Compositions, immune cells containing same, and uses of said immune cells

Abstract

The present invention relates to a composition comprising a first single-stranded nucleic acid molecule comprising a region complementary to a target and a site for transposase recognition, a second single-stranded nucleic acid molecule comprising a region complementary to a target and a site for transposase recognition, and a third single-stranded molecule comprising a sequence encoding TCR CD3ζ and a binding site for the transposase.

Description

[Technical Field]

[0001] The present invention relates to compositions and immune cells comprising the compositions and uses of cells comprising the compositions. [Background technology]

[0002] T lymphocytes are a category of white blood cells that participate in the body's immune defenses by destroying pathogens and tumor cells. However, in certain cancers, these tumor cells have the ability to inactivate their host's immune defenses.

[0003] CAR-T, or chimeric antigen receptor-T cells, are a cellular immunotherapy approach aimed at fighting certain cancers, particularly B-cell leukemias and certain lymphomas, by boosting a patient's immune system.

[0004] This innovative approach consists in genetically modifying T lymphocytes from the patient's own blood or from a matched donor, so that they can effectively recognize and eliminate cancer cells. This ex vivo (outside the patient's body) modification allows for greater control over when the genetic modification takes place, in this case by introducing a gene that enables the production of a surface receptor (CAR) that enables the T cells to attack and destroy cancer cells. Once these cells are produced in sufficient quantities to fight the disease, they are injected into the patient's bloodstream.

[0005] Several genome editing techniques have been adapted to generate these CAR-T cells, including CRISPR / CAS9, zinc finger, and TALEN, although the latter are relatively ineffective at inserting the CAR gene into T cells.

[0006] Other systems such as viral vectors (AAV, lentivirus) and sleeping beauty transposons have also been used, but spurious specific integration of the CAR gene using these approaches can have downstream consequences of their use (e.g., WO 2017180989(A2)).

[0007] Also, given the power of CAR technology, there remains a need for more efficient generation of CAR-T modified T cells.

[0008] The object of the present invention is to overcome these drawbacks.

[0009] One of the aims of the present invention is to propose a composition that can allow the simple and efficient generation of CAR-T modified T cells.

[0010] A further object of the present invention is to provide a method or drug that can treat a disease using the CAR-T modified T cells. Summary of the Invention

[0011] The present invention provides a composition comprising: a first single-stranded nucleic acid molecule comprising or consisting essentially of an A sequence allowing insertion of a complementary sequence of a nucleic acid of interest, or comprising a complementary sequence of a nucleic acid of interest, wherein the complementary sequence is bound at its 5' to a first T-rich sequence of 40 to 60 nucleotides in length and at its 3' to a second T-rich sequence of 40 to 60 nucleotides in length, the first T-rich sequence and the second T-rich sequence comprising a first domain and a second domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of the first domain being complementary to the sequence of the second domain, the first domain and the second domain being located 15 to 52 nucleotides from the A sequence, and the first molecule comprising at its 5' end at least one first sequence oriented 5' to 3' for recognizing a transposase and at its 3' end a second sequence for recognizing the transposase; a second single-stranded nucleic acid molecule comprising or consisting essentially of a B sequence that allows insertion of a complementary sequence of a nucleic acid of interest, or comprising the complementary sequence of a nucleic acid of interest, wherein the complementary B sequence is bound at its 5' to a third T-rich sequence of 40 to 60 nucleotides in length and at its 3' to a fourth T-rich sequence of 40 to 60 nucleotides in length, the third T-rich sequence and the fourth T-rich sequence comprising a third domain and a fourth domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of the third domain being complementary to the sequence of the fourth domain, and the third domain and the fourth domain being located 15 to 52 nucleotides from the B sequence, and the second molecule comprising at its 5' end at least one first sequence oriented 5' to 3' for recognizing the transposase and at its 3' end a second sequence for recognizing the transposase, a second single-stranded nucleic acid molecule, wherein the B sequence is the complement of the nucleic acid of interest, the A sequence is located 5' of the region of interest in the nucleic acid of interest, and the B sequence is located 3' of the region of interest in the nucleic acid of interest; a third single-stranded molecule, * in its 5' portion, at least one complementary sequence of the second sequence for recognizing the transposase of the first molecule; * in its 3' portion, at least one complementary sequence of the first sequence for recognizing the transposase of a second molecule; * a third single-chain molecule comprising an intermediate region located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule, the intermediate region comprising a sequence encoding a fusion protein, the fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, an activation domain of a T lymphocyte, i.e., the CD3ζ chain of a T cell receptor or TCR; The present invention relates to a composition, wherein a first and a third single-stranded nucleic acid molecule are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase, and a second and a third single-stranded nucleic acid molecule are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase.

[0012] The present invention is based on the observation by the inventors that the use of the molecular complexes described herein above with bacterial transposase allows for the targeted and efficient replacement of genomic targets and the generation of CAR-T fusions with sequences of interest, allowing for the custom generation of T cells that target specific tumor cells.

[0013] This is because the present invention provides a molecular complex comprising a first single-stranded nucleic acid molecule, a second single-stranded nucleic acid molecule, and a third single-stranded nucleic acid molecule, wherein the third single-stranded nucleic acid molecule comprises, or essentially consists of, at its 5' end, at least one complementary sequence of the first sequence for recognizing the transposase, and the third single-stranded nucleic acid molecule comprises, or essentially consists of, at its 3' end, at least one complementary sequence of the second sequence for recognizing the transposase; The complex refers to a molecular complex in which the first single-stranded nucleic acid molecule, the second single-stranded nucleic acid molecule, and the third single-stranded nucleic acid molecule are paired according to base complementarity as defined by Watson-Crick to define two double-stranded binding sites for the transposase at the 5' end of the third molecule and two double-stranded binding sites for the transposase at the 3' end of the third molecule.

[0014] The present invention is based on the unexpected observation made by the inventors that the use of specific single-stranded guides that can target regions of a nucleic acid of interest allows for the recruitment of transposases in a controlled, "site-specific" manner, thus making it possible to use the recombination properties of transposases to replace sequences in the molecule of interest.

[0015] The molecular complex described above is actually the basic unit of the technology defined in the present invention.This basic unit is useful for directing recombinase to the specific site where recombination, and therefore sequence replacement, must occur.Unlike the CRISPR / Cas9 system, which requires the presence of PAM type (NGG) sequence, the molecular tool defined herein can be used for any target sequence, regardless of its sequence.

[0016] The aforementioned molecular complex is therefore a basic unit completed by: a homologous region in the 5' region of the target sequence, said homologous region being represented by the A sequence, and - a homologous region in the 3' region of the target sequence, said homologous region being represented by the B sequence.

[0017] It is therefore an intermediate product of the tools described below.

[0018] The molecular complex consists of three single-stranded nucleic acid molecules, which can be DNA molecules, RNA molecules, or mixed RNA and DNA molecules.

[0019] These three molecules are partially complementary to each other, two by two, according to the Watson-Crick rule for nucleic acid base complementarity: adenine pairs with thymidine or uracil, cytosine pairs with guanine, and vice versa.

[0020] More specifically, each of the three molecules forming the complex contains one of the strands of the double-stranded molecule, which corresponds to the binding sequence of the transposase. Each single-stranded molecule therefore contains a transposase binding "half-sequence" and is therefore unable to interact with the corresponding transposase. On the other hand, when the three molecules of the complex interact together through base pairing as defined hereinabove, a double-stranded molecule is thus formed, which reconstitutes the double-stranded binding site of the transposase, and thus allows the latter to interact with the formed molecule.

[0021] Complex In addition to the detailed definition of each of the first, second, and third molecules, each of which is described herein below, a complex according to the present invention comprises two pairs of binding sites for one or more transposases: a first pair of sites for binding to a first transposase resulting from pairing according to Watson-Crick nucleobase complementarity between the first single-stranded nucleic acid molecule and a third nucleic acid molecule; a second pair of sites for binding to a second transposase as a result of pairing according to Watson-Crick defined nucleobase complementarity between the second single-stranded nucleic acid molecule and the third nucleic acid molecule.

[0022] The first transposase and the second transposase can be the same or different.

[0023] Each transposase binding site consists of two double-stranded motifs: the first double-stranded motif consists of a first transposase binding half-site and its complementary sequence, following Watson-Crick base complementarity; and the second double-stranded motif consists of a second transposase binding half-site and its complementary sequence, following Watson-Crick base complementarity.

[0024] In the present invention, the complementary sequence of a binding half-site for a transposase is referred to as the complementary half-site of that site.

[0025] Thus, the site for binding to the transposase is four half-sites: a first transposase binding half-site having a first sequence, a second transposase binding half-site having a second sequence, a third half-site having a sequence complementary to the sequence of the first half-site according to Watson-Crick base complementarity; and - A fourth half-site with a sequence complementary to that of the second half-site according to Watson-Crick base complementarity.

[0026] There are also eight binding sites in the complex, four of which form a first pair of binding sites by pairing the first nucleic acid molecule with the third nucleic acid molecule, and the other four of which form a second pair of binding sites by pairing the second nucleic acid molecule with the third nucleic acid molecule.

[0027] For the first pair of sites for binding to a transposase, the four half-binding sites are: the first half-site and the second half-site are comprised in a first nucleic acid sequence; the complementary portion of the second half-site is contained in a third nucleic acid sequence; Therefore, their position in the complex is defined, and - the complementary portion of the first half-site is either i) comprised in a first nucleic acid molecule or ii) comprised in a third nucleic acid molecule; Therefore, it is not necessary to specify its position in one (first) nucleic acid molecule or the other (third) nucleic acid molecule, but rather it should be noted that when the first nucleic acid molecule and the third nucleic acid molecule pair according to base complementarity defined by Watson-Crick, two half-sites are reconstituted, i.e., the first half-site pairs with the complementary half-site of the first half-site, and the second half-site pairs with the complementary half-site of the second half-site.

[0028] For the second pair of sites for binding to the transposase, the four half-binding sites are the first half-site and the second half-site are comprised in a second nucleic acid sequence; the complement of the first half-site is contained in a third nucleic acid sequence; Therefore, their position in the complex is defined, and - the complementary portion of the second half-site is either i) comprised in a second nucleic acid molecule, or ii) comprised in a third nucleic acid molecule; Therefore, it is not necessary to specify its position in one (second) or the other (third) nucleic acid molecule, but it should also be noted that when the second and third nucleic acid molecules pair according to base complementarity defined by Watson-Crick, two half-sites are reconstituted, i.e., the first half-site pairs with the complementary half-site of the first half-site, and the second half-site pairs with the complementary half-site of the second half-site.

[0029] The first molecule. The first molecule of the complex is a molecule containing a nucleic acid sequence that, when modified, allows specific targeting of a desired region of a nucleic acid molecule of interest. This desired sequence is selected by the user of the system according to the selected target. This desired sequence is inserted into the first molecule of the complex in region A. This region A corresponds to at least two nucleic acids between which a sequence that allows targeting of the target molecule is inserted. Considering the oriented structure of nucleic acids (5' to 3'), it is important that the sequence that allows targeting of the desired region is positioned in the correct direction to enable pairing with the target sequence.

[0030] Advantageously, the A region also contains one or more sites for recognizing a restriction enzyme to facilitate oriented insertion. One or more of the following sites may be present in the A region:

[0031] [Table 1(1)]

[0032] [Table 1(2)]

[0033] [Table 1(3)]

[0034] [Table 1(4)]

[0035] [Table 1(5)]

[0036] Obviously, in the context of chemical synthesis of the first molecule, it is not necessary to have a cloning (or insertion) site for the sequence that allows targeting of the target region, but rather great care must be taken to provide a correctly oriented sequence, although this is certainly possible.

[0037] The first molecule further comprises A / T-rich sequences, or in the case of RNA, A / U-rich sequences, on either side of the A region to allow for a certain flexibility of structure. A / T-rich or A / U-rich is understood in the present invention to mean a sequence containing more than 50% A or T or U, preferably more than 50% T or U, relative to the total number of nucleotides constituting the sequence. These sequences on either side of the A region have a nucleotide size ranging from 10 nucleotides to 60 nucleotides.

[0038] The flexibility of these sequences flanking the A region, due to the presence of numerous A, T, or U bases, may have the effect of allowing poorly regulated recombinase-mediated recombination, perhaps even when the complex has not yet recognized the target molecule.

[0039] To overcome this problem, a G / C-rich sequence is introduced into each of the A / T-rich sequences, particularly the T-rich sequence or the A / U-rich sequence, adjacent to the A region. These G / C-rich regions consist of 6 to 12 nucleotides, and the amount of C or G bases exceeds 50% of the nucleotides contained in the G / C-rich sequence.

[0040] To stabilize the structure of the first molecule and, as described earlier herein, to prevent inadvertent recombination, a G / C-rich region is located 15 to 52 nucleotides from the end of the A region.

[0041] For clarity, if the A region consists of three nucleotides (the central nucleotide corresponds to position 0), the A / T-rich region or the A / U-rich region starts at position −2 on the left and +2 on the right. Thus, the G / C-rich region is located from positions −17 to −54 on the left and from positions +17 to +54 on the right.

[0042] Another important factor is that the G / C-rich sequence to the right (or 5') of the A region is necessarily complementary to the G / C-rich region to the right (or 3') of the A region (according to the Watson-Crick pairing rules). Also, the first single-stranded molecule pairs with itself in the G / C-rich region, preventing any recombination by the transposase unless there is an interaction with the complementary target sequence of the region to be inserted into the A region of the first molecule.

[0043] Finally, the first molecule contains at its 5' end a sequence corresponding to a first site for binding to a transposase and at its 3' end a second site for binding to a transposase.

[0044] The first and second binding sites are advantageously the same, in particular both corresponding to the same strand of the double-stranded binding site of the transposase, which means that only the first transposase binding site present in the 5' region of the first molecule can pair integrally, and therefore stably, with the transposase binding site present in the 3' region.

[0045] The first and second binding sites are preferably the same, but each corresponds to a different strand of the double-stranded transposase binding site. For example, if the first transposase binding site corresponds to the sense strand, the second transposase binding site corresponds to the sequence of the complementary strand. This can then have one of two configurations: i) the second binding site corresponding to the complementary strand is oriented in a 3' to 5' direction (in which case it can pair with the first transposase binding site to form a double-stranded site), or ii) the second binding site corresponding to the complementary strand is oriented in a 5' to 3' direction (in which case it cannot pair with the first transposase binding sequence because their orientations are not complementary). In case i), if the first single-stranded molecule pairs with itself at the first and second binding sites, the aforementioned complex cannot be formed because there are no longer any single-stranded complementary regions available to pair with the second molecule to form two double-stranded transposase binding sites.

[0046] Furthermore, if the first molecule lacks a complementary sequence to the target region in portion A, or if it contains such a target sequence but does not interact (pair) with this target sequence, it forms a three-dimensional structure in which the entire molecule is single-stranded except for the regions corresponding to the G / C-rich regions that pair with each other.

[0047] A schematic diagram of a suitable configuration is shown in Figures 1A-1E.

[0048] The second molecule. The second molecule in the complex is structurally similar to the first molecule, and therefore the above description applies mutatis mutandis, however, in the second molecule the B region (or B sequence) and transposase binding site are configured differently.

[0049] First, the B region is different from the A region of the first molecule, and in fact, for purposes of directed recombination, there may be no competition between the first and second molecules for the same target of interest.

[0050] Thus, the B region must correspond to a second complementary sequence of the target sequence, which is located 3' to the first sequence recognized by the complementary sequence corresponding to the A sequence of the first molecule.

[0051] As a result, when the first and second molecules are paired with a target sequence, the target sequence is adjacent to these two molecules, with the first molecule located 5' and the second molecule located 3'. The region of the target sequence located between the interaction region with the first molecule and the interaction region with the second molecule corresponds to the sequence that will be replaced by the sequence of the third molecule.

[0052] The third molecule The third molecule of the complex is simpler than the first two (the first and second molecules). The third molecule contains a transposase binding site in its 5' portion that is complementary to the binding site for the transposase present in the 3' portion of the first molecule. Thus, when the complex is formed, the 3'-located (single-stranded) transposase binding half-site of the first molecule can pair with the 5'-located (single-stranded) transposase binding half-site of the second molecule to form a double-stranded transposase binding site, a double-stranded site to which the transposase can bind.

[0053] The 3' portion of the third molecule contains a transposase binding site that is complementary to the site for binding to the transposase present in the 5' portion of the second molecule. Thus, when a complex is formed, the 3'-located (single-stranded) transposase binding half-site of the third molecule can pair with the 5'-located (single-stranded) transposase binding half-site of the second molecule to form a double-stranded transposase binding site, a double-stranded site to which a transposase can bind.

[0054] Between the 5' portion containing the transposase binding half-site and the 3' portion containing the transposase binding half-site, the third molecule contains a replacement sequence, i.e., a sequence that ultimately replaces the target sequence. This replacement sequence is flanked 5' by at least one restriction site and 3' by at least one restriction site, where these two restriction sites are different. The third molecule also contains, in the 5' to 3' direction, a transposase binding half-site that is complementary to the transposase binding site of the first molecule, followed by at least one restriction site, followed by the replacement sequence, followed by at least one restriction site that is different from the restriction site upstream of the replacement sequence, followed finally by a transposase binding half-site that is complementary to the transposase binding site of the second molecule.

[0055] The replacement sequence corresponds to a portion of the coding sequence of the CD247 gene that encodes at least the intracellular domain of the CD3ζ (CD3 zeta) protein.

[0056] The zeta chain (or CD3zeta) is one of the components of the T-cell receptor (TCR) complex and is a transmembrane protein involved in signal transduction.

[0057] The ζ chain is characterized by a small extracellular N-terminal portion (9 amino acids) that interacts with the CD3α and CD3β chains, a single transmembrane domain, and a long cytoplasmic chain (120 amino acids) that contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which are involved in interactions with the T cytoskeleton during T cell activation.

[0058] The composition according to the present invention makes it possible to obtain the above-mentioned complex. [Figure 2] shows a schematic diagram of a complex formed between a first molecule, a second molecule, and a third molecule according to the present invention.

[0059] A complex formed from molecules of the composition according to the present invention will contain two pairs of transposase-recognized double-stranded binding sites when the three molecules are correctly paired: a first pair obtained by hybridization of the first molecule with a third molecule, and - a second pair obtained by hybridization of the second molecule with a third molecule; Includes:

[0060] Sequence A in the first molecule is complementary to the same strand of nucleic acid that is complementary to sequence B in the second molecule. In other words, sequence A in the first molecule and sequence B in the second molecule can simultaneously hybridize to the same nucleic acid because these two sequences, A and B, do not recognize the same sequence.

[0061] To further clarify these findings, the present invention proposes a first molecule and a second molecule (both as defined herein above), each of which has sequence A and sequence B capable of recognizing a sequence located 5' of a target sequence of a nucleic acid of interest, and a sequence located 3' of the same target sequence of a nucleic acid of interest, such that sequence A and sequence B are complementary to regions of the nucleic acid molecule of interest that flank the sequence of interest to be replaced.

[0062] Thus, the first and second molecules of interest are required to flank the sequence to be replaced, thereby specifically targeting the molecule of interest.

[0063] The third molecule of the collection then provides a nucleic acid molecule that includes the replacement sequence, ie, the sequence that includes the intracellular portion of activation of CD3ζ.

[0064] From a mechanistic point of view, the complex according to the invention is such that it consists of three molecules, the first and second molecules being structurally organized in space such that their G / C-rich regions are paired.

[0065] On either side of the third molecule, i.e., on either the 5' and 3' sides, two pairs of sites for binding to the transposase by hybridization with the first and second molecules allow the transposase dimer to bind to the assembly when the transposase is present.

[0066] Note that the transposase binding site of the first molecule may be the same as or different from the transposase binding site of the second molecule. If the binding sequences are the same, the transposase at the 5' end of the third molecule (due to hybridization of the 5' portion of the third molecule with the first molecule) and the transposase at the 3' end of the third molecule (due to hybridization of the 3' portion of the third molecule with the second molecule) are the same. Also, for example, if the binding sites are all binding sites for Tn5 transposase, the assembly will associate with two Tn5 transposase dimers.

[0067] It is also possible that the binding sites of the first and second molecules do not recognize the same transposase, in which case, according to the definition provided herein above, the 5' portion of the third molecule, upon hybridization with the first molecule, forms two double-stranded sites for binding to the first transposase, and the 3' portion of the third molecule, upon hybridization with the second molecule, forms two double-stranded sites for binding to the second transposase.

[0068] Next, when the aforementioned complex linked to the two transposase dimers is brought into the presence of a nucleic acid molecule of interest, whose 5' portion is complementary to the A sequence of a first molecule of the assembly and whose 3' portion is complementary to the B sequence of a second molecule of the assembly, the nucleic acid molecule of interest pairs with the assembly at the aforementioned regions A and B. This interaction has the consequence of disrupting the interaction of the two G / C-rich sequences of each of the first and second molecules of the assembly. Thus, the third molecule and the nucleic acid molecule of interest move closely together in space, allowing the transposases to exert their tagmentation activity, so that the sequences of the molecule of interest flanked by the complementary sequences of the A and B sequences are replaced at 5' and 3' by the sequences of the third molecule of the assembly located between the half-sites for binding to the transposase.

[0069] In the present invention, to form a single-stranded DNA molecule, the first molecule, the second molecule and the third molecule of the assembly are advantageously molecules made of deoxyribonucleotides.

[0070] Even more advantageously, the first, second and third molecules of the collection are hybrid DNA / RNA molecules, the "backbone" of the molecules being DNA, and the recognition sequences A and B of the nucleic acid molecule of interest and the central region of the third molecule being RNA. This is particularly advantageous when the sequence substitutions that enable the present invention are made directly to RNA molecules.

[0071] FIG. 2A shows the interaction between a molecule of interest and a first molecule / second molecule / third molecule complex according to the present invention.

[0072] Advantageously, the invention relates to a complex as described above, in which the first molecule or the second molecule, or both molecules, are coupled, in particular via a modified nucleotide, to an enzyme intended to promote the displacement of the molecule of interest. helicases (enzymes capable of opening the two strands of double-stranded molecules that are supercoiled or even associated with proteins such as histones), - topoisomerases (enzymes that act on the topological structure of DNA by generating temporary breaks), - ligase (allows the formation of a phosphodiester bond between the phosphate 5' end of a nucleotide and the OH 3' end of another nucleotide), -polymerase (which synthesizes nucleic acid molecules from an initiation site, a free 3'OH, in a 5' to 3' direction by copying the antiparallel complementary strand according to the Watson-Crick model).

[0073] It is also possible to combine two or more of the enzymes in order to have all the enzyme material necessary to enable the sequence substitutions contemplated within the scope of the present invention.

[0074] In a particular embodiment, the first molecule of the collection can comprise one or more modified nucleotides in its 5' portion, more precisely between the first transposase binding site and region A. Similarly, the third molecule of the collection can comprise one or more modified nucleotides in its 3' portion, more precisely between the first transposase binding site and region A.

[0075] This modified nucleotide is in particular modified by grafting the substituted carbon chain with a protein tag or a molecule allowing a specific interaction, such as streptavidin or biotin.

[0076] This modification then allows the specific binding of enzymes that may be useful for promoting tagmentation and sequence replacement to the first molecule of this assembly. For example, it is particularly advantageous to have a streptavidin graft, which allows the grafting of a biotinylated helicase (or conversely, a helicase grafted to streptavidin and biotinylated nucleotides), which is useful for separating the two strands of a double-stranded molecule. It is also possible to attempt grafting with a biotinylated ligase (or a ligase coupled to streptavidin) to connect the recombinant strand to the 3' side.

[0077] An oligonucleotide can be associated with a first or second molecule of the collection such that the oligonucleotide pairs with a predetermined region of the first or second molecule, and then advantageously coupled to a grafting molecule as described herein above.

[0078] Advantageously, the invention relates to a composition as described above, in which the fusion protein comprises a peptide capable of binding to a membrane protein of a cell fused to the transmembrane part of a membrane protein, the latter being fused to the CD3ζ chain of a TCR.

[0079] In this advantageous embodiment, the third molecule comprises a sequence encoding a fusion protein having the following structure from N-terminus to C-terminus: a peptide capable of binding to a membrane protein of a cell, generally a tumor cell as described below, followed by a transmembrane domain, followed by the intracellular part of the CD3ζ chain of a TCR.

[0080] It is particularly advantageous that the transmembrane portion of the above-mentioned fusion protein is the transmembrane portion of the CD3ζ chain of the TCR, but any other transmembrane domain can be used as long as the fusion protein is such that, when synthesized intracellularly, the binding peptide is exposed to the outside of the cell (extracellular) and the CD3ζ chain of the TCR is exposed inside the cell (intracellular) to enable signal transduction.

[0081] If no other domains are present in the fusion protein, it is called a chimeric receptor or first generation CAR.

[0082] Advantageously, the invention relates to a composition as described above, in which the fusion protein comprises a peptide capable of binding to a membrane protein of a cell fused to the transmembrane part of a membrane protein, the transmembrane domain of the membrane protein being fused to a coactivation domain, the latter being fused to the CD3ζ chain of a TCR.

[0083] Advantageously, the fusion protein also contains a co-activation domain of a protein other than CD3ζ between the transmembrane domain and the activation domain (i.e., the intracellular domain) of the CD3ζ chain of the TCR, which is based on the normal TCR activation mechanism.

[0084] Dual intracellular signaling is a typical feature of T cell activation. Three different types of receptors are involved in this process: T cell antigen receptors, cytokine receptors, and costimulatory receptors. The first signal is a specific signal elicited by the TCR when it recognizes an antigenic peptide-major histocompatibility complex (MHC) complex on the surface of an antigen-presenting cell. The second signal is a costimulatory signal generated by costimulatory molecules such as CD28 / B7, which promotes IL-2 synthesis to complete T cell activation and avoid apoptosis.

[0085] Naive T cells cannot perform their normal role in the absence of costimulatory signals, and the same is true when T cells are stimulated by their antigen.

[0086] CARs containing only the CD3ζ sequence cannot activate cells without recruited endogenous costimulatory signals, which may delay the cellular response.

[0087] To avoid this drawback, it is preferable to group both the CD3ζ chain activation signal and the co-activation signal on the same fusion protein. Upon TCR activation after recognizing antigen-presenting MHC, both signals are activated simultaneously. Therefore, proliferation, cytotoxicity, sustained response, and survival of cells expressing this fusion protein are enhanced.

[0088] It is also possible to include a third activation domain that enables the production of cytokines that stimulate IL-12 production. This third signal can be generated, for example, using the STAT3 factor binding domain of the IL-2 receptor.

[0089] Other examples are possible, and one skilled in the art can adapt the co-activation domain as needed and as CAR cell technology evolves.

[0090] Even more advantageously, the present invention provides a composition as described above, - the A sequence comprises the complement of the sequence of the first region of the locus of the gene encoding the TRAC protein, - the B sequence comprises the complement of the sequence of the second region of the locus of the gene encoding the TRAC protein, The present invention relates to the aforementioned composition, wherein the A sequence and the B sequence are two different sequences, and the first region and the second region of the TRAC locus are advantageously adjacent to the 5' region of the locus of the gene encoding the TRAC protein.

[0091] As previously described herein, to replace a target gene with a third molecule, the sequence of portion A of the first molecule and the sequence of portion B of the second molecule are -capable of recognizing the same target molecule, and It is advantageous to be able to flank the target region to be replaced, which means that the first molecule, via its region A, must recognize the sequence upstream of the sequence to be replaced in the target molecule, and the second molecule, via its region B, must recognize the sequence downstream of the sequence to be replaced in the target molecule, or vice versa, i.e. the first molecule, via its region A, must recognize the sequence downstream of the sequence to be replaced in the target molecule, and the second molecule, via its region B, must recognize the sequence downstream of the sequence to be replaced in the target molecule.

[0092] This means that any target sequence can be replaced with any replacement sequence, as long as the A and B sequences of the first and second molecules, respectively, recognize and flank the target region.

[0093] In the present invention, it is particularly advantageous to insert the fusion encoded by the third molecule into a genetic locus that allows equivalent expression of the CD3ζ chain of the TCR without replacing the endogenous TCR CD3ζ chain required for T cell activity.

[0094] It is therefore possible to insert the fusion (i.e., the sequence of the third molecule encoding the fusion protein) anywhere in the target genome, as long as this insertion does not cause mutations (disruption of the gene or regulatory elements). In particular, it is of interest to propose inserting the fusion into the locus of the TRAC gene, which is the gene encoding the constant part of the TCR alpha domain. Even more particularly, it is advantageous to perform the insertion at the level of the first exon of the TRAC locus, and even more advantageously immediately 5' to the first exon.

[0095] Such insertions in the TRAC locus not only result in basal expression similar to that of the TCR chains, but also provide enhanced anti-tumor efficacy.

[0096] In addition, such an insertion at the TRAC locus - Reduces the occurrence of sustained activation signaling and efficient internalization and balanced re-expression of CAR upon single or repeated exposure to antigen. -Maintains naive / central memory phenotype and delays differentiation and exhaustion of effector T cells, allowing them to become better effector cells. -Improved safety profile (as the absence of a TCR reduces the risk of TCR-induced alloreactivity and autoimmunity, and the precise insertion of the CAR coding sequence reduces the risk of insertional tumorigenesis).

[0097] Advantageously, the present invention provides a method for producing a nucleic acid sequence comprising: at its 5' end, a first sequence oriented 5' to 3' for recognizing a transposase; and at its 3' end, a second sequence oriented 5' to 3' for recognizing said transposase; The present invention relates to the aforementioned composition, wherein the second molecule comprises, at its 5' end, a first complementary sequence of the first sequence for recognizing the transposase, followed by a second complementary sequence of the second sequence for recognizing the transposase.

[0098] More advantageously, the present invention provides a method for producing a nucleic acid sequence comprising the first molecule at its 5' end, a first sequence oriented 5' to 3' for recognizing a transposase, and at its 3' end, a second sequence oriented 5' to 3' for recognizing the transposase; the second molecule comprises at its 5' end a first sequence oriented 5' to 3' for recognizing a transposase and at its 3' end a second sequence oriented 5' to 3' for recognizing the transposase; The present invention relates to a composition as defined herein above, wherein a third molecule comprises, at its 5' end, a first complementary sequence of the first sequence for recognizing the transposase of the first molecule, followed by a second complementary sequence of the second sequence for recognizing the transposase of the first molecule, and at its 3' end, a first complementary sequence of the first sequence for recognizing the transposase of the second molecule, followed by a second complementary sequence of the second sequence for recognizing the transposase of the second molecule.

[0099] Possible formats for the first and second molecules of the present invention are defined herein. This configuration is shown diagrammatically in FIG. 1B. Of course, in the context of this example configuration of the first, second, and third molecules, the replacement sequence is located between two transposase half-sites in the 5' and 3' portions of the third molecule, in this case encoding a fusion protein containing the CD3ζ chain of a TCR.

[0100] Advantageously, the present invention provides a method for the preparation of a nucleic acid sequence comprising a first molecule comprising, at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase, and at its 3' end, a second sequence for recognizing a transposase, followed by a first complementary sequence of the first sequence for recognizing a transposase; It relates to a composition as defined herein above, wherein said second molecule comprises at its 5' end a complementary sequence of said second sequence for recognizing said transposase.

[0101] Even more advantageously, the present invention provides a method for producing a nucleic acid sequence comprising the steps of: at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase; at its 3' end, a second sequence for recognizing the transposase; followed by a first complementary sequence of the first sequence for recognizing the transposase of the first molecule; the second molecule comprises, at its 5' end, a first sequence for recognizing a transposase oriented from 5' to 3', and at its 3' end, a second sequence for recognizing the transposase, the first sequence for recognizing the transposase at the 5' end being preceded by a second complementary sequence of the second sequence for recognizing the transposase of the first molecule; and, The third molecule comprises, at its 5' end, a first complementary sequence of the first sequence for recognizing the transposase of the first molecule, followed by, at its 3' end, a second complementary sequence of the second sequence for recognizing the transposase of the second molecule.

[0102] Further possible formats for the first and second molecules of the present invention are defined herein. This configuration is shown diagrammatically in Figures 1C and 1D. Of course, in the context of this example of a configuration of a first, second, and third molecule, the replacement sequence is located between two transposase half-sites in the 5' and 3' portions of the third molecule, and this sequence encodes a fusion protein comprising the CD3ζ chain of a TCR.

[0103] Advantageously, the present invention provides a method for detecting a transposase comprising the steps of: at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase; at its 3' end, a first complementary sequence of the first sequence for recognizing the transposase of the first molecule; followed by a second sequence for recognizing the transposase; The present invention relates to the aforementioned composition, wherein a third molecule comprises, at its 5' end, a complementary sequence of the second sequence of the first molecule for recognizing the transposase of the first molecule.

[0104] Even more advantageously, the present invention provides a method for detecting a transposase comprising the steps of: (a) providing a first molecule comprising, at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase; and (b) providing, at its 3' end, a first complementary sequence of the first sequence for recognizing the transposase of the first molecule, followed by a second sequence for recognizing the transposase; the second molecule comprises, at its 5' end, a first sequence oriented 5' to 3' for recognizing a transposase and, at its 3' end, a second sequence for recognizing the transposase of the first molecule, the first sequence being followed by a second complementary sequence of the second sequence for recognizing the transposase of the first molecule; and the third molecule comprises, at its 5' end, a complementary sequence of the second sequence for recognizing the transposase of the first molecule and, at its 3' end, a complementary sequence of the first sequence for recognizing the transposase of the second molecule.

[0105] This configuration is shown diagrammatically in Figure 1E. Of course, in the context of this example configuration of a first molecule, a second molecule, and a third molecule, the replacement sequence is located between two transposase half-sites in the 5' and 3' portions of the third molecule, which encodes a fusion protein comprising the CD3ζ chain of a TCR.

[0106] Advantageously, the invention relates to a composition as described above, wherein said transposase is a bacterial transposase, in particular a transposase selected from Tn5, Tn9, Tn10 or Tc1 / mariner.

[0107] Advantageously, said transposase is a bacterial transposase chosen from the transposase of transposon Tn5, the transposase of transposon Tn9, the transposase of transposon Tn10, Tn903, Tn602, or even the transposase of transposon Tc1, or more generally the transposases of the mariner transposon superfamily.

[0108] Other examples of transposases that can be used in the context of the present invention are the Vibrio harveyi transposase (the transposase characterized by Agilent and used in the product SureSelect QXT), the MutA transposase and Mu transposase recognition site including the terminal sequences R1 and R2, the transposase of Staphylococcus aureus transposon Tn552, the transposase of transposon Tn7, the Tn / O and IS10 transposases, and the transposase of transposon Tn3.

[0109] The Tn5 transposase is the best known. It is encoded by the Tnp gene of the transposon Tn5. The transposase initiates transposition by forming a transposase dimer that binds to its target sequence. In association with this complex, the transposase then catalyzes four phosphoryl transfer reactions (DNA cleavage, DNA hairpin formation, hairpin resolution, and strand transfer to the target DNA), resulting in the integration of the transposon into its new DNA site, a process known as "tagmentation."

[0110] The present invention is based on this tagmentation principle: by using the tagmentation properties of transposase, the complex allows for the targeted insertion of one sequence into another sequence.

[0111] Also, in the context of the present invention, when a transposase is mentioned, it refers to one of the aforementioned transposases, namely the transposase of transposon Tn5, Tn9, Tn10, or Tc1 / mariner (or a transposase mutated to increase their transposition or tagmentation activity).

[0112] In the present invention, when several transposases are used simultaneously, a pair of transposases derived from transposons Tn5 and Tn10 is preferred, one of which binds to a complex formed by a first molecule and a third molecule, and the other of which binds to a complex formed by a second molecule and a third molecule.

[0113] Advantageously, the first and second transposase recognition sequences are sequences for recognizing a Tn5 transposase having one of the following sequences: -CTGtCTCTTataCAcAtcT (SEQ ID NO: 29), -CTGACTCTTataCACAagT (SEQ ID NO: 30), and -CTGtCTCTTgatCAgATCT (SEQ ID NO: 31).

[0114] As a result, the corresponding complementary sequence is: AgaTgTGtatAAGAGaCAG (SEQ ID NO: 32), complementary to the sequence SEQ ID NO: 29, ActTGTGtatAAGAGTCAG (SEQ ID NO: 33), which is complementary to the sequence of SEQ ID NO: 30, and AGATcTGatcAAGAGaCAG (SEQ ID NO: 34), complementary to the sequence SEQ ID NO: 31.

[0115] Other transposase recognition sequences are as follows: Tn5MErev, 5'-[phos]CTGTCTCTTATACACATCT-3' (SEQ ID NO: 35) Tn5ME-A (Illumina FC-121-1030), 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', (SEQ ID NO: 36) and Tn5ME-B (Illumina FC-121-1031); 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 37)

[0116] Other transposase recognition sequences are as follows: -sense sequence of SEQ ID NO: i the antisense sequence of SEQ ID NO: i+1, Here, i ranges from 38 to 192.

[0117] This includes, for example, the following pairs of sense and antisense sequences: SEQ ID NO:38 and SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO: 66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO: 99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107, SEQ ID NO:108 and SEQ ID NO:109, SEQ ID NO:110 and SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115, SEQ ID NO:116 and SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123, SEQ ID NO:124 and SEQ ID NO:125, SEQ ID NO:126 and SEQ ID NO:127, SEQ ID NO:128 and SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131, SEQ ID NO:132 and SEQ ID NO:133, SEQ ID NO:134 and SEQ ID NO:135, SEQ ID NO:136 and SEQ ID NO:137, SEQ ID NO:138 and SEQ ID NO:139, SEQ ID NO:140 and SEQ ID NO:141, SEQ ID NO:142 and SEQ ID NO:143, SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:147, SEQ ID NO:148 and SEQ ID NO:149, SEQ ID NO:150 and SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:153, SEQ ID NO:154 and SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157,This means that SEQ ID NO:158 and SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:162 and SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:167, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:170 and SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173, SEQ ID NO:174 and SEQ ID NO:175, SEQ ID NO:176 and SEQ ID NO:177, SEQ ID NO:178 and SEQ ID NO:179, SEQ ID NO:180 and SEQ ID NO:181, SEQ ID NO:182 and SEQ ID NO:183, SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:186 and SEQ ID NO:187, SEQ ID NO:188 and SEQ ID NO:189, SEQ ID NO:190 and SEQ ID NO:191, and SEQ ID NO:192 and SEQ ID NO:193 are considered.

[0118] Advantageously, the first G / C rich domain of the first molecule (or of the second molecule) has the sequence

[0119] [Table 2] (SEQ ID NO: 194), so that the second G / C-rich domain is the same. In fact, as the molecule folds back on itself, the second G / C-rich domain is in a complementary and antiparallel orientation to the first G / C-rich domain, with the interaction occurring in the palindromic region (underlined in the sequence herein above).

[0120] The first and second G / C rich domains also have the sequences

[0121] [Table 3] (SEQ ID NO: 195). The preceding description applies mutatis mutandis.

[0122] Other G / C rich domain sequences of the first or second molecule may be as follows: a first G / C-rich domain of the sequence GGTCGC (SEQ ID NO: 196) and a second G / C-rich domain of the sequence GCGACC (SEQ ID NO: 197).

[0123] These examples are given for illustrative purposes only and cannot limit the scope of the invention.

[0124] In an advantageous embodiment, the A / T-rich sequence of the first molecule of the complex consists essentially of A or T or consists of A or T.

[0125] Even more preferably, the A / T rich sequence of the first molecule of the complex consists of Ts.

[0126] Advantageously, the invention relates to a composition as described above, wherein the intermediate region of the third molecule comprises the sequence SEQ ID NO: 3 or the sequence SEQ ID NO: 1134.

[0127] As previously described herein, the third molecule comprises a sequence encoding the intracellular portion of the CD3ζ chain of the TCR, which sequence is preferentially oriented in a 5' to 3' direction.

[0128] The complete sequence of the CD3ζ protein of the TCR is represented by the sequence of SEQ ID NO: 1, - the peptide portion 1 to 21 of the sequence of SEQ ID NO: 1 corresponds to the propeptide cleaved during CD3ζ maturation, - the peptide portion of positions 22 to 30 of the sequence of SEQ ID NO: 1 corresponds to the extracellular domain of CD3ζ, - the peptide portion from 31 to 51 of the sequence of SEQ ID NO: 1 corresponds to the transmembrane domain of CD3ζ, The peptide portion from 52 to 164 of the sequence of SEQ ID NO: 1 corresponds to the intracellular domain of CD3ζ, i.e., the signal transduction domain.

[0129] The sequence SEQ ID NO:3 corresponds to the DNA encoding the intracellular portion of the sequence SEQ ID NO:2.

[0130] The sequence of SEQ ID NO: 1134 corresponds to the DNA encoding the transmembrane domain and intracellular portion of CD3ζ.

[0131] Advantageously, the invention relates to a composition as described above, in which the peptide capable of binding to a membrane protein is a single-chain variable fragment (scFv) that recognizes a membrane protein, in particular the extracellular part of a membrane protein.

[0132] scFv technology is particularly well suited to the fusions described in this invention, as it allows the variable portion of an antibody to be reproduced by fusing the variable portion of the light chain to the variable portion of the heavy chain (these two variable portions are linked together by a linker). The protein thus produced retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. It is then possible to create nucleic acid molecules that allow the corresponding peptides to be synthesized, thus resulting in simplified "short antibodies." Linkers are generally 10-25 amino acids in size (or 30-75 nucleotides) and are generally glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker may connect the N-terminus of the variable portion of the heavy chain (VH) to the C-terminus of the variable portion of the light chain (VL), or vice versa.

[0133] In the context of the present invention, scFvs of interest are, inter alia, CD19, BCma, CD123, CD20, CD22, CD38, LeY, ROR1, c-MET, CD133, CD171, CD70, CEA, EGFR-VIII, EpCAM, EphA2, FAP, c-KIT, Flt3, VEGFR2, PSMA, PSCA, MUC1, IL13Ra2, HER2, FAP, GD2, GPC3, mesothelin, EIIIB, gp100 / HLA-A2, HLA-A2-WT1Db126 ... *02:01, scFvs against membrane proteins widely expressed in tumors, such as PD-1, PD-L1, CTLA-4, and TGFβ. This list is by no means exhaustive.

[0134] Advantageously, the present invention relates to the aforementioned composition, in which the peptide capable of binding to a membrane protein is a peptide fused to streptavidin. In this way, when a fusion peptide (an extracellular streptavidin peptide and an intracellular activation part comprising the CD3ζ chain of TCR) is used, it is possible to bind a specific antibody whose heavy chain is associated with biotin. Then, streptavidin and biotin specifically interact to create a hybrid chimera that can recognize a cellular target via the antibody and activate a desired signal via the CD3ζ chain of TCR.

[0135] Peptides capable of binding to membrane proteins may also be ligands for membrane receptors found on target cells. This may be, for example, cytokines such as IL-13, which recognize the IL13Ra receptor, but may also be ligands for the receptors Flt3, c-Kit, c-Met, VEGFR2, PD1, HER2, EGFR, TNFRs, NKG2, CD116 (GM-CSF receptor). Again, this list is not exhaustive.

[0136] Advantageously, the invention relates to a composition as defined herein above, in which the co-activation domain corresponds to the co-activation domain of a protein chosen from CD28, 4-1BB, OX40 or ICOS.

[0137] As used herein, the terms "co-activation domain" and "co-stimulatory signaling region" refer to a portion of a chimeric T cell receptor that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an effective response of lymphocytes to antigens. Examples of such molecules include CD28, 4-1BB, DAP-10, and ICOS. For example, -CD28 (SEQ ID NO: 1135), -4-1BB (full sequence provided in SEQ ID NO: 1136), ICOS (full sequence provided in SEQ ID NO: 1137), and - A chimeric TCR comprising the intracellular domain of DAP-10 (SEQ ID NO: 1138) is suitably used in the context of the present invention.

[0138] Even more advantageously, the present invention relates to a composition as defined herein above, wherein the first molecule, the second molecule, and the third molecule are selected from the triplets defined in Table 2 herein below.

[0139] Advantageously, the present invention relates to a composition comprising one of the triplet of a first molecule, a second molecule and a third molecule as set out in Table 2 below.

[0140] [Table 4(1)]

[0141] [Table 4(2)]

[0142] [Table 4(3)]

[0143] [Table 4(4)]

[0144] [Table 4(5)]

[0145] [Table 4(6)]

[0146] Advantageously, the invention also relates to a composition as defined above, said composition comprising a triad selected from the triads #1 to #312 defined above.

[0147] Advantageously, the present invention provides ** a fourth single-stranded nucleic acid molecule comprising, or consisting essentially of, an A sequence that allows insertion of a complementary sequence of a nucleic acid of interest, wherein the complementary sequence is bound at its 5' end to a fifth T-rich sequence 40 to 60 nucleotides in length and at its 3' end to a sixth T-rich sequence 40 to 60 nucleotides in length, the fifth T-rich sequence and the sixth T-rich sequence comprising a fifth domain and a sixth domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of the fifth domain being complementary to the sequence of the sixth domain, and the fifth domain and the sixth domain being located 15 to 52 nucleotides from the A sequence, and the fourth molecule comprising at least one first sequence oriented 5' to 3' for recognizing a transposase and at its 3' end a second sequence for recognizing the transposase; ** a fifth single-stranded nucleic acid molecule comprising, consisting essentially of, or comprising a B sequence that allows insertion of a complementary sequence of a nucleic acid of interest, wherein the complementary B sequence is 5'-linked to a seventh T-rich sequence 40-60 nucleotides in length and 3'-linked to an eighth T-rich sequence 40-60 nucleotides in length, the seventh T-rich sequence and the eighth T-rich sequence comprising a seventh domain and an eighth domain of 6-12 G / C-rich nucleotides, respectively, the sequence of the seventh domain being complementary to the sequence of the eighth domain, and the seventh domain and the eighth domain being located 15-52 nucleotides from the B sequence; and the fifth molecule comprising at its 5'-end at least one first sequence oriented 5'-to-3' for recognizing the transposase and at its 3'-end at least one second sequence for recognizing the transposase; a fifth single-stranded nucleic acid molecule, wherein the B sequence is the complement of the nucleic acid of interest, the A sequence is located 5' of the region of interest in the nucleic acid of interest, and the B sequence is located 3' of the region of interest in the nucleic acid of interest; ** a sixth single-stranded molecule, * in its 5' portion, at least one complementary sequence of the fifth sequence for recognizing the transposase of the first molecule; * in its 3' portion, at least one complementary sequence of the fourth sequence for recognizing the transposase of a second molecule; * a sixth single-chain molecule comprising an intermediate region located between the complementary sequence of the fourth sequence for recognizing the transposase of the first molecule and the complementary sequence of the fifth sequence for recognizing the transposase of the second molecule, wherein the complementary sequence of the sequence encoding the fusion protein, i.e., the complementary sequence of the sequence encoding the CD3ζ chain of TCR, is contained in a third molecule; The present invention relates to a composition as described above, wherein the fourth single-stranded nucleic acid molecule and the sixth single-stranded nucleic acid molecule are paired according to Watson-Crick defined base complementarity to define two double-stranded binding sites for the transposase, and the fifth single-stranded nucleic acid molecule and the sixth single-stranded nucleic acid molecule are paired according to Watson-Crick defined base complementarity to define two double-stranded binding sites for the transposase.

[0148] Advantageously, the composition according to the invention comprises six molecules, i.e. two triplets, that allow double-stranded displacement of the target molecule, which can only be performed if the A, B, A' and B' regions are correctly selected so that the regions of interest to be replaced are correctly adjacent.

[0149] Of course, all provisions provided for compositions containing three molecules apply mutatis mutandis to compositions containing six molecules.

[0150] Advantageously, the present invention provides - the A sequence comprises the complement of the sequence of the first region of the locus of the gene encoding the TRAC protein, - the B sequence comprises the complement of the sequence of the second region of the locus of the gene encoding the TRAC protein, the A sequence and the B sequence are two different sequences, and the first region and the second region are advantageously adjacent to the 5' region of the locus of the gene encoding the TRAC protein; - the A' sequence comprises the complement of the sequence of the third region of the locus of the gene encoding the TRAC protein, - the B' sequence comprises the complement of the sequence of the fourth region of the locus of the gene encoding the TRAC protein, the A' sequence and the B' sequence are two different sequences, and the third and fourth regions of the sequence encoding the TRAC protein are advantageously adjacent to complementary sequences of the 5' region of the locus of the gene encoding the TRAC protein; Sequence A and sequence A' are at most partially complementary, Sequence A and sequence B' are at most partially complementary, Sequence B and sequence A' are at most partially complementary, The present invention relates to the aforementioned composition, wherein the B and B' sequences are at most partially complementary.

[0151] "At most partially complementary" is understood in the present invention to mean that the sequences have sequences that can be paired according to the base complementarity defined by Watson-Crick, but cannot be paired over the entire sequence. In other words, only parts of the sequences are complementary. Advantageously, sequences A, A', B, and B' are partially complementary over less than 50% of the sequences, in particular less than 30% of the sequences, particularly less than 10% of the sequences, and in particular are not completely complementary to each other.

[0152] In this way, it is possible to flank the 5' part of the TRAC locus (immediately upstream of the first exon) in a specific oriented manner on both strands to avoid uncontrolled or inconsistent recombination (i.e., tagmentation) that would result in unpredictable outcomes.

[0153] It is also possible to generate molecules containing the fusion sequence and exon 1 of the TRAC gene, such that the insertion occurs by replacing exon 1 with a sequence containing the coding sequence of a fusion containing TCR CD3ζ, followed by a transcription termination sequence, itself followed by the sequence of TRAC exon 1. An example of recombination at the TRAC locus is shown diagrammatically in [Figure 6].

[0154] Even more advantageously, the aforementioned composition comprises at least one of the sextuplets set forth in Table 3 below.

[0155] [Table 5(1)]

[0156] [Table 5(2)]

[0157] [Table 5(3)]

[0158] The composition according to the invention therefore advantageously proposes 156 sextuplets of molecules that allow the insertion in the locus of the TRAC gene of hybrid sequences encoding fusion proteins comprising the CD3ζ chain of the TCR and comprising, at its N-terminus, a protein sequence that recognizes a peptide expressed on the surface of a target cell.

[0159] This makes it possible to restore functions lost due to mutations and achieve functional complementation depending on the target cell.

[0160] In another aspect, the present invention relates to immune cells, in particular T lymphocytes, comprising a composition as defined herein above, said immune cells being isolated and purified outside their natural context.

[0161] The immune cell in question is in a state in which recombination, i.e., insertion, has not yet occurred, and contains in its cytoplasm or nucleus, or both, a molecular complex formed by a triplet of a first molecule, a second molecule, and a third molecule, and optionally a sextet further comprising a fourth molecule, a fifth molecule, and a sixth molecule, as described herein above.

[0162] Even more preferably, the immune cells are tumor-infiltrating immune cells, in particular infiltrating T cells.

[0163] For example, if it is intended to perform a single-stranded insertion, so that the resulting cell has two different alleles (one allele with the insertion and one allele without the insertion), it is advantageous to use a composition comprising a first molecule, a second molecule, and a third molecule. The same is true when RNA, specifically messenger RNA, is replaced.

[0164] Conversely, if double-stranded insertion is desired, it is advantageous to use a first molecule, a second molecule, a third molecule, a fourth molecule, a fifth molecule, and a sixth molecule.

[0165] The compositions according to the invention are introduced into immune cells using transfection techniques well known to those skilled in the art, in particular electroporation or calcium phosphate precipitation. Those skilled in the art will know how to choose the best technique depending on the nature of the target immune cells.

[0166] It is also advantageous for the cells to contain a composition comprising any one of the triplets in Table 2 or any one of the sextuplets in Table 3.

[0167] In another aspect, the invention relates to a pharmaceutical composition comprising the aforementioned composition or immune cells as described hereinabove, in combination with a pharmaceutically acceptable carrier.

[0168] The aforementioned composition can be used for the treatment of diseases in combination with a pharmaceutically acceptable carrier. The carrier is a commonly accepted carrier known to those skilled in the art, such as distilled water or a physiological buffer solution. The carrier must allow the formation of the complex described above, but at the same time, must be acceptable to cells or organisms.

[0169] The same applies to cells: they are placed in contact with a pharmaceutically acceptable carrier that keeps the cells alive (does not affect the membrane or induce cell death) and has properties that make it compatible with living organisms.

[0170] In another aspect, the present invention relates to the aforementioned composition or the aforementioned immune cells as a medicament.

[0171] It is further advantageous to use a composition comprising any one of the triplets of Table 2 or any one of the sextuplets of Table 3 as a medicament.

[0172] In yet another aspect, the invention relates to the aforementioned composition or the aforementioned immune cells for use in the treatment of cancer.

[0173] Insofar as the aforementioned composition is intended to provide CAR cells, it is particularly suitable to use this composition to treat cancer.

[0174] The composition is adapted to target cancer cells by selecting the extracellular portion of the fusion protein to target specific tumor cells.

[0175] Therefore, the expression by lymphocytes modified by the composition of the present invention ensures specificity to tumor antigens.Then, it is sufficient to inject tens of millions or hundreds of millions of CAR-expressing lymphocytes into patients.Finally, as they proliferate in vivo, the modified lymphocytes persist for several weeks or even months, and help reduce or even destroy tumors by killing cancer cells.

[0176] Advantageously, the present invention relates to a method for treating cancer in a patient in need thereof, comprising administering an effective dose of the composition as described herein before, in particular, said method comprising administering an effective amount of immune cells comprising said composition as described herein before.

[0177] Advantageously, the cancer may be of any type, including any type of solid tumor and hematopoietic tumor. Thus, the cancer may in particular be: -Hematopoietic types: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), acute multiple leukemia (AML), CD19-positive cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), small lymphocytic leukemia leukemia, SLL), and CD20-positive malignancies, or - Solid-state cancers: neuroblastoma (NB), glioma, mesothelioma, lung cancer, ovarian cancer (OC), breast cancer (BC), sarcoma, osteosarcoma (OS), hepatocellular carcinoma (HCC), HER2-positive cancer, EGFRvIII-positive cancer, glioblastoma (GBM), pancreatic cancer (PC), MUC1-positive cancer, prostate cancer (PCa), colorectal adenocarcinoma (COADREAD), colorectal cancer (CRC), melanoma (MEL), medulloblastoma (MB), gastric cancer (GC), liver cancer, neck cancer, head cancer, skin cancer, bone cancer, brain cancer, colorectal cancer, stomach cancer cancer), associated bladder tumors, testicular cancer, thyroid cancer, endometrial cancer, cervical cancer, and endocervical cancer.

[0178] In another aspect, the present invention relates to the use of the aforementioned composition for inserting into a T lymphocyte cell a sequence encoding a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, provided that the use does not include a process for modifying the germline genetic identity of a human being and that the use is not a method for treating the human or animal body by surgery or therapy.

[0179] This insertion is preferentially located at the TRAC locus described earlier herein.

[0180] As previously described herein, the present compositions utilize the tagmentation properties of transposases to allow for specific targeting of a target region and replacement of it with a sequence of interest.

[0181] The compositions according to the invention are particularly advantageous for in vitro CAR cells, i.e. modified T cells that express, in addition to the T cell receptor (TCR), a chimeric T cell receptor that allows specific cell targeting of tumors.

[0182] The present invention further relates to a method for the insertion, in particular in vitro insertion, of a sequence encoding a fusion protein comprising, in the amino-terminal part, a peptide capable of binding to a membrane protein of a cell and, in the carboxy-terminal part, the CD3ζ chain of a TCR, comprising: - placing a composition as defined herein above in contact with a target nucleic acid comprising at least the 5' region of the locus of the gene encoding the TRAC protein, in order to obtain a replacement complex, The composition comprises: the A sequence of the first molecule comprises a complementary sequence to a first region of the locus of a gene encoding a TRAC protein; the B sequence of the second molecule comprises the complement of a second region of the locus of the gene encoding the TRAC protein; and Arranging the third molecule such that it comprises a fusion protein sequence comprising, at its amino-terminal portion, a peptide capable of binding to a cellular membrane protein and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the fusion protein sequence being located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule; - placing the replacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the composition to obtain a recombinant complex; - recombining the combined complex to obtain a sequence encoding a fusion protein inserted into the target nucleic acid between the first and second regions of the locus of the gene encoding the TRAC protein.

[0183] Advantageously, the invention relates to a method for the in vitro insertion of a sequence encoding a fusion protein comprising, in its amino-terminal part, a peptide capable of binding to a membrane protein of a cell and, in its carboxy-terminal part, the CD3ζ chain of a TCR, comprising: placing a composition in contact with a target nucleic acid comprising at least a 5' region of the locus of a gene encoding a TRAC protein, to obtain a replacement complex, wherein the composition comprises a triplet defined in Table 2 or a sextet defined in Table 3; - placing the replacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the composition to obtain a recombinant complex; - recombining the combined complex to obtain a sequence encoding a fusion protein inserted into the target nucleic acid between the first and second regions of the locus of the gene encoding the TRAC protein.

[0184] In another aspect, the present invention provides a method for generating chimeric antigen receptor-T cells, i.e., CAR-T cells, comprising the sequence of a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the method comprising: - placing a composition as defined herein above in contact with purified T cells, the genomic DNA of which comprises the locus of the gene encoding the TRAC protein, The composition or the assembly is the A sequence of the first molecule comprises a complementary sequence to a first region of the locus of a gene encoding a TRAC protein; the B sequence of the second molecule comprises the complement of a second region of the locus of the gene encoding the TRAC protein; and Arranging the third molecule to contain a fusion protein sequence comprising, at its amino-terminal portion, a peptide capable of binding to a cellular membrane protein and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the fusion protein sequence being located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule, to obtain a displacement complex; - placing the displacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the collection to obtain a recombinant complex; - recombining the combination complex to obtain CAR-T cells, the genomic DNA of which contains a sequence encoding the fusion protein in the locus of the gene encoding the TRAC protein; -purifying the CAR-T cells.

[0185] Advantageously, the present invention relates to a method for generating chimeric antigen receptor T cells, i.e. CAR-T cells, comprising the sequence of a fusion protein comprising, at the amino-terminal part, a peptide capable of binding to a membrane protein of a cell and, at the carboxy-terminal part, the CD3ζ chain of a TCR, comprising: placing the composition in contact with purified T cells, the genomic DNA of which comprises the locus of a gene encoding a TRAC protein, to obtain a replacement complex; - placing the displacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the collection to obtain a recombinant complex; - recombining the combination complex to obtain CAR-T cells, the genomic DNA of which contains a sequence encoding the fusion protein in the locus of the gene encoding the TRAC protein; -purifying CAR-T cells.

[0186] The present invention also relates to a modified T lymphocyte cell obtainable by the aforementioned method, wherein the modified T lymphocyte cell comprises an insertion in its genome of a sequence encoding a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a cellular membrane protein and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the insertion being located in the locus of the gene encoding the TRAC protein.

[0187] Due to the "tagmentation" recombination carried out by transposases and the molecular signatures that such recombination produces, i.e., genetic modifications at the transposase-induced DNA cleavage zones, these lymphoid cells can only be more precisely defined by the method by which they are obtained: they can be recognized by recognition zones of regions A and B, and possibly A' and B', which differ from the original sequences.

[0188] These cells are novel and distinct from those that can be obtained using modern genome editing techniques such as, for example, CRISPR / Cas9. [Brief explanation of the drawings]

[0189] The invention will be better understood from a reading of the following examples and figures herein below. [Figure 1A] 1 is a first schematic diagram of a first configuration of pairing a first molecule, a second molecule, and a third molecule of a composition according to the present invention. [Figure 1B] FIG. 2 is a second schematic diagram of a first configuration of pairing a first molecule, a second molecule, and a third molecule of a composition according to the present invention. [Figure 1C] FIG. 2 is a third schematic diagram of a first configuration of pairing a first molecule, a second molecule, and a third molecule of a composition according to the present invention. [Figure 1D] FIG. 4 is a fourth schematic diagram of a first configuration of pairing a first molecule, a second molecule, and a third molecule of a composition according to the present invention. [Figure 1E]FIG. 5 is a fifth schematic diagram of a first configuration of pairing a first molecule, a second molecule, and a third molecule of a composition according to the present invention. [Figure 2A] FIG. 1 shows the principle and steps of single-strand replacement using the first, second, and third molecules of the composition according to the present invention. [Figure 2B] FIG. 1 shows the principle and steps of single-strand replacement using the first, second, and third molecules of the composition according to the present invention. [Figure 2C] FIG. 1 shows the principle and steps of single-strand replacement using the first, second, and third molecules of the composition according to the present invention. [Figure 3A] FIG. 1 shows the principle and steps of double-strand replacement using the first, second, third, fourth, fifth, and sixth molecules of the composition according to the present invention. [Figure 3B] FIG. 1 shows the principle and steps of double-strand replacement using the first, second, third, fourth, fifth, and sixth molecules of the composition according to the present invention. [Figure 3C] FIG. 1 shows the principle and steps of double-strand replacement using the first, second, third, fourth, fifth, and sixth molecules of the composition according to the present invention. [Figure 4] 1 shows the association of molecules contained in Tube 1 in Example 1. The black spheres represent biotin. [Figure 5] 1 shows the association of molecules contained in tube 2 in Example 1. The black spheres represent biotin. [Figure 6A]Figure 1 shows the insertion of an ScFv-coactivation domain-CD3ζ fusion. A shows the TRAC locus near the TRAV and TRAJ loci, and the recognition zone around exon 1 of the complex formed by the first molecule, the third molecule (comprising the TRAC fusion sequence and exon 1), and the second molecule. B shows tagmentation in the presence of transposase. C shows the results of insertion of the fusion sequence at the TRAC locus. D shows a schematic representation of the protein encoded by the fusion sequence inserted into the TRAC locus. * indicates the scFv exposed on the outside of the T cell, ** indicates the coactivation domain (e.g., CD28), and *** indicates the CD3ζ intracellular activation domain. [Figure 6B] Figure 1 shows the insertion of an ScFv-coactivation domain-CD3ζ fusion. A shows the TRAC locus near the TRAV and TRAJ loci, and the recognition zone around exon 1 of the complex formed by the first molecule, the third molecule (comprising the TRAC fusion sequence and exon 1), and the second molecule. B shows tagmentation in the presence of transposase. C shows the results of insertion of the fusion sequence at the TRAC locus. D shows a schematic representation of the protein encoded by the fusion sequence inserted into the TRAC locus. * indicates the scFv exposed on the outside of the T cell, ** indicates the coactivation domain (e.g., CD28), and *** indicates the CD3ζ intracellular activation domain. [Figure 6C] Figure 1 shows the insertion of an ScFv-coactivation domain-CD3ζ fusion. A shows the TRAC locus near the TRAV and TRAJ loci, and the recognition zone around exon 1 of the complex formed by the first molecule, the third molecule (comprising the TRAC fusion sequence and exon 1), and the second molecule. B shows tagmentation in the presence of transposase. C shows the results of insertion of the fusion sequence at the TRAC locus. D shows a schematic representation of the protein encoded by the fusion sequence inserted into the TRAC locus. * indicates the scFv exposed on the outside of the T cell, ** indicates the coactivation domain (e.g., CD28), and *** indicates the CD3ζ intracellular activation domain. [Figure 6D] Figure 1 shows the insertion of an ScFv-coactivation domain-CD3ζ fusion. A shows the TRAC locus near the TRAV and TRAJ loci, and the recognition zone around exon 1 of the complex formed by the first molecule, the third molecule (comprising the TRAC fusion sequence and exon 1), and the second molecule. B shows tagmentation in the presence of transposase. C shows the results of insertion of the fusion sequence at the TRAC locus. D shows a schematic representation of the protein encoded by the fusion sequence inserted into the TRAC locus. * indicates the scFv exposed on the outside of the T cell, ** indicates the coactivation domain (e.g., CD28), and *** indicates the CD3ζ intracellular activation domain. [Example]

[0190] Example 1 - Obtaining B cells expressing a CAR fusion To treat patients suffering from cancers expressing the CD19 marker, it may be advantageous to propose a cell therapy aimed at transplanting into the patient CAR-T cells carrying CD19 scFv-CD3ζ+ that have been modified using a composition according to the invention, and in particular to insert into the patient's T lymphocytes a sequence encoding a fusion protein at the TRAC locus (to limit rejection).

[0191] A- Isolation of T cells from blood Blood samples from healthy donors are collected either fresh or frozen.

[0192] The sample is centrifuged over a sterile density gradient centrifugation medium (eg, Ficoll in the presence of EDTA) at 1000 g for 8 minutes to isolate peripheral blood mononuclear cells (PBMCs).

[0193] The white blood cell ring is collected and washed with PBS (phosphate buffered saline).

[0194] The leukocytes are cultured for 12 hours at 37° C. and 5% CO 2 in a medium specific for hematopoietic cell culture supplemented with IL-2 (20 ng / ml) and 5% human serum.

[0195] T lymphocytes are then isolated in a cell sorter by positive selection using anti-CD3 antibody labeling or by negative selection using a cocktail of anti-CD2, anti-CD34, anti-CD14, anti-CD16, anti-CD19, anti-CD24, anti-CD56n, anti-CD66b, and anti-CD61 antibodies.

[0196] The purified T cells are then cultured under CD3 / 28 and IL-2 activation cocktail (40 ng / ml) at 37° C. and 5% CO 2 for 72 hours.

[0197] The cells are then ready to be transfected with the composition according to the invention.

[0198] B- Preparation of the molecules of the composition according to the invention A construct containing GFP was prepared to target the TRAC gene and verify insertion at the TRAC Chr14 locus: 22,547,500–22,552,358 HG38GR.

[0199] Two tubes were prepared as follows: ** Tube 1 (10 μl): - oligo Great X1 (10 μM) corresponding to molecule 1 of the sequence SEQ ID NO: 198 below 5'-AgaTgTGtatAAGAGaCAGGTAGTGTATTTTTTTTTTTTTTTTATCATCCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTGGCGATCGCTTTTTTTTT TTTTTTTTGCAGACATCCAGCTGGATCCAAAACCAAATTTTTTTTTTTTTTTTGCGATCGCCTTTTTTTTTTTGATACATTTAgaTgTGtatAAGAGaCAGGATGAT-3' - oligo Great X3 (10 μM) corresponding to molecule 4 of sequence SEQ ID NO: 201 below 5'-CACGTGCTGtCTCTTataCAcAtcTTTTTCTCGATCATTATTATTTTTTGGCGATCGCTTTTTTTTTTTTTTCATTGCCGAGGCCACCAGGGCTGGCTCAGCTTTTTTT TTTTTTTTTGCGATCGCCTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGCACGTGTTTTTTTTTTTTTTTTTTCAGCTATACTGtCTCTTataCAcAtcT-3' reverse oligo GREAT X1 Rev (10 μM), corresponding to the 5′ part of molecule 3 of the sequence SEQ ID NO: 1139 below; 5'-TACACTACCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTggttacGCGGCCGCCGTACA-3' reverse oligo GREAT X3 Rev (10 μM), corresponding to the 3′ part of molecule 6 of sequence SEQ ID NO: 1140 below; 5'-TGTACGGCGGCCGCgtaaccTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATAGCTG-3' - a reverse oligo Helix Rev corresponding to an additional strand for binding to the helicase via its biotinylated 3' end (oligo has the sequence SEQ ID NO: 1141 below) 5'-ATAATAATGATCGAGAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[Biot]-3' ** Tube 2 (10 μl): - oligo Great X2 (10 μM) corresponding to molecule 2 of sequence SEQ ID NO: 199 below 5'-CACGTGCTGtCTCTTataCAcAtcTTTTTCTCGATCATTATTATTTTTTGGCGATCGCTTTTTTTTTTTTTT TTGTTGGAGCCACTGACCCTGCCAGAATATGGTTTTTTTTTTTTTTTTGCGATCGCCTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGCACGTGTTTTTTTTTTTTTTTTTTGACGAATACTGtCTCTTataCAcAtcT-3' - oligo Great X4 (10 μM) corresponding to molecule 5 of sequence SEQ ID NO: 202: 5'-AgaTgTGtatAAGAGaCAGAGTATGAATTTTTTTTTTTTTTTATCATCCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTGGCGATCGCTTTTTTTTT TTTTTTTCTGCATGACTCACTAGCACTCTATCACGGCTTTTTTTTTTTTTTGCGATCGCCTTTTTTTTTTTGATACATTTAgaTgTGtatAAGAGaCAGGATGAT-3' reverse oligo GREAT X2 Rev (10 μM), corresponding to the 3′ part of molecule 3 of the sequence SEQ ID NO: 1142 below; 5'-CGATACGGTACCatgttcTTTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATTCGTC-3' reverse oligo GREAT X4 Rev (10 μM), corresponding to the 5′ part of molecule 6 of the sequence SEQ ID NO: 1143 below; 5'-TTCATACTCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAacatGGTACCGTATCG-3' - a reverse oligo Helix Rev (oligo having the sequence SEQ ID NO: 1142 below) corresponding to an additional strand for binding to the helicase via its biotinylated 3' end.

[0200] These two tubes are then heated to 95°C for 5 minutes, then left at room temperature for 1 hour, and then each tube is digested with the corresponding restriction enzyme (NotI for tube 1 and KpnI for tube 2) and purified by PCR purification kit (elution 20 μl).

[0201] Figures 4 and 5 show schematic representations of the contents of tube 1 and tube 2, respectively, prior to enzymatic digestion.

[0202] In parallel, the anti-CD19-CD3ζ-P2A-GFP sequence was obtained by direct amplification of the pSLCAR-CD19-CD3ζ plasmid in DH5α bacteria. This sequence was amplified (using oligos containing NotI and KpnI restriction site structures digested after PCR), then digested with the corresponding restriction enzymes and purified using a PCR purification kit, eluting in a volume of 20 μl. The GFP-CD19-CD3ζ fusion was then provided with flanking ends (i.e., free NotI and KpnI half sites) in tube 3.

[0203] The contents of these three tubes (Tube 1, Tube 2, and Tube 3) are then mixed, and T4 phage ligase (4 μl, 100 U) is added to the mixture in the presence of an appropriate buffer (T4 buffer, 5 μl 10x) and 11 μl of distilled water. The mixture is left at room temperature for 1 hour. The mixture is then gel-purified (elution 30 μl) to isolate the largest GFP-CD19-CD3ζ fragment, which is larger than 1 kb and features four sarcophages, X1, X2, X3, and X4, at its 5' and 3' ends. The purified fragment is ready for use and contains the first, second, fourth, and fifth molecules at its termini, with the third and sixth molecules corresponding to the GFP-CD19-CD3ζ fusion (Tube 4).

[0204] The GFP-CD19-CD3ζ replacement fragment, with its four sarcophages positioned at the 5′ and 3′ ends, is then ready to accept a transposase dimer.

[0205] At this stage, a first molecule containing a biotin-conjugated single-stranded oligonucleotide at its 3' end (biotinylated oligo) and a fourth molecule containing a complementary single-stranded oligonucleotide were added. The biotinylated oligonucleotide is optional.

[0206] C - Transfection of T lymphocytes with a composition according to the invention. The contents of tube 4, optionally containing biotinylated oligos, are then used to transfect isolated T lymphocyte cells by electroporation.

[0207] The cells are centrifuged at 300 g for 5 min, washed with PBS, and then suspended in electrolyte buffer at a cell density of 75,000–100,000 cells per electroporation chamber. The contents of tube 4, along with a plasmid encoding the Tn5 protein and, optionally, a plasmid encoding a helicase-streptavidin fusion (UVRD-mSA), are added to the electroporation chamber according to the "Episomal iPSC Reprogramming Vectors" protocol for Thermofisher's Neon® transfection system.

[0208] The mixture is electroporated at 1650V for three 10 ms sequences.

[0209] The cells are then immediately centrifuged at 300 g for 5 min and suspended in supplemented hematopoietic cell culture medium at 37 °C and 5% CO for 48-96 h.

[0210] Tagmentation (i.e., gene replacement) is then possible. The cells are maintained in a suitable medium.

[0211] D - Verification of transfection Cells transfected with the molecule of interest inserted into the locus are selected using an antibiotic (eg, neomycin, bleomycin, blasticidin S, etc.) that contains a resistance gene in the replacement sequence.

[0212] A simple PCR is performed using a 5' sense oligonucleotide from the insert and a 3' antisense oligonucleotide from the insert (a Single-Tail Adapter / Tag (STAT)-PCR-based method). The PCR product is then sequenced using conventional techniques to detect the presence of at least GFP in the region of interest, a signature of a fusion insert.

[0213] Transfected cells can also be detected by cytometry to detect GFP expression.

[0214] Reverse transcription can also be performed on the isolated cells to obtain a cDNA library. The fusion sequence is then amplified by PCR using oligos for PCR and anti-CD19 sequencing: AGGAGTCCCATCAAGGTTCAGT (SEQ ID NO: 1144) and GFP Rev: CACGAACTCCAGCAGGACCATG (SEQ ID NO: 1145). The amplified fragments are sequenced by the Sanger method and run on an agarose gel. As expected, after analysis (NCBI blast), a very high similarity is found with the theoretical GFP-CD19-CD3ζ fragment at the TRAC locus.

[0215] E- Functional analysis of T cells modified by the composition according to the invention Several tests are performed to verify that the cells have been effectively transfected and that the insert is functional.

[0216] 1. CAR-T cell proliferation test The generated T cells are cultured in 24-well plates in medium not supplemented with IL-2 and in the presence of irradiated Raji cells (1:1 ratio) at 37° C. and 5% CO 2 .

[0217] A fraction of T cells is collected after 4, 7 and 10 days in order to enumerate them by flow cytometry.

[0218] 1. CAR-T cell cytotoxicity test The generated T cells are cultured in 12-well plates in medium not supplemented with IL-2 and in the presence of fluorescent dye-expressing Raji cells (3:1 ratio) at 37° C. and 5% CO 2 .

[0219] After 24 hours of incubation, a fraction of the cells are harvested and quantified for the presence of the fluorescent dye by flow cytometry, and the remaining cells are re-cultured with an additional proportion of Raji cells.

[0220] This process is repeated after 36 and 48 hours of incubation.

[0221] Example 2 - Alternative CD3ζ fusion formats. Another fusion can be performed according to the protocol described in Example 1 herein.

[0222] Tube 1 is prepared as described in the examples for the following sequence: reverse oligo GREAT X1 Rev (10 μM), corresponding to the 5′ part of molecule 3 of the sequence SEQ ID NO: 1146 below; 5'-TACACTACCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTTTTggttacACCGGTCGTACA-3' reverse oligo GREAT X3 Rev (10 μM), corresponding to the 3′ part of molecule 6 of sequence SEQ ID NO: 1147 below; 5'-TGTACGACCGGTgtaaccTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATAGCTG-3' Prepare tube 2 as follows: reverse oligo GREAT X2 Rev (10 μM), corresponding to the 3′ part of molecule 3 of the sequence SEQ ID NO: 1148 below; 5'-CGATACCCGCGGatgttcTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATTCGTC-3' reverse oligo GREAT X4 Rev (10 μM), corresponding to the 5′ part of molecule 6 of the sequence SEQ ID NO: 1149 below; 5'-TTCATACTCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAacatCCGCGGGTATCG-3' - a reverse oligo Helix Rev (oligo having the sequence SEQ ID NO: 1142 below) corresponding to an additional strand for binding to the helicase via its biotinylated 3' end.

[0223] These two tubes are then heated to 95°C for 5 minutes, then left at room temperature for 1 hour, and then each tube is digested with the corresponding restriction enzyme (AgeI for tube 1 and SacII for tube 2) and purified by PCR purification kit (elution 20 μl).

[0224] In parallel, the anti-CD19-CD3ζ-P2A-GFP sequence was obtained by direct amplification of the pSLCAR-CD19-CD3ζ plasmid in DH5α bacteria. This plasmid was then digested via the AgeI and SacII restriction sites, after which the CD19-CD3ζ-P2A-GFP sequence was purified using a gel purification kit in a volume of 20 μl. The CD19-CD3ζ-P2A-GFP fusion was then provided with flanking ends (i.e., free AgeI and SacII half-sites) in tube 3.

[0225] The contents of these three tubes (Tube 1, Tube 2, and Tube 3) are then mixed, and T4 phage ligase (4 μl, 100 U) is added to the mixture in the presence of an appropriate buffer (T4 buffer, 5 μl 10x) and 11 μl of distilled water. The mixture is then left at room temperature for 1 hour. The mixture is then gel-purified (elution 30 μl) to isolate the largest CD19-CD3ζ-P2A-GFP fragment, which is larger than 1 kb and contains four sarcophages, X1, X2, X3, and X4, at its 5' and 3' ends. The purified fragment is ready for use and contains the first, second, fourth, and fifth molecules at its ends, with the third and sixth molecules corresponding to the CD19-CD3ζ-P2A-GFP fusion (Tube 4).

[0226] The CD19-CD3ζ-P2A-GFP replacement fragment, with its four sarcophages positioned at its 5' and 3' ends, is then ready to accept a transposase dimer.

[0227] At this stage, a first molecule containing a biotin-conjugated single-stranded oligonucleotide at its 3' end (biotinylated oligo) and a fourth molecule containing a complementary single-stranded oligonucleotide were added. The biotinylated oligonucleotide is optional.

[0228] Transfection is carried out as described in Example 1.

[0229] Transfection verification is performed as described in Example 1, except for reverse transcriptase detection. The fusion sequence is then amplified by PCR using oligos for PCR and anti-CD19 sequencing: AGGAGTCCCATCAAGGTTCAGT (SEQ ID NO: 1144) and GFP Rev: CACGAACTCCAGCAGGACCATG (SEQ ID NO: 1145), and the amplified fragment is sequenced by Sanger sequencing and run on an agarose gel. As expected, after analysis (NCBI blast), a very high similarity is found with the theoretical CD19-CD3ζ-P2A-GFP fragment at the TRAC locus.

[0230] Example 3 - Obtaining T cells expressing CD3ζ-containing fusions from pluripotent stem cells. Because obtaining T cells from patients can be difficult, induced pluripotent stem cell (iPSc) technology can be used.

[0231] iPSC cells with an immunological profile that matches the patient can be obtained from a cell bank.

[0232] It is also possible to collect differentiated cells (e.g., fibroblasts or epithelial cells) from a patient and force dedifferentiation by expressing the Oct4 and Socs2 genes, as well as other genes such as Klf4, Dub3, c-Myc, Nanog, etc. Such techniques are now well known to those skilled in the art, and protocols described in the prior art can be adapted according to the cell type that one wishes to involve in the dedifferentiation model.

[0233] At this stage, the iPSC cells can be transfected with Lipofectamine in the presence of tube 4 as described in Example 1 or Example 2.

[0234] Transfected cells are selected using the appropriate antibiotic and the insertion is verified as described in Example 1.

[0235] iPSC cells are cultured on plates coated with extracellular matrix gel and co-cultured with inactivated mouse embryonic fibroblasts for 24 hours in complete stem cell culture medium supplemented with bFGF and ROCK inhibitor.

[0236] The iPSC cells are then cultured for 7 days in 24-hour culture medium containing a different differentiation cocktail each day: Days 0-1: medium supplemented with hBMP-4, hVEGF, hWnt3a, and KOSR; Day 2: medium supplemented with hBMP-4, hVEGF, and KOSR; Day 3: medium supplemented with hBMP-4, hVEGF, and bFGF; Days 4-5: medium supplemented with hVEGF and bFGF, Day 6: IMDM medium supplemented with F12, B27, N2, BSA, hVEGF, bFGF, human stem cell factor, and hFlt3 ligand; Day 7: IMDM medium supplemented with F12, B27, N2, BSA, hVEGF, bFGF, human stem cell factor, hFlt3 ligand, TPO, IL-6, hEPOgen, and FICZ (6-formylindolo[3,2-b]carbazole).

[0237] After 7 days, continue culturing the cells for an additional 3-7 days, then continue experiments on the harvested non-adherent cells. Test these cells to verify that they express the CD34+ marker by flow cytometry using an anti-CD34 antibody.

[0238] If the cells have not been transfected at the iPSc stage, they can be transfected at the CD34+ stage as described in Example 1.

[0239] They are then induced to differentiate into T cells as follows: -Differentiation into lymphoid lineage progenitors:

[0240] The resulting CD34+ cells are cultured in hematopoietic cell culture medium supplemented with recombinant human cytokines on plates coated with extracellular matrix gel specifically designed for the culture of lymphoid progenitor cells without feeder cells to specifically promote differentiation into CD7+CD5+ T progenitor (pro-T) cells.

[0241] Continue culturing the cells at 37 °C and 5% CO for 14 days, refreshing half of the medium every 3-4 days. -Maturation of precursor cells into CD4+CD8+ T cells:

[0242] On day 14, the pro-T cells are collected, transferred to new plates coated with extracellular matrix gel, and cultured in fresh hematopoietic cell culture medium supplemented with recombinant human cytokines to specifically promote the maturation of the pro-T cells into mature T cells.

[0243] Continue culturing the cells at 37 °C and 5% CO for 14 days, refreshing half of the medium every 3-4 days.

[0244] T cell maturation to CD8+: The cells obtained in the previous step are collected and transferred to new plates coated with extracellular matrix gel and cultured in the same culture medium as before, supplemented with recombinant human IL-15 and CD3 / 28 activation cocktail.

[0245] Continue culturing the cells at 37 °C and 5% CO for 7 days, refreshing half of the medium on days 3–4.

Claims

1. 1. A composition comprising: a first single-stranded nucleic acid molecule comprising or consisting essentially of an A sequence that allows insertion of a complementary sequence of a nucleic acid of interest, or comprising the complementary sequence of a nucleic acid of interest, wherein the complementary sequence is bound at its 5' to a first T-rich sequence of 40 to 60 nucleotides in length and at its 3' to a second T-rich sequence of 40 to 60 nucleotides in length, the first T-rich sequence and the second T-rich sequence comprising a first domain and a second domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of the first domain being complementary to the sequence of the second domain, the first domain and the second domain being located 15 to 52 nucleotides from the A sequence, and the first molecule comprising at its 5' end at least one first sequence oriented 5' to 3' for recognizing a transposase and at its 3' end a second sequence for recognizing the transposase; a second single-stranded nucleic acid molecule comprising or consisting essentially of a B sequence allowing insertion of a complementary sequence of a nucleic acid of interest, or comprising the complementary sequence of a nucleic acid of interest, wherein said complementary B sequence is bound at its 5' to a third T-rich sequence of 40 to 60 nucleotides in length and at its 3' to a fourth T-rich sequence of 40 to 60 nucleotides in length, said third T-rich sequence and said fourth T-rich sequence comprising a third domain and a fourth domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of said third domain being complementary to the sequence of said fourth domain, said third domain and said fourth domain being located 15 to 52 nucleotides from said B sequence, said second molecule comprising at its 5' end at least one first sequence oriented 5' to 3' for recognizing said transposase and at its 3' end said second sequence for recognizing said transposase, a second single-stranded nucleic acid molecule, wherein the B sequence is a complementary sequence of the nucleic acid of interest, the A sequence is located 5' of the region of interest of the nucleic acid of interest, and the B sequence is located 3' of the region of interest of the nucleic acid of interest; a third single-stranded molecule, ＊ in its 5' portion, at least one complementary sequence of the second sequence for recognizing the transposase of the first molecule; ＊ in its 3' portion, at least one complementary sequence of the first sequence for recognizing the transposase of the second molecule; ＊ a third single-chain molecule comprising: an intermediate region located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule, the intermediate region comprising a sequence encoding a fusion protein, the fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell, and, at its carboxy-terminal portion, an activation domain of a T lymphocyte, i.e., the CD3ζ chain of a T cell receptor or TCR; A composition, wherein the first and third single-stranded nucleic acid molecules are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase, and the second and third single-stranded nucleic acid molecules are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase.

2. The composition of claim 1, wherein the fusion protein comprises the peptide capable of binding to a membrane protein of a cell fused to the transmembrane portion of a membrane protein, the latter being fused to the CD3ζ chain of a TCR.

3. The composition of claim 1, wherein the fusion protein comprises the peptide capable of binding to a membrane protein of a cell fused to a transmembrane portion of a membrane protein, the transmembrane domain of the membrane protein being fused to a coactivation domain, the latter being fused to the CD3ζ chain of a TCR.

4. - said sequence A comprises the complement of the sequence of the first region of the locus of the gene encoding the TRAC protein, - said B sequence comprises the complement of the sequence of the second region of the locus of the gene encoding the TRAC protein, 4. The composition according to any one of claims 1 to 3, wherein the A sequence and the B sequence are two different sequences, and the first region and the second region of the TRAC locus are advantageously adjacent to the 5' region of the locus of the gene encoding the TRAC protein.

5. the first molecule comprises, at its 5' end, a first sequence oriented 5' to 3' for recognizing a transposase, and at its 3' end, a second sequence oriented 5' to 3' for recognizing the transposase; The composition of any one of claims 1 to 4, wherein the second molecule comprises, at its 5' end, a first complementary sequence of the first sequence for recognizing the transposase, followed by a second complementary sequence of the second sequence for recognizing the transposase.

6. the first molecule comprises, at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase, and at its 3' end, a second sequence for recognizing the transposase, followed by a first complementary sequence of the first sequence for recognizing the transposase; The composition of any one of claims 1 to 5, wherein the second molecule comprises, at its 5' end, a complementary sequence of the second sequence for recognizing the transposase.

7. The composition according to any one of claims 1 to 6, wherein the transposase is a bacterial transposase, in particular a transposase selected from Tn5, Tn9, Tn10, or Tc1 / mariner.

8. The composition of any one of claims 1 to 7, wherein the intermediate region of the third molecule comprises the sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 1134.

9. 9. The composition of any one of claims 1 to 8, wherein the first molecule, the second molecule, and the third molecule are selected from the triplets defined in Table 2.

10. ＊＊ a fourth single-stranded nucleic acid molecule comprising, or consisting essentially of, an A sequence that allows insertion of a complementary sequence of a nucleic acid of interest, or comprising the complementary sequence of a nucleic acid of interest, wherein the complementary sequence is bound at its 5' to a fifth T-rich sequence 40 to 60 nucleotides in length and at its 3' to a sixth T-rich sequence 40 to 60 nucleotides in length, the fifth T-rich sequence and the sixth T-rich sequence comprising a fifth domain and a sixth domain of 6 to 12 G / C-rich nucleotides, respectively, the sequence of the fifth domain being complementary to the sequence of the sixth domain, and the fifth domain and the sixth domain being positioned 15 to 52 nucleotides from the A sequence, and the fourth molecule comprising at its 5' end at least one first sequence oriented 5' to 3' for recognizing a transposase and at its 3' end a second sequence for recognizing the transposase; ＊＊ a fifth single-stranded nucleic acid molecule comprising, or consisting essentially of, a B sequence that allows insertion of a complementary sequence of a nucleic acid of interest, wherein the complementary B sequence is 5'-linked to a seventh T-rich sequence 40-60 nucleotides in length and 3'-linked to an eighth T-rich sequence 40-60 nucleotides in length, the seventh T-rich sequence and the eighth T-rich sequence comprising a seventh domain and an eighth domain of 6-12 G / C-rich nucleotides, respectively, the sequence of the seventh domain being complementary to the sequence of the eighth domain, and the seventh domain and the eighth domain being located 15-52 nucleotides from the B sequence; and the fifth molecule comprising at its 5'-end at least one first sequence oriented 5'-to-3' for recognizing the transposase and at its 3'-end the second sequence for recognizing the transposase; a fifth single-stranded nucleic acid molecule, wherein the B sequence is a complementary sequence of the nucleic acid of interest, the A sequence is located 5' of the region of interest of the nucleic acid of interest, and the B sequence is located 3' of the region of interest of the nucleic acid of interest; ＊＊ a sixth single-stranded molecule, ＊ in its 5' portion, at least one complementary sequence of the fifth sequence for recognizing the transposase of the first molecule; ＊ in its 3' portion, at least one complementary sequence of the fourth sequence for recognizing the transposase of the second molecule; ＊ a sixth single-stranded molecule comprising an intermediate region located between the complementary sequence of the fourth sequence for recognizing the transposase of the first molecule and the complementary sequence of the fifth sequence for recognizing the transposase of the second molecule, wherein the complementary sequence of the sequence encoding the fusion protein is contained in the third molecule; The composition of any one of claims 1 to 9, wherein the fourth single-stranded nucleic acid molecule and the sixth single-stranded nucleic acid molecule are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase, and the fifth single-stranded nucleic acid molecule and the sixth single-stranded nucleic acid molecule are paired according to base complementarity defined by Watson-Crick to define two double-stranded binding sites for the transposase.

11. - said sequence A comprises the complement of the sequence of the first region of the locus of the gene encoding the TRAC protein, - said B sequence comprises the complement of the sequence of the second region of the locus of the gene encoding the TRAC protein, the A sequence and the B sequence are two different sequences, and the first region and the second region are advantageously adjacent to the 5' region of the locus of the gene encoding the TRAC protein; - said A' sequence comprises the complement of the sequence of the third region of the locus of the gene encoding the TRAC protein, - said B' sequence comprises the complement of the sequence of the fourth region of the locus of the gene encoding the TRAC protein, the A' sequence and the B' sequence are two different sequences, and the third and fourth regions of the sequence encoding the TRAC protein are advantageously adjacent to complementary sequences of the 5' region of the locus of the gene encoding the TRAC protein, the A sequence and the A' sequence are at most partially complementary to each other; the A sequence and the B' sequence are at most partially complementary; the B sequence and the A' sequence are at most partially complementary to each other; The composition of claim 10, wherein the B sequence and the B' sequence are at most partially complementary.

12. 1. Isolated and purified immune cells, particularly T lymphocyte cells, comprising: An immune cell comprising the composition of any one of claims 1 to 11.

13. 1. A pharmaceutical composition comprising: a composition according to any one of claims 1 to 11, - immune cells according to claim 12, A pharmaceutical composition comprising the compound in combination with a pharmaceutically acceptable carrier.

14. A composition according to any one of claims 1 to 11 for use as a medicament.

15. A composition according to any one of claims 1 to 11 for use in the treatment of cancer.

16. 12. Use of the composition of any one of claims 1 to 11 for inserting into a T lymphocyte cell a sequence encoding a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, with the proviso that said use does not include a process for modifying the germline genetic identity of a human being and that said use is not a method for treating the human or animal body by surgery or therapy.

17. 1. A method for the in vitro insertion of a sequence encoding a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, comprising: - placing a composition according to any one of claims 1 to 11 in contact with a target nucleic acid comprising at least the 5' region of the locus of the gene encoding the TRAC protein, in order to obtain a displacement complex, The composition comprises: the A sequence of the first molecule comprises a complementary sequence to a first region of the locus of a gene encoding a TRAC protein; the B sequence of the second molecule comprises the complement of a second region of the locus of the gene encoding the TRAC protein; and Arranging the third molecule such that it comprises a fusion protein sequence comprising, at its amino-terminal portion, a peptide capable of binding to a cellular membrane protein and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the fusion protein sequence being located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule; - placing the displacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the composition to obtain a recombinant complex; - recombining said combined complex to obtain said sequence encoding said fusion protein inserted into said target nucleic acid between said first region and said second region of the locus of the gene encoding a TRAC protein.

18. A method for generating chimeric antigen receptor-T cells, i.e., CAR-T cells, comprising a sequence of a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the method comprising: - placing a composition according to any one of claims 1 to 11 in contact with purified T cells, the genomic DNA of said purified T cells comprising the locus of the gene encoding the TRAC protein; The composition or the assembly is the A sequence of the first molecule comprises a complementary sequence to a first region of the locus of a gene encoding a TRAC protein; the B sequence of the second molecule comprises the complement of a second region of the locus of the gene encoding the TRAC protein; and Arranging the third molecule so that it contains a fusion protein sequence comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the fusion protein sequence being located between the complementary sequence of the second sequence for recognizing the transposase of the first molecule and the complementary sequence of the first sequence for recognizing the transposase of the second molecule, to obtain a displacement complex; - placing the displacement complex in the presence of a transposase that recognizes the double-stranded binding site of the transposase contained in the collection to obtain a recombinant complex; - recombining the combination complex to obtain CAR-T cells, the genomic DNA of which comprises a sequence encoding said fusion protein in the locus of the gene encoding the TRAC protein; - purifying said CAR-T cells.

19. A modified T lymphocyte cell obtainable by the method of claim 19, wherein the modified T lymphocyte cell comprises an insertion in its genome of a sequence encoding a fusion protein comprising, at its amino-terminal portion, a peptide capable of binding to a membrane protein of a cell and, at its carboxy-terminal portion, the CD3ζ chain of a TCR, the insertion being located in the locus of a gene encoding a TRAC protein.

Images