiment, the subject of the invention is the vector genome as described above, in which said promoter is a constitutive promoter chosen in particular from: ■ SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0047] ■ CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0048] ■ UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0049] ■ the EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6;

[0050] ■ the long EFl-a whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7;

[0051] ■ hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 8;

[0052] ■ CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 9;

[0053] ■ RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10; and

[0054] ■ SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 11, or in which said promoter is an inducible or tissue-specific promoter chosen in particular from:

[0055] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12;

[0056] ■ the 2XAARE TKwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 13;

[0057] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 14;

[0058] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17;

[0059] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16;

[0060] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17; and

[0061] ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18.

[0062] In view of the above, it is understood that the invention implements the vector genome as described above, in which said expression cassette is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 19 to 34. In particular, the subject of the invention is the vector genome as described above, in which said expression cassette is chosen from the sequences SEQ ID NOs: 19 to 34. In other words, the subject of the invention is the vector genome as described above, in which said expression cassette is the sequence SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (see Table 1).

[0063] Table 1. List of expression cassettes

[0064] According to another embodiment, the invention relates to the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) or a Respiratory syncytial virus (RSV).

[0065] According to another embodiment, the subject of the invention is the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN). In particular, the subject of the invention is the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 35. In particular, the subject of the invention is also the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 35.It should be noted that the insertion of said expression cassette into said Self-inactivating Lentivirus (SIN) of sequence SEQ ID NO: 35 can be or is carried out at nucleotides 2229 and 2230 of this SEQ ID NO: 35. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 36 and 37.

[0066] According to another embodiment, the subject of the invention is the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Respiratory syncytial virus (RSV). In particular, the subject of the invention is the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Respiratory syncytial virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 38. In particular, the subject of the invention is also the vector genome as described above, in which said lentiviral genome into which said expression cassette has been introduced is a Respiratory syncytial virus (RSV) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 38.It should be noted that the insertion of said expression cassette into said Respiratory syncytial virus (RSV) of sequence SEQ ID NO: 38 can be or is carried out at nucleotides 1737 and 1738 of this SEQ ID NO: 38. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 39 and 40.

[0067] In view of the above, it is understood that the invention implements the vector genome as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 41 to 72. In particular, the subject of the invention is the vector genome as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 41 to 72. In other words, the subject of the invention is also the vector genome as described above, in which said vector genome is the sequence SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 (see Table 2).

[0068] Table 2. List of vector genomes

[0069] According to another embodiment, the subject of the invention is the vector genome as described above, in which said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level, said coding or non-coding sequence having regulatory properties at the post-transcriptional level being located at the 5' end (upstream) of said toxic or suicide transgene coding a Noxa protein and being in particular chosen from the sequences having at least 90% identity with the sequences SEQ ID NOs: 73 to 99.

[0070] The expression "coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level" designates a nucleic acid sequence which can be either coding (having the capacity to code for proteins) or non-coding (not coding for proteins), and which has regulatory properties acting after the transcription process. That is to say, after the production of an mRNA, the presence of said nucleic acid sequence within it inhibits under certain conditions (for example, normal physiological conditions) the translation of said mRNA and therefore, the production of the protein that the mRNA codes for. Under other conditions (for example, conditions of cellular stress), the presence of said nucleic acid sequence within the mRNA no longer has its role of inhibiting translation. Translation then takes place and the protein coded by said mRNA is produced.

[0071] In the invention, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level is located, in an expression cassette, between a transgene of interest and a promoter, said transgene of interest being located downstream of said promoter. In other words, said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level is located at the 5' end (upstream) of said transgene of interest. This arrangement of said expression cassette (5'-Promoter_ Coding or non-coding sequence having regulatory properties at the asZ-transcriptional level Transgene of interest-3') makes it possible, in the event of unwanted transcription of an mRNA from said promoter, to inhibit its translation using said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level and therefore, to inhibit the production of said transgene of interest.

[0072] According to another embodiment, the subject of the invention is the vector genome as described above, in which said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level located at the 5' end (upstream) of said toxic or suicide transgene encoding a Noxa protein is chosen from the sequences having at least 90% identity with the sequences SEQ ID NOs: 73 to 99. In particular, the subject of the invention is the vector genome as described above, in which said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level located at the 5' end (upstream) of said toxic or suicide transgene encoding a Noxa protein is chosen from the sequences SEQ ID NOs: 73 to 99.In other words, the invention also relates to the vector genome as described above, in which said coding or non-coding sequence having regulatory properties at the / ?as7-transcriptional level is the sequence SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 (see Table 3).

[0073] Table 3. List of 5'UTR sequences with regulatory properties at the post-transcriptional level

[0074] Advantageously, the subject of the invention is the vector genome as described above, in which said coding or non-coding sequence having regulatory properties at the post-transcriptional level is the 5'UTR sequence of ATF4 of sequence SEQ ID NO: 73.

[0075] In view of the above, it is understood that the invention implements the vector genome as described above, in which said expression cassette is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 100 to 531. In particular, the subject of the invention is the vector genome as described above, in which said expression cassette is chosen from the sequences SEQ ID NOs: 100 to 531 (see Table 4).

[0076] Table 4. List of expression cassettes comprising a 5'UTR

[0077] In view of the above, it is also understood that the invention implements the vector genome as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 532 to 1395. In particular, the subject of the invention is the vector genome as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 532 to 1395 (see Table 5).

[0078] Table 5. List of vector genomes including a 5'UTR

[0079] Interestingly, it should be noted that to optimize the production of said vector genome as described above and to add safety, the 5' to 3' (5' > 3') orientation of the transcription of said expression cassette as described above is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome. Also and according to another embodiment, the subject of the invention is the vector genome as described above, in which the 5' to 3' orientation of the transcription of said expression cassette is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, in particular said expression cassette further comprising at the 3' end (downstream) of said toxic or suicide transgene encoding a Noxa protein a nucleic acid sequence allowing the transcription of a polyA tail.

[0080] When this embodiment is implemented, the vector genome as described above, comprises in particular at the 3' end of said transgene of interest a polyA sequence (eg SEQ ID NO: 1396). Indeed, the 5' to 3' orientation of the transcription of said expression cassette as described above being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, the addition of this polyA sequence (eg SEQ ID NO: 1396) to the 3' end of the nucleic acid coding for said toxic or suicide transgene makes it possible to promote the transcription and stability of an mRNA, which authorizes the translation of said transgene of interest in particular when the inhibition of translation by said coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level is lifted.

[0081] According to another embodiment, the subject of the invention is therefore the vector genome as described above, said expression cassette further comprising a polyA tail whose nucleic acid sequence is at least 80% identical to the sequence SEQ ID NO: 1396. In particular, the subject of the invention is the vector genome as described above, said expression cassette further comprising a polyA tail of sequence SEQ ID NO: 1396.

[0082] In view of the above, it is understood that the invention implements the vector genome as described above, in which said expression cassette further comprises a polyA tail and is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 1397 to 1412. In particular, the subject of the invention is the vector genome as described above, in which said expression cassette further comprises a polyA tail and is chosen from the sequences SEQ ID NOs: 1397 to 1412 (see Table 6).

[0083] Table 6. List of expression cassettes containing a polyA tail

[0084] In view of the above, it is also understood that the invention implements the vector genome as described above, in which said expression cassette further comprises a polyA tail and the 5' to 3' (5' > 3') orientation of the transcription of which is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said vector genome being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 1845 to 1876. In particular, the subject of the invention is the vector genome as described above, in which said expression cassette further comprises a polyA tail and the 5' to 3' (5' > 3') orientation of the transcription of which is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, said vector genome being chosen from SEQ ID NOs: 1845 to 1876. 876 (see Table 7).

[0085] Table 7. List of “inverted” vector genomes including a polyA tail

[0086] In view of the above, it is also understood that the invention implements the vector genome as described above, wherein said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the post-transcriptional level and a polyA tail, and is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 1413 to 1844. In particular, the subject of the invention is the vector genome as described above, wherein said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the / w.s7-transcriptional level and a polyA tail, and is chosen from the sequences SEQ ID NOs: 1413 to 1844 (see Table 8).

[0087] Table 8. List of expression cassettes comprising a 5'UTR and a polyA tail

[0088] In view of the above, it is also understood that the invention implements the vector genome as described above, in which said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the post-transcriptional level and a polyA tail, and whose 5' to 3' (5' > 3') orientation of transcription is reversed with respect to the 5' to 3' orientation of transcription of said lentiviral genome, said vector genome being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 1877 to 2740.In particular, the subject of the invention is the vector genome as described above, in which said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the post-transcriptional level and a polyA tail, and whose 5' to 3' (5' > 3') orientation of transcription is reversed with respect to the 5' to 3' orientation of transcription of said lentiviral genome, said vector genome being chosen from SEQ ID NOs: 1,877 to 2,740 (see Table 9).

[0089] Table 9. List of “inverted” vector genomes comprising a 5’UTR and a polyA tail

[0090] According to another aspect, the invention relates to a method for producing lentiviral vectors comprising a vector genome as described above, said method comprising at least the steps of: a. co-transfecting an animal eukaryotic cell with: i. a plasmid comprising a vector genome as described above and the means for expressing it; ii. a plasmid comprising a viral envelope protein and the means for expressing it; and iii. a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b. culturing said transfected animal eukaryotic cell to allow the production of lentiviral vectors comprising a vector genome as described above; and c. harvesting and purifying said lentiviral vectors.

[0091] Such a method is illustrated in the examples below and is accessible to those skilled in the art in view of the literature at their disposal (eg Zennou et al. Nat Biotechnol. 2001 May; 19(5):446-50)). Nevertheless, and for the purposes of illustration, it is specified that:

[0092] ■ said animal eukaryotic cell may be a cell belonging to a cell line chosen from: HEK 293T, HEK 293, CHO, GS-CHO, Free style 293-F cells (FS293), Viral Production Cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8 and PS46.2;

[0093] ■ said plasmid comprising a viral envelope protein and the means for expressing it may be chosen from the plasmids: pHCMV-VSV (SEQ ID NO: 4595), pHCMV-Mokola, pHCMV-RabiesG, pHCMV-AmphoEnv, pHCMV-LCMV-WE, pHCMV-LCMV-Arm53b and pHCMV-10Al (Sena-Esteves M et al. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. 2004 Dec 15;122(2):131-9); and

[0094] ■ said plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it can be chosen from the plasmids: p8.92 (SEQ ID NO: 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).

[0095] For illustrative purposes and without limitation, the following protocol is also provided: Cells (e.g. HEK 293 T) were seeded on supports suitable for cell culture and so as to reach a confluence of 50 to 60% after 24 hours of culture. The next day, the cells were co-transfected, by the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome and two transcomplementing plasmids encoding the enzymes and structural proteins of HIV-1 (p8.9) and the envelope glycoprotein of the vesicular stomatitis virus (pVSVg). Co-transfection was carried out at a plasmid ratio of 2:2:1. The transfected cells were cultured for 5 hours in contact with the precipitate and then the culture medium was renewed. The supernatant containing the lentiviral particles was harvested 48 hours later.The lentiviral suspension was treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 µm) and then ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PBS) corresponding to a concentration of a factor of 1,000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0096] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell belongs to a cell line chosen from: HEK 293T, HEK 293, CHO, GS-CHO, Free style 293-F cells (FS293), Viral Production Cells (VPC and VPC 2.0), COS-1, HeLa, PS5.8 and PS46.2. Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell belongs to the HEK 293T cell line.

[0097] As mentioned above, while the Noxa protein is a toxic or suicide transgene, i.e. its expression induces cell death in the cell that expresses it, its expression in the context of production of the lentiviral vector of the invention surprisingly does not interfere with it. That is to say, it does not cause the death of the producing cell (e.g. HEK 293T) of the lentiviral vector of the invention, whereas it does cause the death of the genetically modified target cell (e.g. T lymphocyte). With the tools of the invention developed by the inventors, the current gaps identified by the medical profession in terms of gene therapy tools are therefore fully addressed. On this point, the invention constitutes a real technological breakthrough.

[0098] In the invention, it is of course understood that "lentiviral vector" designates a recombinant lentiviral particle obtained using the plasmid comprising a vector genome as described above and the means for expressing it, which makes it possible to produce the (lenti-)viral particle of the invention. This comprises said encapsidated and / or enveloped vector genome. It is effective for introduction into the cell, it is non-replicative and it leads either to the integration (targeted or not) of said vector genome into the genome of an infected cell, or to the presence of an episome not integrated into the cellular genome.

[0099] The term "plasmid" designates a double-stranded circular DNA molecule, which necessarily has a bacterial origin of replication and, incidentally, the SV40 virus, so that it can replicate autonomously, e.g., in E. coli for its amplification and, e.g., in the HEK 293T cell after transfection, and a selection gene so that it is not lost during its bacterial amplification during cell multiplications. In the invention, the one mentioned in i. further comprises the nucleic acid of said vector genome of the invention and the means for expressing it. That is to say, the genetic elements necessary for the expression of the vector genome of the invention and the production of a lentiviral vector according to the invention. The same applies to the other plasmids ii. and iii. which respectively allow the expression of a viral envelope protein, and the 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 mentioned in i. is reversed with respect to the 5' to 3' orientation of the transcription of said expression cassette.

[0100] This plasmid comprising a vector genome as described above and the means for expressing it may be a pLV-SIN plasmid or a pLV-RSV plasmid.

[0101] If it is a pLV SIN plasmid, the nucleic acid thereof has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2741. In particular, the pLV SIN plasmid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2741. Note that the insertion of said vector genome into said pLV-SIN plasmid of sequence SEQ ID NO: 2741 can be or is carried out at nucleotides 4939 and 4940 of this SEQ ID NO: 2741. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 2742 and 2743.

[0102] If it is a pLV RSV plasmid, the nucleic acid thereof has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2744. In particular, the pLV RSV plasmid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2744. Note that the insertion of said vector genome into said pLV-RSV plasmid of sequence SEQ ID NO: 2744 can be or is carried out at nucleotides 234 and 235 of this SEQ ID NO: 2744. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 2745 and 2746.

[0103] In view of the above, it is understood that according to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid i. as described above is chosen from the sequences having 80% identity with the sequences SEQ ID NOs: 2747 to 2778, 2779 to 3642, 3643 to 3674 and 3675 to 4538. Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid i. as described above is chosen from the sequences SEQ ID NOs:

[0104] ■ 2,747 to 2,778 (see Table 10 - “sense” plasmid without 5'UTR);

[0105] ■ 2,779 to 3,642 (see Table 11 - “sense” plasmid with 5'UTR);

[0106] ■ 3,643 to 3,674 (see Table 12 - “inverted” plasmid without 5'UTR); and

[0107] ■ 3,675 to 4,538 (see Table 13 - “inverted” plasmid with 5'UTR).

[0108] It should be noted that the plasmids as such cited below also form an integral part of the invention and constitute one of its objects, as does their use for producing a lentiviral vector comprising a vector genome as described above.

[0109] Table 10. List of “sense” plasmids

[0110] Table 11. List of “sense” plasmids + 5'UTR

[0111] Table 12. List of “inverted” plasmids

[0112] Table 13. List of “inverted” plasmids + 5'UTR

[0113] In order to facilitate and optimize the production of the lentiviral vectors of the invention, it is also possible to use an innovative eukaryotic cell which expresses a ribonucleoprotein complex constructed specifically to repress the expression of the toxic or suicide transgene of the lentiviral vector to be produced. According to another embodiment, the invention therefore relates to the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell expresses at least one ribonucleoprotein complex comprising:

[0114] ■ a fusion protein comprising the fusion:

[0115] - an inactive Cas endonuclease (dCas); and

[0116] - a KRAB transcriptional repression domain or a methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L; or a fusion protein comprising the fusion:

[0117] - a KRAB transcriptional repression domain;

[0118] - an inactive Cas endonuclease (dCas); and

[0119] - a methylation domain chosen from: DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L and MeCP2; and

[0120] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said inducible promoter, said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA, dCas-DNMT3B-L / gRNA, KRAB-dCas- DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular expressed stably.

[0121] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3 A, DNMT3B, DNMT3 AL and DNMT3B-L is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain or said methylation domain chosen from: DNMT3 A, DNMT3B, DNMT3A-L and DNMT3B-L is in the C-terminal position of said inactive Cas endonuclease (dCas).

[0122] The expression "animal eukaryotic cell expressing at least one ribonucleoprotein complex" refers to the fact that the animal eukaryotic cell can express a single ribonucleoprotein complex (the interference one dCas-KRAB / gRNA; or one of the methylation ones dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA or dCas-DNMT3B-L / gRNA; or one of the bifunctional ones KRAB-dCas-DNMT3A, gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3 AL / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA) as it can express several and in particular at least two, namely:

[0123] ■ a dCas-KRAB / gRNA interference ribonucleoprotein complex (CRISPRi); and

[0124] ■ a ribonucleoprotein methylation complex (called CRISPRm) chosen from: dCas- DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3A-L / gRNA and dCas- DNMT3B-L / gRNA.

[0125] The CRISPR interference (CRISPRi) and CRISPR-targeted methylation (CRISPRm) strategies according to the invention are based on the fusion of an inactive or "dead" CRISPR-Cas endonuclease (dCas) and a transcriptional repression domain KRAB (Krüppel-Associated Box) or methylation domain DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L (DNA methyltransferase). The dCas, thanks to its guide RNA (gRNA), makes it possible to target a specific sequence. This targeting allows the KRAB domain to sterically block transcription and the DNMT3 A, DNMT3B, DNMT3A-L or DNMT3B-L domains to block transcription by de novo methylation of the targeted DNA. The implementation of a CRISPRi and / or CRISPRm strategy on a sequence internal to the lentiviral vector during the production of recombinant particles requires the co-expression of at least one CRISPRi and / or CRISPRm complex and a gRNA in the transfected animal eukaryotic cells.Alternatively, the invention implements a bifunctional CRISPRi / m complex comprising the fusion of an inactive or "dead" CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3 A, DNMT3B, DNMT3 AL, DNMT3B-L or MeCP2 methylation domain, and a gRNA in the transfected animal eukaryotic cells.

[0126] The term "gRNA (guide RNA)" refers to an RNA that associates with an enzyme or protein complex, which, when paired with a complementary RNA or DNA sequence, allows the enzyme or protein complex to position itself on this complementary RNA or DNA.

[0127] The term "dCas-KRAB / gRNA ribonucleoprotein complex" refers to an interference system comprising the association of a dCas-KRAB fusion protein and a guide RNA (gRNA) specifically recognizing a succession of 17 to 24 bases followed by a single or repeated "PAM" (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These two elements can work together, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce lentiviral vector titers carrying a transgene of interest (in particular a toxic or suicide transgene) that have never been achieved before. In this sense, this embodiment is a real revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0128] The term “dCas-DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA or dCas-DNMT3B-L / gRNA ribonucleoprotein complex” refers to a methylation system comprising the association of a dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3 AL or dCas-DNMT3B-L fusion protein and a guide RNA (gRNA) specifically recognizing a succession of 17 to 24 bases followed by a single or repeated “PAM” (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These two elements can work in concert, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce lentiviral vector titers carrying a transgene of interest (notably a toxic or suicide transgene) never before achieved.In this sense, this method is a real revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0129] The term “KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA ribonucleoprotein complex” refers to a bifunctional interference and methylation system comprising the association of a KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 fusion protein and a guide RNA (gRNA) specifically recognizing a sequence of 17 to 24 bases followed by a single or repeated “PAM” (Protospacer Adjacent Motif) motif in the promoter sequence of a promoter present in the genome of the lentiviral vector to be produced. These three elements can work together, in synergy with the inversion of said expression cassette, and unexpectedly make it possible to produce lentiviral vector titers carrying a transgene of interest (in particular a toxic or suicide transgene) never before achieved.In this sense, this method is a real revolution since it finally makes it possible to offer patients the safe and effective cell therapies they need.

[0130] The term "dCas-KRAB fusion protein" refers to the combination of two amino acid sequences from different proteins or their fragments to create an artificial and functional protein. In the invention, these two fused components are, as mentioned, an inactive Cas endonuclease (dCas) and a KRAB transcriptional repression domain, and a nuclear import signal (NLS) peptide is added to ensure its nuclear localization (e.g. encoded by a nucleic acid of sequence SEQ ID NO: 4,543). 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 their fragments to create an artificial and functional protein. In the invention, these two fused components are, as mentioned, an inactive Cas endonuclease (dCas) and a DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L methylation domain.A nuclear import signal peptide (NLS) is also added to ensure its nuclear localization (eg encoded by a nucleic acid of sequence SEQ ID NO: 4543).

[0131] The term “KRAB-dCas-DNMT3A, KRAB-dCas-DNMT3B, KRAB-dCas-DNMT3A-L, KRAB-dCas-DNMT3B-L or dCas-KRAB-MeCP2 fusion protein” refers to the combination of three amino acid sequences from different proteins or fragments thereof to create an artificial and functional protein. In the invention, these three fused components are, as mentioned, a KRAB transcriptional repression domain, an inactive Cas endonuclease (dCas) and a DNMT3A, DNMT3B, DNMT3A-L or DNMT3B-L methylation domain. A nuclear import signal (NLS) peptide is also added to ensure its nuclear localization (e.g. encoded by a nucleic acid of sequence SEQ ID NO: 4543).

[0132] The expression "inactive Cas endonuclease (dCas)" or the equivalent expression "dead" Cas endonuclease (dCas) refers to endonucleases whose activity of cleaving one or two DNA strands does not function. To date, several Cas endonucleases (Cas9, Casl2a, Casl2b, CasX or Cas 12e, Casl2f or Cas 14, Casl2j or Cas®) from different hosts have been identified and characterized, or even modified (e.g. improvement of their function), which can be implemented by the invention after their inactivation. According to another embodiment, the subject of the invention is therefore the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease (dCas) is chosen from:

[0133] ■ inactive Cas9 endonucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, '

[0134] ■ inactive Caslla endonucleases from Lachnospiraceae bacterium and Acidaminococcus sp, '

[0135] ■ inactive Casllb endonucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, '

[0136] ■ inactive CasX or Casl2e endonucleases from Deltaproteobacteria and Planctomycetes, '

[0137] ■ inactive type V CRISPR endonucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans,'

[0138] ■ inactive Cas 12j or Cas® endonucleases of the Biggiephage clade, -

[0139] ■ inactive Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and

[0140] ■ Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or inactive variants derived from these organisms. The term “orthologs” refers to similar Cas endonucleases present in two or more different species. These, due to evolution, may be inactive and if this is not the case, it is possible to render them non-functional by mutation in order to exploit them for the purposes of the invention.

[0141] The term "mutants" refers to a Cas endonuclease into which one or more mutations have been introduced, including the deletion, substitution, and / or addition of one or more amino acids. "Inactive mutants" therefore refers to Cas proteins modified by human intervention (e.g., by genetic engineering) to inactivate their DNA cleavage activity.

[0142] The term "inactive variants" refers to naturally evolving Cas endonucleases that lack DNA cleavage activity.

[0143] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9). This is a ribonucleoprotein complex dCas9-KRAB / gRNA, dCas9-DNMT3A / gRNA, dCas9-DNMT3B / gRNA, dCas9-DNMT3A-L / gRNA, dCas9-DNMT3B-L / gRNA, KRAB-dCas9-DNMT3A / gRNA, KRAB-dCas9-DNMT3B / gRNA, KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA or dCas9-KRAB-MeCP2 / gRNA. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4546 or whose nucleic acid encodes an inactive Cas9 endonuclease (dCas9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4547.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said inactive Cas endonuclease is an inactive Cas9 endonuclease (dCas9) whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4546 or whose nucleic acid codes for an inactive Cas9 endonuclease (dCas9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 4547.

[0144] The expression "KRAB transcriptional repression domain" refers to the KRAB (Kruppel-associated suppression box) domain of the human zinc finger protein Koxl (or zinc finger protein 10) or Zim3, which causes specific inhibition of RNA polymerases II and III. In doing so, this KRAB domain inhibits the expression of the gene(s) present on the DNA where it is recruited. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4544 or 4552, or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4545 or 4553.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said KRAB transcriptional repression domain is encoded by the nucleic acid of sequence SEQ ID NO: 4544 or 4552, or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4545 or 4553.

[0145] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein. In particular, the invention relates to the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4549 or 4555.In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said dCas-KRAB fusion protein is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4549 (encoded by the plasmid pLV-EFla-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 4551) or 4555.

[0146] The expression "methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L and MeCP2" refers to the functional domain of DNA methyltransferase, which catalyzes the covalent modification of DNA by transfer of S-adenosyl-1-methionine (SAM) by converting cytosine into 5-methylcytosine. In doing so, this DNMT3A, DNMT3B, DNMT3A-L, DNMT3B-L or MeCP2 domain inhibits the expression of the gene(s) present on the DNA where it is recruited. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said methylation domain:

[0147] ■ DNMT3 A is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4556 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4557;

[0148] ■ DNMT3B is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4568 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4569;

[0149] ■ DNMT3A-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4560 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4561; or

[0150] ■ DNMT3B-L is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4570 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4571.

[0151] In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which said methylation domain:

[0152] ■ DNMT3A is encoded by the nucleic acid of sequence SEQ ID NO: 4556 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4557; ■ DNMT3B is encoded by the nucleic acid of sequence SEQ ID NO: 4568 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4569;

[0153] ■ DNMT3A-L is encoded by the nucleic acid of sequence SEQ ID NO: 4560 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4561; or

[0154] ■ DNMT3B-L is encoded by the nucleic acid of sequence SEQ ID NO: 4570 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4571.

[0155] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said MeCP2 methylation domain is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4572 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4573. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said MeCP2 methylation domain is encoded by the nucleic acid of sequence SEQ ID NO: 4572 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4573.

[0156] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3A, dCas-DNMT3B, dCas-DNMT3A-L or dCasDNMT3B-L fusion protein is respectively a dCas9-DNMT3 A, dCas9-DNMT3B, dCas9-DNMT3 AL or dCas9-DNMT3B-L fusion protein. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0157] ■ said dCas-DNMT3A fusion protein is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4562 or whose nucleic acid encodes a dCas9-DNMT3A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4563;

[0158] ■ said dCas-DNMT3B fusion protein is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4574 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4575;

[0159] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4566 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4567; or

[0160] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4576 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4577. In particular, the invention also relates to the method for producing lentiviral vectors as described above, in which

[0161] ■ said 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 a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4563;

[0162] ■ said 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 a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4575;

[0163] ■ said dCas-DNMT3A-L fusion protein is a dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4566 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4567; or

[0164] ■ said dCas-DNMT3B-L fusion protein is a dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4576 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4577.

[0165] Interestingly, if the DNMT3L enzyme is catalytically inactive, it stimulates the activity of DNMT3A and DNMT3B and thus promotes the silencing of a promoter by methylation. Hence the DNMT3 AL and DNMT3B-L methylation domains where respectively DNMT3A or DNMT3B have been combined with DNMT3L. This being the case, it is possible to envisage the addition of a dCas-DNMT3L / gRNA ribonucleoprotein complex in the animal eukaryotic cell of the invention. According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell further expresses another ribonucleoprotein complex comprising:

[0166] ■ a fusion protein comprising the fusion:

[0167] - an inactive Cas endonuclease (dCas); and

[0168] - a domain promoting DNMT3L methylation (dCas-DNMT3E fusion) ■ and

[0169] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said inducible promoter, said other dCas-DNMT3L / gRNA ribonucleoprotein complex being in particular stably expressed.

[0170] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said DNMT3L methylation-promoting domain is in the N-terminal or C-terminal position of said inactive Cas endonuclease (dCas). Advantageously, the subject of the invention is the use of an animal eukaryotic cell as described above, in which said DNMT3L methylation-promoting domain is in the C-terminal position of said inactive Cas endonuclease (dCas). According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said domain promoting DNMT3L methylation is encoded by a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4558 or whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4559.In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said domain promoting DNMT3L methylation is encoded by the nucleic acid of sequence SEQ ID NO: 4558 or whose amino acid sequence corresponds to the sequence SEQ ID NO: 4559.

[0171] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4565. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said dCas-DNMT3L fusion protein is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a fusion protein dCas9-DNMT3L whose amino acid sequence corresponds to the sequence SEQ ID NO: 4565.

[0172] As mentioned above, the invention also implements a bifunctional CRISPRi / m complex comprising:

[0173] ■ the fusion of an inactive or “dead” CRISPR-Cas endonuclease (dCas), a KRAB transcriptional repression domain and a DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L or MeCP2 methylation domain; and

[0174] ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said inducible promoter (that of the expression cassette of the invention).

[0175] In particular, this bifunctional complex (advantageously expressed in a stable manner) is chosen from: KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA and dCas-KRAB-MeCP2 / gRNA, and is advantageously chosen from: KRAB-dCas9-DNMT3A-L / gRNA, KRAB-dCas9-DNMT3B-L / gRNA and dCas9-KRAB-MeCP2 / gRNA. Also, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0176] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4,578 or 4,584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4,579 or 4,585;

[0177] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4581 or 4587; or

[0178] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4583 or 4589.

[0179] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0180] ■ said KRAB-dCas-DNMT3 AL fusion protein is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4578 or 4584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4579 (encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 4590) or 4585;

[0181] ■ said KRAB-dCas-DNMT3B-L fusion protein is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4581 or 4587; or

[0182] ■ said dCas-KRAB-MeCP2 / gRNA fusion protein is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4583 or 4589.

[0183] Advantageously, the subject of the invention is the method for producing lentiviral vectors as described above, in which said ribonucleoprotein complex dCas-KRAB / sgRNA, dCas-DNMT3A / sgRNA, dCas-DNMT3B / sgRNA, dCas-DNMT3L / sgRNA, dCas-DNMT3A-L / sgRNA, dCas-DNMT3B-L / sgRNA, KRAB-dCas-DNMT3A / sgRNA, KRAB-dCas-DNMT3B / sgRNA, KRAB-dCas-DNMT3A-L / sgRNA, KRAB-dCas-DNMT3B-L / sgRNA or dCas-KRAB-MeCP2 / sgRNA is stably expressed via targeted integration into the genome of said animal eukaryotic cell.

[0184] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4541 (which specifically recognizes the constitutive SV40 promoter (SEQ ID NO: 3)). In particular, the invention relates to the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 4541. According to another embodiment, the invention therefore relates to the method for producing lentiviral vectors as described above, in which:

[0185] ■ said fusion protein:

[0186] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4549 or 4555,

[0187] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4562 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4563,

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

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

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

[0191] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4565,

[0192] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4578 or 4584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4579 or 4585,

[0193] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4581 or 4587, and / or

[0194] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4583 or 4589;

[0195] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4541; and

[0196] ■ said constitutive promoter is SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3.

[0197] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0198] ■ said fusion protein:

[0199] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4549 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 4551) or 4555,

[0200] - dCas-DNMT3A 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 a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4563,

[0201] - dCas-DNMT3B 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 a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4575,

[0202] - 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 a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4567,

[0203] - 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 a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4577, - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4565,

[0204] - 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 4584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4579 (encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 4590) or 4585,

[0205] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4581 or 4587, and / or

[0206] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4583 or 4589;

[0207] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 4,541; and

[0208] ■ said constitutive promoter is SV40 whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 3.

[0209] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 19, 100 to 126, 1397 and 1412 to 1439, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 41, 57, 532 to 558, 964 to 990, 1845, 1861, 1877 to 1903 and 2309 to 2335, which can be encoded by a plasmid chosen from the sequences SEQ ID NOs: 2747, 2763, 2779 to 2805, 3211 to 3237, 3643, 3659, 3675 to 3 701 and 4107 to 4133.

[0210] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4,540 (which specifically recognizes the inducible promoter 2XAARE YB TATA(SEQ ID NO: 12)). In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 4,540.

[0211] According to another embodiment, the invention therefore relates to the method for producing lentiviral vectors as described above, in which:

[0212] ■ said fusion protein: - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4549 or 4555,

[0213] - dCas-DNMT3A is a dCas9-DNMT3A fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4562 or whose nucleic acid encodes a dCas9-DNMT3 A fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4563,

[0214] - dCas-DNMT3B is a dCas9-DNMT3B fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4574 or whose nucleic acid encodes a dCas9-DNMT3B fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4575,

[0215] - dCas-DNMT3A-L is a dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4566 or whose nucleic acid encodes a dCas9-DNMT3A-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4567,

[0216] - dCas-DNMT3B-L is a dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4576 or whose nucleic acid encodes a dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4577,

[0217] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4565,

[0218] - KRAB-dCas-DNMT3A-L is a KRAB-dCas9-DNMT3A-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4578 or 4584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3 AL fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4579 or 4585,

[0219] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4581 or 4587, and / or

[0220] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 4583 or 4589;

[0221] ■ said gRNA comprises or consists of a nucleic acid having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4,540; and

[0222] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12.

[0223] In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which:

[0224] ■ said fusion protein:

[0225] - dCas-KRAB is a dCas9-KRAB fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4548 or 4554, or whose nucleic acid encodes a dCas9-KRAB fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4549 encoded by the plasmid pLV-EF la-dCas9-KRAB-P2A-BlastR of sequence SEQ ID NO: 4551) or 4555,

[0226] - dCas-DNMT3A 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 a dCas9-DNMT3A fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4563,

[0227] - dCas-DNMT3B 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 a dCas9-DNMT3B fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4575,

[0228] - 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 a dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4567,

[0229] - 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 a dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4577,

[0230] - dCas-DNMT3L is a dCas9-DNMT3L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4564 or whose nucleic acid encodes a dCas9-DNMT3L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4565,

[0231] - 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 4584, or whose nucleic acid encodes a KRAB-dCas9-DNMT3A-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4579 (encoded by the plasmid pLV-EF la-KRAB l-dCas9-DNMT3a-l-P2A-BlastR of sequence SEQ ID NO: 4590) or 4585,

[0232] - KRAB-dCas-DNMT3B-L is a KRAB-dCas9-DNMT3B-L fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4580 or 4586, or whose nucleic acid encodes a KRAB-dCas9-DNMT3B-L fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4581 or 4587, and / or

[0233] - dCas-KRAB-MeCP2 / gRNA is a dCas9-KRAB-MeCP2 / gRNA fusion protein whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 4582 or 4588, or whose nucleic acid encodes a dCas9-KRAB-MeCP2 / gRNA fusion protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 4583 or 4589;

[0234] ■ said gRNA comprises or consists of a nucleic acid corresponding to the nucleic acid of sequence SEQ ID NO: 4,540; and

[0235] ■ said inducible promoter is 2XAARE YB TATA whose nucleic acid corresponds to the nucleic acid of sequence SEQ ID NO: 12.

[0236] According to this embodiment, it is therefore possible to produce a lentiviral vector comprising an expression cassette chosen from the sequences SEQ ID NOs: 28, 343 to 369, 1406 and 1656 to 1682, and / or whose vector genome can be chosen from the sequences SEQ ID NOs: 50, 66, 775 to 801, 1207 to 1233, 1854, 1870, 2120 to 2146 and 2552 to 2578, which can be encoded by a plasmid chosen from the sequences SEQ ID NOs: 2756, 2772, 3022 to 3048, 3454 to 3480, 3652, 3668, 3918 to 3,944 and 4,350 to 4,376.

[0237] Since a method for producing a lentiviral vector is described, it is understood that another aspect of the invention relates to a lentiviral vector comprising a vector genome as described above. According to this same aspect, the invention also relates to a lentiviral vector capable of being obtained by the method for producing lentiviral viral vectors as described above.

[0238] It is also understood that another aspect of the invention relates to the in vitro use of a lentiviral vector as described above to transduce an animal eukaryotic cell, in particular said animal eukaryotic cell being chosen from: ■ a cancer cell, in particular a cancer cell forming a solid tumor or being involved in a blood cancer;

[0239] ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell;

[0240] ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process;

[0241] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and

[0242] ■ an induced stem cell (iPS).

[0243] According to another embodiment, the subject of the invention is the in vitro use of a viral vector as described above, in which said animal eukaryotic cell is different from a pluripotent or totipotent stem cell, or from a stem cell isolated from a tissue of the organism.

[0244] The term "cancer cell" refers to an abnormal cell that has acquired specific characteristics that cause cancer (or malignant tumor), which is a disease characterized by uncontrolled and abnormally large cell (tumor) proliferation within normal (healthy) tissue of the body, such that the survival of the latter is threatened. These tumors include solid tumors and liquid tumors.

[0245] According to another embodiment, the invention therefore relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a cancer cell, in particular a cancer cell forming a solid tumor or being involved in a blood cancer. In particular, the invention also relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a cancer cell forming a solid tumor. In particular, the invention also relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a cancer cell being involved in a blood cancer.

[0246] The expression "an immune cell" designates a cell involved in the functioning of the immune system. Many examples exist such as lymphocytes (e.g. T, B, NK and NKT), phagocytes (e.g. macrophage and dendritic cell) and granulocytes (e.g. neutrophil, eosinophil and basophil) and the precursor cells of these cells. These cells can be of autologous, syngeneic, allogeneic or xenogeneic origin. Finally, these cells can be genetically modified. It should be noted that in the invention, a T lymphocyte and an NK cell are particularly used. By "T lymphocyte", the invention refers in particular to a naive, effector or memory T lymphocyte, with auxiliary, cytotoxic or regulatory properties. By "NK cell", the invention refers in particular to a primary, cancerous or immortalized NK cell. I ll

[0247] According to another embodiment, the invention therefore relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is an immune cell chosen in particular from: the T lymphocyte and the NK cell. In particular, the invention also relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a T lymphocyte or an NK cell.

[0248] The term "myofibroblast" refers to a cell characterized by matrix protein production, proliferation, migration, and contraction properties, activated in repair and healing processes. These myofibroblasts are found in certain organs such as the liver, lung, heart, or kidney during chronic diseases. The process of tissue replacement by myofibroblasts is called fibrosis.

[0249] According to another embodiment, the invention therefore relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a myofibroblast. In particular, the invention also relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is a myofibroblast involved in a fibrosis process.

[0250] The term "astrocyte" refers to a glial cell in the central nervous system. Typically star-shaped, it performs a variety of important functions, centered on supporting and protecting neurons.

[0251] According to another embodiment, the invention therefore relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is an astrocyte, in particular an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration. In particular, the invention also relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is an astrocyte activated during brain or spinal cord trauma forming a glial scar preventing regeneration.

[0252] The term "stem cell" refers to a cell characterized by its undifferentiation and its ability to differentiate and generate or regenerate tissue, such as hematopoietic tissue, muscle, brain (neurons), retina, liver (hepatocytes), pancreatic beta cells, kidney, cartilage, bone, or skin. These cells, taken from an adult organism or induced from differentiated cells (iPS), can be of autologous, syngeneic, allogeneic, or xenogeneic origin.

[0253] According to another embodiment, the invention therefore relates to the in vitro use of a lentiviral vector as described above, in which said animal eukaryotic cell is an induced stem cell (iPS). According to another aspect of the invention, the invention relates to an animal eukaryotic cell transduced by a lentiviral vector as described above, said transduced animal eukaryotic cell being in particular chosen from:

[0254] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in a blood cancer;

[0255] ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell;

[0256] ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process;

[0257] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and

[0258] ■ an induced stem cell (iPS).

[0259] According to another embodiment, the invention relates to the transfected / transduced animal eukaryotic cell as described above, in which said transduced animal eukaryotic cell is different from a pluripotent or totipotent stem cell, or from a stem cell isolated from a tissue of the organism.

[0260] According to another embodiment, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell, in particular a cancer cell forming a solid tumor or being involved in a blood cancer. In particular, the subject of the invention is also the transfected / transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell forming a solid tumor. In particular, the subject of the invention is also the transfected / transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell being involved in a blood cancer.

[0261] According to another embodiment, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is an immune cell chosen in particular from: the T lymphocyte and the NK cell. In particular, the subject of the invention is also the transfected / transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is a T lymphocyte or an NK cell.

[0262] According to another embodiment, the invention relates to the transfected / transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast. In particular, the invention also relates to the transfected / transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast involved in a fibrosis process.

[0263] According to another embodiment, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is an astrocyte, in particular an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration. In particular, the subject of the invention is also the transfected / transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is an astrocyte activated during brain or spinal cord trauma forming a glial scar preventing regeneration.

[0264] According to another embodiment, the invention relates to the transfected / transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is an induced stem cell (iPS).

[0265] According to another aspect of the invention, the subject of the invention is a transduced animal eukaryotic cell as described above for its use in cell therapy, in particular for treating a pathology chosen from: cancer, leukemia and fibrosis.

[0266] According to another embodiment, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above for its use as described above for treating a pathology chosen from: cancer, leukemia and fibrosis. In particular, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above for its use as described above for treating cancer. In particular, the subject of the invention is the transfected / transduced animal eukaryotic cell as described above for its use as described above for treating leukemia.

[0267] The term "cell therapy" refers to a method known as "biotherapy" which aims to treat an organ or organism by providing therapeutic cells to replace, supplement or kill defective cells.

[0268] Alternatively and according to this same aspect, the subject of the invention is a method for treating a pathology chosen from: cancer, leukemia and fibrosis, said method comprising the administration of a transfected / transduced animal eukaryotic cell according to the invention (or of a pharmaceutical composition comprising at least one transfected / transduced animal eukaryotic cell according to the invention and a pharmaceutically acceptable excipient) to a patient in need thereof.

[0269] With the help of the tools of the invention, it allows the implementation of effective and safe cell therapies. In particular, these can exploit the so-called safety switch technology, which under the action of the right stimulus (and only the right stimulus) allows the activation of the inducible promoter of said expression cassette and / or the lifting of the inhibition of translation, making possible the expression of the toxic or suicide transgene. In this case and by way of example, the therapeutic use of an immune cell:

[0270] ■ modified and armed to target and destroy cancer cells; and

[0271] ■ transduced according to the invention with a toxic or suicide transgene, will, after its administration to a patient who needs it, play its role of targeting and destroying cancer cells. Only if this same immune cell becomes dysfunctional, it will then be possible to destroy it in order to avoid adverse effects, thanks to the safety systems provided by the invention (control of the induction of transcription and / or control of the inhibition of translation).

[0272] In any respect, it should be noted that the various aspects of the invention, as well as the various embodiments thereof, are interdependent. The latter can therefore be combined with each other as much as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This is also valid for all the definitions provided in this description, which applies to all aspects of the invention and its embodiments.

[0273] Further, the present invention is illustrated, but not limited to, the following figures and examples.

[0274] LIST OF FIGURES

[0275] Figure 1. Production of a pLV vector expressing Noxa in HEK-293T cells.

[0276] Recombinant particles of pLV-2xAARE vectors YB TATA -GFP-p2a-Luc (GFP) (SEQ ID NO: 4,591) and pLV-2xAARE YB - TATA-GFP-p2a-Noxa (Noxa) (SEQ ID NO: 4592) were produced and titrated in HEK-293T cells. Concentrated supernatants were titrated by measuring the integrated genomes in HEK-293T cells transduced with dilutions of these vectors. The graph indicates that the titers of each of the two vectors are equivalent. Each point represents an independent production. Statistics, t-test.

[0277] Figure 2. Study of cell death induction by Noxa expression in NK-92 cells.

[0278] (a) Measurement of the death rate in NK-92 cells transduced with the vectors, pLV- 2xAARE YB TATA -GFP-p2a-Luc (GFP) (SEQ ID NO: 4,591) or pLV-2xAARE YB TATA -GFP- p2a-Noxa (Noxa) (SEQ ID NO: 4592), 24h after treatment with DMSO or artesunate (2 pM). (b) Measurement of the death rate in NK-92 cells transduced with the vectors, pLV- 2xAARE YB TATA -GFP-p2a-Luc (GFP) (SEQ ID NO: 4,591) or pLV-2xAARE YB TATA-GFP- p2a-Noxa (Noxa) (SEQ ID NO: 4592), 48h after treatment with DMSO or artesunate (2 pM). Each point represents an independent experiment measuring the cell death rate by cytometry after labeling with annexin V and propidium iodide. Statistical, one-way ANOVA. Figure 3. Study of cell death induction by Noxa expression in human T lymphocytes.

[0279] Measurement of the mortality rate in lymphocytes transduced with vectors, pLV- 2xAARE YB TATA -GFP-p2a-Luc (GFP) (SEQ ID NO: 4,591) or pLV-2xAARE YB TATA -GFP- p2a-Noxa (Noxa) (SEQ ID NO: 4592), 24h after treatment with DMSO or artesunate (2 pM). Each point represents the measurement of cell death rate by cytometry after labeling with Fannexin V and propidium iodide on a different donor. Statistics, oneway ANOVA.

[0280] EXAMPLES - PRODUCTION OF TOXIC VECTORS

[0281] MATERIALS & METHODS

[0282] Cloning of lentiviral plasmids

[0283] The lentiviral plasmids used in the experiments were cloned by enzymatic linearization and homology recircularization using the NEBuilder HiFi DNA Assembly protocol (New England Biolabs) by inserting synthesized sequences (Twist Bioscience) into a plasmid carrying the second-generation self-inactivating lentiviral genome (pLV(SIN); SEQ ID NO: 2741).

[0284] Cell culture

[0285] Cells were cultured in an incubator with a humidified and controlled atmosphere at 37°C and 5% CO2.

[0286] HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) were cultured in High Glucose Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL Penicillin / Streptomycin. Stress induction was achieved by treatment with tunicamycin (Sigma Aldrich).

[0287] NK-92 cells are a suspension-cultured cell line derived from NK lymphocytes from a patient 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, Ref: 61870-01), 20% FCS and 1% penicillin / streptomycin. They were passaged every 3 to 4 days.

[0288] Primary T lymphocytes isolated from the blood of healthy donors were cultured in serum-free, feeder-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL Penicillin / Streptomycin.

[0289] Production of lentiviral vectors

[0290] Non-replicating lentiviral particles were obtained using the second-generation production protocol described by Zennou et al. in 2001 (NatBiotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded onto media suitable for cell culture and grown to 50-60% confluence after 24 hours of culture. The next day, the cells were co-transfected, by the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome (pLV or pLVi) and two transcomplementing plasmids encoding the HIV-1 enzymes and structural proteins (p8.92; SEQ ID NO: 4596 or 4597), and the envelope glycoprotein of the vesicular stomatitis virus (pVSVg; SEQ ID NO: 4595). Co-transfection was carried out at a plasmid ratio of 2:2:1. The transfected cells were cultured for 5 hours in contact with the precipitate and then the culture medium was renewed.The supernatant containing the lentiviral particles was harvested 48 hours later. The lentiviral suspension was treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 μm) and then ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1,000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0291] Titration

[0292] The concentration of lentiviral particles was determined by quantification of the lentiviral capsid protein p24. For this, an aliquot of lentiviral suspension was thawed, lysed and diluted according to the supplier protocol of the HIV-1 p24 Antigen ELISA 2.0 titration kit (Zeptometrix ref. 0801008). The concentration of p24 obtained was expressed in pg / pL, the equivalent of which is 1 pg of p24 for 104 VSVg-pseudotyped physical lentiviral particles (Dull, T et al. J Virol. 1998 Nov;72(l 1 ):8463-71 ). Tests can specify these values ​​but, on average, 1% of the physical particles obtained are infectious, i.e. 1 pg ~ 100 transduction units (TU).

[0293] Transduction

[0294] To integrate a transgene by infection of a lentiviral vector, the quantity of particles necessary to obtain the desired multiplicity of infection (vector:target ratio) was diluted in culture medium. This lentiviral suspension was brought into contact with the target cells. The infected cells were cultured for at least 24 hours. Depending on the integrated promoter / gene cassette, transgene expression could be observed or measured the following day by microscopy, cytometry, or biochemical analysis.

[0295] Establishment of a KRAB line (CRISPRi)

[0296] HEK 293T cells were transduced with a lentiviral vector (SEQ ID NO: 4551) carrying the constitutive expression cassettes:

[0297] ■ the dCas9-KRAB fusion protein (SEQ ID NO: 4548) fused by a 2A peptide (SEQ ID NO: 4601) to the blasticidin resistance gene (BlastR) (SEQ ID NO: 4550) under the control of the eF-1a minimal promoter (SEQ ID NO: 4542); and

[0298] ■ of guide RNA specific to the targeted promoter (SEQ ID NO: 4540) under the control of the U6 promoter (SEQ ID NO: 4539).

[0299] Two days later, transduced cells were selected by treatment with blasticidin (2 μg / mL). Cells were cloned by limiting dilution and each clone was characterized. Establishment of a KRAB (CRISPRi) and DNMT (CRISRPm) line

[0300] HEK 293T cells are transduced with a lentiviral vector (SEQ ID NO: 4590) carrying the constitutive expression cassettes:

[0301] ■ the KRAB-dCas9-DNMT3 AL fusion protein (SEQ ID NO: 4578) fused by a 2A peptide (SEQ ID NO: 4601) to the blasticidin resistance gene (BlastR) (SEQ ID NO: 4550) under the control of the eF-1a minimal promoter (SEQ ID NO: 4542); and

[0302] ■ guide RNA specific to the targeted promoter (SEQ ID NO: 4540) under the control of the U6 promoter (SEQ ID NO: 4539).

[0303] Two days later, transduced cells were selected by treatment with blasticidin (2 pg / mL). HEK-CRISPRi / m cells SV40 are cloned by limiting dilution and each clone is characterized.

[0304] Flow cytometry analysis

[0305] At the end of the experiment, the cultured cells were harvested and rinsed with PB S. They were then incubated for 30 minutes with surface labeling antibodies and the viability marker Live-or-Dye 405 / 452 (Biotium) according to the respective manufacturers' recommendations. The cells were rinsed and then fixed in PBS with 1% paraformaldehyde for 15 minutes at 4°C. After rinsing, transgene expression was analyzed using a MACSQuant Analyzer 10 cytometer (Miltenyi).

[0306] RESULTS

[0307] Two lentiviral vectors, pLV-2xAARE YB TATA -GFP-p2a-nLuc (SEQ ID NO: 4,591) and pLV-2xAARE YB TATA -GFP-p2a-Noxa (SEQ ID NO: 4592) were produced in HEK-293T cells. These vectors carried the dual reporter transgene GFP-p2a-nLuc (SEQ ID NO: 4602), or the reporter and proapoptotic transgene GFP-p2a-Noxa (SEQ ID NO: 4603), respectively, under the control of the 2XAARE promoter YB TATA(SEQ ID NO: 12). In three independent vector production experiments, equivalent recombinant particle titers were measured with each of the two pLV-2xAARE vectors YB TATA -GFP-p2a- nLuc (SEQ ID NO: 4,591) and pLV-2xAARE YB TATA -GFP-p2a-Noxa (SEQ ID NO: 4,592) (Fig-1)

[0308] Human Natural Killer NK-92 cells (ATCC CRL 2407 and 2408) were then transduced at MOI 10 and treated with artesunate or DMSO vehicle. After 24 hours of treatment, it was observed that artesunate induced a significant increase in the death of NK-92 cells transduced with the pLV-2xAARE vector. YB TATA -GFP-p2a-Noxa (SEQ ID NO: 4592), but not NK-92 cells transduced with the control vector pLV-2xAARE YB TATA -GFP-p2a-nLuc (SEQ ID NO: 4,591) (Fig. 2a). In addition, a doubling of the death of NK-92 cells transduced with the pLV-2xAARE vector YB TATA-GFP-p2a-Noxa (SEQ ID NO: 4592) was observed after 48h of artesunate treatment compared to only 24h of treatment, while no effect of the same treatment could be measured in these DMSO-treated cells, nor in NK-92 cells transduced with the control vector pLV-2xAARE YB TATA -GFP-p2a-nLuc (SEQ ID NO: 4,591) (Fig. 2b).

[0309] The induction of Noxa expression in human primary T lymphocytes was also studied. To this end, donor T cells were transduced with pLV-2XAARE vectors YB TATA -GFP-p2a-nLuc (SEQ ID NO: 4,591) or pLV-2xAARE YB TATA -GFP-p2a- Noxa (SEQ ID NO: 4592) at MOI 10. In cells transduced with the control vector pLV-2xAARE YB TATA -GFP-p2a-nLuc (SEQ ID NO: 4591), no difference in cell death rate was measured after treatment with artesunate or DMSO. In contrast, transduction of T lymphocytes with the pLV-2xAARE vector YB TATA-GFP-p2a-Noxa (SEQ ID NO: 4592) showed a doubling of cell death rate under artesunate treatment but not after DMSO treatment (Fig. 3).

[0310] These results thus demonstrated that, surprisingly, while the induction of the expression of the Noxa protein in HEK-293T cells does not induce the death of the cells producing the lentiviral vector of the invention, this same expression in NK-92 cells and in T lymphocytes effectively induced the death of the genetically modified cells.

Claims

CLAIMS 1. Vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising a promoter upstream of a toxic or suicide transgene encoding a Noxa protein, said toxic or suicide transgene encoding a Noxa protein being chosen from sequences having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1 or whose nucleic acid encodes a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO:

2.

2. Genome vector according to claim 1, in which said promoter is a constitutive promoter notably chosen from: ■ SV40 whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3; ■ CMV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4; ■ UbC whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5; ■ the EFl-a core whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; ■ the long EFl-a whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7; ■ hPGK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 8; ■ CAGG whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 9; ■ RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10; and ■ SFFV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 11, or in which said promoter is an inducible or tissue-specific promoter chosen in particular from: ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12; ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 13; ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 14; ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17; ■ the GRP78 YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16; ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 17; and ■ the miniCHACl YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO:

18.

3. Genome vector according to claim 1 or 2, wherein said expression cassette further comprises a coding or non-coding sequence having regulatory properties at the / ?o5t-transcriptional level, said coding or non-coding sequence having regulatory properties at the post-transcriptional level being located at the 5' end of said toxic or suicide transgene encoding a Noxa protein and being in particular chosen from the sequences having at least 90% identity with the sequences SEQ ID NOs: 73 to 99.

4. Genome vector according to any one of claims 1 to 3, wherein the 5' to 3' orientation of the transcription of said expression cassette is reversed with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, in particular said expression cassette further comprising at the 3' end of said toxic or suicide transgene encoding a Noxa protein a nucleic acid sequence allowing the transcription of a polyA tail.

5. A method for producing lentiviral vectors comprising a vector genome according to any one of claims 1 to 4, said method comprising at least the steps of: a. co-transfecting a eukaryotic cell with: i. a plasmid comprising a vector genome according to any one of claims 1 to 4 and the means for expressing it; ii. a plasmid comprising a viral envelope protein and the means for expressing it; and iii. a plasmid comprising the structural proteins and enzymes of HIV-1, and the means for expressing it, to obtain a transfected animal eukaryotic cell; b. culturing said transfected animal eukaryotic cell to enable the production of lentiviral vectors comprising a vector genome according to any one of claims 1 to 4; and c. harvesting and purifying said lentiviral vectors.

6. Method for producing lentiviral vectors according to claim 5, wherein said animal eukaryotic cell expresses at least one ribonucleoprotein complex comprising: ■ a fusion protein comprising the fusion: - an inactive Cas endonuclease (dCas); and - a KRAB transcriptional repression domain or a methylation domain selected from: DNMT3A, DNMT3B, DNMT3A-L and DNMT3B-L; or a fusion protein comprising the fusion: - a KRAB transcriptional repression domain; - an inactive Cas endonuclease (dCas); and - a methylation domain chosen from: DNMT3A, DNMT3B, DNMT3 AL, DNMT3B-L and MeCP2; and ■ a guide RNA (gRNA) specifically recognizing the promoter sequence of said inducible promoter, said at least one ribonucleoprotein complex dCas-KRAB / gRNA, dCas- DNMT3A / gRNA, dCas-DNMT3B / gRNA, dCas-DNMT3 AL / gRNA, dCas-DNMT3B- L / gRNA, KRAB-dCas-DNMT3A / gRNA, KRAB-dCas-DNMT3B / gRNA, KRAB-dCas-DNMT3A-L / gRNA, KRAB-dCas-DNMT3B-L / gRNA or dCas-KRAB-MeCP2 / gRNA being in particular expressed stably.

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

8. In vitro use of a lentiviral vector according to claim 7 for transducing an animal eukaryotic cell, in particular said animal eukaryotic cell being chosen from: ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in a blood cancer; ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell; ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process; ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and ■ an induced stem cell (iPS).

9. Animal eukaryotic cell transduced by a lentiviral vector according to claim 7, said transduced animal eukaryotic cell being in particular chosen from: ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in a blood cancer; ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell; ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process; ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and ■ an induced stem cell (iPS).

10. Transduced animal eukaryotic cell according to claim 9 for its use in cell therapy, in particular for treating a pathology chosen from: cancer, leukemia and fibrosis.

Images