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 to significantly reduce the expression of the transgene in the packaging cells and thus to produce high titers of lentiviral vectors carrying a 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 invention relates to a vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising an inducible promoter upstream of a toxic or suicide transgene, said inducible promoter being chosen from:

[0015] ■ the 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1;

[0016] ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2;

[0017] ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3;

[0018] ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4;

[0019] ■ the GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5;

[0020] ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and

[0021] ■ the miniCHACl YB TATA the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7, and the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

[0022] Unexpectedly, the combination of an inducible promoter, notably exemplified below by the 2XAARE promoter YB - TATA (SEQ ID NO: 1), with the inversion of the transgene expression cassette, has proven to be an efficient system for producing lentiviral vectors carrying any cytotoxic (toxic or suicide) transgene in wild-type cells at high yield, which was not possible until now. Moreover, and in addition to reducing the expression of the cytotoxic (toxic or suicide) transgene, the use of an inducible promoter upstream of it has the advantage of preventing the production of complementary RNAs that would cause the destruction of the transcripts of the lentiviral genome by RNA interference.

[0023] By “vector genome” is meant a nucleic acid sequence comprising both:

[0024] ■ 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

[0025] ■ the nucleic acid comprising the genetic information of an expression cassette comprising an inducible promoter upstream of a toxic or suicide transgene, the nucleic acid encoding said toxic or suicide transgene being able to comprise at its 3' end a nucleic acid sequence allowing the transcription of a polyA tail, said expression cassette having been introduced into said lentiviral genome such that its transcription is reversed compared to that of said lentiviral genome.

[0026] 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.

[0027] The term "inducible promoter" means a nucleic acid located upstream of a gene (or transgene) and which controls its expression, in particular by regulating its transcription. In the invention, this promoter is described as inducible because the expression of the gene (or transgene) that it controls is not constitutive and is only induced after its activation in response to the right stimulus (small molecule, hormone, etc.) or the right cellular environment. Thus, the expression "inducible promoter" also includes tissue-specific promoters.

[0028] In the invention, these are the 2XAARE YB - TATA , the 2xAARE TK , the 4xSRE YB - TATA , the 9XHRE YB TATA , the GRP78 YB TATA , the miniCHACl YB TATA or the miR223 promoter. The 2XAARE promoters YB - TATA , 2xAARE TK and miniCHACl YB - TATAare 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 and the miR223 promoter is specifically induced in myeloid cells and is an example of a tissue-specific promoter not expressed in HEK-293T cells. Interestingly, the aforementioned inducible promoters are dependent on the expression of cellular transcription factors that are poorly or not expressed under vector production conditions in HEK 293T cells.

[0029] As mentioned above, the nucleic acid of the latter has a sequence having respectively at least 80% identity with the nucleic acid of sequence SEQ ID NOs: 1 to 5, 7 and 6.

[0030] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 1.

[0031] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 2xAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2.

[0032] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 4xSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3.

[0033] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 9xHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4. In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is GRP78 YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5.

[0034] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6.

[0035] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the miniCHAClYB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7.

[0036] 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.

[0037] By "toxic or suicide transgene" is meant a nucleic acid located downstream of said inducible or tissue-specific promoter and which codes for a toxic, cytotoxic or suicide protein in the genetically modified cell. That is to say, after the activation of said inducible or tissue-specific promoter controlling the expression of said toxic or suicide transgene and this, only in response to the right stimulus or the right cellular environment, the product (i.e. the toxic or suicide protein) resulting from the transcription and translation of this nucleic acid causes (leads to) the cell death of the cell expressing it.

[0038] In the invention, the latter may be chosen from the genes coding: inducible Caspase-9 (iC9), Bax (S 184 del), Noxa (the wild-type protein or one of its mutants, in particular the S 13 A mutant), Gasdermin B (N-ter), constitutively active caspase-3 (V266E), an M2 ion channel (H37A) of the influenza virus, the A subunit of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens). These are non-secreted toxic or suicide proteins, i.e. whose expression after activation of said inducible or tissue-specific promoter upstream of the nucleic acid encoding them is not found in the extracellular medium and therefore only results in the cell death of the cell expressing them.By "the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome" is meant, as illustrated in Figure 1, that the transcription of said expression cassette is inverted with respect to that of said lentiviral genome. With this innovative construction, the inventors have thus developed a novel and efficient system for the high-titer production of lentiviral vectors encoding toxic or suicide transgenes.

[0039] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from:

[0040] ■ the 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1; 2XAARE TKwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2; the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3; 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4; GRP78 YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5; the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and the miniCHACl YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0041] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1.

[0042] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 2xAARE TK whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2.

[0043] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 4xSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3.

[0044] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 9xHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4.

[0045] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is GRP78 YB TATAwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5.

[0046] In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6. In particular, the subject of the invention is the vector genome as described above, in which said inducible promoter is the miniCHACl YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 7.

[0047] According to another embodiment, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D).

[0048] According to another embodiment, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is chosen from:

[0049] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid encodes an inducible Caspase-9 (iC9) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 9;

[0050] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 11;

[0051] ■ an influenza virus M2 (H37A) ion channel whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 (H37A) ion channel whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 13;

[0052] ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 15;

[0053] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 19;

[0054] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 21;

[0055] ■ a Noxa protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 23;

[0056] ■ a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 334 or whose nucleic acid encodes a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 335; and

[0057] ■ an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 336 or whose nucleic acid encodes an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 337.

[0058] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid codes for an inducible Caspase-9 (iC9) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 9.

[0059] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Bax protein (S 184 del) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid codes for a Bax protein (S 184 del) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 11.

[0060] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an M2 ion channel (H37A) of the influenza virus whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid codes an M2 ion channel (H37A) of the influenza virus whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 13.

[0061] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid codes for a constitutively active caspase-3 protein (V266E) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 15.

[0062] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 17; or an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 19.

[0063] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Gasdermin B (N-ter) protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid codes for a Gasdermin B (N-ter) protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 21.

[0064] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Noxa protein whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid codes for a Noxa protein whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 23.

[0065] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a streptolysin O whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 334 or whose nucleic acid codes for a streptolysin O whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 335.

[0066] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an enterotoxin whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 336 or whose nucleic acid codes for an enterotoxin whose amino acid sequence is at least 80% identical to the sequence SEQ ID NO: 337.

[0067] According to another embodiment, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is chosen from:

[0068] ■ an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid encodes an inducible Caspase-9 (iC9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 9;

[0069] ■ a Bax protein (S 184 del) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid encodes a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 11;

[0070] ■ an influenza virus M2 ion channel (H37A) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid encodes an influenza virus M2 ion channel (H37A) whose amino acid sequence corresponds to the sequence SEQ ID NO: 13; ■ a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid encodes a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 15;

[0071] ■ a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 17, and an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence corresponds to the sequence SEQ ID NO: 19;

[0072] ■ a Gasdermin B (N-ter) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid encodes a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 21;

[0073] ■ a Noxa protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid encodes a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 23;

[0074] ■ a streptolysin O whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 334 or whose nucleic acid encodes a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 335; and

[0075] ■ an enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 336 or whose nucleic acid encodes an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 337.

[0076] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an inducible Caspase-9 (iC9) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 8 or whose nucleic acid codes for an inducible Caspase-9 (iC9) whose amino acid sequence corresponds to the sequence SEQ ID NO: 9.

[0077] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Bax protein (S 184 del) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 10 or whose nucleic acid codes for a Bax protein (S 184 del) whose amino acid sequence corresponds to the sequence SEQ ID NO: 11.

[0078] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an M2 ion channel (H37A) of the influenza virus whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 12 or whose nucleic acid codes for an M2 ion channel (H37A) of the influenza virus whose amino acid sequence corresponds to the sequence SEQ ID NO: 13. In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a constitutively active caspase-3 protein (V266E) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 14 or whose nucleic acid codes for a constitutively active caspase-3 protein (V266E) whose amino acid sequence corresponds to the sequence SEQ ID NO: 15.

[0079] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a diphtheria toxin A subunit (DTA) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 16 or whose nucleic acid encodes a diphtheria toxin A subunit (DTA) whose amino acid sequence corresponds to the sequence SEQ ID NO: 17; or an attenuated mutant aDTA (G128D) whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 18 or whose nucleic acid encodes an attenuated mutant aDTA (G128D) whose amino acid sequence corresponds to the sequence SEQ ID NO: 19.

[0080] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Gasdermin B (N-ter) protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 20 or whose nucleic acid codes for a Gasdermin B (N-ter) protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 21.

[0081] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a Noxa protein whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 22 or whose nucleic acid codes for a Noxa protein whose amino acid sequence corresponds to the sequence SEQ ID NO: 23.

[0082] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is a streptolysin O whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 334 or whose nucleic acid codes a streptolysin O whose amino acid sequence corresponds to the sequence SEQ ID NO: 335.

[0083] In particular, the subject of the invention is the vector genome as described above, in which said toxic or suicide transgene is an enterotoxin whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 336 or whose nucleic acid codes for an enterotoxin whose amino acid sequence corresponds to the sequence SEQ ID NO: 337.

[0084] According to another embodiment, the subject of the invention is the vector genome as described above, in which the nucleic acid coding for said toxic or suicide transgene comprises at its 3' end a polyA sequence (eg SEQ ID NO: 24). Insofar as the 5' to 3' orientation of the transcription of said expression cassette is inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome, the addition of this polyA sequence (eg SEQ ID NO: 24) to the 3' end of the nucleic acid coding for said toxic or suicide transgene promotes, after (and only after) induction of said inducible promoter, the transcription and stability of an mRNA, which authorizes the translation of said toxic or suicide transgene. According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSREYB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is an inducible Caspase-9 (iC9) protein.

[0085] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is a Bax protein (S 184 del).

[0086] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK, the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is an M2 ion channel (H37A) of the influenza virus.

[0087] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is a constitutively active caspase-3 protein (V266E).

[0088] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is a diphtheria toxin A subunit (DTA) or an attenuated aDTA mutant (G128D).

[0089] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA; and wherein said toxic or suicide transgene is a Gasdermin B (N-ter) protein.

[0090] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant).

[0091] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA, the miR223 promoter and miniCHACl YB - TATA ; and wherein said toxic or suicide transgene is a streptolysin O (in particular that of S. pyogenes).

[0092] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is chosen from: 2XAARE YB TATA , the 2XAARE TK , the 4XSRE YB - TATA , the 9xHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; and in which said toxic or suicide transgene is an enterotoxin (in particular that of C. perfringens).

[0093] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is 2XAARE YB TATA; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0094] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 2xAARE TK; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0095] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 4xSRE YB TATA; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0096] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is the 9xHRE YB TATA; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes and an enterotoxin (in particular that of C. perfringens).

[0097] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is GRP78 YB TATA; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0098] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is the promoter of miR223; and in which said toxic or suicide transgene is chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens).

[0099] According to another embodiment, the subject of the invention is the vector genome as described above, in which said inducible promoter is miniCHACl YB TATA; and wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O (in particular that of S. pyogenes) and an enterotoxin (in particular that of C. perfringens). According to another embodiment, the subject of the invention is the 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: 25 to 80. 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: 25 to 80.In particular, the invention also relates to the vector genome as described above, in which said expression cassette is the sequence SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 (see Table 1).

[0100] Table 1. List of expression cassettes

[0101] According to another embodiment, the subject of the invention is 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: 25 to 32. 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: 25 to 32.

[0102] According to another embodiment, the subject of the invention is 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: 33 to 40. 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: 33 to 40.

[0103] According to another embodiment, the subject of the invention is 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: 41 to 48. 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: 41 to 48. According to another embodiment, the subject of the invention is 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: 49 to 56. 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: 49 to 56.

[0104] According to another embodiment, the subject of the invention is 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: 57 to 64. 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: 57 to 64.

[0105] According to another embodiment, the subject of the invention is 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: 65 to 72. 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: 65 to 72.

[0106] According to another embodiment, the subject of the invention is 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: 73 to 80. 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: 73 to 80.

[0107] According to another embodiment, the subject of the invention is 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: 25, 33, 41, 49, 57, 65 and 73. 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: 25, 33, 41, 49, 57, 65 and 73.

[0108] According to another embodiment, the subject of the invention is 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: 26, 34, 42, 50, 58, 66 and 74. 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: 26, 34, 42, 50, 58, 66 and 74.

[0109] According to another embodiment, the subject of the invention is 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: 27, 35, 43, 51, 59, 67 and 75. 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: 27, 35, 43, 51, 59, 67 and 75.

[0110] According to another embodiment, the subject of the invention is 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: 28, 36, 44, 52, 60, 68 and 76. 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: 28, 36, 44, 52, 60, 68 and 76.

[0111] According to another embodiment, the subject of the invention is 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: 29, 37, 45, 53, 61, 69 and 77. 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: 29, 37, 45, 53, 61, 69 and 77.

[0112] According to another embodiment, the subject of the invention is 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: 30, 38, 46, 54, 62, 70 and 78. 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: 30, 38, 46, 54, 62, 70 and 78.

[0113] According to another embodiment, the subject of the invention is 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: 31, 39, 47, 55, 63, 71 and 79. 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: 31, 39, 47, 55, 63, 71 and 79.

[0114] According to another embodiment, the subject of the invention is 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: 32, 40, 48, 56, 64, 72 and 80. 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: 32, 40, 48, 56, 64, 72 and 80.

[0115] 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).

[0116] 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: 81. 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: 81.It should be noted that the insertion of said expression cassette into said Self-inactivating Lentivirus (SIN) of sequence SEQ ID NO: 81 can be or is carried out at nucleotides 2229 and 2230 of this SEQ ID NO: 81. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 82 and 83.

[0117] 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: 84. 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: 84.It should be noted that the insertion of said expression cassette into said Respiratory syncytial virus (RSV) of sequence SEQ ID NO: 84 can be or is carried out at nucleotides 1737 and 1738 of this SEQ ID NO: 84. That is to say that said expression cassette is located in this case between the sequences SEQ ID NOs: 85 and 86.

[0118] 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:

[0119] ■ a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 81; or

[0120] ■ a Respiratory syncytial virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 84; and wherein said expression cassette comprises:

[0121] ■ an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4XSRE YB - TATA , the 9XHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; And

[0122] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), streptolysin O (in particular that of S. pyogenes and an enterotoxin (in particular that of C. perfringens).

[0123] According to another embodiment, the subject of the invention is 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: 87 to 198. 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: 87 to 198. In particular, the subject of the invention is also the vector genome as described above, in which said vector genome is the sequence SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,

[0124] 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,

[0125] 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,

[0126] 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197 or 198 (see Table 2).

[0127]

[0128] Table 2. List of vector genomes

[0129] It should also be noted that due to the inversion of the expression cassette as described above with respect to the orientation of the transcription of the vector genome as described above, all of the sequences provided relating to said expression cassette as described above are found on the complementary strand of said vector genome as described above of which only the sense sequences (5' > 3') are provided.

[0130] According to another embodiment, the subject of the invention is 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: 87 to 94. 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: 87 to 94.

[0131] According to another embodiment, the subject of the invention is 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: 95 to 102. 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: 95 to 102.

[0132] According to another embodiment, the subject of the invention is 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: 103 to 110. 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: 103 to 110.

[0133] According to another embodiment, the subject of the invention is 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: 111 to 118. 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: 111 to 118.

[0134] According to another embodiment, the subject of the invention is 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: 119 to 126. 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: 119 to 126. According to another embodiment, the subject of the invention is 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: 127 to 134. 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: 127 to 134.

[0135] According to another embodiment, the subject of the invention is 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: 135 to 142. 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: 135 to 142.

[0136] According to another embodiment, the subject of the invention is 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: 87, 95, 103, 111, 119, 127 and 135. 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: 87, 95, 103, 111, 119, 127 and 135.

[0137] According to another embodiment, the subject of the invention is 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: 88, 96, 104, 112, 120, 128 and 136. 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: 88, 96, 104, 112, 120, 128 and 136.

[0138] According to another embodiment, the subject of the invention is 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: 89, 97, 105, 113, 121, 129 and 137. 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: 89, 97, 105, 113, 121, 129 and 137.

[0139] According to another embodiment, the subject of the invention is 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: 90, 98, 106, 114, 122, 130 and 138. 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: 90, 98, 106, 114, 122, 130 and 138.

[0140] According to another embodiment, the subject of the invention is 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: 91, 99, 107, 115, 123, 131 and 139. 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: 91, 99, 107, 115, 123, 131 and 139.

[0141] According to another embodiment, the subject of the invention is 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: 92, 100, 108, 116, 124, 132 and 140. 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: 92, 100, 108, 116, 124, 132 and 140.

[0142] According to another embodiment, the subject of the invention is 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: 93, 101, 109, 117, 125, 133 and 141. 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: 93, 101, 109, 117, 125, 133 and 141.

[0143] According to another embodiment, the subject of the invention is 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: 94, 102, 110, 118, 126, 134 and 142. 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: 94, 102, 110, 118, 126, 134 and 142.

[0144] According to another embodiment, the subject of the invention is 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: 143 to 150. 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: 143 to 150.

[0145] According to another embodiment, the subject of the invention is 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: 151 to 158. 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: 151 to 158.

[0146] According to another embodiment, the subject of the invention is 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: 159 to 166. 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: 159 to 166.

[0147] According to another embodiment, the subject of the invention is 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: 167 to 174. 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: 167 to 174.

[0148] According to another embodiment, the subject of the invention is 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: 175 to 182. 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: 175 to 182.

[0149] According to another embodiment, the subject of the invention is 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: 183 to 190. 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: 183 to 190.

[0150] According to another embodiment, the subject of the invention is 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: 191 to 198. 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: 191 to 198.

[0151] According to another embodiment, the subject of the invention is 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: 143, 151, 159, 167, 175, 183 and 191. 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: 143, 151, 159, 167, 175, 183 and 191.

[0152] According to another embodiment, the subject of the invention is 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: 144, 152, 160, 168, 176, 184 and 192. 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: 144, 152, 160, 168, 176, 184 and 192.

[0153] According to another embodiment, the subject of the invention is 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: 145, 153, 161, 169, 177, 185 and 193. 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: 145, 153, 161, 169, 177, 185 and 193.

[0154] According to another embodiment, the subject of the invention is 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: 146, 154, 162, 170, 178, 186 and 194. 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: 146, 154, 162, 170, 178, 186 and 194.

[0155] According to another embodiment, the subject of the invention is 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: 147, 155, 163, 171, 179, 187 and 195. 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: 147, 155, 163, 171, 179, 187 and 195. According to another embodiment, the subject of the invention is 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: 148, 156, 164, 172, 180, 188 and 196. 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: 148, 156, 164, 172, 180, 188 and 196.

[0156] According to another embodiment, the subject of the invention is 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: 149, 157, 165, 173, 181, 189 and 197. 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: 149, 157, 165, 173, 181, 189 and 197.

[0157] According to another embodiment, the subject of the invention is 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: 150, 158, 166, 174, 182, 190 and 198. 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: 150, 158, 166, 174, 182, 190 and 198.

[0158] According to a second aspect, the invention relates to a plasmid comprising a vector genome as described above and the means for expressing it, in particular in which the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed with respect to the 5' to 3' orientation of the transcription of said expression cassette.

[0159] By "plasmid" is meant a double-stranded circular DNA molecule, which necessarily has a bacterial origin of replication and, secondarily, the SV40 virus, so that it can replicate autonomously, e.g., in coli for its amplification and, eg, 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, this further comprises the nucleic acid of said vector genome of the invention and the means for expressing it.

[0160] By "the means of expressing it" is meant the genetic elements necessary for the expression of the vector genome of the invention and the production of a lentiviral vector according to the invention.

[0161] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-SIN plasmid or a pLV-RSV plasmid.

[0162] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-SIN plasmid. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-SIN plasmid whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 199. In particular, the subject of the invention is also the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-SIN plasmid whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 199.It should be noted that the insertion of said vector genome into said plasmid pLV-SIN of sequence SEQ ID NO: 199 can be or is carried out at nucleotides 4939 and 4940 of this SEQ ID NO: 199. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 200 and 201.

[0163] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-RSV plasmid. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-RSV plasmid whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 202. In particular, the subject of the invention is also the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being a pLV-RSV plasmid whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 202.It should be noted that the insertion of said vector genome into said plasmid pLV-RSV of sequence SEQ ID NO: 202 can be or is carried out at nucleotides 234 and 235 of this SEQ ID NO: 202. That is to say that said vector genome is located in this case between the sequences SEQ ID NOs: 203 and 204.

[0164] By "the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed relative to the 5' to 3' orientation of the transcription of said expression cassette" is meant that the orientation of said expression cassette is reversed relative to that of said cryptic promoter of the bacterial origin of replication.

[0165] In particular, the subject of the invention is therefore the plasmid as described above comprising a vector genome as described above and the means for expressing it, in which the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed with respect to the 5' to 3' orientation of the transcription of said expression cassette.

[0166] According to another embodiment, the subject of the invention is the plasmid as described above, said plasmid into which a vector genome as described above has been introduced and the means for expressing it is:

[0167] ■ a plasmid pLV-SIN whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 199; or

[0168] ■ a plasmid pLV-RSV whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 202; said lentiviral genome into which said expression cassette has been introduced being: ■ a Self-inactivating Lentivirus (SIN) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 81; or

[0169] ■ a Respiratory syncytial virus (RSV) whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 84; and said expression cassette comprising:

[0170] ■ an inducible promoter chosen from: 2XAARE YB - TATA , the 2xAARE TK , the 4XSRE YB - TATA , the 9XHRE YB - TATA , the GRP78 YB - TATA , the miR223 promoter and miniCHACl YB - TATA ; And

[0171] ■ a toxic or suicide transgene chosen from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein (the wild-type protein or one of its mutants, in particular the S 13 A mutant), a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an M2 ion channel (H37A) of the influenza virus, a subunit A of diphtheria toxin (DTA) and an attenuated aDTA mutant (G128D), streptolysin O (in particular that of S. pyogenes and an enterotoxin (in particular that of C. perfringens).

[0172] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205 to 316. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 205 to 316. In particular, the subject of the invention is also the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid having the sequence SEQ ID NO: 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,

[0173] 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,

[0174] 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,

[0175] 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,

[0176] 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,

[0177] 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 or 316 (see Table 3).

[0178] Table 3. List of plasmids

[0179] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205 to 212. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 205 to 212.

[0180] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 213 to 220. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 213 to 220.

[0181] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 221 to 228. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 221 to 228.

[0182] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 229 to 236. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 229 to 236.

[0183] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 237 to 244. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 237 to 244.

[0184] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 245 to 252. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 253 to 260.

[0185] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205, 213, 221, 229, 237, 245 and 253. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 205, 213, 221, 229, 237, 245 and 253.

[0186] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 206, 214, 222, 230, 238, 246 and 254. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 206, 214, 222, 230, 238, 246 and 254.

[0187] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 207, 215, 223, 231, 239, 247 and 255. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 207, 215, 223, 231, 239, 247 and 255.

[0188] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 208, 216, 224, 232, 240, 248 and 256. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 208, 216,

[0189] 224, 232, 240, 248 and 256.

[0190] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 209, 217, 225, 233, 241, 249 and 257. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 209, 217,

[0191] 225, 233, 241, 249 and 257.

[0192] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 210, 218, 226, 234, 242, 250 and 258. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 210, 218,

[0193] 226, 234, 242, 250 and 258.

[0194] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 210, 218, 226, 234, 242, 250 and 258. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 210, 218, 226, 234, 242, 250 and 258. According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 210, 218, 226, 234, 242, 250 and 258. identity with the sequences SEQ ID NOs: 211, 219, 227, 235, 243, 251 and 259.In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 211, 219,.

[0195] 227, 235, 243, 251 and 259.

[0196] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 212, 220, 228, 236, 244, 252 and 260. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 212, 220,

[0197] 228, 236, 244, 252 and 260.

[0198] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 261 to 268. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 261 to 268.

[0199] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 269 to 276. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 269 to 276.

[0200] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 277 to 284. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 277 to 284.

[0201] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 285 to 292. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 285 to 292. According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 293 to 300.In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 293 to 300.

[0202] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 301 to 308. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 301 to 308.

[0203] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 309 to 316. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 309 to 316.

[0204] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 261, 269, 277, 285, 293, 301 and 309. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 261, 269,

[0205] 277, 285, 293, 301 and 309.

[0206] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 262, 270, 278, 286, 294, 302 and 310. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 262, 270,

[0207] 278, 286, 294, 302 and 310.

[0208] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 263, 271, 279, 287, 295, 303 and 311. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 263, 271,

[0209] 279, 287, 295, 303 and 311. According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 264, 272, 280, 288, 296, 304 and 312. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 264, 272,

[0210] 280, 288, 296, 304 and 312.

[0211] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 265, 273, 281, 289, 297, 305 and 313. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 265, 273,

[0212] 281, 289, 297, 305 and 313.

[0213] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 266, 274, 282, 290, 298, 306 and 314. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 266, 274,

[0214] 282, 290, 298, 306 and 314.

[0215] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 267, 275, 283, 291, 299, 307 and 315. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 267, 275,

[0216] 283, 291, 299, 307 and 315.

[0217] According to another embodiment, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 268, 276, 284, 292, 300, 308 and 316. In particular, the subject of the invention is the plasmid as described above comprising a vector genome as described above and the means for expressing it, said plasmid being chosen from the sequences SEQ ID NOs: 268, 276,

[0218] 284, 292, 300, 308 and 316. According to a third aspect, the invention relates to the use of a plasmid as described above for producing a lentiviral vector comprising a vector genome as described above.

[0219] The term "lentiviral vector" means a recombinant lentiviral particle obtained using the plasmid of the invention, which makes it possible to produce the (lenti-)viral particle of the invention. This comprises said encapsidated and / or enveloped vector genome. It is efficient for introduction into the cell, it is non-replicative and it leads either to the integration (targeted or not) of said vector genome into the genome of an infected cell, or to the presence of a non-integrated episome in the cellular genome.

[0220] According to another embodiment, the subject of the invention is the use of a plasmid as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 87 to 198. In particular, the subject of the invention is the use of a plasmid as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 87 to 198.

[0221] According to another embodiment, the subject of the invention is the use of a plasmid as described above, in which said plasmid is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205 to 316. In particular, the subject of the invention is also the use of a plasmid as described above, in which said plasmid is chosen from the sequences SEQ ID NOs: 205 to 316. It should be noted that the plasmids of sequences SEQ ID NOs: 205 to 316 respectively comprise the vector genomes of sequences SEQ ID NOs: 87 to 198.

[0222] According to a fourth 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 as described above; 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. 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:

[0223] ■ 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;

[0224] ■ said plasmid comprising a viral envelope protein and the means for expressing it may be chosen from the plasmids: pHCMV-VSV (SEQ ID NO: 328), 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

[0225] ■ 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: 329), p8.92 (IN D64V) (SEQ ID NO: 330), psPAX2 (SEQ ID NO: 331), pMDLg / pRRE (SEQ ID NO: 332) and pRSV-Rev (SEQ ID NO: 333).

[0226] 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 1000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0227] According to another embodiment, the invention relates to the method for producing lentiviral vectors as described above, in which said animal eukaryotic cell is a cell belonging to the HEK 293 T line.

[0228] According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 87 to 198. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 87 to 198. According to another embodiment, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid ai is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 205 to 316. In particular, the subject of the invention is the method for producing lentiviral vectors as described above, in which said plasmid ai is chosen from the sequences SEQ ID NOs: 205 to 316.

[0229] According to a fifth aspect, 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 vectors as described above.

[0230] According to another embodiment, the subject of the invention is the lentiviral vector as described above, in which said vector genome is chosen from the sequences having at least 80% identity with the sequences SEQ ID NOs: 87 to 198. In particular, the subject of the invention is the lentiviral vector as described above, in which said vector genome is chosen from the sequences SEQ ID NOs: 87 to 198.

[0231] According to a sixth aspect, the subject of the invention is the in vitro use of a lentiviral vector as described above for transducing an animal eukaryotic cell, in particular said animal eukaryotic cell being chosen from:

[0232] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in a blood cancer;

[0233] ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell;

[0234] ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process;

[0235] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and

[0236] ■ an induced stem cell (iPS).

[0237] According to another embodiment, the subject of the invention is the in vitro use of a lentiviral 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.

[0238] By "a cancer cell" is meant an abnormal cell that has acquired specific characteristics that cause cancer (or malignant tumor), which is a disease characterized by uncontrolled and abnormally significant cell proliferation (tumor) within normal (healthy) tissue of the body, such that the survival of the latter is threatened. Among these tumors, there are solid tumors and liquid tumors. According to another embodiment, the subject of the invention is 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 subject of the invention is also 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.

[0239] By "an immune cell" is meant 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.

[0240] According to another embodiment, the invention 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.

[0241] A "myofibroblast" is a cell characterized by the properties of matrix protein production, proliferation, migration, and contraction, activated in repair and healing processes. These myofibroblasts are found in certain organs such as the liver, lung, heart, or kidney during chronic diseases. The process of tissue replacement by myofibroblasts is called fibrosis.

[0242] According to another embodiment, the invention 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.

[0243] By "an astrocyte" is meant a glial cell of the central nervous system. Generally stellar in shape, it provides a variety of important functions, centered on the support and protection of neurons. According to another embodiment, the subject of the invention is 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 subject of the invention is also 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.

[0244] A "stem cell" means 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.

[0245] According to another embodiment, the invention 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).

[0246] According to another aspect, 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:

[0247] ■ a cancer cell, including a cancer cell forming a solid tumor or being involved in a blood cancer;

[0248] ■ an immune cell chosen in particular from: the T lymphocyte and the NK cell;

[0249] ■ a myofibroblast, in particular a myofibroblast involved in a fibrosis process;

[0250] ■ an astrocyte, particularly an astrocyte activated during brain or spinal cord trauma and forming a glial scar preventing regeneration; and

[0251] ■ an induced stem cell (iPS).

[0252] According to another embodiment, the invention relates to the 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.

[0253] According to another embodiment, the subject of the invention is the transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell, in particular a cancer cell forming a solid tumor or being involved in a blood cancer. In particular, the subject of the invention is also the 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 transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a cancer cell being involved in a blood cancer.

[0254] According to another embodiment, the subject of the invention is the 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 transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is a T lymphocyte or an NK cell.

[0255] According to another embodiment, the subject of the invention is the transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast. In particular, the subject of the invention is also the transduced animal eukaryotic cell as described above, wherein said animal eukaryotic cell is a myofibroblast involved in a fibrosis process.

[0256] According to another embodiment, the subject of the invention is the 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 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.

[0257] According to another embodiment, the invention relates to the transduced animal eukaryotic cell as described above, in which said animal eukaryotic cell is an induced stem cell (iPS).

[0258] According to another aspect, the invention relates to 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.

[0259] According to another embodiment, the subject of the invention is the 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 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 transduced animal eukaryotic cell as described above for its use as described above for treating leukemia. By "cell therapy" is meant a method called "biotherapies" which aims to treat an organ or an organism by providing therapeutic cells to replace, supplement or kill defective cells.

[0260] 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 transduced animal eukaryotic cell according to the invention (or of a pharmaceutical composition comprising at least one transduced animal eukaryotic cell according to the invention and a pharmaceutically acceptable excipient) to a patient who needs it.

[0261] Beyond enabling the production at high titers of lentiviral vectors encoding toxic (cytotoxic) or suicide transgenes, hitherto unattainable, the invention allows the implementation of effective and safe cell therapies. These in fact 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, making possible the expression of the toxic or suicide transgene, causing the death of the cell. Also and for example, the therapeutic use of an immune cell:

[0262] ■ modified and armed to target and destroy cancer cells; and

[0263] ■ transduced according to the invention, will, after its administration to a patient who needs it, play its role of targeting and destroying cancer cells. Only if this same immune cell becomes dysfunctional, it will then be possible to destroy it in order to avoid adverse effects, thanks to the safety system provided by the invention (i.e. expression cassette comprising an inducible promoter upstream of a toxic or suicide transgene).

[0264] 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.

[0265] Further, the present invention is illustrated, but not limited to, the following figures and examples. LIST OF FIGURES

[0266] Figure 1. Schematics of forward (pLV; top panel) and reverse (pLVi; bottom panel) lentiviral vectors carrying a transgene under the control of the 2XAARE promoter YB - TATA (SEQ ID NO: 1).

[0267] Figure 2. Production of lentiviral particles carrying a toxic transgene.

[0268] The titers of lentiviral particles (lVPs), carrying an inducible toxic gene, measured in quantity of p24 (pg / pL), are significantly lower compared to the control sense vector expressing GFP (experiments n>3 productions; mean ± standard deviation; 1-way ANOVA statistics with Dunnett's test for multiple comparison to the GFP control; *** P<0.0002).

[0269] Figure 3. Incremental titers of lentiviral vectors by inversion of the 2xAARE cassette YB - TATA -suicide gene (GS).

[0270] A) Reversing the 2xAARE cassette YB TATA-GFP-GS (GS: suicide gene) in a lentiviral vector (pLVi, gray columns) significantly increased the titers of recombinant particles in HEK-293T cells compared to a sense vector (pLV, black columns). Statistics: Two-way ANOVA and Sidak multiple comparison test, (ns) p>0.05; (*) p<0.05; (**) p<0.01; (***) p<0.001; (****) p0.001.

[0271] Figure 4. Functionality of pLVi vectors carrying a cytotoxic transgene on melanoma lines and human lymphocytes.

[0272] AB) Impedance measurement (xCELLigence) represented by the normalized cell index from the start of treatment. A) Human melanoma line EST 109 transduced with the pLVi-2XAARE vector YB TATA-GFP (SEQ ID NO: 327); puromycin treatment was toxic and caused detachment of EST 109 cells, visible by a drop in cell index; treatments with the stress inducer tunicamycin (Tm, 4pg / mL) or its solvent DMSO did not induce toxicity. B) Human melanoma cell line EST 109 transduced with the pLVi-2xAARE vector YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205). Puromycin treatment caused EST109 cell detachment. Treatments with the stress inducer tunicamycin (Tm, 4pg / mL) or its solvent DMSO did not induce toxicity. Only the double treatment with Tm and FAP1903 (4pM) induced iC9 transgene toxicity and caused cell detachment. C) Flow cytometry confirmation of 2xAARE cassette toxicity YB TATA -GFP-P2A-iC9 (SEQ ID NO: 25) in EST 109 cells. Dual treatment with Tm and AP 1903 had no effect on the population transduced with pLVi- 2XAARE YB TATA-GFP (SEQ ID NO: 327). In contrast, the combination of Tm and FAP1903 caused significant disappearance of cells transduced with pLVi-2xAARE YB - TATA -GFP-P2A-iC9 (SEQ ID NO: 205). D) Cell index measurement of EST 109 cells transduced with the pLVi-2xAARE vector YB TATA -GFP-P2A-aDTA (SEQ ID NO: 210). Tm treatment induced the transgene, the toxicity of which caused cell detachment. EF) flow cytometry analysis of human CD3+ lymphocytes transduced with pLVi-2xAARE vectors YB TATA -GFP (SEQ ID NO: 327), pLVi-2xAARE YB TATA -GFP-P2A- iC9 (SEQ ID NO: 205) or pLVi-2xAARE YB TATA-GFP-P2A-M2 (SEQ ID NO: 207). E) Tm treatment induced the transgene in these cells (GFP signal); double treatment with Tm and FAP1903 activated iC9 toxicity and thus caused the disappearance of GFP-positive cells. F) Double treatment with Tm and Amantadine (Amt) induced the expression of the GFP and M2 transgenes but protected the cells from M2 ion channel toxicity and thus increased the proportion of GFP-positive cells; in the absence of Amt, Tm treatment caused the disappearance of GFP-expressing cells.

[0273] EXAMPLES - PRODUCTION OF TOXIC VECTORS

[0274] MATERIALS & METHODS

[0275] Cloning of lentiviral plasmids

[0276] The lentiviral plasmids used in the experiments were cloned by enzymatic linearization and homology recircularization using the NEBuilder HiFi DNA Assembly protocol (New England Biolabs) by inserting synthesized sequences (Twist Bioscience) into a plasmid carrying the second-generation self-inactivating lentiviral genome (pLV(SIN); SEQ ID NO: 199).

[0277] Cell culture

[0278] Cells were cultured in a humidified and controlled atmosphere incubator at 37°C and 5% CO2. HEK-293T (Human Embryonic Kidney, ATTC-CRL-11268) were cultured in High Glucose Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer) containing 10% fetal bovine serum (FCS) and 100 U / mL Penicillin / Streptomycin. Stress induction was performed by treatment with tunicamycin (Tm, 4 pg / mL, Sigma Aldrich). EST109 cells (published as UKRV-Mel-2 [ECACC 13012436]; Artuc M et al. Biochem Biophys Res Commun. 213(2):699-705) were cultured under the same conditions but in Roswell Park Memorial Institute medium (RPMI 1640, Thermo Fischer) containing 10% FCS and 100 U / mL Penicillin / Streptomycin.Primary T lymphocytes isolated from the blood of healthy donors were cultured in serum-free, feeder-free medium (SFM) GIBCO OpTmizer™ CTS™ (Thermo Fischer) supplemented with Glutamax (Thermo Fischer) and 100 U / mL Penicillin / Streptomycin.

[0279] Production of lentiviral vectors

[0280] Non-replicating lentiviral particles were obtained using the second-generation production protocol described by Zennou et al. in 2001 (NatBiotechnol. 2001 May;19(5):446-50). HEK 293T cells were seeded onto suitable cell culture media and grown to 50-60% confluence after 24 hours of culture. The following day, cells were co-transfected, using the calcium phosphate precipitation method, with the plasmid encoding the lentiviral genome (pLV or pLVi) and two transcomplementing plasmids encoding HIV-1 enzymes and structural proteins (p8.92; SEQ ID NO: 329 or 330), and the vesicular stomatitis virus envelope glycoprotein (pVSVg; SEQ ID NO: 328). Co-transfection was performed at a plasmid ratio of 2:2:1. The transfected cells were cultured for 5 hours in contact with the precipitate and then the culture medium was renewed.The supernatant containing the lentiviral particles was harvested 48 hours later. The lentiviral suspension was treated with DNAse I to remove persistent plasmids, filtered (membrane porosity 0.2 μm) and then ultra-centrifuged at 60,000g for 90 minutes at 4°C. The particle pellets were taken up in a volume of phosphate buffered saline (PB S) corresponding to a concentration of a factor of 1000. The suspensions obtained were aliquoted and stored at -80°C until use.

[0281] Titration

[0282] 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).

[0283] Transduction

[0284] 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.

[0285] Flow cytometry analysis

[0286] 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).

[0287] Real-time cell adhesion analysis

[0288] Cells (3.5×105 EST109 per well) were seeded onto E-plates (Agilent) and placed on the xCELLigence Real-Time Cell Analysis device (Agilent) in an incubator. Cells were transduced and processed directly in the E-plates. The xCELLigence device continuously measures and normalizes impedance changes, a value directly related to the number, size, and morphology of cells adhering to the E-plate surface. RESULTS

[0289] Increased titers of reversed lentiviral vectors expressing a cytotoxic gene.

[0290] In order to produce high titers of lentiviral vectors carrying cytotoxic (or suicide) genes, the expression of which is inducible by ATF4, the following were obtained and used:

[0291] ■ seven sense lentiviral vectors (pLV); and

[0292] ■ seven inverted lentiviral vectors (pLVi), to produce recombinant particles carrying a control reporter transgene, or a toxic transgene (Fig. 1). In each vector the transgene was placed under the control of the 2XAARE promoter YB - TATA (SEQ ID NO: 1). The control vectors (SEQ ID NO: 320 and 327) carry the reporter transgene “enhanced Green Fluorescent Protein” (eGFP; SEQ ID NO: 317), and the twelve toxic vectors (SEQ ID NOs: 205 to 210 and 321 to 326) respectively carry the eGFP gene associated by a P2A peptide (SEQ ID NO: 318) with the cytotoxic genes chosen from:

[0293] ■ inducible Caspase-9 (iC9; SEQ ID NO: 8);

[0294] ■ Bax (S 184 del; SEQ ID NO: 10);

[0295] ■ the M2 ion channel (H37A; SEQ ID NO: 12) of the influenza virus;

[0296] ■ constitutively active caspase-3 (V266E; SEQ ID NO: 14); And

[0297] ■ the diphtheria toxin A subunit (DTA; SEQ ID NO: 16) or an attenuated aDTA mutant (G128D; SEQ ID NO: 18).

[0298] The lentiviral vector bank thus produced includes:

[0299] ■ the following seven sense lentiviral vectors: pLV-2xAARE YB TATA -GFP (SEQ ID NO: 320), pLV-2xAARE YB TATA -GFP-P2A-iC9 (SEQ ID NO: 321), pLV-2xAARE YB TATA - GFP-P2A-Bax (SEQ ID NO: 322), pLV-2xAARE YB TATA -GFP-P2A-Casp3 (SEQ ID NO: 323), pLV-2xAARE YB TATA -GFP-P2A-M2 (SEQ ID NO: 324), pLV-2xAARE YB TATA -GFP-P2A-aDTA (SEQ ID NO: 325), and pLV-2xAARE YB TATA -GFP- P2A-DTA (SEQ ID NO: 326); And

[0300] ■ the following seven reverse lentiviral vectors: pLVi-2xAARE YB TATA -GFP (SEQ ID NO: 327), pLVi-2xAARE YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205), pLVi- 2xAARE YB TATA -GFP-P2A-Bax (SEQ ID NO: 206), pLVi-2xAARE YB TATA -GFP- P2A-Casp3 (SEQ ID NO: 208), pLVi-2xAARE YB TATA-GFP-P2A-M2 (SEQ ID NO: 207), pLVi-2xAARE YB TATA -GFP-P2A-aDTA (SEQ ID NO: 210), and pLVi- 2XAARE YB TATA -GFP-P2A-DTA (SEQ ID NO: 209).

[0301] After production of the sense lentiviral particles, it was observed by quantification of p24 in the culture supernatant that the six sense vectors carrying a toxic gene were produced at significantly lower titers than the control sense vector (Fig. 2). This demonstrates that the transgene was expressed during transfection in HEK-293T cells, despite the use of an inducible 2XAARE promoter YB TATA, and that the leakage of the expression of a toxic transgene affected the production process of vector particles and caused the titers to drop. Furthermore, these results indicate that among the different toxic genes used, Bax appeared to be the least toxic and DTA the most toxic, if it is considered that the average titer of each vector is inversely proportional to the toxicity of the expression of its transgene on the viability of HEK-293T cells (Fig. 2). After production of the inverted lentiviral particles, it was observed by quantification of p24 in the culture supernatant that the inversion of the expression cassette between the lentiviral 5' and 3' LTRs significantly increases the titers of lentiviral particles carrying the toxic transgenes iC9, Bax, Casp3, M2 and aDTA, but not the toxic transgene DTA (Fig. 3).In detail, this modification of the orientation of the expression cassette allowed to significantly increase the titers by 3x for Bax, 26x for M2, 28x for iC9, 77x for Casp3 and more than 5000x for aDTA.

[0302] Inversion of the toxic transgene expression cassette resulted in high lentiviral vector titers suitable for transducing tissues in vivo.

[0303] The produced reverse lentiviral vectors are functional.

[0304] The abilities to transduce cells in culture and to make them disappear under stress conditions were measured for recombinant particles produced from pLVi-2XAARE inverted vectors. YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205), pLVi-2xAARE YB TATA -GFP-P2A-M2 (SEQ ID NO: 207) or pLVi-2xAARE YB TATA-GFP-P2A-aDTA (SEQ ID NO: 210). For this purpose, human melanoma cells (EST 109) or human primary lymphocytes were used; and the toxicity of the transgenes was correlated either with the detachment of EST 109 cells by measuring the impedance estimated on xCELLigence (Agilent) by the “cell index” (Hamidi, H., Lilja, J. & Ivaska, J. Bio-Protoc. 7, e2646 (2017)); or with the disappearance of cells expressing GFP by flow cytometry.

[0305] In a first experiment, adherent EST109 cells were transduced with a pLVi-2xAARE vector YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205) or a control vector pLVi-2xAARE YB TATA-GFP (SEQ ID NO: 327). Expression of the GFP and iC9 transgenes was then induced with 4 μg / mL of Tunicamycin (Tm), in the presence or absence of the dimerizer AP1903 (4 μM). EST109 cells treated with 5 μg / mL of puromycin served as a positive toxicity control. Cell detachment (drop in cell index) was observed only with EST 109 cells transduced with the pLVi-2xAARE vector YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205) in the presence of Tm and AP1903, but not in control cells (Fig. 4A-B). In addition, flow cytometry analysis of the same EST109 cells transduced with a pLVi-2xAARE vector YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205) or a control vector pLVi-2xAARE YB TATA -GFP (SEQ ID NO: 327), showed a significant reduction in the proportion of cells expressing GFP only for cells transduced with the pLVi-2xAARE vector YB - TATA-GFP-P2A-iC9 (SEQ ID NO: 205) treated with Tm and AP 1903, but not on controls, expressing only GFP or treated only with Tm (Fig. 4C).

[0306] The efficacy of another cytotoxic vector on EST 109 cells was tested and for this, these cells were transduced with the pLVi-2xAARE vector YB TATA -GFP-P2A-aDTA (SEQ ID NO: 210). Under these conditions, massive cell detachment was observed following induction of transgene expression at Tm, but not in the absence of induction (Fig. 4D). These results were complemented by the evaluation of the capacity of the two vectors, pLVi- 2xAARE YB TATA -GFP-P2A-iC9 (SEQ ID NO: 205) and pLVi-2xAARE YB TATA-GFP-P2A-M2 (SEQ ID NO: 207) to transduce and eliminate human primary lymphocytes under induced conditions. The iC9 and M2 proteins are not toxic in the absence of AP1903 or in the presence of Amantadine (Amt), respectively. This conditional toxicity allowed monitoring of cells modified by their expression of GFP under induced and non-toxic conditions. Thus, CD3+ lymphocytes were transduced with the pLVi-2xAARE vectors YB - TATA -GFP-P2A-iC9 (SEQ ID NO: 205) or pLVi-2xAARE YB TATA -GFP (SEQ ID NO: 327), then treated with Tm, with or without AP 1903. Treatment of these lymphocytes with Tm alone resulted in an equivalent number of cells expressing GFP with both vectors. However, dual treatment with Tm and FAP1903 significantly reduced the number of lymphocytes transduced with the pLVi-2xAARE vector YB - TATA -GFP-P2A-iC9 (SEQ ID NO: 205), but not by the pLVi-2xAARE vector YB TATA-GFP (SEQ ID NO: 327) (Fig. 4E). Similarly, in CD3+ lymphocytes transduced with the pLVi-2xAARE vectors YB - TATA -GFP-P2A-M2 (SEQ ID NO: 207) or pLVi-2xAARE YB TATA -GFP (SEQ ID NO: 327), GFP expression was induced in an equivalent proportion of lymphocytes in the presence of Amt, whereas in the absence of this drug, the number of lymphocytes expressing M2 was significantly reduced (Fig. 4F).

[0307] These experiments demonstrated that cytotoxic genes exert their deleterious functions on cell survival only when ATF4 is induced by stress; and that recombinant lentiviral particles carrying the inverted 2xAARE cassettes YB - TATA -GFP-P2A-iC9 (SEQ ID NO: 25), 2xAARE YB TATA -GFP-P2A-M2 (SEQ ID NO: 27) and 2xAARE YB TATA - GFP-P2A-DTA (SEQ ID NO: 29), and produced in HEK-293T cells, are functional.

Claims

CLAIMS 1. Vector genome comprising a lentiviral genome into which an expression cassette has been introduced comprising an inducible promoter upstream of a toxic or suicide transgene, said inducible promoter being chosen 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: 1; ■ the 2XAARE TK whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 2; ■ the 4XSRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 3; ■ the 9XHRE YB TATA whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 4; ■ the GRP78 YB TATAwhose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 5; ■ the miR223 promoter whose nucleic acid has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 6; and ■ the miniCHACl YB TATA the nucleic acid of which has a sequence having at least 80% identity with the nucleic acid of sequence SEQ ID NO: 7, and the 5' to 3' orientation of the transcription of said expression cassette being inverted with respect to the 5' to 3' orientation of the transcription of said lentiviral genome.

2. Genome vector according to claim 1, in which said inducible promoter is chosen from: ■ the 2XAARE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 1; ■ the 2XAARE TKwhose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 2; ■ the 4XSRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 3; ■ the 9XHRE YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 4; ■ the GRP78 YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 5; ■ the miR223 promoter whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO: 6; and ■ the miniCHACl YB TATA whose nucleic acid has a sequence corresponding to the nucleic acid of sequence SEQ ID NO:

7.

3. Vector genome according to claim 1 or 2, wherein said toxic or suicide transgene is selected from: an inducible Caspase-9 protein (iC9), a Bax protein (S 184 del), a Noxa protein, a Gasdermin B protein (N-ter), a constitutively active caspase-3 protein (V266E), an influenza virus M2 ion channel (H37A), a diphtheria toxin A subunit (DTA) and an attenuated aDTA mutant (G128D), a streptolysin O and an enterotoxin.

4. Plasmid comprising a vector genome according to any one of claims 1 to 3 and the means for expressing it, in particular in which the 5' to 3' orientation of the cryptic promoter of the bacterial origin of replication is reversed with respect to the 5' to 3' orientation of the transcription of said expression cassette.

5. Use of a plasmid according to claim 4 for producing a lentiviral vector comprising a vector genome according to any one of claims 1 to 3.

6. A method for producing lentiviral vectors comprising a vector genome according to any one of claims 1 to 3, said method comprising at least the steps of: a. co-transfecting an animal eukaryotic cell with: i. a plasmid according to claim 4; 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 3; and c. harvesting and purifying said lentiviral vectors.

7. Lentiviral vector comprising a vector genome according to any one of claims 1 to 3.

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