WO2016016358A1 - Targeting of non-viral integrative vectors in the nucleolar dna sequences of eukaryotes - Google Patents

Abstract

The invention relates to a system for integrating a nucleic acid molecule of interest in a chromosome region associated with the nucleolus. Said system includes at least: - one non-viral integrative vector located in the nucleolus of the cell nucleus and lacking a functional recombinase source; and - one functional recombinase source that is located in the nucleolus of the cell nucleus and provided via transfer from said non-viral integrative vector.

Description

 TARGETING NON-VIRAL INTEGRATED FS VECTORS IN NUCLEOLAI RES DNA SEQUENCES IN EUKARYOTICS

Numerous exogenous nucleotide sequence transfer techniques have been proposed in the field of gene therapy, or for making cells and organisms genetically modified for the production of proteins of economic or therapeutic interest, or for basic research.

These techniques include systems comprising non-viral integrative vectors coupled to specific enzymes. The nonviral integrative vectors are vectors comprising sequences that can be integrated into the genome. This integration generally requires the intervention of enzymes capable of catalyzing or stimulating the integration of the sequence of interest into the DNA. There are several types of non-viral vector / integrative enzyme systems, which operate according to various mechanisms. By way of example, it is possible to cite the transposable elements (TE), including transposons and retro-transposons, which allow the integration of sequences of interest when they are coupled to recombinases such as transposases or integrases. . Among the nonviral vector / integration enzyme systems are also the plasmid / nuclease systems in which the enzyme stimulates the integration of the sequence of interest by homologous recombination.

In general, transposable elements (TE) or mobile genetic elements (EGMs) are small DNA fragments that are able to move from one chromosomal site to another. There are various classes of TE depending on the mechanisms of integration in the DNA put into play by them. In the case of transposons, which are so-called class II TEs, these DNA fragments are characterized by inverted repeat sequences (ITRs) located at the 5 'and 3' terminal positions. An enzyme encoded by the TEs themselves, the transposase, catalyzes the transposition process of the latter.

Transposons have been identified in both prokaryotes and eukaryotes. In prokaryotes, a large number of transposons have been listed to date. Include, for example, insertion sequences such as IS1, and transposons, such as Tn5. In eukaryotes, class II elements comprise ten families: P, PiggyBac (including the Pokey transposon), hAT, helitron, Harbinger, En / Spm, Mutator, Transib, Pogo and IS630-Tc1 -mariner (including Sleeping Beauty).

Plasmid / nuclease systems are also routinely used to transfer genes of interest into the genome of eukaryotic cells in a targeted manner. These systems are, like the transposon / transposase systems, bipartite systems which generally comprise a plasmid whose sequence is defined so as to favor the transposition of a transgene by homologous recombination, and a nuclease of the meganuclease type, Zinc finger nuclease, TALEN, or CRISPR / Cas9 to specifically target integration to a chromosomal site at a high frequency.

The principle of targeting these latter vectors is therefore based on the ability of cleavage of DNA at specific sites by the nucleases in question. This cleavage stimulates homologous recombination close to these sites, thus allowing the integration of exogenous sequences. In addition, the molecular bases governing the link between the structure of these nucleases and the sites they recognize have been well studied. It is now possible with these systems to produce nucleases in a manner specifically targeting the DNA sequences in which integration is desired.

However, the transfer of an exogenous DNA using these known means, whether it is systems derived from transposons or systems based on nucleases, is not without difficulties, in particular because of an efficiency, particularly with respect to the size of the fragment to be integrated, and a specificity of integration of the transgene which remain nevertheless very limited.

Indeed, these systems do not exclude random insertions. However, these are likely to have deleterious effects by mutating genes for which there is only one copy in the genome. In addition, it has been shown that random insertions are 95% extinct in HeLa cells (Garisson et al., 2007). Thus, it would appear that some transgenes, depending on the site in which they were inserted, are eventually recognized by the cell as belonging to the non-self and extinct. These results emphasize the importance of the insertion site as such for the subsequent stability of transgene expression. Finally, the properties of the integrated DNA pair / integration site are essential to obtain a sustainable and controllable expression of a transgene.

However, research has shown that the coding sequences for ribosomal RNAs are privileged sites for optimal expression of transgenes in eukaryotes. In addition, these sequences have the advantage of being grouped spatially at the level of the nucleolus, in specific genomic regions comprising, inter alia, clusters of repeated sequences (genes coding for the 5S, 5.8S, 18S and 28S rRNAs). . Thus, the insertion of nucleic acid molecules into these regions is less likely to give rise to deleterious mutations. Such an integration site has already been evaluated in the context of techniques without any link with the implementation of a transposable tool. It has thus been shown that the inactivation by DNA integration of 50% of the genes encoding the 18S-5.8S-28S rRNA does not affect the viability of the cell or organism. Indeed, although their number may vary by a factor of 1 to 2 in the genome of the same species, these sequences are highly repeated because they are essential for protein synthesis and, therefore, the viability of the body. Using tandem repeats containing the 18S-5.8S-28S rRNA genes as targets for integration also makes it possible to limit or even eliminate the unwanted spread of transgenes to non-target crops and natural plant and animal populations.

Despite this interest in the integration of transgenes at the level of the nucleolus, and particularly in the regions comprising genes encoding the 18S-5.8S-28S rRNAs, that is to say for the chromosomal regions associated with the nucleolus, it is not There is currently no effective system guaranteeing a good integration efficiency at these sites, in particular in animal and plant genomes.

Expression of integrated transgenes at these sites has been confirmed in mammalian cells in a gene therapy context using homologous recombination (Wen et al., 2008). According to the reported work, by homologous recombination, passive and random integration mechanism, the integration efficiency reaches 10 ^"4 - 10 ^{" 5} , that is to say that 1 cell out of 10,000 or 1 One in 100,000 cells has the transgene correctly integrated into the target chromosomal site (Wen et al., 2008, Liang et al., 2007). It is therefore clear that the levels of integration efficiency likely to be obtained by the various methods described until now are still too weak for the applications concerned, and that there is indeed a need for means and methods allowing integration of nucleic acid molecules specifically in these sites of interest, namely in chromosomal regions associated with the nucleolus.

The means and methods of the invention make it possible to solve this technical problem in a simple and elegant manner, easy to implement and with integration rates in the targeted sites of particular interest.

LEGEND OF FIGURES

Figure 1: Analysis of the quality of PNA plasmid complexes by agarose gel delay. All digestions were deposited on 1% agarose gel, 1 XTAE, 1 X GelRed. Well 1: Hindlll digested λ DNA size marker; Well 2: BamHI / SpeI digested pBS-PB-PNABS-pPol1 h-NeoR-pASV40; Well 3: [pBS-PB-PNABS-pPol1h-NeoR-pASV40 / PNA-NoLS] complex hybridized in excess of X60 to oligonucleotide and then digested with BamHI / SpeI.

Figure 2: Epifluorescence microscopy analysis of HeLa cells transfected for 24 hours with YOYO-1 stained [plasmid / PNA-NoLS] complexes. A: epifluorescence observation of DNA-PEI complexes; B: white light localization of nucleoli in nuclei; C: the fusion of the two images (epifluorescence and white light) makes it possible to visualize the concentration of fluorescence in the nucleoli.

Figure 3: Epifluorescence microscopy analysis of HeLa cells transfected for 24 hours with PB-GFP. A: The nucleoli appear as dark areas in the nucleus. B: nucleolar localization controls fused to DsREd. C: The fusion of the two images reveals that the transposase PB is actively internalized in the nuclei but is excluded from the nucleoli.

Figure 4: Epifluorescence microscopy analysis of HeLa cells transfected for 24 hours with NoLS PB-GFP. A: The nucleoli appear as zones fluorescent in the nucleus due to the presence of NoLS PB-GFP. B: Nucleolar localization controls fished at DsREd. C: The fusion of the two images reveals that the NoLS-PB transposase is actively internalized in the nucleoli. Figure 5: Results of transposition assays performed without transposase (control) or with (PB and NoLS-PB) and two vectors respectively containing two cassettes of resistance to neomycin dependent on two different transcription enzymes: pSV40 = RNA polymerase II and pPoll = RNA polymerase I. The ordinate indicates the number of stable NeoR colonies obtained after 2 weeks of selection. This number is proportional to the efficiency of the transposition. The controls were obtained with an empty plasmid pCS2 as a negative source of transposase.

Figure 6: Percentage of different vector insertion events located in the chromosomal DNA of the nucleoplasmic nuclear compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 7: Percentage of different vector insertion events located in the chromosomal DNA of the LAD nuclear compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 8: Percentage of different vector insertion events located in the chromosomal DNA of the nucleoplasmic nuclear compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration.

Figure 9: Percentage of different vector insertion events located in the chromosomal DNA of the LAD nuclear compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 10: Percentage of different vector insertion events located in the chromosomal DNA of the nucleolar compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 1 1: Percentage of different vector insertion events located in the NAD regions contained in the nucleolar compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration.

Figure 12: Percentage of different vector insertion events located in the flanking regions of the boundary rDNAs in the nucleolar compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 1 3: Percentage of different vector insertion events located in the rDNAs contained in the nucleolar compartment. Two nucleic qualities are used for each vector as a source of transposase and transposon: pDNA / pDNA and mRNA / pDNA respectively. The combinations of plasmids and RNA are indicated on the ordinate. The gray bars indicate the percentage of loci affected by a vector insertion in this compartment. The black bars indicate the percentage of sequences describing a vector insertion in this compartment. The two graphs at the top (A) describe results obtained by IPCR while those at the bottom (B) were obtained by LAM-PCR. In each graph, the two gray and black bars indicate the theoretical percentages expected in the nuclear compartment under consideration. Figure 14: Efficacy of the IPCR to detect vectors inserted in the 3 main nuclear compartments, nucleoplasm, LAD, and nucleoli. The ordinate shows the results obtained with 8 molecular vector combinations. Figure 15: Efficacy of LAM-PCR to detect vectors inserted in the 3 major nuclear compartments, nucleoplasm, LAD, and nucleoli. The ordinate shows the results obtained with 8 molecular vector combinations.

DESCRIPTION

The inventors have developed a bi-partite molecular system for the integration of nucleic acid molecules of interest in the regions of the genome forming the nucleolus. The molecular system of the inventors allows specific site integration in eukaryotic host cells, more particularly in the chromosomal regions associated with the nucleolus. Since the nucleolus is the most active transcription site of the nucleus, the sequences integrated thanks to the molecular system of the invention are stably expressed in time. The molecular system of the invention comprises on the one hand a non-viral integrative vector, devoid of enzymatic activity allowing its integration into the genome, and, on the other hand, an enzyme allowing this integration.

This enzyme can thus be, for example, and depending on the type of integrative vector chosen, a recombinase, a transposase, an integrase, a nuclease.

In addition, the molecular system of the invention is particularly original in that both of these elements, both the non-viral integrative vector and the functional recombinase source, both possess the ability to localize near the targeted sites. Indeed, the inventors have developed molecular tools, in particular non-viral integrative vectors and functional recombinase sources (ie recombinases as well as their functional equivalents), localizing in the cell nucleus, and even in the nucleolus. . This specific localization of the means of the molecular system of the invention makes it possible to dramatically increase the rate of integration into chromosomal regions associated with the nucleolus.

To obtain this result, the inventors have designed in particular specific non-viral integrative vectors, whose ability to localize the nucleus and more particularly the nucleolus relies on the use of NoLS peptide localization signals. This technical solution is particularly surprising. Indeed, NoLS localization signals are peptides conventionally used to allow the localization of peptides and proteins. It is therefore surprising to use this type of tool to locate molecules such as vectors, which are polynucleotides. The use of such a technical solution may seem counterintuitive. Indeed, its implementation requires the development of additional connecting elements, for grafting the integrative non-viral vector to the NoLS location signal, which can make it a priori complex. However, against all odds, the nonviral integrative vectors of the invention are found to be particularly effective at integrating nucleic acid sequences of interest into the chromosomal regions constituting the nucleolus.

Thus, the invention firstly relates to a molecular system for integrating a nucleic acid molecule of interest into a chromosomal region associated with the nucleolus, comprising at least:

 a non-viral integrative vector locating in the nucleolus of the cell nucleus, and lacking a source of functional recombinase; and

 a source of functional recombinase located in the nucleolus of the cell nucleus, provided in trons of said non-viral integrative vector.

By "molecular system" is meant in the sense of the invention a set of molecular biology means. Within the meaning of the invention, these means are in particular adapted to the integration of nucleic acid molecules of interest within a chromosomal region. More particularly, within the meaning of the invention, the means of the molecular system according to the invention comprise at least (a) a non-viral integrative vector and (b) a functional recombinant enzyme source or a source of a functional equivalent of it. In general terms, the various enzymes referred to by the invention will be referred to hereinafter by their functional character, by grouping them under the terms "source of functional recombinase", it being understood that this source may consist of a recombinase. as such, or a functional equivalent, such as for example an integrase, a transposase or a nuclease.

For the purposes of the invention, the term "functional recombinase source" is intended to mean an enzyme source capable of fulfilling the same function as a recombinase. The terms "activity", "function", "biological activity" and "biological function" are equivalent and correspond to the usual meaning in the technical field of the invention. In particular, the recombinase included in the molecular system according to the invention is considered "functional" if it is able to catalyze the process of integration of the exogenous nucleotide sequence (s) of interest in the target chromosomal region.

Those skilled in the art can easily verify that the recombinase of interest is "functional" within the meaning of the invention, by in vitro tests well known in the art. This type of assay consists of performing a biochemical analysis of the binding and cleavage activities at the ends of the DNA by the recombinase as well as the analysis of the capacity of the recombinase to transfer DNA strands. In the case of a nuclease, one skilled in the art can easily verify that the enzyme of interest is functional by in vitro tests of specificity of DNA cleavage. The functional recombinase source is preferably selected from transposases, nucleases, integrases and recombinases. In particular, one skilled in the art will be able to choose, from among the various available enzymes, the one that will be best adapted to the integration of the sequence of interest, for example as a function of the non-viral integrative vector used. For example, when the non-viral integrative vector is a transposon or pseudo-transposon, those skilled in the art can preferably choose a transposase to allow the transposition of the sequence of interest. Preferably, the functional recombinase source is a transposase.

Advantageously, the transposase according to the invention is chosen from the family of transposases DD [E / D], which notably comprises the transposases resulting from the transposons Sleeping Beauty, Prince Frog, MuA, Mos-1, Pokey, PiggyBac, Tol2, Tn3, Tn5, Tn7, Tn10, and Hermes. This family of transposases has been described in detail in Nesmelova et al. (Adv Drug Deliv Rev.; 30; 62 (12): 1,187-95; 2010). Advantageously, the integrase according to the invention is chosen from integrases of lentivirus or retrovirus. Advantageously, the recombinase according to the invention is chosen from phage recombinases (such as for example PhiC31). Advantageously, the nuclease according to the invention is chosen from the TALEN nucleases (for "Activator-Like Transcription (TAL) Effector Nudeoses"), or the zinc finger nucleases (ZFNs, for "Zinc-finger nucleases"), the meganuclease , or the Cas9 nucleases (used in the CRISPR / Cas9 system).

By "functional recombinase source located in the nucleolus of the cell nucleus" is meant within the meaning of the invention that the source of recombinase according to the invention is associated with a molecular system for its location in the nucleolus of the cell nucleus. Thus, "locating in the nucleolus of the cell nucleus" means that the "functional recombinase source" molecules, whether transposases, nucleases, recombinases or integrases, are actively located in the eukaryotic cell of so that a very large majority of them, if not all, are in the nucleolus of the cell nucleus. This addressing of the molecules is easily followed, in particular by standard fluorescence microscopy techniques.

The cell nucleus is easily detectable in eukaryotic cells, and can in particular be observed by microscopy via a large number of usual techniques. The nucleolus is an organelle devoid of membrane. Despite this, the nucleolus is individualized in the cell nucleus and the molecular exchanges are active and strictly regulated, and refractory to the diffusion of biological macromolecules. The nucleolus is most often basophilic and therefore visible in light microscopy via basic dyes (Pyronine in red and Giemsa blue). It can be observed more precisely by using electron microscopy. The localization of the recombinase in the nucleolus of the cell nucleus is thus easily evaluable.

Molecular systems for the targeted localization of proteins in the nucleolus of the cell nucleus are well known to those skilled in the art, and do not require not to be detailed. These molecular systems include nucleotide localization signal peptides NoLS. These signal peptides are well known in the field of molecular biology and many sequences have been described in the literature (see in particular Emmott and Hiscox, 2009, Christophe et al, 2000).

Preferably, the functional recombinase source according to the invention comprises a nucleolar localization pattern or signal, or NoLS, which allows its localization to the nucleolus.

By "provided in trans of said non-viral integrative vector" is meant within the meaning of the invention that the functional recombinase source is provided in addition to the non-viral integrative vector, that is to say that it is provided under a distinct form of the nonviral integrative vector. For example, the functional recombinase source is provided in trans via an expression plasmid that will express the protein in the cell, or via a messenger RNA. Alternatively, the source of functional recombinase can be provided in trans in the form of a protein, for example an isolated native protein, or, preferably, a purified recombinant protein. Expression plasmids and / or recombinant proteins can be produced according to methods of molecular engineering and production of conventional recombinant proteins, well known to those skilled in the art.

For the purposes of the present invention, the term "non-viral integrative vector" is intended to mean a vector in which all or part of the nucleotide sequence is intended to integrate into the genome. For the purposes of the invention, non-viral integrative vectors comprise, for example, derivatives of mobile genetic elements such as transposons or pseudo-transposons, but also plasmids whose sequence is defined so as to favor the transposition of a transgene. by homologous recombination stimulated by a nuclease whose cut is specific. In the case of transposons, the integration of the nucleotide sequence is generally stimulated by the transposase for which they encode. In the case of plasmids for homologous recombination, the integration of the nucleotide sequence is generally stimulated by a meganuclease, Zinc finger nuclease, TALEN, or CRISPR / Cas9 nuclease. In the case of non-viral integrative vectors within the meaning of the invention, the integration of the nucleotide sequence is stimulated by the recombinase provided in trans.

For the purposes of the invention, the term "pseudo-transposon" is intended to mean a transposon devoid of the transposase activity. Most often, the pseudo-transposon is a transposon whose gene encoding the original transposase has been deleted or mutated by molecular biology techniques. A pseudo-transposon therefore has the ends ITR and UTR but is devoid of the transposase activity. It has therefore lost the ability to transpose, except to be associated with an external transposase activity (provided in trans).

Preferably, the pseudo-transposon of the invention is a transposon devoid of the transposase activity, and chosen from the transposons Sleeping Beauty, Frog Prince, piggy Bac, MuA, Mos-1, Himarl, Hsmarl, Pokey, Tol2, Tn3, Tn5, Tn7, Tn10, and Hermes and their derivatives. Preferably, the pseudo-transposon of the invention is a transposon devoid of the transposase activity chosen from piggyBac and its derivatives.

By "non-viral integrative vector located in the nucleolus of the cell nucleus" is meant within the meaning of the invention that the non-viral integrative vector according to the invention has, or is associated with, a molecular system allowing its active localization in the nucleolus of the cell nucleus. Thus, by "located in the cell nucleus" it is meant that the non-viral integrative vector molecules are localized in the eukaryotic cell such that a majority or all of them are in the core nucleolus. cellular. This majority localization of the molecules is easily identifiable, in particular by standard fluorescence microscopy techniques.

The inventors have determined that the most effective molecular signal for targeting the non-viral integrative vector into the nucleolus of the cell nucleus, and thus to promote the integration of the nucleic acid molecule of interest into an associated chromosomal region. to the nucleolus is, in a particularly surprising manner, the nuclear localization signal NoLS. Preferably, the non-viral integrative vector according to the invention comprises a nucleolar localization pattern, or NoLS, which allows its active localization in the nucleolus of the cell nucleus. Thus, according to one embodiment of the invention, said non-viral integrative vector is localized to the nucleolus and / or said source of recombinase is localized to the nucleolus via a NoLS nucleolar localization signal. According to one embodiment of the invention, the nucleolar localization signal NoLS is a peptide of sequence SEQ ID No.1: RQARRNRRRRWRERQRQI.

By "via a NoLS localization signal" is meant within the meaning of the invention that the non-viral integrative vector according to the invention has, or is associated with, a NoLS peptide.

The NoLS location signal is a peptide; it is therefore composed of amino acids. The non-viral integrative vector is conventionally a molecule consisting of nucleic acids. Thus, in practice, the association of the non-viral integrative vector according to the invention with the NoLS localization signal is effected by means of a link element allowing a chemical or biochemical anchoring. Within the meaning of the invention, the molecular system of the invention further comprises a binding element of said NoLS localization signal to said non-viral integrative vector.

Preferably, within the meaning of the invention, this connecting element is coupled to the NoLS location signal. In addition, the binding element according to the invention is capable of hybridizing to the non-viral integrative vector, in particular to form a triple helix. Within the meaning of the invention, any molecule capable of hybridizing to a DNA molecule and capable of being coupled to a peptide may be used as a binding element.

By hybridization is meant the process in which, under appropriate conditions, two polynucleotides, for example a polynucleotide and an oligonucleotide, bind with stable and specific hydrogen bonds to form a double-stranded complex. In the context of the invention, a molecule is "able to hybridize with a DNA molecule" if said molecule forms a complex with the DNA molecule under stringent conditions, as defined generally accepted in the art. The hybridization of two polynucleotides can be complete (the double-stranded complex obtained during this hybridization comprises only AT bonds and CG bonds), or partial (the double-stranded complex obtained comprises bases not linked to a complementary base) . hybridization between two polynucleotides depends on the operating conditions. All these data are well known and the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the polynucleotides that it is desired to hybridize, the hybridization temperature is between about 20 and 70 ° C, in particular between 35 and 65 ° C in a saline solution at a concentration of about 0, 5 to 1 M.

Numerous DNA binding protein domains have already been described, such as, for example, LexA DNA binding domains (Schnarr et al., 1988, Oertel-Buchheit et al., 1993), N57SB10 (Izsvak). et al., 2002), and pZFD ^{bcr abl} (Me Namara et al., 2000).

However, the inventors have discovered, quite surprisingly, that the most effective molecules as a linkage element of the NoLS localization signal to said non-viral integrative vector are the molecules chosen from the triplex forming oligonucleotides (OFTs). , in particular peptide nucleic acids and their derivatives. Preferably, the non-viral integrative vector according to the invention comprises a sequence for the hybridization of the binding element. Advantageously, the binding element according to the invention is a triplex-forming oligonucleotide (OFT), preferably chosen from the group of PNAs (peptide nucleic acids), LNAs (locked nucleic acids), PNA hybrids: PNA ("bisPNA") and LNA hybrids: LNA ("Zorro LNA"). In practice, the inventors have determined that the use of bisPNA oligonucleotide-type binding elements enables the formation of non-viral integrative vector / highly stable NoLS location signal complexes. Advantageously, the link element is a bisPNA coupled to the NoLS localization signal.

The peptide nucleic acids (PNAs for "peptide nucleic acids") and their derivatives have the particular advantage of having a high affinity for DNA or RNA. This high affinity makes it possible to obtain a strong hybridization with the target DNA, in this case the pseudo-transposon DNA, including with the small peptide nucleic acids. In addition, peptide nucleic acids are synthetic molecules that do not exist in the natural state, and are not recognized by nucleases or proteases. These molecules are therefore particularly resistant to enzymatic degradation.

According to one embodiment of the invention, the molecular system for locating the non-viral integrative vector to the nucleolus comprises a bisPNA binding element coupled to a NoLS location signal, said bisPNA link element coupled to said NoLS location signal having sequence: KKKLLTTCTTCTTTTLLLTTTTCTTCTTLLLKKKRQARRNRRRRWRERQRQI, wherein K, L, I, R, Q, A, N, W and E are amino acids, C and t are nucleotides.

The coupling between the linking element and the localization signal can easily be achieved via molecular biology techniques known to those skilled in the art, such as, for example, hybridization by TACH (for "Temperature-Assisted Cyclic Hybridization"). ", Detailed in Oprea et al., Mol e / oted7no /.; 45 (2): 171 -9; 2010).

For the purposes of the invention, the term "chromosomal region" is intended to mean a chromosome portion of variable size. The chromosomal regions within the meaning of the invention may in particular comprise all or part of the chromosomal regions p and q (otherwise called "p-arm" and "q-arm" of the chromosomes) as conventionally defined in cell biology, although are not limited to these.

It is well known in the technical field of the invention that the nucleolus comprises different types of chromosomal regions, and especially NORs (for Nucleolus Organizer Re ion) which are chromosomal regions which participate in the formation of the nucleolus. NORs include several tandem copies of genes encoding ribosomal RNAs, as well as nucleolus associated domains (or Nucleolus-Associated Chromatin Domains, NADs). These domains and regions have been described in Nemeth et al. (PLoS Genet 6: e1000889, 2010) and in Stults et al. (Genome Res 18: 13-18, 2008).

For the purposes of the invention, the term "chromosomal region associated with the nucleolus" is preferably understood to mean a region comprising an organizing region of the NOR nucleolus. NORs have been identified in a large number of mammals. In humans, NORs contain genes for 5.8S, 18S and 28S rRNAs and are localized on the short arms of chromosomes 13, 14, 15, 21 and 22 (in other words, on the p-arms of acrocentric chromosomes). Sequences coding for 18S ribosomal RNA from more than 270,000 living species are listed in the Ribosomal dotabase project database and can be consulted at rdp.cme.msu.edu.

The nucleolus-associated regions can therefore easily be visualized by microscopy, according to well-established conventional cytogenetic techniques. For example, NORs can be localized indirectly, by identifying these regions by the technique of in situ hybridization (FISH) on mitotic chromosomes. In addition, NORs can be identified by karyotype following silver staining. In addition, it is relatively easy to locate by this same technique sequences of interest that we tried to integrate into the genome of the cell.

Thus, those skilled in the art can easily identify the chromosomal regions associated with the nucleolus and verify the effectiveness of the molecular system of the invention, regardless of the species of origin of the eukaryotic cell in which it is desired to perform the insertion. of the sequence of interest.

For the purposes of the invention, the term "integration" means the recombination process leading to the insertion of one molecule of nucleic acid into another. Thus, within the meaning of the invention, the molecular system is capable of leading to the integration of said nucleic acid molecule of interest into the chromosomal target region of said eukaryotic host cell, namely in a chromosomal region associated with the nucleolus.

For the purposes of the invention, the term "eukaryotic host cell" is intended to mean a host cell comprising an individualized nucleus. In the context of the present invention, the eukaryotic host cell is chosen from fungi, plant and animal cells. For the purposes of the present invention, the term "animals" includes members of the animal kingdom, and in particular mammals, in particular humans.

By "nucleic acid molecule of interest" is meant within the meaning of the invention any molecule of nucleic acid whose introduction into the genome of the cell Ekaryote according to the invention is desirable, such as for example a nucleic acid molecule coding for an RNA or a peptide whose expression is desired, preferably stable, or a nucleic acid molecule inducing a mutation of interest. .

According to a preferred embodiment, the nucleic acid molecule is a gene, in particular a therapeutic gene, functional (we can speak of "transgene"). By "therapeutic gene" is meant that the gene itself or its expression product is of therapeutic interest. Within the meaning of the invention, a gene is said to be "functional" if the corresponding nucleotide sequence codes for an RNA, such as, for example, a structural RNA, a nucleic enzyme (RNAseP, ribozyme), an RNA involved in the mechanisms of the Interfering RNA (miRNA, shRNA), or code for a peptide. According to one embodiment, the therapeutic gene encodes a peptide if it comprises at least one open reading frame (ORF), that is to say a coding sequence, capable of giving rise to an amino acid sequence presenting a therapeutic activity. The functional therapeutic gene may be a gene of wild or mutated interest, that is to say a wild-type gene comprising one or more mutations, provided that the product of the mutated gene retains a therapeutic activity. Thus, in this definition of "functional gene", coding sequences devoid of their promoter are also encompassed. By way of example, the nucleic acid molecule of interest may be an antibiotic resistance gene, devoid of its promoter or not (for example, the neomycin resistance gene), or any other appropriate selection marker. The nucleolus is the preferred site for the synthesis of ribosomal RNA molecules, and essentially comprises type I RNA polymerase molecules. Thus, to facilitate the expression of the sequence of interest, when the coding sequences of the functional genes of interest are accompanied by a promoter, it is preferentially a promoter dependent on the type I RNA polymerase.

For the purposes of the invention, a "mutation" is in accordance with the usual meaning of biotechnology. Thus, a mutation may be a substitution, addition or deletion of one or more bases in a nucleotide sequence, or one or more amino acids in a protein sequence. A "mutation" can in particular designate a substitution of at least one base of a codon of a nucleotide sequence, said substitution resulting for example, during the translation of the nucleotide sequence in question, the incorporation of a different amino acid instead and place of the native amino acid in the resulting protein sequence. As a general rule, in the case of a functional gene, it will be preferred that the mutation (s) cause (s) no loss of the biological function of the mutated product. On the other hand, a decrease in activity may possibly be tolerated. In general, a "genetic modification" is equivalent to one or more mutations. If a coding sequence is genetically modified, then, typically, it contains one or more mutations.

It has been shown that in the absence of counter-selection, the integration of vectors into the tandem repeats encoding the rRNAs is labile in germ cells. This phenomenon is due to a control mechanism called restoration, which eliminates, in the germ line (the cells of the reproduction), in 2 to 5 generations, the copies of the genes containing integrations or deletions.

This lability can be used to prevent an untimely spread of transgenes. However, it may be desirable in some cases to stabilize transgene integration over time.

For this, it may be useful to make selections from the clonal organisms obtained through the molecular system of the invention, according to the techniques and methods conventionally used in the field. The nucleolus is the only transcriptional site by RNA polymerase I and RNA polymerase II is absent, its location being reduced to the nucleoplasm (Malyavantham et al 2008, Németh et al 2010). This feature is of particular interest in the context of the use of targeted vectors in the nucleolar regions.

The inventors have used certain aspects of the location of the target chromosomal sequences to determine embodiments of the molecular systems of the invention which are particularly advantageous. In one embodiment, the non-viral integrative vector according to the invention comprises a reporter gene whose expression depends on a promoter of the type I RNA polymerase. The expression of this first reporter gene, which depends on type I RNA polymerase, may subsequently be used to select cells in which the molecular system of the invention has effectively integrated the target site, namely the nucleolus. Thus, the expression of this first reporter gene, which depends on the type I RNA polymerase, may later be used to perform a "positive selection".

In another embodiment, the non-viral integrative vector according to the invention comprises a reporter gene whose expression depends on a type II RNA polymerase promoter.

The expression of this second reporter gene, which depends on type II RNA polymerase, may later be used to remove cells in which the transgene has integrated in a region other than the nucleolus. In this perspective, the expression of this second reporter gene, which depends on type II RNA polymerase, may subsequently be used to perform a "negative selection".

By way of example, it will be preferable to use a gene which makes it possible to select positive genes for resistance to specific antibiotics or genes coding for easily observable proteins (fluorescence, detectable enzymatic activity). The gene encoding the thymidine kinase of human herpesvirus 1, which will make the clone susceptible to selection by ganciclovir, or else a gene encoding a fluorescent protein, which will make it possible to sort the gene, may be used as a negative selection gene. clones by flow cytometry.

According to a particularly advantageous embodiment, the non-viral integrative vector according to the invention comprises a reporter gene whose expression depends on a type I RNA polymerase promoter and a reporter gene whose expression depends on a promoter of type II RNA polymerase. The inventors have developed a method, implementing the transposition systems of the invention, and making it possible to increase very significantly the level of eukaryotic cells actually genetically modified by the integration of sequences of interest at the level of the nucleolus. The method of the invention is based on the selection of characteristics of interest by simple methods that are simple and quick to implement, and which contribute to stabilizing the integration of the transgene over time. The implementation of the method of the invention thus makes it possible to obtain a population in which 99.99% of the cells have a specific genetic modification. By "specific genetic modification" is meant in the sense of the application that the genetic modification is specifically located in a chromosomal region associated with the nucleolus of the host cell.

The invention also relates to a method of genetic modification of at least one eukaryotic host cell, comprising:

 a) introducing into said cell at least one system according to the invention, comprising said non-viral integrative vector and said functional recombinase source, as described above;

 b) The selection of cells of step a), in which the nucleic acid molecule of interest is expressed;

 c) The selection of cells of step b), wherein the non-viral integrative vector is specifically integrated into a chromosomal region associated with the nucleolus.

The method of the invention refers to the host cell as previously defined in the present application.

The introduction of the transposition system into the eukaryotic host cell can be done according to any usual technique known to those skilled in the art. By way of example, the usual techniques notably include calcium phosphate transfection, liposome transfection, polycationic agent transfection, DEAE-Dextran, electroporation, and the particular properties of reagents such as GeneCellin and PEI Jet. or the copolymer blocks. In practice, the eukaryotic host cell may be transfected simultaneously or separately with the non-viral integrative vector and the recombinase, and preferably with a linker coupled to the NoLS location signal. For example, the non-viral integrative vector (with or without the linking element coupled to the NoLS localization signal) and the recombinase may be transfected together or separately.

Furthermore, it is possible to assemble the non-viral integrative vector and the linking element coupled to the NoLS localization signal prior to transfection.

The selection of step a) can be carried out according to conventional methods, for example if the non-viral integrative vector comprises a reporter gene, by selecting the cells expressing this reporter gene. By way of example, it will be preferable to use, as the reporter gene, genes for resistance to specific antibiotics, or genes encoding easily observable proteins (fluorescence, detectable enzymatic activity). Thus, preferably, for this step, the cells will be selected according to the expression of a gene allowing positive selection. In this case, the cells selected at the end of step b) are those that express the gene allowing positive selection.

In the case where the reporter gene is an antibiotic resistance gene, the use of this antibiotic will allow the selection of clones in which the nucleotide sequence of interest is expressed.

The selection of step b) can be carried out again according to conventional methods, for example if the non-viral integrative vector comprises a reporter gene whose expression depends on a type II RNA polymerase promoter and allowing a negative selection. . In this case, the cells selected at the end of step c) are those not expressing the reporter gene. Cells expressing the reporter gene whose expression depends on a type II RNA polymerase promoter and allowing negative selection are eliminated. Depending on the embodiments, the integration of the nucleic acid molecule of interest is carried out in vitro or ex vivo or in vivo. In vivo applications, the host cell is a eukaryotic cell preferably selected from fungi cells, plants, animal models.

Thus, the subject of the invention is also the use of at least one transposition system according to the invention for the genetic modification of at least one eukaryotic host cell, in particular for the controlled integration of at least one molecule. of nucleic acid of interest in the genome of a eukaryotic host cell.

By "controlled integration" is meant within the meaning of the invention that the integration of the nucleic molecule of interest is controlled spatially. In particular, "controlled integration" means that the integration is controlled to intervene in a chromosomal region associated with the nucleolus.

In addition, the invention also relates to a genetically modified eukaryotic host cell that can be obtained by the process of the invention.

 In addition, the invention also relates to a genetically modified eukaryotic host cell:

 characterized in that it contains at least one molecular system according to the invention; and or

 in which at least one nucleotide molecule of interest is integrated into a chromosomal region associated with the nucleolus, according to the method described above. In particular, such a eukaryotic host cell will express the nucleic acid molecule of interest.

The invention also relates to a non-human transgenic eukaryotic organism, in particular a transgenic animal, of which at least one cell is a genetically modified cell according to the invention. By "eukaryotic organism" is meant here a fungus, a plant or an animal. This definition does not purport to refer specifically to plant varieties and animal breeds as such, but may include individuals belonging to plant varieties or animal breeds. Such an organism will be, in particular, an animal model particularly useful as an animal biomarker (applicable, for example, in chemical analyzes environmental or materials). Examples of animal models are, without limitation: mammals (eg, cattle, pigs, goats, sheep, horses, rodents such as mice, rats, hamsters, domestic animals such as cats, dogs), birds (birds, poultry), fish.

The invention also relates to a kit for the integration of at least one nucleic acid molecule of interest into the genome of a eukaryotic host cell for the purpose of its expression, characterized in that it comprises at least:

 - a molecular system, and / or

- a genetically modified host cell, and / or

 a non-human transgenic eukaryotic organism,

according to the invention.

In addition, such a kit may comprise one or more additional elements, such as, in particular, a buffer solution compatible with the recombinase or its functional equivalent, one or more control DNAs (reaction controls), oligonucleotides useful for sequencing allowing check the effectiveness of the reaction, the competent bacteria, instructions for use. The kit according to the present invention may for example be used to modify the genome of eukaryotic cells. These modifications may, for example, make it possible to modify the metabolism of the target cells, in order to increase their yield in bioproduction. Alternatively, the modifications made may have therapeutic effects.

The kit according to the present invention may in particular be used to produce a protein of interest, in particular a protein of cosmetic interest, a protein of therapeutic interest or a cell expressing such a protein. The "protein of interest" is here the product of expression of the nucleic acid molecule of integrated interest, thanks to the means of the invention, in the genome of the host cell. A "cell expressing a protein of therapeutic interest" can be described as an implant, useful in particular in regenerative therapy.

The invention also provides a pharmaceutical composition comprising at least: a molecular system according to the invention, and / or a host cell genetically modified according to the invention

in combination with a pharmaceutically acceptable carrier.

In any case, the numerous applications of the means of the invention, for which these are of considerable interest and whose list provided here is far from being exhaustive, call upon conventional techniques of molecular biology well. known to those skilled in the art.

The following examples are provided for purely illustrative purposes and in no way limit the subject of the present invention.

EXAMPLES

The work to obtain the following results concerning the systems of the invention was carried out on the human cell HeLa line.

1. Principle of targeting in the nucleolus of the two components of the piggybac vector

The physical source of transposase may be a plasmid or a messenger RNA synthesized from this plasmid. Both types of source can be transfected into the cells with a transfection agent. The use of JetPEl makes it possible to have a single transfection protocol for these two types of source. To be targeted in the nucleolus, the gene encoding the piggybac transposase was fused to an N-terminal with a particular oligonucleotide encoding a NoLS (RQARRNRRRRWRERQRQI, Cochrane et al 1990).

The physical source of transposon vector is a pBS plasmid containing the ends of a piggybac vector with a cassette encoding markers and a hybridization site for a bis-PNA oligonucleotide coupled to a NoLS (PNA-NoLS; KKK- LLTTCTTCTTTTLLLTTTTCTTCTT-LLL-KKK-RQARRNRRRRWRERQRQI wherein K, L, I, R, Q, A, N, W and E are amino acids, C and T are nucleotides). Hybridization of PNA-NoLS is done using a protocol similar to a PCR. It is called TACH (Temperature-assisted cyclic hybridization, Oprea et al 2010). The purification of [plasmid / PNA-NoLS] complexes is carried out using plasmid mini-purification kits or Qiagen-type PCR products. Masherey-Nagel, or Promega. The hybridized NoLS on the plasmid allows the plasmid to be addressed to the nucleus and the nucleolus. These [plasmid / PNA-NoLS] complexes are transfected into cells with a transfection agent (jetPE1). Hybridization of a bis-PNA oligonucleotide on the DNA forms a structure resulting from a double hybridization on a DNA strand that is much more stable than that resulting from the simple hybridization of an oligonucleotide PNA. This double hybridization makes it possible to produce [Plasmid / bis-PNA] complexes which are much more stable and specific at the time of hybridization than the [Plasmid / PNA] complexes.

2. Verification of the functioning of targeting in the nucleolus of the two components of piggybac vectors

The sequence of the pBS plasmids having a binding site for the PNA-NoLS oligonucleotide and containing a piggybac vector into which a transgene is inserted corresponds to the sequences SEQ ID No.2 and SEQ ID No.3.

2.1. Hybridization protocol for bis-PNA on the transposon source plasmid a-Hybridization

 The bis-PNA-NoLS is added 20, 40 or 60 times in excess of the plasmid for hybridization. The hybridization is carried out in 0.5mL PCR tubes:

T x20 x40 x60

 PBSK Neo (0.5Mg / ML final) 5.26 5.26 5.26 5.26

 Bis-PNA 400μΜ 0 0.5 1 1.5

 0.1M Phosphate Buffer pH 5.8 4 4 4 4

 EtOHabs 4 4 4 4

 H20 6.74 6.24 5.74 5.24

 Vf 20 [il 20μί 20μί 20μί

A drop of mineral oil is added to the surface of each mix to prevent evaporation during the hybridization reaction. The hybridization reaction is carried out in a thermocycler. The program used consists of 20 cycles of two phases. - 30s 80 ° C (de-hybridization)

 - 2min 60 ° C (hybridization) At the end of the program, the whole of the mixture contained in the 0.5 ml tube is deposited on a piece of parafilm in order to separate the mineral oil from the aqueous phase containing the DNA . The two phases are separated by pouring the sample on the parafilm. The oil then separates from the aqueous phase, which can be recovered with a cone and then into a 1.5 ml micro-tube. b - Purification of bis-PNA / plasmid complexes

 Purification is performed with the reagents Using the Wizard SV Gel Kit or the Promega PCR Clean-Up System: - 200μί Membrane Binding Solution (for 2.5 μg DNA)

 Swirl gently

 Place on the column equipped with a collector tube

 Incubate on the column for 1 min at room temperature

 Centrifuge 1 min at 1000 rpm at room temperature

- Remove the eluate contained in the collecting tube and put the column back on the collector tube

 Add to column 700 μί Membrane Wash Solution

 Centrifuge 1 min at 14,000 rpm

 Eliminate the eluate contained in the collector tube and then put the column back into the collector tube

 Deposit on the column 500 μί Membrane Wash Solution

 Centrifuge 5 min at 14,000 rpm

 Eliminate the eluate contained in the collector tube and then put the column back into the collector tube

- Centrifuge 1 min at 14,000 rpm to dry the column (removal of traces of EtOH is crucial)

 Transfer the column to a new 0.5 ml microtube 1

 Elute the bis-PNA / DNA complexes by incubating the column for 1 min at room temperature with 50 μί "Nuclease-free" water

- Centrifuge 1 min at 14000 rpm Re-elute with the first eluate reiterating the last two stages

 Assay of the concentration of the RNAs with BioSpec Nano with 2 μΙ_ c - Quality control of bis-PNA / plasmid complexes

The bis-PNA / DNA complexes are stable for at least 2-3 weeks at 20 ° C. At any time, the quality of a complex stock can be verified using the procedure described below.

 PNABS-560 sites (250 bp fragments) being cloned between two unique sites in our BamHI-SpeI constructs, agarose gel electrophoresis delayed gels can be performed to verify the quality of specific bis-PNA hybridization. on the plasmid.

Step I: 5μί Digestion of Elua by BamHI-Spel

In a microtube of 1.5 ml, put:

- 0.5 g of complex

 1 μί Promega 10x E Buffer

 1 [he BSA 1 mg / mL

 0.5 μί. Bam

 0.5 ML Spel

- <¾; ρ Η2Ο έ 10 μί

 Incubate 1 h 37 ° C then leave at 4 ° C

All digestions are deposited on 1% agarose gel, 1 XTAE, 1 X GelRed Well 1: Hindi digested λ DNA size marker 11

Well 2: BamHI / Spel digested pBS-PB-PNABS-pPol1 h-NeoR-pASV40

 Well 3: [pBS-PB-PNABS-pPol1h-NeoR-pASV40 / PNA-NoLS] complex hybridized in excess of X60 to oligonucleotide and then digested with BamHI / Spel

The results are shown in Figure 1. CONCLUSIONS: Comparison of the central and right profiles (B and C) shows the appearance of the delay when PNA is hybridized on the plasmid DNA (disappearance of the 250 bp band and appearance of dragging the vector from the place of deposit of the sample). The method of assembling and purifying the complexes is therefore reliable. 2.2. Verification of the Functionality of Transposon Source Plasmid Targeting Plasmid / PNA-NoLS complexes stained with YOYO-1 intercalating fluorochrome were used to transfect HeLa cells for the purpose of testing whether they have the property of be actively located in the nucleoli.

The results are shown in Figure 2.

Conclusions: Our results made it possible to verify that [plasmid / PNA-NoLS] complexes are actively located in the nucleoli. However, this verification could only be performed under conditions where small amounts of complexes are transfected (50 ng). When large amounts are used, the system is saturated and [plasmid / PNA-NoLS] complexes are visualized in all the compartments of the nucleus. 2.3. Preparation of transposase sources

 Two types of transposases were used (PB and HIV-NoLS PB) from two supports: an expression vector plasmid (Appendix 3) or a messenger RNA. Plasmic DNAs were prepared using a Masherey-Nagel purification kit (endotoxin-free). a - Preparation of plasmids

 The messenger RNAs to be prepared are cloned into the plasmid expression vector pCS2 + (see map below). To be used as a transcription matrix, the plasmid must be linearized by enzymatic cleavage with a restriction enzyme cutting downstream of the ORF. For transcription to be effective, particularly its termination, cleavage must produce fragments with blunt or outgoing 5 'outward ends. Here, the enzymes used are located in the most proximal multi-site cloning (MCS) of the CMV promoter: NotI and Acc651 (KpnI isoschizomer with 5 'outward ends). The enzyme chosen for digestion should not cut into the ORF.

Linearization protocol of the plasmid.

 10 g of plasmid DNA

10 μl of 10X of enzymatic digestion buffer (here, NEB 3) 1lb of BSA 10mg / ml (final: 100mg / ml)

 5 μl of restriction enzyme (NotI or Acc651 according to the plasmid), ie 50 units (5-fold excess)

 ς5ρ 100 μΙ_

Incubate for 1 hour 30 hours at 37 ° C, then take a 5 μl aliquot to control digestion on minigel (1 X TAE, 0.8% agarose, 1 X GelRed, controls: 500 ng undigested plasmid and 1 cut). - If digestion is saturated, incubate digestion for 5 min at 65 ° C, then 1 min in ice.

Deprotenization of the linearized plasmid

 5 μί. SDS 20%

- ML ML proteinase K (final 100 Mg / ml)

 qs 200 it with sterile water

Incubate 1 hour at 37 ° C and then 5 minutes in the ice

 Extract the proteins in volume / volume conditions with saturated phenol (pH 6 to 8) / chloroform (100 μl phenol + 100 μl chloroform / isoamyl alcohol 24/1): vortex 1 minute, then centrifuge for 5 minutes at 13500 rpm (15000 G), and finally recover the aqueous phase.

 Extract traces of phenol under volume / volume conditions with chloroform (+200 μl chloroform / isoamyl alcohol 24/1): vortex 1 minute, then centrifuge for 5 minutes at 1 3500 rpm (15000 G), and finally recover the aqueous phase .

 Precipitate the nucleic acids by adjusting to 0.3 M sodium acetate (stock solution at 3 M, thus more than 20 μl.) And 3 volumes of absolute ethanol. Incubate for 1 hour at -80 ° C or overnight at -20 ° C, then centrifuge for 15 minutes at 1 3500 rpm (1 5000 G), 4 ° C. Decant the pellet, wash with 750 μl.) EtOH 70% (The pellet is washed with 250 μl of EtOH 70 prepared with absolute EtOH + H20 Nuclease free (kit)) and then lamp-dried on the bench or 10 min in the vacuum chamber.

 Re-suspend the plasmid pellet in 20 μl of RNase-Free water

Quantify the plasmid on the BioSpec Nano with 2 μί. b - Synthesis of messenger RNAs (mRNA)

Step 1: Transcription and styling of RNAs

 1 μg of linearized plasmid

 2 [he mix ATP / CTP / GTP1 / 5 / UTP

- 2 [he ARCA (tube independent of synthesis kit)

 2 μΙ. 10X Buffer reaction

 2 [he enzyme mix

 qsp 20 μl with H20 RNase-Free Incubation 5 hours at 37 ° C.

 1 μΐ Turbo DNAse

Incubation 15 min at 37 ° C

 Take 1 μΐ for subsequent control on minigel (RNA synthesis control) Step 2: Polyadenylation of the RNAs

- 37 μί Η20

 20 it 5X-E-PAP Buffer

 10 [25 mM MnCl2]

 10 [ATP 10 mM

 Μΐ E-PAP (E. coli Poly (A) Polymerase I)

νί = 100 Ι_

 Incubation 2h at 37 ° C

 add 60 [LiCl 7.5M (2.8125M final)

Precipitation on the night at -20 ° C

 Centrifuge 1 5 min at 13500 rpm (15000 G), 4 ° C.

- Decant the pellet

 The pellet is washed with 250 μl of 70% EtOH prepared with absolute EtOH + H20 nuclease "free" (kit)

 Dry with a lamp on the bench or 10 min in the vacuum bell.

 The pellet is taken up in 20 μl of H20 nuclease free (transcription kit) Step 3: Quantification of the mRNAs

 Assay of the concentration of the RNAs at BioSpec Nano with 2 [he

Step 4: Quality control of mRNAs

Prepare a minigel in 1 X TAE (special RNA), 0.8% Argarose, 1 X GelRed Prepare RNA samples for electrophoresis: 1 μΐ extract + 5 μΐ H20 nuclease free + 6 μΐ Loading Buffer II gel (2X, Ambion). Incubate for 5 min at 65 ° C and then 1 min on ice

 Deposit the samples in the gel

- To migrate for 1 hour at 90 V, a marker for PM DNA and RNA if available in parallel of the 2 samples: 1 μΐ RNA Synthesis Control and 1 μΐ of final RNA. Step 5: Preservation of mRNA at -80 ° C

 Aliquot mRNAs in 4 samples of 4 ί

 Store at -80 ° C

2.2. Verification of the Functionality of Transposon Source Plasmid Targeting a - Functionality of Targeting in Nucleoli

 Plasmids pCS2 expressing PB-GFP, HIV-NoLS-PB-GFP and DSRed-NoLS fusions (nucleolar localization marker (Becherel et al., 2006) were used to transfect HeLa cells to test for HIV-NoLS protein. -PB has the property of being actively localized in the nucleoli.

The results are presented in Figures 3 and 4. Conclusions: Our results made it possible to verify that the HIV-NoLS-PB transposase is actively localized in the nucleoli. However, this verification could only be performed under conditions of low NoLS-PB expression. When strong expressions are used, the system is saturated and NoLS-PB is displayed in all nuclear compartments. b - Functionality of the HIV-NoLS-PB transposase

Many transposases (Sleeping Beauty, Tol2, Mos1, ...) and integrases have a recombination activity which is diminished or canceled when they are fused with a peptide or an N-terminal and / or C-terminal protein domain. . The activity of N-terminal HIV-NoLS-PB fusion was compared with that of native PB transposase in HeLa cell transposition assays (see protocol in the following section; 2 plasmids are used in each test condition: a source of transposase and a source of transposon). Two types of transposon vector sources have been used, the sequences of which correspond to the sequences of SEQ ID No.3 and No.4. The results are presented in figure 5.

 Conclusions: Our results indicate that the transposition activity of the two transposases is not different regardless of the selection cassette used in transposon vectors.

3.3. Transposition conditions used.

Two types of transposition tests were used. The first test involves two plasmids (a transposase source and a transposon source). In this test, expression of the transposase takes place for 7 to 8 days, the time that the transposase source plasmid is naturally eliminated by the cell. The second type of test involves 1 mRNA (as a transposase source) and a plasmid (as a transposon source). In this assay, expression of the transposase takes place for 16 hours, a shorter duration than the freshly transfected HeLa cell division time.

The details of the characteristics of these two sources of PB transposases and the conditions of the two types of transposition tests are described in Bire et al (2013). Briefly, for each test, a transposase source (~ 200 ng) and a transposon vector source (-200 ng) are transfected for 4 hours in 50,000 HeLa cells using jetPE1 as the transfection agent. From the perspective where the targeting works very precisely, we have retained non-optimal transposition conditions (in terms of the amount of transposon vector source) so that we can observe alongside the targeting of background noise in control. The cells are then cultured for 48 hours before being subjected to G418 selection for two weeks. At the end of the selection, cloned cells of G418-resistant cells (ie stably expressing the integrated transgene via the transposon vector in chromosomes) are fixed and stained before being enumerated or trypsinized, washed and pelleted to purify their DNA. genomics. Each test is triplicate. The conditions used make it possible to obtain genetically modified cells by means of an average integration of 2 to 3 transposon vectors / haploid genome of cellular clone.

4. Verification of the insertion specificity of piggybac vectors in tandem repeat units containing the genes encoding 18S-5.8S-28S rRNAs 4.1. Production of genetically modified HeLa cell populations

 Fourteen transposition tests (indicated in Table 1) were performed with the sources whose sequences correspond to the sequences SEQ ID No.2 to 4.

Table 1 Tests 1 to 4 are transposition controls. An average of ~ 30 clones was observed for the tests N ° 1, 3, 5 and 7. An average of ~ 0 to 2 clones was observed for the tests N ° 2, 4, 6 and 8. These witnesses correspond to integration tests of transposon source plasmids by random recombination. In our experimental conditions, they indicate that the cryptic promoters of RNA polymerase 2 in the RNA polymerase 1 (pPoll h) promoter are ineffective in expressing a transgene in the nucleoplasm.

Mean -1000 and 2000 clones were respectively obtained with the tests No. 9 and 10. Mean -50, 300, 200 and 300 clones were respectively obtained with the tests No. 1 1, 12, 13 and 14. These The results indicate that the integration of vectors into transposing chromosomes probably lasts longer when transposition is performed with two plasmids rather than mRNA and plasmid. This difference between the two transposition tests had already been observed in the non-targeted transposition tests described in Bire et al (2014).

4.2. Verification of the specificity of insertion of the targeted vectors. a - Purification of genomic DNAs from clone populations

 Lots of 1000 clones of cells were harvested from transposition tests Nos. 9, 10, 11, 12, 13 and 14. The genomic DNA of these clone populations was then purified by avoiding commercial kits and using the procedure described in Ahmad et al. 1995. b - Determination of insertion site populations in each population of clones

I PCR. The IPCR protocol used is that described in Wang et al. 2012a, the detailed protocol having been at our request transmitted by the authors (Wang et al., 2012b). Novel oligonucleotides adapted to piggybac vectors have been defined used for the amplification of insertion site populations on their 5 'end, then their labeling. For each sample, 3 types of restriction enzyme digestions were performed: [Tail], [BsaW1 + BsrF1 + Xmal] (BBX) and [Sau3AI]. After purification of the digests, the DNA fragments at the concentration of -1 ng / L were circularized overnight at 16 ° C by T4 DNA ligase. of the Oligonucleotides were designed and synthesized to amplify the PCR products and then label them for Illumina sequencing (Table 2).

 Sequence name SEQ ID

oligonucleotide

 ITR-UTR PB 5sens ATAAACCTCGATATACAGACC SEQ ID No.5

 ITR-UTR PB 5'rev CGATAAMCACATGCGTCAAT SEQ ID No.6

 TABLE 2 Table 3 summarizes the oligonucleotides for Illumina labeling of 5 'insertion site populations of piggybac vectors.

Sequence name SEQ ID

the oligonucléot

ide

 Mega- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCC SEQ ID No.7

 Linkerlllumin TGCTGAACCGCTCTTCCGATCTAGTGGCACAGCAGTTAGG

 Ά

 01igoITR5oute AATGATAC G GAC GAC CAC C GAGATCTACACTCTTTC C CTAC SEQ ID NO.8 rBCN ° l ACGACGCTCTTCCGATCTACGCGATAAAACACATGCGTCA

 AT

 01igoITR5oute AATGATAC G GAC GAC CAC C GAGATCTACACTCTTTC C CTAC SEQ ID No.9 rBCN ° 2 ACGACGCTCTTCCGATCTCTACGATAAAACACATGCGTCA

 AT

 01igoITR5out AATGATAC G GC GAC CAC C GAGATCTACACTCTTTC C CTAC SEQ ID NO.10 rBCN ° 3 ACGACGCTCTTCCGATCTGATCGATAAAACACATGCGTCA

 AT

 01igoITR5oute AATGATAC G GAC GAC CAC C GAGATCTACACTCTTTC C CTAC SEQ ID NO.11 rBCN ° 4 ACGACGCTCTTCCGATCTTGCCGATAAAACACATGCGTCA

 AT

TABLE 3 LAM-PCR. The LAM-PCR protocol used is that described in Bartholomae et al. 2012. This protocol has been defined to amplify lentivirus insertion sites. New oligonucleotides adapted to piggybac vectors have been defined used for the amplification of insertion site populations on their 5 'end, followed by their labeling. For each sample, 3 types of restriction enzyme digestions were performed: [Dpnll], [Pcil + Ncol + BspHI] (PNB) and [Spel + Avril + Nhel + Xbal] (SANX). Oligonucleotides for each of these cleavage types have been designated and synthesized.

Table 4 summarizes the biotynylated oligonucleotides (B) or not for the 3 amplifications nested at the 5 'end of the piggybac vectors.

Sequence name SEQ ID

oligonucleotide

 (B) -ITR-UTR 5 'PB (B) - SEQ ID No.12

pSV40 I GACTTTCCACACCCTAACTGACAC

 (B) -ITR-UTR 5 'PB (B) - SEQ ID No.13

pPollh I GAT C C AT G AATT CGTCGACATCG

 ATACCA

 (B) -ITR-UTR PB 5ΊΙ (B) - SEQ ID No.14

 ATAAAC CTC GATATAC AG AC C

TABLE 4 Table 5 summarizes the oligonucleotides used for each type of restriction enzyme cleavage

 Sequence name SEQ ID

the oligonucléotid

 BOY WUT

 Oligo I GACCCGGGAGATCTGAATTCAGTGGCACAGCAGT SEQ ID No.15

 Tagg

 Oligo II Dpnll GATCCCTAACTGCTGTGCCACTGAATTCAGATC SEQ ID No.16

Oligo II PNB CATGCCTAACTGCTGTGCCACTGAATTCAGATC SEQ ID No.17

 TABLE 5

Table 6 summarizes the oligonucleotides for lllumina labeling of 5 'insertion site populations of piggybac vectors. Sequence name SEQ ID

oligonucleotide

 Mega- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGC SEQ ID No.1 8

 Linkerlllumina ATTCCTGCTGAACCGCTCTTCCGATCTAGTGGCA

 CAGCAGTTAGG

 01igoITR5outerB AATGATACGGCGACCACCGAGATCTACACTCTTT SEQ ID No.1 9

 CN ° 1 CCCTACACGACG CTCTTCC GATCTAC GC GATAAA

 ACACATGCGTCAAT

 01igoITR5outerB AATGATACGGCGACCACCGAGATCTACACTCTTT SEQ ID No.20

 CN ° 2 CCCTACACGACGCTCTTCCGATCTCTAC GATAAA

 ACACATGCGTCAAT

 01igoITR5outerB AATGATACGGCGACCACCGAGATCTACACTCTTT SEQ ID No.21

 CN ° 3 CCCTACACGACGCTCTTCCGATCTGATCGATAAA

 ACACATGCGTCAAT

 01igoITR5outerB AATGATACGGCGACCACCGAGATCTACACTCTTT SEQ ID No.22

 CN ° 4 CCCTACACGACGCTCTTCCGATCTTGCCGATAAA

 ACACATGCGTCAAT

 Oligo II SANX CTAGCCTAACTGCTGTGCCACTGAATTCAGATC SEQ ID No.23

 TABLE 6

Sequencing of fragment populations corresponding to piggybac vector insertion sites was performed in Illumina MiSeq, 300-bp per reading. Approximately 2 million readings were obtained per sample. c - Sequence analysis procedure Mi-Seq

Preamble on the limits of the AML-PCR.

LAM-PCR is an approach whose objective is to determine the insertion profile of a vector in chromosomes. It is a qualitative approach and not very quantitative. In fact, the amplifications made by PCR systematically lead to the preferential amplification of fragments which represent for the most numerous 90% of the readings. This amplification bias is constant. Therefore, if a strict quantification of vector targeting efficiency can not be performed, the comparison of LAM-PCR results makes it possible to determine whether there are differences between samples taken from vector-based transposition assays. integrative with targeted insertion or not. Preamble on the chromosomal targets of targeting.

 The targeting of the vectors of the invention is directed to the chromosomal regions associated with the nucleoli. In a haploid genome containing a maximum of 500 repeating units of the 18S-5.8S-28S rRNA genes, these represent -0.69% of the genome of a HeLa cell. Their flanking regions and NADs represent respectively -0.55% and -2.92% of the genome. Nuclear membrane-bound sequences, called lamina-associated domains (LADs), represent -36.83% of the genome (Guelen et al 2008). Their characteristic of being compacted on the nuclear membrane makes these sequences, in theory, less accessible to transposition. They were therefore retained as internal witnesses to our targeting experiments.

In a transposition test in which the insertion is random (non-targeted), it is therefore expected that 4.4 to 4.5% of the insertions will occur in these nucleolar chromosomal sequences, if and only if these sequences are as accessible as n any other sequence contained in the nucleoplasm. However, as the entry and exit of macromolecules in the nucleolus are highly controlled and require active transport mechanisms, it is expected that the vector has little or no ability to diffuse into this organelle. Therefore, it is expected that the level of insertion of the non-targeted vectors into the nucleolar chromosomal regions is low, or even zero, at least in tests whose transposition time is shorter than that of a cell division cycle.

Preamble on the properties of populations of insertion sites obtained by PCR. Impact of the molecular approach. The LAM-PCR approach (same for IPCR) produces qualitative results on the ability or not of a vector to integrate a target. This approach is limited by inherent defects (sensitivity to the GC level of the sequences to be amplified, the rDNA being very rich in GC% (60 to 80%), compared to the other sequences whose average GC% is 45%; Aird et al 201 1, Oyola et al 2012, van Heesch et al 2013) as soon as it is necessary to obtain qualitative results. In the case of the vectors of the invention, where the targeted sequences are nucleolar (in total -4.16% of the genome), the results will strictly be able to answer only the following questions: 1 - Are the different combinations of vector sources, targeted or not, targeted in the nucleolar sequences?

 2 - Are targeted combinations more effective for targeting in nucleolar sequences?

The answers to these two questions are, however, necessary and sufficient to demonstrate the effectiveness of targeting.

Impact of computer analysis. A complete chain of DNA sequence library analysis describing insertion site populations obtained by LAM-PCR or IPCR is available (HASTI, Arens et al 2012). However, HASTI does not allow the automation of the sequence separation according to the tags (specific to each condition) and does not have a quality of locus mapping sufficient for our work. A homemade processing chain was created to process our data.

First, in steps 1 to 3, the following fragments are removed from the sequences: tag: 8 possibilities (acg cta gat tgc atc this gta tgc) - mandatory (the sequences that do not possess it are excluded from the subsequent processing)

· Primer: 2 possibilities (5 'or 3') - obligatory

 • fragment piggybac (PB): 1 possibility but different according to the type of primer (5 'or 3') - obligatory

 TSD: PB's canonical TSD is TTAA

 • the linker illuminates 3 ': may be present

Once these fragments are removed, the remaining sequences (the sequence flanking the insertion locus) are mapped to the human genome using bwa (Li and Durbin, 2010). The mapped loci are then filtered: only the loci for which the alignment of the sequence covers at least 95% of it are conserved. d - Results of the qualitative analysis of the targeting of vector prototypes

Assays were performed with both population types of sequences produced by IPCR and LAM-PCR from amplification made on the 5 'end of the vector. The results are presented in graphical form. For each vector prototype, the results are expressed in percentages of locus (black bars) and in percentages of mapped sequences (gray bars) in each of the 6 following nuclear categories: Nucleolasm, "Lamina-associated domains", "Nucleolus-associated domains", "rDNA repeats", "rDNA flanking regions" and Total Nucleol. It was considered that the two percentages should have the same tendency in the 4 trials to be taken into account. In reference, the percentages expected in each of the compartments were used if the transposition had taken place in a non-targeted manner and in an equivalent manner in all the nuclear compartments. The prototype vector totally targeted for its two components and whose marker gene depends on a nucleolar promoter (Hair) is indicated in italics. d1 - Control of the aptitude of our experimental approaches to detect tropisms of insertion of the piggyBac vectors according to the accessibility to the chromosomal DNA.

 LADs are nuclear compartments that contain highly heterochromatinized chromosomal domains. It is therefore expected that the genomic DNA regions contained in the LADS are less accessible to the integration of integrative vectors, in particular during the phases GO and G1 of the cell cycle. The insertion profiles of 6 vector prototypes were therefore first analyzed in this perspective (see Figure 6 and 7).

The first observation is that the six vector prototypes tend to significantly fit into the DNA contained in the nucleoplasm. The integration rates in the nucleoplasm are systematically higher than the expected rate by the simple effect of chance.

The second observation is that the six vector prototypes tend to significantly less fit into the DNA contained in the LADs.

These results were confirmed using not the amount of DNA present in each nuclear compartment, but the amount of potential insertion sites for our piggyBac (tetranucleotide "TTAA") vectors.

Proportions of nuclear categories / amount of DNA or number of TTAA motifs in the hg19 version of the human genome. % Compartments in gDNA% of motifs # of units Total nuclear density TTAA totals TTAA TTAA / Mpb

Nucleoplasm 59.0057 53.0563 38092044 12304.85

LADs 36.8351 43.5537 16590508 1 540.64

NADs 2.9292 3.0033 1144020 12608.90

Flanking rDNA 0.5359 0.1898 72290 4354.82 rDNA 0.6941 0.1969 75000 3488.45

Total Nucleolus 4,1591 3,3899 1291310 12028.86

 TABLE 7

The results obtained (see Figures 8 and 9) taking into account the number of TTAA sites by nuclear domain category confirm and even amplify the conclusions drawn from the first analysis on the difference in accessibility to integrative vectors between LADs and nucleoplasm. These controls made it possible to verify that the predictions made on the accessibility of the chromosomal DNA to integrative vectors as a function of its location in the nucleoplasm or the LADs are systematically verified in the results of our experimental approach. Integration rates are consistently lower than the expected random rate in the nucleoplasm. d2 - Presence of insertion in the genomic DNA contained in the set of nucleolar regions.

The nucleolar regions were represented by the "Nucleolus-associated domains", the "rDNA repeats", and the "rDNA flanking regions".

The analysis of the results (Figure 10) provided four pieces of information.

 1 - The genomic DNA contained in the nucleolus is difficult to access for an integrative vector, even if targeted.

2 - The integrative vector prototype whose two components are targeted in the nucleolus (pCS2-HIV-PB x pPol1 h-Neo-PNA) and whose expression of the marker is dependent on a nucleolar promoter (RNA polymerase I) is the most effective to integrate into the nucleolar sequences (relative ratio of 5 to 300 times, according to the test). 3 - A vector targeted or not in the nucleolus and containing a selection marker whose expression is placed under the control of a non-nucleolar nuclear promoter (pSV40 - RNA polymerase II) is incapable of being retained by the process of cell selection. This inability arises from the absence of RNA polymerase II in the nucleolus.

 4 - Transfections made with vectors whose two components are plasmidic would be a priori a little less effective for targeting in the nucleolar sequences. d3 - Nucleus regions accessible to integrative vectors.

For these analyzes, the nucleolar regions were subdivided into three categories: "Nucleolus-associated domains", "rDNA repeats", and "rDNA flanking regions". The analysis of the results (Figure 1 1) gives access to two information concerning the accessibility of the three categories of nucleolar regions to the integrative vectors used, the impact of the transfection method.

It is in the NAD (nucleolar region category) regions that the targeted vectors, whose selection marker depends on RNA polymerase I, fit best and at a higher level than other vector prototypes. In these sequences, the results obtained in IPCR suggest that the difference in the efficiency of the nucleolar targeting is more to the advantage of the targeted vector when the prototype consists of an mRNA and a plasmic than of two plasmid components.

The conclusions drawn from the results on the flanking chromosomal regions (FIG. 12) of the genes encoding the 18S-5.8S-28S ribosomal RNAs are identical to those of the NADs. Within the efficiency limits of the preparation of IPCR or LAM-PCR libraries of vector insertion sites, and the sensitivity of our in silico analyzes, the results (Figure 13) indicate that our vector prototypes are unable to access to the tandem repeat genes encoding 18S-5.8S-28S ribomosomal RNAs and their non-transcribed spacers. Three non-exclusive hypotheses make it possible to explain this phenomenon:

1 - The genes encoding ribosomal RNA 18S-5.8S-28S are much less accessible than other nucleolar sequences, because of their essential role in transcription by RNA polymerase I. 2 - The polymerase DNAs used in PCR are much less effective for amplification on DNA templates having a GC level of 60 to 80% than on matrices with a GC level of -45%. In fact, rDNA insertion site amplifications are dramatically underrepresented in the final products of IPCR and LAM-PCR.

In this perspective, an analysis of PCR amplification efficiency levels was carried out. The results (FIGS. 14 and 15) indicate that the IPCR in our experimental conditions amplifies the sequences homogeneously, regardless of their nucleolar location and the sample. However, the advantage of this quality of amplification is reduced by the fact that only 0.1 to 1% of the sequences produced do not come from artefactual amplification products. In contrast, the results indicate that LAM-PCR in our experimental conditions biased the sequences according to their nucleolar localization and the sample. This property is to be compared with the fact that the sequences recovered from LAM-PCR products are 70 to 99% mapping in silico.

3 - The composition in repetitive sequences (in particular in sequence Alu \ (300-bp) of the non-transcribed spacers (themselves at 60 to 80% of GC level) present between the transcribed regions in ribosomal RNA 18S-5.8S- 28S in the rDNA does not allow to map them correctly in silico On the other hand the exact sequence of the regions containing the repeated units coding for the rRNA is not known 4. Conclusions on the effectiveness of the targeted vectors

 Experimental results allow us to draw different conclusions.

The integrative vectors of the invention can be efficiently targeted in the nucleolar DNA.

 2 - The use of the targeted vectors can be done with two plasmid components or 1 mRNA and a plasmid.

5. Additional assays on the ability of the targeted vectors to integrate into the genes encoding 18S-5.8S-28S ribomosomal RNAs The impossibility of detecting vector insertions in the genes encoding ribosomal RNAs 18S-5.8S-28S may have two origins. The first would be that the vectors can not access these chromosomal sequences because they would be protected in the nucleoli and would require a specific mechanism of access to these sequences. The second is related to certain amplification properties of the polymerases used in PCR and which are to amplify with greater efficiency the richest sequences of AT at the expense of those rich in GC. The second hypothesis was tested using a LAM-PCR strategy targeted to the transcribed region of the genes encoding the 18S-5.8S-28S ribosomal RNAs of the specifically anchored oligonucleotides on the one hand in this region and, on the other hand, in the 5 'and 3' ends of the vectors used.

Characteristics of oligonucleotides

 Primers anchored in rDNAs: Each pair of primers has been defined to allow nested amplification in rDNAs. Thus in the 18S, the first amplification is carried out with the 18F or the 18R, the second being respectively made with 18S F2 and 18S R2.

18S R - SEQ ID NO.24

 5 'GCTGAACGCCACTTGTCCCTCTAAGAAGT 3'

18S F - SEQ ID No.25

 5 'GGACACGGACAGGATTGACAGATTGATAGC 3'

18S R2 - SEQ ID No.26

5 'GCTATCAATCTGTCAATCCTGTCCGTGTCC 3'

18S F2 - SEQ ID No.27

 5 'ACTTCTTAGAGGGACAAGTGGCGTTCAGC 3'

28S Rmid - SEQ I D No.28

5 'GGGCTAGTTGATTCGGCAGGTGAGTTG 3'

28S Fmid - SEQ ID No.29

5 'CTAGTAGCTGGTTCCCTCCGAAGTTTCCCT 3'

28S Rmid2 - SEQ I D No.30

5 'GGGAAACTTCGGAGGGAACCAGCTACTA 3' 28S Fmid2 - SEQ ID No.31

 5 'GTAACAACTCACCTGCCGAATCAACTAGCC 3'

28S Rend - SEQ I D No.32

5 'CGACCCAGAAGCAGGTCGTCTACGAAT 3'

28S Fend - SEQ ID No.33

 5 'GGCGAAGCTACCATCTGTGGGATTATGACTG 3'

28S Rend2 - SEQ I D No.34

5 'CAGTCATAATCCCACAGATGGTAGCTTCGC 3'

28S Fend2 - SEQ ID No.35

5 'CGCTAAACCATTCGTAGACGACCTGCTTCTG 3'

Primers anchored to the ends of the piggybac vector

ITR 5 '

 ITR5a - SEQ ID No.36 - 5 'GAATTCGTCGACATCGATACCAAAAGTTTTGTTAC 3'

ITR5b - SEQ I D No.37 - 5 'CCTCGATATACAGACCGATAAAACACATGCGTCA 3'

ITR 3 '

 ITR3a - SEQ ID No.38 - 5 'GCGGCGACTGAGATGTCCTAAATGCAC 3'

ITR3b - SEQ ID No.39 - 5 'GCGACGGATTCGCGCTATTTAGAAAGAGAG 3'

Samples used

 Two pairs of genomic DNA samples from non - targeted and targeted vectors were used: # 9 and # 10, and # 2 and # 14.

Amplification procedure of integrated vectors in rDNAs

at. Linear PCR (1 sample = 2 linear PCR in 5 'and 3')

Dilute the oligos (100 μΜ) to 1/100 = 1 μΜ 50 gDNA to 100 ng

 5X GC One Taq 10 μΐ buffer

dNTPs (10 mM each) 1 μΐ

 Primer (B) transposon I (1 μΜ) 4, 18 μΐ (83.5 nM)

 One Taq polymerase (NEB 51Ι / μΙ) 0.5 μί

H20 QSP 50 μΐ Notes:

 -Primer (B) transposon I

-Make a control with gDNA from non-transfected cells

95 ^° C 2 min

2 95 ^° C 45 sec l

3 60 ^° C 45 sec l x50

68 ^° C 6min

5 68 ^° C 10 min

Add 2.5 U (0.5 μΐ) of Taq polymerase per tube and repeat the entire PCR cycle b. Magnetic catch

 - Expose 20 μl of magnetic particles (10 μ / μΙ) (Dynabeads M-280 Streptavidin (Invitrogen, Carlsbad, USA) 60 seconds to the magnetic field of the Single Place Magnetic Stand (Invitrogen, Carlsbad, USA) at RT.

 - Remove the supernatant in the presence of the magnetic field.

 - Resuspend the particles in 40 μΐ PBS-0.1% BSA (pH 7.5). Remove the supernatant in the presence of the magnetic field. Repeat this step a second time. (=

2 washes magnetic beads in PBS-0.1% BSA)

 - Wash the particles with 20 μΐ LiCl 3M BW buffer (same magnetic procedure as washing with PBS-0.1% BSA). - Resuspend the beads in 50 μΐ of LiCl 6M BW buffer.

Note: From the following steps, proceed with the Side Skirted Magnetic device. - Transfer 50 μΐ of magnetic particle solution into each linear PCR product (the PCR product ratio and the LiCl solution should always be 1: 1). Mix gently.

 - Incubate on an agitator at 300 rpm at RT (between 20 ° C and 43 ° C) OVN (8h-48h).

- Expose the 60 sec sample to a magnetic field, remove the supernatant in the presence of the magnetic field and wash the beads once with 100 μl of distilled water. c - 1st Amplification

 Each linear PCR is amplified by 6 sets of oligonucleotides (1 end of the transposon and then 6 primers rDNA)).

 By samples, it is therefore expected to harvest 12 PCR products (6 in 5 'and 6 in 3'). With 4 samples we will have at this stage 48 PCR tubes. The first PCR is performed on 5 μί of linearized and purified DNA. Resuspend the beads in 70 μl of distilled water

Primer 1 = ITR5a or ITR3a

 Primer 2 = 18SF, 18SR, 28SFmid, 28SRMid, 28S Fend, 28SRend

Thermal Cycling Program

Hold at 94 ° C

X 35 cycles

6 min at 68 ° C 5min to 68 ° C

 Hold at 4 ° C

Gel control of the quality of the PCR products (5 ° l) with a gel 1.5% Agarose, 1 X TBE, 1X Gelred.

 vs. 2nd Amplification

 Each PCR tube has its primer mix nested

Primer 1 = ITR5b or ITR3b

 Primer 2 = 18SF2, 18SR2, 28SFmid2, 28SRMid2, 28S Fend2, 28SRend2

 Thermal Cycler Program

 Hold at 94 ° C

X 35 cycles

 6 min at 68 ° C

 5min to 68 ° C

 Hold at 4 ° C

Gel control of the quality of the PCR products (5 μl) with a gel 1.5% Agarose, TBE, 1X Gelred. d. Purification of PCR products and sequencing

For each pair of primers, a comparison of the amplification products was performed between samples 9 and 10 on the one hand and samples on the other hand 12 and 14. The amplification fragments present in the products from samples 10 or 14, respectively absent in the products from samples 9 or 12, were purified and sequenced by a provider by the Sanger method. e. Results and conclusion

 Thirty amplification products have been sequenced. Their sequence has demonstrated that the vector is capable of integrating into different TTAA motifs contained in the genes encoding the ribosomal RNAs 18S-5.8S-28S.

These results indicate that the target vectors in the nucleolus with the invention are capable of integrating into all nucleolar sequences.

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Claims

1. Molecular system for integrating a nucleic acid molecule of interest into a chromosomal region associated with the nucleolus, comprising at least:  a non-viral integrative vector comprising a nucleolar localization motif and lacking a source of functional recombinase; and  a functional recombinase source comprising a nucleolar localization motif, provided in trans of said non-viral integrative vector. 2. System according to claim 1, characterized in that the non-viral integrative vector is a pseudo transposon. System according to claim 1 or 2, characterized in that said non-viral integrative vector and said functional recombinase source is localized to the nucleolus comprising a nucleolar locating motif. System according to any one of claims 1 to 3, characterized in that said nucleolar localization pattern is a nucleolar locating motif NoLS. 5. System according to any one of claims 1 to 4, characterized in that it further comprises a connecting element of said location signal to said integrative vector non-viral. 6. System according to claim 5, characterized in that said linking element is a triplex forming oligonucleotide (OFT), preferably selected from the group of PNAs (peptide nucleic acids), LNAs ( locked nucleic acids, "locked nucleic acids"), PNA hybrids: PNA ("bisPNA") and LNA hybrids: LNA ("Zorro LNA"). 7. System according to claim 6, characterized in that said connecting element is a bisPNA. 8. System according to any one of claims 1 to 7, characterized in that said non-viral integrative vector is a pseudo-transposon selected from the transposons Sleeping Beauty, Prince Frog, piggyBac, MuA, Mos-1, Pokey, Tol2 , Tn3, Tn5, Tn7, Tn10, Hermes and their derivatives. 9. System according to any one of claims 4 to 8, characterized in that said nucleolar localization signal NoLS is a peptide of sequence SEQ ID No.1: RQARRNRRRRWRERQRQ.I. The system of claim 9, characterized in that said bisPNA link member coupled to said NoLS location signal is sequenced by the sequence: KKKLLTTCTTCTTTTLLLTTTTCTTCTTLLLKKKRQARRNRRRRWRERQRQI, wherein K, L, I, R, Q, A, N, W and E are amino acids, C and T are nucleotides. 1 1. In vitro method for genetically modifying at least one eukaryotic host cell, comprising:  a) introducing into said cell at least one system according to one of claims 1 to 10;  b) selecting cells of step a), in which the nucleic acid molecule of interest is expressed;  c) selecting cells from step b), wherein the non-viral integrative vector is integrated into a chromosomal region associated with the nucleolus. 12. Eukaryotic host cell genetically modified, characterized in that it contains at least one system according to any one of claims 1 to 10. A genetically modified eukaryotic host cell obtainable by the process according to claim 11, characterized in that at least one nucleic acid molecule of interest is integrated into a chromosomal region associated with the nucleolus. 1. Non-human transgenic eukaryotic organism, in particular a transgenic animal, of which at least one cell is a cell according to claim 12 or 1 3. A kit for integrating at least one nucleic acid molecule of interest into the genome of a eukaryotic host cell for expression thereof, characterized in that it comprises at least: a molecular system according to any one of claims 1 to 10; and / or a host cell according to claim 12 or 13; and or a transgenic non-human eukaryotic organism according to claim 1.