Compositions for the treatment of genetic disorders and uses thereof

Abstract

The present invention provides a composition comprising: a first single-stranded nucleic acid molecule comprising a region complementary to a target and a transposase recognition site; a second single-stranded nucleic acid molecule comprising a region complementary to the target and a transposase recognition site; and a third single-stranded molecule comprising a sequence encoding a portion of the interleukin-2 gamma receptor (IL2-Rγ) or encoding the entire IL2-Rγ receptor, and a transposase binding site.

Description

[Technical Field]

[0001] The present invention relates to compositions and uses thereof for the treatment of genetic disorders.

[0002] X-linked severe combined immune deficiency (SCID-X) is caused by mutations in the IL2Rγ gene that induce immune system dysfunction, leading to severe infections, fever, and rashes.

[0003] The gamma subunit of the interleukin-2 receptor (IL2-Rγ), regulated by the IL2-Rγ gene, is common to many interleukin receptors (IL-2, IL-4, IL-7, IL-9, and IL-15) and is present on the surface of hematopoietic stem cells. This protein, along with other proteins, is essential for lymphocyte formation. Therefore, mutations in the IL2Rγ gene that block production of the IL2-Rγ receptor also block cell differentiation into lymphocytes, primarily T lymphocytes and natural killer (NK) lymphocytes. Similarly, the absence of T lymphocytes and NK lymphocytes in the body leads to the inactivation of formed B lymphocytes.

[0004] The IL2Rγ gene is located on the X chromosome. Men who have only one copy of this chromosome, and therefore the IL2Rγ gene, are automatically affected by the disease if the gene is mutated. On the other hand, women who have two copies of the gene are said to be "normal carriers" and only show symptoms of the disease in very rare cases.

[0005] Symptoms of SCID-X generally appear between the ages of 3 and 6 months. Most affected children suffer from failure to thrive, oral and genital rashes, and multiple persistent infections that, if left untreated, lead to death within one or two years.

[0006] The prevalence of SCID-X is very difficult to assess due to lack of diagnosis, but is estimated to be approximately 1 in 50,000 live births.

[0007] The gold standard treatment is immune reconstitution via bone marrow transplantation. The success rate of this approach is directly related to the level of match between the bone marrow donor and the sick child. When the match is high (usually bone marrow from the patient's brother or sister), complete remission is achieved in 20% of cases. However, in 80% of cases, patients develop various severe symptoms of GVHD (Graft Versus Host Disease), which means that immune cells in the transplanted bone marrow attack the patient.

[0008] An alternative treatment is gene therapy, based on the use of viruses (lentiviruses or AAVs), in which the IL2Rγ gene is reintroduced into a sample taken from the patient's hematopoietic stem cells and then reinjected to repopulate the immune system. This approach, which is still undergoing clinical trials, claims to be effective in 80% of cases, but requires several sessions of very expensive treatment and in 20% of cases leads to the development of leukemia due to the fact that this viral approach is not targeted and does not introduce a new IL2Rγ gene into its original locus.

[0009] US Patent Application Publication No. 20150152436(A1) describes a new therapeutic development aimed at adapting CRISPR / CAS9 genome editing approaches to the treatment of SCID-X. However, polymorphisms associated with this pathology, as well as the possible insertion of IL2Rγ cDNA by more than 600 base pairs, remain significant technical obstacles to clinical use.

[0010] Therefore, there remains a need to provide effective treatments.

[0011] One of the aims of the present invention is to overcome the drawbacks of the prior art.

[0012] One object of the present invention is to propose a composition that can allow replacement of the IL2Rγ gene in an efficient and complete manner.

[0013] A further object of the present invention is to provide a method or drug capable of treating pathologies associated with defects in said genes. Summary of the Invention

[0014] The present invention provides 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, and 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-stranded molecule comprising an intermediate region located between the complementary sequence of the second recognition sequence of the transposase of the first molecule and the complementary sequence of the first recognition sequence of the transposase of the second molecule, the intermediate region comprising a sequence encoding a portion of the interleukin-2 receptor gamma, i.e., IL2-Rγ, or the entire IL2-Rγ receptor; 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.

[0015] The present invention is based on the inventors' observation that the use of the above-described complex molecule encoding IL2-Rγ together with a bacterial transposase allows for targeted and efficient replacement of the IL2Rγ gene.

[0016] 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 consists essentially of, at its 5' end, at least one complementary sequence of the first recognition sequence of the transposase, and the third single-stranded nucleic acid molecule comprises, or consists essentially of, at its 3' end, at least one sequence complementary to the second recognition sequence of 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] It is therefore an intermediate product of the tools described below.

[0021] The molecular complex consists of three single-stranded nucleic acid molecules, which can be DNA molecules, RNA molecules, or mixed RNA and DNA molecules.

[0022] These three molecules are partially complementary to each other in pairs according to the base complementarity of nucleic acids defined by Watson-Crick, i.e., adenine pairs with thymidine or uracil, cytosine pairs with guanine, and vice versa.

[0023] 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.

[0024] Complex In addition to the detailed definition of each of the first, second, and third molecules, each of which is described below, a complex according to the present invention comprises two pairs of binding sites for one or more transposases: a first pair of binding sites for a first transposase resulting from pairing according to Watson-Crick nucleic acid base complementarity between the first single-stranded nucleic acid molecule and a third nucleic acid molecule; a second pair of binding sites for a second transposase as a result of pairing according to Watson-Crick defined nucleic acid base complementarity between the second single-stranded nucleic acid molecule and the third nucleic acid molecule.

[0025] The first transposase and the second transposase can be the same or different.

[0026] Each transposase binding site consists of two double-stranded patterns: the first double-stranded pattern consists of a first transposase binding half-site and its complementary sequence, following Watson-Crick base complementarity; and the second double-stranded pattern consists of a second transposase binding half-site and its complementary sequence, following Watson-Crick base complementarity.

[0027] In the present invention, the complementary sequence of a transposase binding half-site is referred to as the complementary half-site of that site.

[0028] Thus, the transposase binding site consists of 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.

[0029] Thus, there are 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.

[0030] For the first pair of transposase binding sites, 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) or the other (third) nucleic acid molecule, but rather it should be noted that when the first and third nucleic acid molecules are paired according to base complementarity as defined by Watson-Crick, two half-sites are reconstituted, i.e., the first half-site is paired with the half-site complementary to the first half-site, and the second half-site is paired with the half-site complementary to the second half-site.

[0031] For the second pair of transposase binding sites, 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 half-site complementary to the first half-site, and the second half-site pairs with the half-site complementary to the second half-site.

[0032] The first molecule.

[0033] 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.

[0034] 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:

[0035] [Table 1(1)]

[0036] [Table 1(2)]

[0037] [Table 1(3)]

[0038] [Table 1(4)]

[0039] [Table 1(5)]

[0040] 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.

[0041] 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.

[0042] 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.

[0043] To overcome this problem, a GC-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, G, or G+C bases exceeds 50% of the nucleotides contained in the G / C-rich sequence.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] A schematic diagram of the pairing morphology is shown in Figures 1A to 1E.

[0052] The second molecule.

[0053] The second molecule in the complex is structurally similar to the first molecule, and the above description applies mutatis mutandis, however, in the second molecule, the B region (or B sequence) and transposase binding site are configured differently.

[0054] 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.

[0055] Thus, the B region must correspond to a second sequence complementary to the target sequence, which second sequence complementary to the target sequence is located 3' to the first sequence recognized by the complementary sequence corresponding to the A sequence of the first molecule.

[0056] 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 is replaced by the sequence of the third molecule.

[0057] The third molecule The third molecule in the complex described above 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 site for binding to a transposase present in the 3' portion of the first molecule. Thus, when the complex is formed, the (single-stranded) transposase binding half-site located at 3' of the first molecule can pair with the (single-stranded) transposase binding half-site located at 5' of the second molecule to form a double-stranded transposase binding site, a double-stranded site to which a transposase can bind.

[0058] 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.

[0059] 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.

[0060] The replacement sequence corresponds to all or part of the gene encoding the IL2-Rγ protein, ie, all or part of the ILR2γ gene.

[0061] The common gamma chain (γc) (or CD132) is also known as the interleukin-2 receptor gamma subunit, i.e., IL-2RG, or IL2-Rγ. It is a cytokine receptor subunit common to the receptor complexes of at least six different interleukin receptors: interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-15 (IL-15), and interleukin-21 receptors. This chain is a glycoprotein belonging to the type I cytokine receptor family expressed on most lymphocyte populations. The ILR2γ gene is located on the mammalian X chromosome.

[0062] IL2-Rγ is expressed on the surface of immature blood cells in the bone marrow. One end of the protein is on the outside of the cell where it binds to cytokines, and the other end of the protein is on the inside of the cell where it transmits a signal to the cell nucleus. The common gamma chain associates with other proteins to direct hematopoietic cells to form lymphocytes. This receptor also directs the growth and maturation of lymphocyte subtypes: T lymphocytes, B lymphocytes, and natural killer (NK) cells.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Thus, the first and second molecules of interest are required to flank the sequence to be replaced, thereby specifically targeting the molecule of interest.

[0068] The third molecule in the collection then provides the replacement sequence, ie, a nucleic acid molecule containing a sequence corresponding to all or part of the gene encoding IL2-Rγ.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 sequence A of a first molecule of the assembly and whose 3' portion is complementary to sequence B 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 sequence of the molecule of interest flanked by the complementary sequences of sequences A and B is replaced at 5' and 3' by the sequence of the third molecule of the assembly located between the half-sites for binding to the transposase.

[0074] 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.

[0075] Even more advantageously, the first molecule, the second molecule, the third molecule, and the assembly of third molecules of the assembly are hybrid DNA / RNA molecules, in which the "backbone" of the molecules is DNA, and the recognition sequences A and B of the nucleic acid molecule of interest and the central region of the third molecule are RNA. This is particularly advantageous when the sequence substitutions that enable the present invention are made directly to RNA molecules.

[0076] [FIG. 2]-A shows the interaction between a molecule of interest and the assembly according to the present invention.

[0077] 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 (forming a phosphodiester bond between the 5' phosphate end of a nucleotide and the 3' OH 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).

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 dissociating the two strands of a double-stranded molecule. It is also possible to consider grafting with a biotinylated ligase (or one coupled to streptavidin) to connect the recombinant strand to the 3' side.

[0082] 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.

[0083] Advantageously, the present invention relates to a composition as described above, wherein said sequence A comprises a sequence complementary to the sequence of a first region of the gene encoding IL2-Rγ and said sequence B comprises a sequence complementary to the sequence of a second region of said gene encoding IL2-Rγ, said sequence A and said sequence B being two different sequences, and said first region and said second region of said gene encoding IL2-Rγ flank a region comprising a sequence encoding a portion of IL2-Rγ or encoding the complete IL2-Rγ receptor.

[0084] As explained above, 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 frame 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.

[0085] Thus, any target sequence can be replaced with any replacement sequence, so long as the A and B sequences of the first and second molecules, respectively, recognize and frame the target region.

[0086] It is particularly advantageous in the present invention to replace all or part of the gene encoding IL2-Rγ with the sequence of a third molecule, especially when the target sequence contains one or more mutations compared to the wild-type reference sequence, such as a substitution of one or more adjacent or spaced nucleotides, a deletion of one or more adjacent or spaced nucleotides, or an insertion of one or more adjacent or spaced nucleotides.

[0087] The entire gene encoding IL2-Rγ is taken to mean the entire sequence of the gene, including the 5′, 3′ regulatory elements, exons and introns.

[0088] In the context of the present invention, a "part of the gene encoding IL2-Rγ" is understood to mean a part of the gene that does not allow the complete coding of the IL2-Rγ protein. This may include, but is not limited to, an intron, an exon, several introns, several exons, etc., in which the entire translation unit is not complete. It may be particularly advantageous to replace only a part of the gene encoding IL2-Rγ, for example, to replace only one exon in which a mutation is present. In this case, it may be advantageous to select a sequence complementary to the A region of the first molecule in the intron preceding the sequence to be replaced and the complementary part of the B region in the intron following the replaced exon. Of course, depending on the desired replacement, a person skilled in the art can choose between the entire gene or a part of the gene and can therefore define the most relevant A and B regions.

[0089] 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 a composition as defined herein above, wherein 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 a second complementary sequence of the second sequence for recognizing the transposase of the first molecule.

[0090] Even more advantageously, the present invention provides a method for producing a nucleic acid sequence encoding a nucleic acid fragment, the first molecule 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 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 sequence complementary to the first sequence for recognizing the transposase of the second molecule, followed by a second sequence complementary to the second sequence for recognizing the transposase of the second molecule.

[0091] Possible formats for the first and second molecules of the present invention are defined herein. This configuration is shown in FIG. 1B. Of course, in this example of a configuration of the first, second, and third molecules, there is a replacement sequence, in this case all or part of the gene encoding IL2-Rγ, between the two transposase half-sites in the 5' and 3' portions of the third molecule.

[0092] Advantageously, the present invention provides a method for detecting 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 said transposase, followed by a first complementary sequence of said first sequence for recognizing said transposase of the first molecule; 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.

[0093] 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 oriented from 5' to 3' for recognizing a transposase, and at its 3' end, a second sequence for recognizing a transposase, the first sequence for recognizing the transposase at the 5' end being preceded by a second sequence complementary to the second sequence for recognizing the transposase of the first molecule; and, The present invention relates to the aforementioned composition, wherein a 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.

[0094] Another possible format for the first and second molecules of the present invention is defined herein. This configuration is shown in Figures 1C and 1D. Of course, in this example of a configuration of the first, second, and third molecules, there is a replacement sequence, in this case all or part of the gene encoding IL2-Rγ, between the two transposase half-sites in the 5' and 3' portions of the third molecule.

[0095] Advantageously, the present invention provides a method for detecting a transposase comprising the step of: at its 5' end, a first sequence oriented from 5' to 3' for recognizing a transposase; at its 3' end, a first sequence complementary to 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.

[0096] 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; (b) providing a first sequence at its 3' end, the first sequence being complementary to the first sequence for recognizing the transposase of the first molecule; and (c) providing a second sequence for recognizing the transposase; the second 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 of the first molecule, the first sequence being followed by a second sequence complementary to the second sequence for recognizing the transposase of the first molecule; The present invention relates to the aforementioned composition, wherein 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.

[0097] This configuration is illustrated in Figure 1E. Of course, in this example configuration of a first molecule, a second molecule, and a third molecule, there is a replacement sequence, in this case all or part of the gene encoding IL2-Rγ, between the two transposase half-sites in the 5' and 3' portions of the third molecule.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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).

[0104] 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.

[0105] 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).

[0106] 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.

[0107] Other recognition sequences for transposases 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) 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.

[0108] 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.

[0109] Advantageously, the first G / C rich domain of the first molecule (or of the second molecule) has the sequence

[0110] [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).

[0111] The first and second G / C rich domains also have the sequences

[0112] [Table 3] (SEQ ID NO: 195). The preceding description applies mutatis mutandis.

[0113] Other sequences of the G / C rich domain 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).

[0114] These examples are given for illustrative purposes only and cannot limit the scope of the invention.

[0115] 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.

[0116] Even more preferably, the A / T rich sequence of the first molecule of the complex consists of Ts.

[0117] Advantageously, the invention relates to a composition as described above, in which the gene encoding IL2-Rγ comprises the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2 or the sequence SEQ ID NO: 3.

[0118] The sequence of SEQ ID NO: 1 corresponds to the complete human IL2Rγ gene, referenced under GenBank number: AY692262.1. This gene is also known by the following names: P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, and SCIDX1. It corresponds to 7130 bases.

[0119] The exons of this gene correspond to the following delimited sequences: exon 1: 1956 (first nucleotide of the ATG start codon) to 2070, and intron 1: 2071 to 2448; Exon 2: 2449–2602, and intron 2: 2603–2810; Exon 3: 2811–2995, and intron 3: 2996–3203; exon 4:3204–3343, and intron 4:3344–4108; Exon 5:4109–4271, and intron 5:4272–4803; exon 6:4804–4900, and intron 6:4901–5152; Exon 7:5153-5222 and intron 7:5223-5577, and Exon 8: 5578–5763 (last nucleotide of the stop codon).

[0120] The sequence SEQ ID NO:2 corresponds to a fragment of SEQ ID NO:1, since it starts at position 1956 of the sequence SEQ ID NO:1 and ends at the stop codon at position 5763 of SEQ ID NO:1.

[0121] The sequence of SEQ ID NO: 3 corresponds to the coding region (or CDS) of the gene, which corresponds to the combination of exons described above. This sequence is referenced under GenBank number: AK314932.1.

[0122] Considering the above-mentioned breakdown, those skilled in the art can select the part of the gene defined above to achieve partial gene replacement.For example, but not limited to, if it is desired to replace exon 1, it is possible to target intron 1 with the first molecule, target intron 2 with the second molecule, while the third molecule contains exon 2 sequence.

[0123] Even more advantageously, the present invention relates to a composition as defined above, wherein the first molecule, the second molecule and the third molecule are selected from the triplets defined in Table 2 below.

[0124] 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.

[0125] [Table 4(1)]

[0126] [Table 4(2)]

[0127] [Table 4(3)]

[0128] [Table 4(4)]

[0129] [Table 4(5)]

[0130] [Table 4(6)]

[0131] 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.

[0132] 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 at its 5' end and a second sequence for recognizing the transposase at its 3' end; **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 third domain and a fourth 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 positioned 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-stranded molecule comprising an intermediate region located between the complement of the fourth recognition sequence of the transposase of the first molecule and the complement of the fifth recognition sequence of the transposase of the second molecule, the intermediate region comprising a sequence that is antiparallel to and complementary to a sequence encoding interleukin-2 receptor gamma, i.e., IL2-Rγ, contained in a third molecule of the first composition; The present invention relates to the composition described above, 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.

[0133] 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 regions A, B, A', and B' are correctly selected so as to correctly frame the region of interest to be replaced.

[0134] Of course, all provisions provided for three-molecule compositions apply mutatis mutandis to six-molecule compositions.

[0135] Advantageously, the present invention provides - said sequence A comprises a sequence complementary to the sequence of a first region of the gene encoding IL2-Rγ, - said B sequence comprises a sequence complementary to the sequence of a second region of the gene encoding IL2-Rγ, the sequence A and the sequence B are two different sequences, and the first and second regions of a gene encoding IL2-Rγ frame a region containing a sequence encoding a portion of IL2-Rγ or the entire IL2-Rγ receptor; - said A' sequence comprises a sequence complementary to the sequence of the third region of the gene encoding IL2-Rγ, - the B' sequence comprises a sequence complementary to the sequence of the fourth region of the gene encoding IL2-Rγ, the A' sequence and the B' sequence are two different sequences, and the third and fourth regions of a gene encoding IL2-Rγ frame a region containing a sequence encoding a portion of IL2-Rγ or the entire IL2-Rγ receptor; 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 composition as described above, wherein the B and B' sequences are at most partially complementary.

[0136] "At most partially complementary" means in the present invention 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 sequence, in particular less than 30% of the sequence, particularly less than 10% of the sequence, and in particular are not complementary to each other at all.

[0137] In this way, it is possible to frame the gene encoding the IL2-Rγ receptor in a specific, oriented manner for both chains to avoid uncontrolled or inconsistent recombination (i.e., tagmentation) that would result in unpredictable outcomes.

[0138] Even more advantageously, the above-mentioned composition comprises at least one of the sextuplets set out in Table 3 below.

[0139] [Table 5(1)]

[0140] [Table 5(2)]

[0141] [Table 5(3)]

[0142] Thus, the composition according to the invention advantageously provides 156 sextuplets of molecules that allow potentially mutated or aberrant sequences at the locus of the IL2R gene gamma to be replaced by a reference wild-type sequence.

[0143] This makes it possible to restore functions lost due to mutations and achieve functional complementation depending on the target cell.

[0144] In another aspect, the present invention relates to a pharmaceutical composition comprising the above-described composition in combination with a pharmaceutically acceptable vehicle.

[0145] The above-mentioned composition can be used for the treatment of pathologies in combination with a pharmaceutically acceptable vehicle. The vehicle is a commonly accepted vehicle known to those skilled in the art, such as distilled water or a physiological buffer solution. This vehicle must allow the formation of the above-mentioned complex, but at the same time, must be acceptable to cells or organisms.

[0146] In another aspect, the present invention relates to a composition as described above for use as a medicament.

[0147] Furthermore, compositions comprising any of the triplets of Table 2 or any of the sextets of Table 3 may advantageously be used as medicines.

[0148] In yet another aspect, the present invention relates to a composition for use in the treatment of pathologies associated with mutations in the gene encoding IL2-Rγ.

[0149] Mutations in the IL2Rγ gene have been widely described in the literature and can correspond to substitutions, insertions or deletions affecting either the exons, introns or the 5′ or 3′ regulatory regions of the gene.

[0150] More specifically, the present invention relates to a composition for use in the treatment of a pathology associated with a mutation in the gene encoding IL2-Rγ, said composition comprising a triplet as set forth in Table 2 or a sextet as set forth in Table 3.

[0151] Advantageously, the invention relates to a composition for the aforementioned use, wherein the disease associated with a mutation in the gene encoding IL2-Rγ is SCID-X disease or Omenn's syndrome.

[0152] Severe combined immunodeficiency due to gamma chain deficiency (SCID) T-B+, also known as SCIDX1 or SCID-X, is a form of SCID characterized by severe recurrent infections associated with diarrhea and growth failure.

[0153] During the first month of life, SCIDX1 manifests as severe, often fatal, viral, bacterial, or fungal infections (e.g., Pneumocystis jirovecii pneumonia, disseminated BCG infection secondary to vaccination) and failure to thrive. Chronic diarrhea is a frequent feature. Some patients experience skin rash and abnormal liver function. Graft-versus-host disease, related to maternal-fetal transmission, is also associated with the disease. Immunological findings show lymphopenia with absence of T or NK lymphocytes, hypogammaglobulinemia, and normal or elevated B lymphocyte counts.

[0154] Omenn syndrome is an inflammatory disorder associated with severe combined immunodeficiency characterized by erythroderma, desquamation, alopecia, chronic diarrhea, growth retardation, adenopathy, and hepatosplenomegaly. Omenn syndrome manifests in the first year of life and is accompanied by signs characteristic of severe combined immunodeficiency, including chronic diarrhea, pneumonia, and growth retardation. Patients also present with inflammatory symptoms such as adenopathy, hepatosplenomegaly, and generalized erythroderma, which often result in alopecia and loss of eyebrows and eyelashes. Protein loss can cause generalized edema and metabolic disorders. Signs and symptoms may evolve over time and may manifest separately. Some patients exhibit only certain symptoms and may be considered cases of atypical Omenn syndrome. Rather than a distinct form of severe combined immunodeficiency, this is an inflammatory phenotype that may be associated with different types of severe combined immunodeficiency. Most cases reported to date are characterized by hypomorphic mutations in the RAG1 and RAG2 (11p13) genes. Other mutations occur in the RMRP, ADA, and IL2Rγ genes, as well as other genes.

[0155] In another aspect, the present invention relates to a method of treating SCID-X or Omenn's syndrome, the method comprising administering to an individual in need thereof an effective amount of the aforementioned composition, wherein the composition specifically comprises a triplet defined in Table 2 or a sextet defined in Table 3.

[0156] In yet another aspect, the present invention relates to the use of a composition as defined above for the replacement in somatic cells of a mutated sequence of a gene encoding IL2-Rγ with the wild-type sequence of a gene encoding wild-type IL2-Rγ, with the proviso that the use does not include a method 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.

[0157] As previously described herein, the aforementioned compositions utilize the transposase tagmentation properties to specifically target a target region and replace it with a sequence of interest.

[0158] The compositions according to the invention are particularly advantageous for performing targeted gene modifications (and in particular gene replacements, or replacement of non-coding sequences) in humans, in particular for replacing a mutated IL2Rγ gene with its wild-type reference sequence.

[0159] In yet another advantageous embodiment, the present invention relates to a method for replacing a mutated sequence of a gene encoding IL2-Rγ with a wild-type sequence encoding IL2-Rγ, in particular an in vitro method, which method comprises the steps of: contacting a composition as defined above with a nucleic acid comprising a mutated sequence encoding the IL2-Rγ gene to obtain a replacement complex, The composition comprises: the A sequence of the first molecule comprises a complementary sequence to the region immediately 5' to the mutant sequence encoding the IL2-Rγ gene; the B sequence of the second molecule comprises the complement of the region immediately 3' to the mutant sequence encoding the IL2-Rγ gene; and contacting the third molecule such that the third molecule comprises a wild-type sequence of a gene encoding IL2-Rγ located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a 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 collection to obtain a recombination complex; - recombining the combined complex to obtain a hybrid nucleic acid molecule containing the wild-type sequence of the gene encoding IL2-Rγ instead of the mutated sequence of the gene encoding IL2-Rγ.

[0160] Advantageously, the present invention relates to a method for replacing a mutated sequence of a gene encoding IL2-Rγ with a wild-type sequence encoding IL2-Rγ, in particular an in vitro method, comprising the steps of: contacting a composition comprising a triplet as defined in Table 2 or a sextet as defined in Table 3 with a nucleic acid comprising a mutated sequence encoding the IL2-Rγ gene 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 recombination complex; - recombining the combined complex to obtain a hybrid nucleic acid molecule containing the wild-type sequence of the gene encoding IL2-Rγ instead of the mutated sequence of the gene encoding IL2-Rγ.

[0161] In yet another aspect, the present invention provides a method for the in vitro replacement of a mutated double-stranded sequence of a gene encoding IL2-Rγ in stem cells, in particular hematopoietic stem cells, with a wild-type double-stranded sequence encoding IL2-Rγ, said method comprising: contacting a composition as defined above with hematopoietic stem cells comprising a mutated sequence encoding the IL2-Rγ gene to obtain a replacement complex, The assembly is the A sequence of the first molecule comprises a complementary sequence to the region immediately 5' to the mutant sequence encoding the IL2-Rγ gene; the A' sequence of the fourth molecule comprises a complementary sequence to the region immediately 5' to the mutated complementary sequence encoding the IL2-Rγ gene; the B sequence of the second molecule comprises a complementary sequence to the region immediately 3' to the mutant sequence encoding the IL2-Rγ gene; the B' sequence of the second molecule comprises the complement of the region immediately 3' to the mutated complement encoding the IL2-Rγ gene; and a third molecule comprising a wild-type sequence of a gene encoding IL2-Rγ, located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a complementary sequence of the first sequence for recognizing the transposase of the second molecule; contacting the sixth molecule such that the sixth molecule comprises a sequence complementary to a wild-type sequence of a gene encoding IL2-Rγ, the sequence being located between the complement of the second sequence for recognizing the transposase of the first molecule and the complement 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 hybrid nucleic acid molecule containing the wild-type sequence of the gene encoding IL2-Rγ instead of the mutated sequence of the gene encoding IL2-Rγ; - possibly purifying hematopoietic stem cells carrying a mutant gene replacement.

[0162] Advantageously, the present invention relates to a method for the in vitro replacement of a mutated double-stranded sequence of a gene encoding IL2-Rγ in stem cells, in particular hematopoietic stem cells, by a wild-type double-stranded sequence encoding IL2-Rγ, said method comprising the steps of: - contacting a composition as defined above with hematopoietic stem cells comprising a mutated sequence encoding the IL2-Rγ gene to obtain a replacement complex, said composition comprising a triplet as defined in Table 2 or a sextet as 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 hybrid nucleic acid molecule containing the wild-type sequence of the gene encoding IL2-Rγ instead of the mutated sequence of the gene encoding IL2-Rγ; - possibly purifying hematopoietic stem cells carrying a mutant gene replacement.

[0163] In yet another aspect, the present invention provides a method for inserting, particularly in vitro, into a cell, particularly a stem cell, particularly a hematopoietic stem cell, a double-stranded sequence of a wild-type gene encoding IL2-Rγ at the locus of the gene encoding IL2-Rγ, wherein said locus of the gene encoding IL2-Rγ contains a mutation that affects the expression, function, or both of IL2-Rγ, said double-stranded sequence of the wild-type gene encoding IL2-Rγ corresponds to the cDNA of said gene, in particular to SEQ ID NO: 3; The method comprises: - contacting a composition as defined in any one of claims 1 to 9 with a cell containing a mutation in the locus of the gene encoding IL2-Rγ that affects the expression, function, or both of IL2-Rγ to obtain a replacement complex, The composition comprises: the sequence A of the first molecule comprises a complementary sequence to the 5' region of exon 1 of the gene encoding IL2-Rγ; the A' sequence of the fourth molecule comprises a complementary sequence to a complementary sequence of a region 5' to exon 1 of the gene encoding IL2-Rγ; the B sequence of the second molecule comprises a sequence complementary to the 3' side of the region 5' side of exon 1 of the gene encoding IL2-Rγ; the B' sequence of the fifth molecule comprises a sequence 3' complementary to a sequence complementary to a region 5' of exon 1 of the gene encoding IL2-Rγ; and a third molecule comprising a wild-type cDNA sequence of a gene encoding IL2-Rγ, located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a complementary sequence of the first sequence for recognizing the transposase of the second molecule; contacting a sixth molecule such that the sixth molecule comprises a wild-type cDNA sequence of a gene encoding IL2-Rγ located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a 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 collection to obtain a recombination complex; - recombining the combined complex to obtain an insertion of the cDNA of the gene encoding IL2-Rγ 5' to exon 1 of the gene encoding IL2-Rγ; - optionally purifying the inserted cells. [Brief explanation of the drawings]

[0164] The invention will be better understood on reading the following examples and figures. [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 2] FIG. 1 shows the principles and steps involved in single-strand displacement using a first molecule, a second molecule, and a third molecule of a composition according to the invention. [Figure 3] FIG. 1 shows the principles and steps involved in double-stranded displacement using the first, second, third, fourth, fifth, and sixth molecules of the composition according to the present invention. [Figure 4] 1 shows a combination of molecules contained in tube 1 in Example 1. The black spheres represent biotin. [Figure 5] 1 shows the combination of molecules contained in tube 2 in Example 1. The black spheres represent biotin. [Example]

[0165] Example 1 - Obtaining B cells expressing the wild-type ILR2G gene To treat patients suffering from SCID-X associated with a mutation in the ILR2G gene, it may be advantageous to propose a cell therapy aimed at transplanting CD34+ hematopoietic stem cells modified using the composition according to the invention into the patient, in particular replacing the mutated ILR2G gene with the wild-type sequence of said gene.

[0166] A- Purification of CD34+ hematopoietic stem cells (HSCs) from blood Purification of CD34+ cells is well known to those skilled in the art and can be summarized as follows: 1- Collect a donor blood sample (adult blood or umbilical cord blood enriched with CD34+ cells) fresh or frozen (it is also possible to purify CD34+ cells from patients with DISC-X), 2- The sample is centrifuged on a sterile density gradient centrifugation medium (e.g., Ficoll in the presence of EDTA) at 1000 g for 8 minutes without braking to isolate peripheral blood mononuclear cells (PBMCs); 3- Collect the white blood cell ring and wash with PBS (phosphate buffered saline); 4 - Hematopoietic stem cells (i.e., CD34+ cells) are then isolated in a cell sorter by positive selection using anti-CD34 antibody labeling or by negative selection using a cocktail of anti-CD2, anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD24, anti-CD56n, anti-CD66b, and anti-CD61 antibodies; 5- Purified CD34+ cells are centrifuged at 300g for 5 minutes, and the cell pellet is suspended at a cell density of 100,000-300,000 cells / mL. Culture the cells in a 24-well plate at 37°C and 5% CO2 for 48 hours at 1 mL / well in a culture medium specifically designed for stem cell culture and supplemented with a cocktail containing human IL-3, human IL-6, recombinant thrombopoietin, recombinant hCSF (ligand kit), and recombinant Flt-3 ligand at 100 ng / mL each.

[0167] 6-The CD34+ stem cells are then ready to be transfected with the composition according to the invention.

[0168] B- Preparation of the molecules of the composition according to the invention A construct containing GFP was prepared to target the IL2R gene γ and verify insertion at the IL2R locus γchrX:70,327,254-70,331,958 GRCh37 / hg19.

[0169] 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 TTTTTTTGCTAGAAAGAGTACTGTTCTGGAAACTGACTTTTTTTTTTTTTTGCGATCGCCTTTTTTTTTTTGATACATTTAgaTgTGtatAAGAGaCAGGATGAT-3' - oligo Great X3 (10 μM) corresponding to molecule 4 of sequence SEQ ID NO: 201 below 5'-CACGTGCTGtCTCTTataCAcAtcTTTTTCTCGATCATTATTATTTTTTGGCGATCGCTTTTTTTTTTTTTTAGCAAATTGGTAATACTCCTGCCTCCACAGTTTTTTT TTTTTTTTTGCGATCGCCTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGCACGTGTTTTTTTTTTTTTTTTTTCAGCTATACTGtCTCTTataCAcAtcT-3' GREAT X1 Rev reverse oligo (10 μM) corresponding to the 5′ portion of molecule 3 having the sequence SEQ ID NO: 1134 below 5'-TACACTACCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTggttacATGCATCGTACA-3' GREAT X3 Rev reverse oligo (10 μM) corresponding to the 3′ part of molecule 6 having the sequence SEQ ID NO: 1135 below 5'-TGTACGATGCATgtaaccTTTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATAGCTG-3' - a reverse oligo Helix Rev (oligo having the sequence SEQ ID NO: 1136 below) corresponding to an additional strand for binding to the helicase via its biotinylated 3' end. 5'-ATAATAATGATCGAGAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[Biot]-3' ** Tube 2 (10 μL): - oligo Great X2 (10 μM) corresponding to molecule 2 of sequence SEQ ID NO: 199 below 5'-CACGTGCTGtCTCTTataCAcAtcTTTTTCTCGATCATTATTATTTTTTGGCGATCGCTTTTTTTTTTTTTTGTTGAGAATGGTGCTAGTGGTAGTGAACAGTTTTTTT TTTTTTTTTGCGATCGCCTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGCACGTGTTTTTTTTTTTTTTTTTTGACGAATACTGtCTCTTataCAcAtcT-3' - oligo Great X4 (10 μM) corresponding to molecule 5 of sequence SEQ ID NO: 202: 5'-AgaTgTGtatAAGAGaCAGAGTATGAATTTTTTTTTTTTTTTATCATCCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTTTTTTGGCGATCGCTTTTTTTTT TTTTTTTCCTTCTCCTCTAAATCATTACCTTCTATAATTTTTTTTTTTTTTTTGCGATCGCCTTTTTTTTTTTGATACATTTAgaTgTGtatAAGAGaCAGGATGAT-3' GREAT X2 Rev reverse oligo (10 μM) corresponding to the 3′ part of molecule 3 having the sequence SEQ ID NO: 1137 below 5'-CGATACgcggccgcatgttcTTTTTTTTTTTTTTTTTTTTTAgaTgTGtatAAGAGaCAGTATTCGTC-3' GREAT X4 Rev reverse oligo (10 μM) corresponding to the 5′ part of molecule 6 having the sequence SEQ ID NO: 1138 below 5'-TTCATACTCTGtCTCTTataCAcAtcTTTTTTTTTTTTTTTTgaacatgcggccgcGTATCG-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: 1136 below) 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 (NsiI for tube 1 and NotI for tube 2) and purified by PCR purification kit (20 μL elution).

[0170] Figures 4 and 5 show schematic representations of the contents of tube 1 and tube 2, respectively, prior to enzymatic digestion.

[0171] In parallel, amplified GFP-ILR2γ sequences were obtained by lysis of HEK293T cells and genomic DNA extraction, followed by amplification of the locus of interest by hyperprocessive Taq PCR. This sequence was amplified using oligonucleotides containing a 5' NsiI and a 3' NotI restriction site. The PCR product was digested with these two enzymes and purified with a PCR purification kit, eluting in a volume of 20 μL. The GFP-ILR2γ fusion was then flanked in tube 3 (i.e., free NsiI and NotI half sites).

[0172] The contents of these three tubes (Tube 1, Tube 2, and Tube 3) are then mixed, and T4 phage ligase is added to the mixture (4 μL, 100 U) 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 (30 μL elution) to isolate the largest fragment, GFP-ILR2γ, which is over 1 kilobase in size.

[0173] 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 IL2Rγ fusion (tube 4).

[0174] 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 modified single-stranded oligonucleotide complementary to the first molecule were added. The biotinylated oligonucleotide is optional.

[0175] C—Transfection of CD34+ cells with a composition according to the invention. The contents of tube 4, containing either the biotinylated or non-biotinylated oligos, are then used to transfect isolated CD34+ cells by electroporation.

[0176] 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® Transfer System transfection system.

[0177] The mixture is electroporated at 1650V for three 10 ms sequences.

[0178] The cells are then immediately centrifuged at 300 g for 5 min and suspended in supplemented stem cell culture medium at 37 °C and 5% CO for 48-96 h.

[0179] Tagmentation (i.e., gene replacement) is then possible. The cells are maintained in a suitable medium.

[0180] D - Verification of transfection Cells transfected with the molecule of interest inserted into the gene locus are selected using an antibiotic (eg, neomycin, bleomycin, blasticidin S, etc.) that contains a resistance gene in the replacement sequence.

[0181] Genomic DNA was extracted from transfected resistant cells, fragmented by sonication, and then sequencing adapters were added. PCR amplification of all genomic DNA fragments was performed. The library was sequenced using a sequencer (HiSeq 2500 paired-end, 50 x 8 x 50 reads), and the results were compiled by mapping and alignment via the bwa-mem module (hg19 bank). On-target / off-target analysis was performed by specific selection and clustering of reads by comparison with the reference genome (hg19) using the Bwa-mem module in short read mode and mapping with the minimap2 module. Peak insertion data were collected using the macs2 module.

[0182] A simple PCR can also be performed using a sense oligonucleotide 5' from the insert and an antisense oligonucleotide 3' 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 the fusion insert.

[0183] E- Functional analysis of CD34+ cells modified by the composition according to the invention Differentiation of the modified CD34+ cells will be monitored in vitro using OP9-DL1 or DL4 mouse stromal cell co-culture models as described in the literature.

[0184] Briefly, OP9-DL1 or DL4 cells are cultured with modified CD34 cells in MEM (Minimum Essential Medium) supplemented with 10% SVF (fetal bovine serum), a cytokine cocktail required for CD34 cell culture (described above), and a cytokine cocktail promoting activation of myeloid erythroid and lymphoid differentiation pathways at 37°C, 5% CO2, and 10% O2 for 14 days.

[0185] After 2 weeks of culture, the differentiated CD34+ cells are collected and analyzed by flow cytometry to observe their hematopoietic differentiation profile using anti-CD19 (B lymphoid lineage) antibodies, anti-CD3 (T lymphoid lineage), anti-CD56 (NK lineage), anti-CD16 (monocyte / macrophage lineage), anti-CD14 (macrophage / neutrophil lineage), anti-CD11c (myeloid lineage), and anti-CD235a (erythroid lineage).

[0186] The differentiation of modified CD34+ cells will also be monitored in vivo using NSG or NOD SCID mouse models (immunodeficient mice).

[0187] The modified CD34+ cells are injected IH (intrahepatic) or IF (intrafemoral) into 3-4 day-old or 6-8 week-old mice.

[0188] Differentiation is monitored in the bone marrow, spleen, and blood of mice at 8, 12, and 16 weeks after injection. Human cells are analyzed by flow cytometry using anti-CD45 and anti-HLA ABC antibodies, and their cellular profile is analyzed using the same panel of antibodies used in vitro.

[0189] For each cell type, GFP expression can be detected by flow cytometry.

[0190] In the case of differentiated cells, reverse transcription can also be performed on the isolated cells to obtain a cDNA library. The GFP and ILR2γ sequences are then amplified by PCR and the PCR oligos for IL2Rγ sequencing: ACTGGCATATGCTTGTACAGCTCGTCCAT (SEQ ID NO: 1139) and GFP Rev: CACGAACTCCAGCAGGACCATG (SEQ ID NO: 1140). The amplified fragments are sequenced by the Sanger method and run on an agarose gel. As expected, after analysis (NCBI blast), we find a very high similarity to the theoretical substitution of the GFP-IL2Rγ fragment at the IL2Rγ locus.

[0191] These results demonstrate that the technique according to the present invention allows the endogenous IL2Rγ gene to be efficiently replaced by an exogenous sequence.

[0192] Of course, the above examples are intended to demonstrate the effectiveness of the technology and use genes such as GFP to facilitate insertion detection.

[0193] Cell therapy does not use the gene encoding GFP, but rather uses receptor sequencing or functional detection to verify efficacy.

[0194] Example 2 - Obtaining B cells expressing the wild-type ILR2G gene from pluripotent stem cells. Because it can be difficult to obtain CD34+ stem cells from SCID-X patients, induced pluripotent stem cell (iPSC) technology can be used.

[0195] iPSC cells with an immunological profile that matches the patient can be obtained from a cell bank.

[0196] 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 those skilled in the art can adapt the protocols described in the prior art according to the cell type they wish to involve in the dedifferentiation model.

[0197] At this stage, the iPSC cells can be transfected with Lipofectamine in the presence of tube 4 as described in Example 1.

[0198] Transfected cells are selected using the appropriate antibiotic and the insertion is verified as described in Example 1.

[0199] iPSC cells are grown on plates coated with cell 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.

[0200] 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).

[0201] After 7 days, cells are continued in culture for an additional 3-7 days, after which experiments are continued on harvested non-adherent cells, which are tested for expression of the CD34+ marker by flow cytometry using an anti-CD34 antibody.

[0202] If the cells have not been transfected at the iPSc stage, they can be transfected at the CD34+ stage as described in Example 1. Their differentiation is then induced as described in Example 1.

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 the 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-stranded molecule comprising an intermediate region located between the complementary sequence of the second recognition sequence of the transposase of the first molecule and the complementary sequence of the first recognition sequence of the transposase of the second molecule, the intermediate region comprising a sequence encoding a portion of interleukin-2 receptor gamma, i.e., IL2-Rγ, or the complete IL2-Rγ receptor; 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. 2. The composition of claim 1, wherein sequence A comprises a sequence complementary to a sequence of a first region of a gene encoding IL2-Rγ, sequence B comprises a sequence complementary to a sequence of a second region of a gene encoding IL2-Rγ, sequence A and sequence B are two different sequences, and the first region and second region of the gene encoding IL2-Rγ flank a region comprising a sequence encoding a portion of IL2-Rγ or encoding the entire IL2-Rγ receptor.

3. 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 claim 1 or 2, wherein 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 a second complementary sequence of the second sequence for recognizing the transposase of the first molecule.

4. 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 3, wherein the third molecule comprises, at its 5' end, a complementary sequence of the second sequence for recognizing the transposase.

5. The composition according to any one of claims 1 to 4, wherein the transposase is a bacterial transposase, in particular a transposase selected from Tn5, Tn9, Tn10, or Tc1 / mariner.

6. The composition of any one of claims 1 to 5, wherein the gene encoding IL2-Rγ comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

7. 7. The composition of any one of claims 1 to 6, wherein the first molecule, the second molecule, and the third molecule are selected from the triplets defined in Table 2.

8. ＊＊ 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 third domain and a fourth 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 positioned 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 the complement 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 recognition sequence of the transposase of the first molecule and the complementary sequence of the fifth recognition sequence of the transposase of the second molecule, the intermediate region comprising a sequence antiparallel to and complementary to a sequence encoding interleukin-2 receptor gamma, i.e., IL2-Rγ, contained in the third molecule of the first composition; The composition of any one of claims 1 to 7, 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.

9. - said sequence A comprises a sequence complementary to the sequence of a first region of the gene encoding IL2-Rγ, - said B sequence comprises a sequence complementary to the sequence of a second region of the gene encoding IL2-Rγ, the A sequence and the B sequence are two different sequences, and the first and second regions of a gene encoding IL2-Rγ frame a region containing a sequence encoding a portion of IL2-Rγ or the complete IL2-Rγ receptor; - said sequence A' comprises a sequence complementary to the sequence of the third region of the gene encoding IL2-Rγ, - said B' sequence comprises a sequence complementary to the sequence of the fourth region of the gene encoding IL2-Rγ, the A' sequence and the B' sequence are two different sequences, and the third and fourth regions of a gene encoding IL2-Rγ frame a region containing a sequence encoding a portion of IL2-Rγ or the complete IL2-Rγ receptor; 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; 9. The composition of claim 8, wherein the B sequence and the B' sequence are at most partially complementary.

10. A pharmaceutical composition comprising the composition of any one of claims 1 to 9 in combination with a pharmaceutically acceptable carrier.

11. A composition according to any one of claims 1 to 9 for use as a medicament.

12. A composition according to any one of claims 1 to 9 for use in the treatment of a disease associated with a mutation in the gene encoding IL2-Rγ.

13. The composition for use according to claim 12, wherein the disease associated with a mutation in the gene encoding IL2-Rγ is SCID-X disease or Omenn's syndrome.

14. 10. Use of the composition of any one of claims 1 to 9 for the replacement of a mutated sequence of a gene encoding IL2-Rγ with the wild-type sequence of a gene encoding wild-type IL2-Rγ in somatic cells, with the proviso that said use does not include a method for modifying the germline genetic identity of a human and that said use is not a method for treating the human or animal body by surgery or therapy.

15. 1. An in vitro method for replacing a mutated sequence of a gene encoding IL2-Rγ with a wild-type sequence encoding IL2-Rγ, said method comprising: - contacting a composition as defined in any one of claims 1 to 9 with a nucleic acid encoding the IL2-Rγ gene and comprising said mutated sequence to obtain a substitution complex, The composition comprises: the A sequence of the first molecule comprises a complementary sequence to a region immediately 5' to the mutant sequence encoding the IL2-Rγ gene; the B sequence of the second molecule comprises the complement of the region immediately 3' to the mutant sequence encoding the IL2-Rγ gene; and contacting the third molecule such that the third molecule comprises the wild-type sequence of the gene encoding IL2-Rγ located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a 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 collection to obtain a recombination complex; - recombining said combined complex to obtain a hybrid nucleic acid molecule containing said wild-type sequence of the gene encoding IL2-Rγ in place of said mutated sequence of the gene encoding IL2-Rγ.

16. 1. A method for the in vitro replacement of a mutated double-stranded sequence of a gene encoding IL2-Rγ in stem cells, in particular hematopoietic stem cells, with a wild-type double-stranded sequence encoding IL2-Rγ, said method comprising: - contacting a composition as defined in any one of claims 1 to 9 with said hematopoietic stem cells comprising said mutated sequence encoding the IL2-Rγ gene to obtain a replacement complex, The assembly is the A sequence of the first molecule comprises a complementary sequence to a region immediately 5' to the mutant sequence encoding the IL2-Rγ gene; the A' sequence of the fourth molecule comprises a complementary sequence of a region immediately 5' to the mutated complementary sequence encoding the IL2-Rγ gene; the B sequence of the second molecule comprises a complementary sequence to the region immediately 3' of the mutant sequence encoding the IL2-R gene gamma; the B' sequence of the second molecule comprises the complement of the region immediately 3' to the mutated complement sequence encoding the IL2-Rγ gene; and the third molecule comprises the wild-type sequence of the gene encoding IL2-Rγ, located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a complementary sequence of the first sequence for recognizing the transposase of the second molecule; contacting the sixth molecule such that the sixth molecule comprises the wild-type sequence of the gene encoding IL2-Rγ located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a 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 collection to obtain a recombination complex; - recombining said combined complex to obtain a hybrid nucleic acid molecule of said wild-type sequence of the gene encoding IL2-Rγ in place of said mutated sequence of the gene encoding IL2-Rγ; - possibly purifying hematopoietic stem cells carrying the mutant gene replacement.

17. 1. A method for inserting, in particular in vitro, into cells, in particular stem cells, in particular hematopoietic stem cells, a double-stranded sequence of a wild-type gene encoding IL2-Rγ into the locus of the gene encoding IL2-Rγ, wherein said locus of the gene encoding IL2-Rγ contains a mutation that affects the expression, function, or both of IL2-Rγ, the double-stranded sequence of the wild-type gene encoding IL2-Rγ corresponds to the cDNA of the gene; The method comprises: - contacting a composition as defined above with said cells containing a mutation in the locus of the gene encoding IL2-Rγ that affects the expression, function, or both, of IL2-Rγ, in order to obtain a replacement complex, The composition comprises: the sequence A of the first molecule comprises a complementary sequence to a region 5' of exon 1 of a gene encoding IL2-Rγ; the A' sequence of the fourth molecule comprises a complementary sequence to the complementary sequence of the 5' region of exon 1 of the gene encoding IL2-Rγ; the B sequence of the second molecule comprises a sequence complementary to the 3' side of the region 5' side of exon 1 of the gene encoding IL2-Rγ; the B' sequence of the fifth molecule comprises the 3' complement of the complement of the region 5' of exon 1 of the gene encoding IL2-Rγ; and the third molecule comprises the wild-type cDNA sequence of the gene encoding IL2-Rγ, located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a complementary sequence of the first sequence for recognizing the transposase of the second molecule; contacting the sixth molecule such that the sixth molecule comprises the wild-type cDNA sequence of the gene encoding IL2-Rγ located between a complementary sequence of the second sequence for recognizing the transposase of the first molecule and a 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 collection to obtain a recombination complex; - recombining said combined complex to obtain an insertion of the cDNA of the gene encoding IL2-Rγ 5' to exon 1 of the gene encoding IL2-Rγ; - optionally purifying said inserted cells.

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