New peptides and use thereof as delivery systems for internalization of molecules of interest into target cells

Abstract

The invention relates to new cell-penetrating peptides comprising or consisting of an amino acid sequence selected from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The invention further relates to fusion proteins comprising said peptides and a molecule of pharmaceutical, diagnostic or biotechnological interest.

Description

Description Title of the invention: Novel peptides and their use as transporters for the internalization of molecules of interest into target cells Technical field of the invention

[0001] The invention relates to the field of peptide transporters for the internalization of molecules of interest into target cells. It relates more particularly to new peptides or nucleic acids encoding such peptides, and their uses as transporters for the internalization of molecules of interest into target cells. It also relates to the combination of such peptides with molecules of interest, in particular polypeptides of interest, and the use of such a combination in the treatment, diagnosis or prevention of pathologies, in particular cancer.The invention further relates to a fusion protein comprising a novel peptide having the property of cell penetration (called "cell-penetrating peptide" or "CPP") which is the subject of the present invention and a polypeptide of interest, as well as a nucleic acid encoding such a fusion protein, an expression vector comprising nucleic acids encoding such a fusion protein, but also a host cell comprising such an expression vector. Finally, the invention also relates to a pharmaceutical composition comprising a novel peptide which is the subject of the present invention and a polypeptide of interest. Prior art

[0002] Biotechnology-derived drugs, also known as biomedicines or biological drugs, play an important role in the treatment of human pathologies. They include therapeutic proteins (enzymes, growth hormones, monoclonal antibodies, growth factors, recombinant vaccines, etc.), nucleic acids (DNA, siRNA, mRNA, oligonucleotides, etc.), peptides and derivatives such as peptide nucleic acids (PNA).

[0003] In some cases, it is necessary to use transporters to internalize these biomedicines into target cells. The internalization of therapeutic molecules into cells is the subject of much research with the aim of finding new transporters, improving the efficiency of known transporters but also of the therapeutic molecules transported, improving their targeting at the cellular level and reducing the effective dose of certain treatments in order to reduce the risk of toxicity.

[0004] Several families of transporter peptides have already been identified. These natural or synthetic peptides, called PTD (for "Protein Transduction Domain" in English terminology) or CPP (for "Cell-Penetrating Peptides" in English terminology), have the ability to transport and transfer molecules such as peptides or nucleic acids into cells using various possible cellular internalization mechanisms such as endocytosis, macropinocytosis or the formation of membrane pores, for example.

[0005] In their 2008 publication “Expression and purification of Zebra Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells” in the journal Current Protocols in Protein Science, 54: 18.11.1 - 18.11.29, Lenormand and Rothe describe a method for producing fusion proteins comprising a segment of the ZEBRA protein and either eGFP or β-galactosidase. These fusion proteins are capable of being internalized into HeLa cells at a concentration of 0.01 pM to 0.3 pM.

[0006] The ZEBRA protein is a transcriptional activator derived from the Epstein-Barr virus. It is a 245-amino acid protein comprising an N-terminal transactivation region (TAD), a DNA-binding domain (DBD), and a leucine zipper dimerization region (DIM). The C-terminal domain of the protein interacts with the leucine zipper, leading to the formation of a hydrophobic pocket that stabilizes the ZEBRA protein / DNA complex.

[0007] Until now, the internalization pathways used by transport peptides, known to those skilled in the art, such as endocytosis and macropinocytosis, required significant energy expenditure in order to achieve this intracellular penetration mechanism. In addition, the mechanism Endocytosis leads to the internalization of molecules via endosomes, the contents of which undergo partial degradation. Only a small fraction of the transported peptides is released into the cytosol, after rupture of the endosome membrane, allowing them to exert their action at the intracellular level. Therefore, on an industrial production scale, in order to ensure the efficiency of transduction of polypeptides of interest, it is necessary to produce a large quantity of transporter and polypeptides of interest. This sometimes requires a cumbersome production or purification procedure that is not feasible for all types of polypeptides of interest. Furthermore, for certain polypeptides of interest, it is necessary to limit the quantity administered due to the risk of toxicity. It therefore appears necessary to use transport polypeptides that are more efficient in their capacity for cellular penetration in order to limit these risks.

[0008] Patent application WO2011135222 discloses the use of a peptide fragment from the ZEBRA protein (from amino acid position 170 to amino acid position 220) as a carrier peptide for internalizing polypeptides of interest at low concentrations and avoiding degradation of these polypeptides of interest by a direct penetration mechanism independent of endosomes.

[0009] The publication “MD11 mediated delivery of recombinant elF3f induces melanoma and colorectal carcinoma cell death” by R. MARCHIONE et al. in 2015 discloses the use of a complex formed by the elF3f protein fused with a cell-penetrating peptide to increase the available intracellular amount of elF3f protein in cancer cells and activate their apoptosis. The cell-penetrating peptide used is a sequence fragment of the ZEBRA protein (from amino acid position 178 to amino acid position 220) called MD11.

[0010] There is still a need today to find new carrier peptides with improved cellular penetration potential and easier production, which should enable molecules of interest to be transported at low concentration, with high efficiency and better affinity into target cells while ensuring good stability and low toxicity in said cells. Presentation of the invention

[0011] The present invention aims to provide new peptides as transporters intended for the internalization of molecules of interest in target cells. These new peptides are mutants of a peptide fragment of the ZEBRA protein. This fragment, the sequence of which is the sequence SEQ ID NO: 1 corresponding to the peptide sequence of the ZEBRA protein ranging from the amino acid in position 178 to the amino acid in position 220, is known and will be called MD11 in the remainder of this description. It is a cell-penetrating peptide (CPP) also called a “transport peptide” or “transport peptide” or more simply a “transporter” or “transport vector” (“Delivery system” in English terminology).

[0012] To this end, according to a first aspect, the present invention relates to a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0013] Such peptides can be described as transporter peptides and are intended for the internalization of molecules of interest into target cells. They have the advantage of having a cell penetration capacity at least equivalent to or greater than that of the transport peptide MD11. They are also present in cells even when they are treated with low concentrations of the order of nanomolar, which corroborates a direct and unmediated penetration into cells for the molecules they can transport. Finally, no toxicity of the peptides alone for the target cells could be demonstrated.

[0014] Cell penetration capacity refers to the efficiency of internalization of molecules of interest, i.e. the percentage of treated cells containing the molecules of interest in their cytoplasm. This internalization efficiency is based on the detection of the molecules of interest in the transduced cells by means, for example, of microscopy or flow cytometry or any other imaging techniques allowing the visualization of internalization at the molecular level, for example, the transfer of energy by resonance of bioluminescence (BRET for "Bioluminescence Resonance Energy Transfer" in Anglo-Saxon terminology), Fôrster resonance energy transfer (FRET for "Forster Resonance Energy Transfer" in Anglo-Saxon terminology), electron microscopy, atomic force microscopy (AFM for "Atomic Force Microscopy" in Anglo-Saxon terminology), optogenetics,....

[0015] The term "transporter" refers to a molecule capable of transporting and transferring another different molecule across the plasma membrane to allow it to enter the cell.

[0016] The expression "internalization of a molecule of interest into target cells" refers to the passage of a molecule of interest from outside a target cell to inside it.

[0017] According to a second aspect, the present invention relates to a nucleic acid molecule encoding a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0018] The nucleic acid sequences encoding the peptides represented by the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 as described above can be deduced from these amino acid sequences according to the principle of degeneracy of the genetic code known to those skilled in the art.

[0019] According to a third aspect, the present invention relates to an expression vector comprising a nucleic acid molecule encoding a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. Such an expression vector may be of any type known per se for implementation in genetic engineering, in particular a plasmid, a cosmid, a virus, a bacteriophage, containing the elements necessary for the transcription and translation of the sequence encoding the peptide according to the invention.

[0020] According to a particular embodiment of the present invention, the vector further comprises the genetic engineering elements, in particular the origins replication and promoters, allowing to control the autonomous replication of the vector in the host organism and the specific expression of the peptides as described above.

[0021] According to a fourth aspect, the present invention relates to a host cell comprising a nucleic acid molecule encoding a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or an expression vector comprising such a nucleic acid molecule. The host cells may be, for example, prokaryotic cells such as E. co t or Bacillus, or eukaryotic cells such as yeasts, in particular Saccharomyces cerevisiae and Pichia pastoris, filamentous fungi, in particular Trichoderma reesei and Aspergillus niger, insect cells using Baculoviruses, or cell lines such as CHO, HEK 293, Cos or Per.C6.

[0022] According to a fifth aspect, the present invention relates to the use of a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or of a nucleic acid molecule coding for such a peptide or of an expression vector comprising such a nucleic acid molecule or of a host cell comprising such a nucleic acid molecule or such an expression vector, for obtaining a transporter intended for the internalization of a molecule of interest in target cells.

[0023] The cells that may be the target cells of an internalization process implemented by a transporter that is the subject of the present invention are preferably chosen from eukaryotic cells, in particular human cells. These human cells may be tumor cells, such as melanoma cells, breast cancer cells, glioblastoma cells, colon cancer cells, lymphoma cells. These human cells may also be normal cells including fibroblasts, epithelial cells, lymphocytes, dendritic cells, muscle cells such as myocytes, myoblasts and myotubes for example.... These cells Human cells can also be differentiated somatic cells that will be brought to dedifferentiation to form induced pluripotent stem cells (iPSCs). To target certain cell lines, peptide sequences such as guide peptides or targeting peptides (respectively "homing peptides" and "target peptides" in English terminology), nuclear localization signals or NLS (for "nuclear localization signal" in English terminology), can be grafted onto the transporter according to the invention.

[0024] According to a sixth aspect, the present invention relates to a combination comprising a transporter and a molecule of interest, said transporter being a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0025] In particular embodiments of the present invention, the molecule of interest is preferably a molecule of biotechnological, diagnostic or therapeutic interest. Such a molecule of interest is preferably chosen from polypeptide, DNA, RNA, oligonucleotides, siRNA, shRNA miRNA, antisense RNA, or peptide nucleic acids (PNA).

[0026] According to a seventh aspect, the present invention relates to a fusion protein comprising a transporter which is a peptide comprising or consisting of an amino acid sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and a molecule of interest which is a polypeptide of interest.

[0027] A "fusion protein" means a recombinant or synthetic polypeptide containing at least two peptides, derived from two different proteins, one linked to the other directly by peptide bond, or by a peptide linker, for example GSGG. The polypeptide of interest may be linked to the N-terminal or C-terminal part of the transporter.

[0028] According to a preferred embodiment of the present invention, the fusion protein comprises or consists of an amino acid sequence selected from the sequences SEQ ID NO: 23 to SEQ ID NO: 29, SEQ ID NO: 31 to SEQ ID NO: 37, SEQ ID NO: 39 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 53 and SEQ ID NO: 55 to SEQ ID NO: 61.

[0029] According to an eighth aspect, the present invention relates to a nucleic acid molecule coding for the fusion protein which is the subject of the present invention.

[0030] According to a ninth aspect, the present invention relates to an expression vector comprising a nucleic acid molecule coding for the fusion protein which is the subject of the present invention. Such an expression vector may be of any type known per se for implementation in genetic engineering, in particular a plasmid, a cosmid, a virus, a bacteriophage, containing the elements necessary for the transcription and translation of the sequence coding for the fusion protein according to the invention.

[0031] According to a particular embodiment of the present invention, the vector further comprises the genetic engineering elements, in particular the origins of replication and the promoters, making it possible to control the autonomous replication of the vector in the host organism and the specific expression of the fusion proteins as described above.

[0032] According to a tenth aspect, the present invention relates to a host cell comprising a nucleic acid molecule encoding the fusion protein which is the subject of the present invention or an expression vector comprising such a nucleic acid molecule. The host cells may be, for example, prokaryotic cells such as E. co t or Bacillus, or eukaryotic cells such as yeasts, in particular Saccharomyces cerevisiae and Pichia pastoris, filamentous fungi, in particular Trichoderma reesei and Aspergillus niger, insect cells using Baculoviruses, or cell lines such as CHO, HEK 293, Cos, Per.C6.

[0033] In particular embodiments of the combination or fusion protein that is the subject of the present invention, the molecule of interest is linked to the transporter by a covalent or non-covalent bond, such as an ionic bond, a hydrogen bond, or a hydrophobic bond. When the molecule of interest is a polypeptide of interest, according to an advantageous embodiment of the invention, the polypeptide of interest is linked to the transporter according to the invention by a direct peptide bond.

[0034] According to an eleventh aspect, the present invention relates to a pharmaceutical composition comprising a combination or a fusion protein which is the subject of the present invention. Preferably, the composition comprises a pharmaceutically acceptable excipient and / or vehicle. The choice of a excipient and / or a pharmaceutically acceptable vehicle is known to those skilled in the art.

[0035] According to a twelfth aspect, the present invention relates to the combination or fusion protein or pharmaceutical composition which is the subject of the present invention for its use in the treatment, diagnosis or prevention of cancers such as melanomas, breast cancer, brain tumors, glioblastomas, colon cancer, lymphomas.

[0036] For any of the aspects of the invention cited above, according to particular embodiments of the invention, the molecule of interest comprises or consists of a polypeptide of interest chosen from a polypeptide encoding the eGFP protein represented by the sequence SEQ ID NO: 17, a polypeptide encoding the elF3f protein represented by the sequence SEQ ID NO: 18, a polypeptide encoding the FERM protein represented by the sequence SEQ ID NO: 19, a polypeptide encoding all or part of the MDA-7 protein represented respectively by the sequences SEQ ID NO: 20 (entire MDA-7 protein) and SEQ ID NO: 21 (truncated MDA-7 protein) and a polypeptide represented by a sequence having 80%, in particular 90%, particularly 95% sequence identity with one of the sequences SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0037] The percent identity of peptide sequences is determined by direct comparison of two polypeptide molecule sequences, determining the number of identical amino acid residues in the two sequences, then dividing it by the number of amino acid residues in the longer sequence of the two, and multiplying the result by 100.

[0038] The nucleic acid sequences encoding the polypeptides represented by the sequences SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 as described above can be deduced from these amino acid sequences of the peptides according to the principle of degeneracy of the genetic code known to those skilled in the art. Brief description of the figures

[0039] The invention will be better understood by reading the following description, given as a non-limiting example, and made with reference to the figures which represent:

[0040] [Fig. 1] Figure 1 illustrates a histogram showing the percentages of fluorescent HEK, B16-Ova and beta-TC-6 cells after treatment with fluorescent fusion proteins which are the subject of the present invention;

[0041] [Fig. 2] Figure 2 illustrates images of HEK and B16-Ova cells observed under a fluorescence microscope after treatment with fluorescent fusion proteins which are the subject of the present invention;

[0042] [Fig. 3] Figure 3 illustrates viability curves of B16-Ova and HEK cells as a function of the treatment dose after 24h and 48h allowing the calculation of the IC50 of the potentially therapeutic fusion proteins which are the subject of the present invention. Description of the embodiments

[0043] In the remainder of the description, the nucleic acid sequences encoding the peptides and polypeptides represented by the amino acid sequences as described below can be deduced from these amino acid sequences according to the principle of the degeneracy of the genetic code known to those skilled in the art.

[0044] Production of MDmut and MD11 plasmids

[0045] From the original sequence of the cell penetrating peptide (CPP) MD11, the inventors created 7 sequence modifications numbered from #2 to #8 depending on the distance from the original sequence: - SEQ ID NO: 2 called MDmutl DelCys which corresponds to the sequence KRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLLK, - SEQ ID NO: 3 called MDmut2 which corresponds to the sequence RREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLLLK, - SEQ ID NO: 4 called MDmut2DelCys which corresponds to the sequence RREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLLK, - SEQ ID NO: 5 called MDmut3 which corresponds to the sequence KRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHKLEQL, - SEQ ID NO: 6 called MDmut3DelCys which corresponds to the sequence KRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKLEQL, - SEQ ID NO: 7 called MDmut4 which corresponds to the sequence RREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHKLEQL, - SEQ ID NO: 8 called MDmut4DelCys which corresponds to the sequence RREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKLEQL.

[0046] The DNA fragments encoding each MDmut mutant CPP are obtained by PCR and inserted into the pET15b expression vector which allows expression in bacteria of the peptides whose N-terminal end is linked to a 6-histidine tag, all plasmids being constructed according to the same organization of genetic engineering elements surrounding the CPP sequence.

[0047] The MD11 sequence numbered #1 and corresponding to the sequence KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQ (SEQ ID NO: 1) is also inserted into an expression plasmid pET_15b comprising the same organization of genetic engineering elements as the plasmids comprising the MDmut mutants.

[0048] The organization of the genetic engineering elements is as follows in each expression plasmid in the 5' to 3' direction: Ndel Res Site - His Tag - TEV seq - MMP2 cleavage site - MD Seq - Xhol Res Site in which: Ndel Res Site = Ndel restriction site His Tag = polyhistidine tag TEV seq = Tev protease cleavage sequence MMP2 cleavage site = metalloproteinase 2 cleavage sequence (tumor targeting) MD Seq = the MD11 or MDmut sequence Xhol Res Site = Xhol restriction site

[0049] All plasmids were verified by double-direction sequencing at the insertion sequence level of the genetic elements encoding the fusion proteins of interest.

[0050] With this organization of genetic engineering elements, the following sequences of cell penetrating peptides (CPP) are obtained: - SEQ ID NO: 9 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQ for the CPP comprising the sequence MD11, - SEQ ID NO: 10 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYREV AAAKSSENDRLRLLLK for the CPP including the sequence MDmutl DelCys, - SEQ ID NO: 11 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLK for the CPP including the Mdmut2 sequence, - SEQ ID NO: 12 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLK for the CPP comprising the sequence Mdmut2DelCys, - SEQ ID NO: 13 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQL for the CPP comprising the sequence Mdmut3, - SEQ ID NO: 14 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQL for the CPP comprising the sequence Mdmut3DelCys, - SEQ ID NO: 15 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQL for the CPP comprising the sequence Mdmut4, - SEQ ID NO: 16 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLISE IDLLRKQNEQLKHKLEQL for the CPP comprising the sequence Mdmut4DelCys.

[0051] Production of plasmids of the polypeptides of interest

[0052] To obtain the sequences of the reporter molecules of interest, here polypeptides of the eGFP proteins (for "enhanced green fluorescent protein" in English terminology) for biotechnological use and elF3f (for "eukaryotic Translation Initiation Factor 3 subunit F" in English terminology), FERM (corresponding to the FERM domain) and MDA-7 (for "Melanoma Differentiation Associated 7" in English terminology) whole or truncated, also known as IL-24, for therapeutic use, to be fused with our CPP-mutants Mdmut, 5 plasmids, called donors, containing the inserts of interest whose sequences of the polypeptides of interest are as follows were used: - SEQ ID NO: 17 corresponding to the polypeptide MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTKFIC TTGKLPVPWPTLVTTLTYGVQCFSRYPDDHMQHDFFKSAMPEGYVQE RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK of the eGFP protein, - SEQ ID NO : 18 corresponding to the polypeptide ATPAVPVSAPPATPTPVPAAAPASVPAPTPAAAPVPAAAPASSSDPA AAAAAATAAPGQTPASAQAPAPALPGPALPGPFPPGGRWRLHPVI LASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHNESEDEVA VDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIHEYYSREAPN PIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTPLTVKYAYDTE RIGVDLIMKTCCFSPNRVIGLSSDDLQQVGGASARIQDALSTVLQYAEDVLS GKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNSNINDLLMVTYLANLT QSQIALNEKLVNL of the elF3f protein, - SEQ ID NO : 19 correspondant au polypeptide MPKPINVRVTTMDAELEFAIQPNTTGKQLFDQWKTIGLREVWYFGLHY VDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVAEELIQDIT QKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNKEVHKSGYL SSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDNAMLEYLKIA QDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDDKLTPKIGFPWSEI RNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLCMGNHELYMRRR KPDTIEVQQMKAQAREEKHQKQLER de la protéine FERM, - SEQ ID NO: 20 corresponding to polypeptide NFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSG AQGQEFHFGPCQVKGWPQKLWEAFWAVKDTMQAQDNNTSCRLLQQ EGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSFSTLANN FVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGE VDILLTWMQKFYKL de la protéine MDA-7, - SEQ ID NO: 21 corresponding to polypeptide GQEFHFGPCQVKGWPQKLWEAFWAVKDTMQAQDNITSARLLQQEVL QNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLI VSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDIL LTWMQKFYKL of the MDA-7 tronquée protein.

[0053] The inventors have these sequences (SEQ ID NO: 17 to SEQ ID NO: 21) flanked by the Xhol and BamHI restriction sites in a plasmid pET 15b. They were previously sequenced using the commercial primer pET_RP and their restriction profile was also analyzed by single and double digestions to ensure the possibility of extracting them without impacting the sequence that will have to be translated.

[0054] Creation of the plasmid bank

[0055] First, the 8 Mdmut plasmids and the 5 donor plasmids of the polypeptides of interest were transformed into E. coli XL10 Gold bacteria. For this, 25 μl of bacteria were transformed with 50 ng of plasmids by heat shock 30 min in ice then 40 sec at 42°C before passage in ice. This step was followed by liquid culture in 100 μL of SOC culture medium at 37°C for 1 h then plating on Petri dishes containing LB (Lysogeny Broth) - Agar medium - Ampicillin and incubation at 37°C overnight. The next day, an isolated colony was inoculated into 5 ml of LB-Ampicillin medium and incubated at 37°C - 150 rpm overnight. 2 ml of culture were then centrifuged at 11000g and the plasmids were purified and eluted in 25 pL of water using the Macherey Nagel Miniprep kit according to the manufacturer's protocol.

[0056] The plasmids thus obtained were prepared for subcloning after double digestion with the restriction enzymes Xhol and BamHI. 2 pg of plasmid were digested with 5 units of each restriction enzyme for 3 hours at 37°C. Migration of the digestion products in a 0.6% agarose gel - TAE 0.5X - Gel Red 1 X for 1 hour 30 minutes at 75V was followed by purification of the bands of interest using the Macherey Nagel PCR and Gel clean Up kit according to the manufacturer's protocol.

[0057] The plasmids MDmut and MD11, which can be described as recipients, gave their skeleton containing the CPP-mutant or MD11 sequences and the plasmids comprising the polypeptides of interest, described as donors, gave their insert containing the sequence of the polypeptides of interest.

[0058] The inserts corresponding to the polypeptides of interest could be subcloned into each of the 8 MDmut plasmids, thus creating a new library of 40 recombinant plasmids allowing the synthesis of fusion proteins. For this, a ligation was carried out with 3 molar fold of each insert of polypeptides of interest and 50 ng of mutant plasmid using a T4 DNA ligase in 10 min at 22°C. This step was followed by the transformation of 25 pL of XL10 Gold bacteria with half of the ligation product according to the same protocol as previously mentioned.

[0059] Colonies transformed by recombinant plasmids were preselected using colony PCR. To achieve this, a ready-to-use 2X commercial enzyme was used by diluting 10 μL of forward and reverse primers by half in sterile water. All colonies in the dish were picked with a cone to contaminate 20 μL of PCR mix before starting the thermocycler according to a program adapted to the primers (Lid = 110°C; 1: 3 min - 94°; 2: 30 sec - 94°; 3: 30 sec - 60°C; 4: 1 min - 72°C; 5: GOTO 2 x 25 repeats; 6: 5 min - 72°C; 7: Hold - 16°C). Subsequently, the migration of the PCR products in a 1.5% agarose gel - TAE 0.5X - Gel Red 1X for 40 min at 100V was carried out.

[0060] After migration of the PCR products on agarose gel, the samples from the colony pickings were compared to the positive control samples (donor plasmid) or negative control (water). One or two colonies having given an amplicon of size and appearance comparable to the positive control were then cultured in order to amplify and purify the plasmids using a commercial "miniprep" kit to send them to double-direction sequencing (T7 and pET_RP primers) to verify the alignment of their sequence with the theoretical sequence.

[0061] New E. coli XL10 Gold bacteria were then retransformed from the plasmid samples validated by sequencing. This new transformation allowed the constitution of a glycerol stock (freezing at -80°C of 750pJ of overnight culture of XL10 bacteria with 25% sterile glycerol) and a stock of concentrated recombinant plasmids, purified by the use of the Macherey Nagel "midiprep" kit according to the manufacturer's protocol.

[0062] In each recombinant plasmid the organization of the genetic engineering elements is as follows in the 5' to 3' direction: Ndel Res Site - His Tag - TEV seq - MMP2 cleavage site - MD Seq - Xhol - PolyPep - BamHI in which: Ndel Res Site = Ndel restriction site His Tag = polyhistidine tag TEV seq = Tev protease cleavage sequence MMP2 cleavage site = metalloproteinase 2 cleavage sequence (tumor targeting) MD Seq = the MD11 sequence (SEQ ID NO: 1) or an MDmut sequence (SEQ ID NO: 2 to SEQ ID NO: 8) XhoI = XhoI restriction site PolyPep = the sequence of the polypeptide of interest (SEQ ID NO: 17 to SEQ ID NO: 21) BamHI = BamHI restriction site

[0063] The following sequences of fusion proteins encoded by the recombinant plasmids thus created are thus obtained: - SEQ ID NO: 22 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQLEMVSKGEELFTGVVPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITLGMDELYK for the CPP (SEQ ID NO: 9) to comprenant the genetic elements and the sequence MD11, En fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 23 to correspond in the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYREV AAAKSSENDRLRLLLKLEMVSKGEELFTGVVPILVELDGDVNGHKFSVS GEGEGDATYGKLTLKFICTTGKLPVPWPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV TAAGITLGMDELYK for the CPP (SEQ ID NO: 10) to comprenant the genetic elements and the sequence MDmutl DelCys in fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 24 to correspond in the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLKLEMVSKGEELFTGWPILVELDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITLGMDELYK for the CPP (SEQ ID NO: 11) Compare the genetic elements and the sequence MDmut2 in Fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 25 to correspond in the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLKLEMVSKGEELFTGVVPILVELDGDVNGHKFSVS GEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV TAAGITLGMDELYK pour le CPP (SEQ ID NO : 12) comprenant les éléments de génie génétique et la séquence MDmut2DelCys in fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 26 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQLLEMVSKGEELFTGWPILVELDGDVNGHKF SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK for the CPP (SEQ ID NO: 13) comprising the genetic engineering elements and the MDmut3 sequence in fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 27 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQLLEMVSKGEELFTGWPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITLGMDELYK for the CPP (SEQ ID NO : 14) comprenant The generation elements and the sequence MDmut3DelCys in fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 28 to correspond in the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQLLEMVSKGEELFTGWPILVELDGDVNGHKF SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK pour le CPP (SEQ ID NO : 15) comprenant les éléments de génie génétique et la Séquence MDmut4 into fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 29 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLISE IDLLRKQNEQLKHKLEQLLEMVSKGEELFTGWPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITLGMDELYK for CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence in fusion with the eGFP protein (SEQ ID NO: 17). - SEQ ID NO: 30 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQLEATPAVPVSAPPATPTPVPAAAPASVPAPT PAPAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALP GPALPGPFPGGRVVRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKH SVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYAT GHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPG RTMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGG ASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDF ETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 31 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYREV AAAKSSENDRLRLLLKLEATPAVPVSAPPATPTPVPAAAPASVPAPTPA PAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGP ALPGPFPGGRWRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSV EVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGH DITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRT MGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGAS ARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFET MLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 10) to comprenant the genetic elements and the sequence of MDmutl DelCys in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 32 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLKLEATPAVPVSAPPATPTPVPAAAPASVPAPTP APAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPG PALPPGPFPGGRVVRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHS VEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATG HDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGR TMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGA SARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFE TMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 11) to comprenant the genetic elements and the sequence MDmut2 in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 33 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLKLEATPAVPVSAPPATPTPVPAAAPASVPAPTPA PAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGP ALPGPFPGGRWRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSV EVTNCFSVPHNESEDEVAVDMMEFAKNMYELHKKVSPNELILGWYATGH SEQ ID NO: 12) Comprenent les Genetic elements and the sequence MDmut2DelCys by fusion with the protein elF3f (SEQ ID NO: 18). - SEQ ID NO: 34 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQLLEATPAVPVSAPPATPTPVPAAAPASVPAP TPAPAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPAL PGPALPGPFPGGRWRLHPVILASIVDSYERRNEGAARVIGTLLGTVDK HSVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYA TGHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVP GRTMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVG GASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDD FETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 13) comprising the genetic engineering elements and the MDmut3 sequence in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 35 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQLLEATPAVPVSAPPATPTPVPAAAPASVPAPT PAPAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALP GPALPGPFPGGRWRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKH SVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYAT GHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPG RTMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGG ASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDF ETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 14) comprising the genetic engineering elements and the MDmut3DelCys sequence in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 36 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQLLEATPAVPVSAPPATPTPVPAAAPASVPAP TPAPAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPAL PGPALPGPFPGGRWRLHPVILASIVDSYERRNEGAARVIGTLLGTVDK HSVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYA TGHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVP GRTMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVG GASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDD FETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO : 15) comprising the genetic engineering elements and the MDmut4 sequence in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 37 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLISE IDLLRKQNEQLKHKLEQLLEATPAVPVSAPPATPTPVPAAAPASVPAPT PAPAAAPVPAAAPASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALP GPALPGPFPGGRVVRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKH SVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYAT GHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPG RTMGVMFTPLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGG ASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDF ETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence in fusion with the elF3f protein (SEQ ID NO: 18). - SEQ ID NO: 38 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQLEPKPINVRVTTMDAELEFAIQPNTTGKQLFD QVVKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFK FRAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAV QAKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHA EHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLN IYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINK RILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 39 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYREV AAAKSSENDRLRLLLKLEPKPINVRVTTMDAELEFAIQPNNTTGKQLFDQV VKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFR AKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQA KFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEH RGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIY EKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRI LQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 10) to comprenant the genetic elements and the sequence of MDmutl DelCys in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 40 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLKLEPKPINVRVTTMDAELEFAIQPNNTTGKQLFDQ WKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKF RAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQ AKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAE HRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNI YEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINK RILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 11) to comprenant the genetic elements and the sequence MDmut2 in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 41 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLKLEPKPINVRVTTMDAELEFAIQPNNTTGKQLFDQV VKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFR AKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQA KFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEH RGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIY EKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRI LQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for le CPP (SEQ ID NO : 12) comprenant the genetic elements and the sequence MDmut2DelCys in fusion with the protein FERM (SEQ ID NO : 19). - SEQ ID NO: 42 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQLLEPKPINVRVTTMDAELEFAIQPNNTTGKQLF DQWKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQF KFRAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYA VQAKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWH AEHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGL NIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRIN KRILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 13) to comprenant the genetic elements and the sequence MDmut3 in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 43 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQLLEPKPINVRVTTMDAELEFAIQPNNTTGKQLFD QVVKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFK FRAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAV QAKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHA EHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLN IYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINK RILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 14) to comprenant the genetic elements and the sequence MDmut3DelCys in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 44 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQLLEPKPINVRVTTMDAELEFAIQPNNTTGKQLF DQWKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQF KFRAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYA VQAKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWH AEHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGL NIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRIN KRILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for CPP (SEQ ID NO: 15) comprising the genetic engineering elements and the MDmut4 sequence in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 45 which corresponds to the sequence MMHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLI SEIDLLRKQNEQLKHKLEQLLEPKPINVRVTTMDAELEFAIQPNTTGKQL FDQWKTIGLREVWYFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQ FKFRAKFYPEDVAEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSY AVQAKFGDYNKEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVW HAEHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDAL GLNIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRL RINKRILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence in fusion with the FERM protein (SEQ ID NO: 19). - SEQ ID NO: 46 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQLENFQQRLQSLWTLARPFCPPLLATASQMQ MVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGWPQKLWEAFWA VKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKN YHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLL FRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence in fusion with the MDA-7 protein (SEQ ID NO: 20). - SEQ ID NO: 47 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYREV AAAKSSENDRLRLLLKLENFQQRLQSLWTLARPFCPPLLATASQMQMV VLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKD TMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutl DelCys sequence in fusion with the MDA-7 protein (SEQ ID NO: 20). - SEQ ID NO: 48 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLKLENFQQRLQSLWTLARPFCPPLLATASQMQM WLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGWPQKLWEAFWAVK DTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYH NRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFR RAFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 11) comprising the genetic engineering elements and the MDmut2 sequence in fusion with the MDA-7 protein (SEQ ID NO: 20). - SEQ ID NO: 49 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLKLENFQQRLQSLWTLARPFCPPLLATASQMQMV VLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKD TMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence in fusion with the MDA-7 protein (SEQ ID NO: 20). - SEQ ID NO: 50 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQLLENFQQRLQSLWTLARPFCPPLLATASQM QMWLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFW AVKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFK NYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFL LFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 13) to comprenant the generation elements Genetic and the sequence MDmut3 in combination with the protein MDA-7 (SEQ ID NO: 20). - SEQ ID NO: 51 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQLLENFQQRLQSLWTLARPFCPPLLATASQMQ MVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGWPQKLWEAFWA VKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKN YHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLL FRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL pour le CPP (SEQ ID NO: 14) Comprenant of genetic elements and the sequence MDmut3DelCys in fusion with the protein MDA-7 (SEQ ID NO: 20). - SEQ ID NO: 52 to correspond to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQLLENFQQRLQSLWTLARPFCPPLLATASQM QMWLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFW AVKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFK NYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFL LFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 15) comprising the genetic engineering elements and the MDmut4 sequence in fusion with the MDA-7 protein (SEQ ID NO : 20). - SEQ ID NO: 53 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLISE IDLLRKQNEQLKHKLEQLLENFQQRLQSLWTLARPFCPPPLLATASQMQ MVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGWPQKLWEAFWA VKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKN YHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLL FRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence in fusion with the MDA-7 protein (SEQ ID NO: 20). - SEQ ID NO: 54 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQLLQHYRE VAAAKSSENDRLRLLLKQLEGQEFHFGPCQVKGWPQKLWEAFWAVK DTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 55 which corresponds to the sequence MMHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQLLQHYRE VAAAKSSENDRLRLLLEGQEFHFGPCQVKGWPQKLWEAFWAVKD TMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNR TVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAF KQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutl DelCys sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 56 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNLLQHYRE VAAAKSSENDRLRLLLKLEGQEFHFGPCQVKGWPQKLWEAFWAVKD TMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNR TVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAF KQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 11) comprising the genetic engineering elements and the MDmut2 sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 57 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNLLQHYREV AAAKSSENDRLRLLLKLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDT MQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRT VEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFK QLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 58 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQKLIS EIDLLRKQNEQLKHKLEQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVK DTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for CPP (SEQ ID NO: 13) comprising the genetic engineering elements and the MDmut3 sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 59 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKRAKFKQAETQKLISE IDLLRKQNEQLKHKLEQLLEGQEFHFGPCQVKGWPQKLWEAFWAVK DTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 14) comprising the genetic engineering elements and the MDmut3DelCys sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 60 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKCRAKFKNAETQKLIS EIDLLRKQNEQLKHKLEQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVK DTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 15) comprising the genetic engineering elements and the MDmut4 sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21). - SEQ ID NO: 61 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLISE IDLLRKQNEQLKHKLEQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVK DTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHN RTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRR AFKQLDVEAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence in fusion with the truncated MDA-7 protein (SEQ ID NO: 21).

[0064] Fusion protein production and purification protocols

[0065] The following protocols were developed using the "trial and error" technique on each of the following parameters: - Production: E. coli bacterial strain, time, temperature, culture volume, stress condition (1%, 3% or 5% ethanol addition), optical density of production induction, concentration of production inducer (IPTG, isopropyl 0-D-1-thiogalactopyranoside); - Lysis: chemical and / or mechanical method, time, power, repetition; - Purification: solubilization or not, buffer, affinity, purity, column type, renaturation conditions, dialysis.

[0066] Production of fusion proteins comprising a CPP and the eGFP polypeptide

[0067] The optimal conditions for production and purification of fusion proteins comprising a mutant CPP (MDmut) or MD11 and the eGFP polypeptide (SEQ ID NO: 22 to SEQ ID NO: 29) were defined as described below.

[0068] The best production yield was observed after culturing E. coli BL21 (DE3) bacteria transformed with recombinant plasmids encoding sequences SEQ ID NO: 22 to SEQ ID NO: 29 overnight at 16°C after induction of production with 0.5 mM IPGT added at D026o=1.

[0069] Bacterial lysis was optimal in 2 stages in a Tris - NaCl - imidazole buffer. First, a chemical lysis with lysozyme at 1 mg / ml for 30 min on a wheel for gentle agitation was carried out at 4°C in order to preserve the integrity of the proteins. Then a mechanical lysis by 6 cycles of 30 sec of sonication first 3 cycles at 30% power then 3 cycles at 70% power followed, the bacterial lysates having been put back on ice at least 30 sec between each sonication.

[0070] After centrifugation, the soluble fraction was passed through a nickel column and the proteins could be eluted by competition with 500 mM imidazole.

[0071] Protein purity, quality and quantity were analyzed by SDS-PAGE and Western blotting. For this, 15 μl of sample were mixed with 5 μL of 4X reducing buffer and migrated in a 12% acrylamide gel for 1 h at 30 mA-150 V. The gels were either stained with Coomassie blue for 2 h at room temperature (RT) with shaking and then destained with water, or transferred onto a 0.2 μm nitrocellulose membrane by the Bio-Rad Mini-protean system according to the manufacturer's protocol on the mixed molecular weight program. The membranes were then blocked with 5% skimmed milk - TBS 1X tween 0.1% for 2 hours at RT with shaking then incubated with an anti-His HRP antibody (for "HorseRadish Peroxidase" in English terminology) diluted 1 / 50000 in the blocking solution.The anti-His antibody binds to the polyhistidine tag of the fusion proteins and HRP allows its detection by the ChemiDoc Imaging System (BioRad) by chemiluminescence.

[0072] Coomassie blue staining of acrylamide gels allowed us to observe that the higher the production yield, the less efficient the solubilization of proteins. However, the amounts of solubilized proteins remain very important and sufficient for the purification of several milligrams of proteins according to the protocol.

[0073] Western blotting, on the other hand, indicated the presence of protein aggregates in the samples which could be eliminated by the addition of a very high-speed centrifugation step.

[0074] Production of fusion proteins comprising a CCP and the elF3f polypeptide

[0075] The optimal conditions for production and purification of fusion proteins comprising a mutant CPP (MDmut) or MD11 and the elF3f polypeptide (SEQ ID NO: 30 to SEQ ID NO: 37) are defined below.

[0076] The best production yield was observed after culturing E. coli BL21 (DE3) bacteria transformed by the recombinant plasmids encoding the sequences SEQ ID NO: 30 to SEQ ID NO: 37 overnight at 16°C after induction of production at 0.5 mM IPGT added to OD=1.

[0077] Bacterial lysis was optimal in 2 stages in a MOPS - NaCl - imidazole buffer. First, a chemical lysis with lysozyme at 1 mg / ml for 30 min on a wheel for gentle agitation was carried out at 4°C to preserve protein integrity. Then a mechanical lysis by 5 cycles of 30 sec of sonication at 70% power followed, the bacterial lysates having been returned to ice for at least 30 sec between each sonication.

[0078] None of the protocols tested allowed obtaining a sufficient quantity of proteins in soluble form. After centrifugation, the soluble fraction was therefore eliminated and the inclusion bodies were washed in 3 different washing buffers (MOPS 20mM - NaCI 2M - Cysteine ​​15mM; MOPS 20mM - NaCI 250mM - Cysteine ​​15mM - Tritton X100 2%; MOPS 20mM - NaCI 250mM - Cysteine ​​15mM) then solubilized in denaturing conditions (MOPS 20mM - NaCI 250mM - Cysteine ​​15mM - Urea 8M). Proteins were purified on a Nickel column by imidazole competition (MOPS 20mM - NaCI 250mM - Cysteine ​​15mM - Urea 8M - Imidazole 500mM), proteins were renatured by the dropwise dilution method (Na Citrate 50mM - MgCl22mM - DTT 2mM - NaCI 50mM - L-Argine 500mM - Tween 200.5%) then dialyzed in 2 steps (Na Citrate 50mM - MgCl22mM - DTT 2mM - NaCI 50mM - L-Argine 50mM - N-Lauryl Sarcosine 0.01%; Na Citrate 50mM - NaCI 50mM - Glycerol 10%) to be in solution in a buffer compatible with in cellulo and in vivo experiments.

[0079] Protein purity and quality were analyzed by SDS-PAGE and Western blotting using the same protocol as that used for fusion proteins including eGFP protein and described in the previous section. Treatment samples were assayed by the microBCA method according to the manufacturer's protocol.

[0080] In the elution, refolding and dialysate samples, a single band, labeled by the anti-histidine antibody in Western blot, of 50 kDa was highlighted by Coomassie blue staining of the acrylamide gel. The treatment samples used for in vitro experiments are therefore pure and of good quality. Indeed, the absence of a supernumerary band in Western blot demonstrated the absence of degraded and / or aggregated protein in the samples.

[0081] Production of fusion proteins comprising a CCP and the MDA-7 polypeptide or truncated MDA-7

[0082] The optimal conditions for production and purification of fusion proteins comprising a mutant CPP (MDmut) or MD11 and the MDA-7 polypeptide (SEQ ID NO: 46 to SEQ ID NO: 53) or the truncated MDA-7 polypeptide (SEQ ID NO: 54 to SEQ ID NO: 61) are defined below.

[0083] The best production yield was observed after culturing E. coli BL21 (DE3) bacteria transformed by the recombinant plasmids encoding the sequences SEQ ID NO: 46 to SEQ ID NO: 61 overnight at 16°C after induction of production with 1 M IPGT added at D026o>1.

[0084] Bacterial lysis was optimally performed in 2 stages in a 50 mM Tris - 250 mM NaCl - 5 mM imidazole buffer. First, a chemical lysis with lysozyme at 1 mg / ml for 30 min on a wheel for gentle agitation was performed at 4°C to preserve protein integrity. Then a mechanical lysis by 5 cycles of 30 sec of sonication at 70% power followed, the bacterial lysates having been returned to ice for at least 30 sec between each sonication.

[0085] None of the protocols tested allowed to obtain a sufficient concentration of proteins in soluble form. After centrifugation, the soluble fraction was therefore eliminated and the inclusion bodies were washed in 3 different washing buffers (Tris 50 mM - p-mercaptoethanol 20 mM - Tritton X100 2%; Tris 20 mM - [3-mercaptoethanol 20 mM; Tris 50 mM - DTT 20 mM - Guanidine Hydrochloride 6 M) then solubilized in denaturing condition of Guanidine. Proteins were purified on a Nickel column by imidazole competition (Tris 50 mM - Guanidine HydroChloride 6M - Imidazole 500mM) and renatured by the dropwise dilution method (Tris 50 mM - Guanidine HydroChloride 1.4M - NaCI 50mM - L-Argine 500mM - Titton X100 0.5%) then dialyzed in 2 steps to be in solution (Tris 50 mM - Guanidine HydroChloride 0.75M - NaCI 50mM - L-Argine 50mM - Tween20 0.5%; Tris 50mM - NaCI 50 mM - Glycerol 10%) in a buffer compatible with in cellulo and in vivo experiments.

[0086] The purity and quality of the proteins were analyzed by SDS-PAGE and Western blot, and then the latter were assayed by the microBCA method. A single band, labeled by the anti-histidine antibody in Western blot, was highlighted by staining the acrylamide gel with Coomassie blue. The samples of CPP fusion proteins coupled to the full or truncated MDA7 polypeptides are therefore pure and of good quality. Indeed, the absence of supernumerary bands in Western blot demonstrated the absence of degraded and / or aggregated proteins in the samples.

[0087] Analysis of the cellular penetration potential of CPPs

[0088] To demonstrate the high intracellular penetration potential of MDmut mutant CPPs, flow cytometry and fluorescence microscopy analyses were performed.

[0089] Treatments with fusion proteins comprising a mutant CPP and the eGFP polypeptide (SEQ ID NO: 23 to SEQ ID NO: 29) were compared to treatment with eGFP alone, not linked to a transporter, and to treatments with fusion proteins comprising either the MD11 CPP (SEQ ID NO: 1) without the novel genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 17), or comprising the MD11 CPP with the novel genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 22).

[0090] To enumerate the number of fluorescent cells after treatment, HEK293, B16-Ova and beta-TC-6 cells were seeded and then treated at approximately 60% confluence with 200 nM of the respective fusion proteins in the absence of bovine serum for 4 h. They were then supplemented with 5% bovine serum and incubated overnight. The next day, the cells were trypsinized, then washed with 1X PBS and passed through FACS for analysis.

[0091] An untreated cell sample supplemented with Propridium iodide at 1 μg / ml was used to calibrate the blank and live cell range. For treated samples, a minimum of 25,000 live cells were analyzed for green fluorescence.

[0092] The analysis results indicate the percentage of GFP-positive cells after treatment and are presented in Figure 1 in which: - MD11 -eGFP corresponds to the control fusion protein comprising the MD11 CPP sequence but without the genetic engineering elements (SEQ ID NO: 1) and the eGFP polypeptide (SEQ ID NO: 17); - #1 A-eGFP corresponds to the fusion protein comprising the sequence of CPP MD11 with the genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 22); - #2B-eGFP corresponds to the fusion protein comprising the sequence of the CPP MDmutl DelCys and the eGFP polypeptide (SEQ ID NO: 23); - #3C-eGFP corresponds to the fusion protein comprising the MDmut2 CPP sequence and the eGFP polypeptide (SEQ ID NO: 24); - #4D-eGFP corresponds to the fusion protein comprising the sequence of the CPP MDmut2DelCys and the eGFP polypeptide (SEQ ID NO: 25); - #5E-eGFP corresponds to the fusion protein comprising the MDmut3 CPP sequence and the eGFP polypeptide (SEQ ID NO: 26); - #6F-eGFP corresponds to the fusion protein comprising the CPP MDmut3DelCys sequence and the eGFP polypeptide (SEQ ID NO: 27); - #7G-eGFP corresponds to the fusion protein comprising the MDmut4 CPP sequence and the eGFP polypeptide (SEQ ID NO: 28); - #8H-eGFP corresponds to the fusion protein comprising the CPP MDmut4DelCys sequence and the eGFP polypeptide (SEQ ID NO: 29).

[0093] Flow cytometry showed us that overall, under current treatment conditions with a number of replicates between 3 and 5 per condition, the penetration power of proteins is greater in human non-tumor HEK293 cells than in B16-Ova (B16) tumor cells and human beta-TC-6 (bTC6) tumor cells. For the MD11-eGFP and #5E-eGFP fusion proteins, we are at the limit of significance p=0.057. The same penetration defect, compared to MD11-eGFP, is observed for the #4D-eGFP mutant in all three cell lines.

[0094] In HEK293 cells, fusion proteins comprising a mutant CPP and the eGFP polypeptide have preserved cell penetration capacity, including a non-significant upward trend for #3C-eGFP, #5E-eGFP, #6F-eGFP and #7G-eGFP compared to the control (MD11 -eGFP) with a rate of GFP-positive cells between 73% and 82% versus 57%.

[0095] In B16-Ova (B16) cells, the penetration capacity of mutants #3C-eGFP, #5E-eGFP, #6F-eGFP and #7G-eGFP is equivalent to that of MD11 - eGFP. In contrast, the cell penetration capacity of mutants #2B-eGFP, #4D-eGFP and #8H-eGFP is significantly lower between 15% and 20% compared to more than 41%.

[0096] In bTC6 cells, the penetration capacity of #1A-eGFP, #5E-eGFP and #7G-eGFP mutants is significantly higher between 21% and 30% compared to 15% for MD11-eGFP. In contrast, the penetration capacity of #2B-eGFP, #3C-eGFP, #4D-eGFP, #6F-eGFP and #8H-eGFP mutants is significantly lower between 1% and 8% compared to 15%.

[0097] In parallel, the same cells (HEK293 and B16-Ova) were also seeded on glass slides equipped with culture chambers and then treated with MD11-eGFP, #1 A-eGFP, #3C-eGFP, #5E-eGFP, #7G-eGFP fusion proteins or buffer for 4 hours in the absence of serum. The cells were then washed with 1X PBS then heparin and fixed with Paraformaldehyde and observed under a fluorescence microscope. 5 successive focal planes spaced 2pm apart were captured to highlight the intracellular localization of the proteins.

[0098] Representative images of cell fluorescence after treatment are given in Figure 2.

[0099] Since the cell penetration efficiency of the different mutants was unequal, some fluorescent signals had to be "forced" to be visible, but all were identified as intracellular. However, the protein aggregates visualized in some images appear to be attached to the membranes.

[0100] Analysis of the antitumor therapeutic potential of the fusion protein comprising CPP and elF3f polypeptide

[0101] In order to demonstrate the therapeutic potential of MDmut mutant CPPs fused to the translation regulatory factor elF3f (SEQ ID NO: 31 to SEQ ID NO: 37) on melanoma, the study continued with HEK293 and B16-Ova cells.

[0102] Viability was tested using Prestoblue®. For this, cells were seeded in 96-well plates with black edges and transparent bottoms and, the next day, treated at 60% confluence with 0.25X fetal calf serum (FCS). In order to calculate the median inhibitory concentration (IC50) of each fusion protein, they were diluted according to a wide concentration range between 15 and 100 nM and incubated for 24 or 48 hours.

[0103] The viability curves of B16-Ova (B16) and HEK293 cells after 24h and 48h of treatment with the fusion proteins are illustrated in Figure 3 in which: - 1 E corresponds to the fusion protein comprising the sequence of CPP MD11 and the polypeptide elF3f (SEQ ID NO: 30); - 3E corresponds to the fusion protein comprising the sequence of the CPP MDmut2 and the polypeptide elF3f (SEQ ID NO: 32); - 5E corresponds to the fusion protein comprising the sequence of the CPP MDmut3 and the polypeptide elF3f (SEQ ID NO: 34); - 7E corresponds to the fusion protein comprising the sequence of the CPP MDmut4 and the polypeptide elF3f (SEQ ID NO: 36).

[0104] These tests showed that the 3E mutant CPP was the least effective on both B16 and HEK293 cells after 24 or 48 hours. The 24-hour IC50 of the other mutant CPPs was identical at approximately 50 nM for B16 cells and 30 nM for HEK293. The 48-hour IC50 was also not significantly different and was between 25 and 35 nM, an extremely low concentration that corroborated a direct and unmediated action of the fusion proteins in the cells.

[0105] These tests also showed that the fusion proteins #1A-eGFP, #5E-eGFP and #7G-eGFP (and by extension the mutant peptides that form them) have a better cell penetration capacity than that of the fusion protein formed with MD11. This increased capacity was confirmed in at least two different cell lines, namely the human non-tumor line HEK and the human tumor line bTC6.

[0106] More generally, it should be noted that the modes of implementation and embodiment of the invention considered above have been described as non-limiting examples and that other variants are consequently conceivable.

Claims

Claims Claim 1. Peptide comprising or consisting of an amino acid sequence selected from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:

8. Claim 2. Nucleic acid molecule encoding a peptide according to claim 1. Claim 3. An expression vector comprising a nucleic acid molecule encoding a peptide according to claim 1. Claim 4. A host cell comprising a nucleic acid molecule according to claim 2 or an expression vector according to claim 3. Claim 5. Use of a peptide according to claim 1 or of a nucleic acid molecule according to claim 2 or of an expression vector according to claim 3 or of a host cell according to claim 4, for obtaining a transporter intended for the internalization of a molecule of interest in target cells. Claim 6. Combination comprising a transporter and a molecule of interest, said transporter being a peptide according to claim 1. Claim 7. A fusion protein comprising a transporter which is a peptide according to claim 1, and a molecule of interest which is a polypeptide of interest. Claim 8. Nucleic acid molecule encoding the fusion protein according to claim 7. Claim 9. An expression vector comprising a nucleic acid molecule encoding the fusion protein of claim 7. Claim 10. A host cell comprising a nucleic acid molecule according to claim 8 or an expression vector according to claim 9. Claim 11. A pharmaceutical composition comprising a combination according to claim 6 or a fusion protein according to claim 7 together with a pharmaceutically acceptable excipient and / or carrier. Claim 12. Combination according to claim 6 or fusion protein according to claim 7 or pharmaceutical composition according to claim 11, for its use in the treatment, diagnosis or prevention of pathologies, in particular cancers such as melanomas, breast cancer, brain tumors, glioblastomas, colon cancer, lymphomas. Claim 13. Combination according to claim 6, fusion protein according to claim 7, nucleic acid molecule according to claim 8, expression vector according to claim 9, host cell according to claim 10 or pharmaceutical composition according to claim 11, wherein the molecule of interest comprises or consists of a polypeptide of interest selected from a polypeptide encoding the eGFP protein represented by the sequence SEQ ID NO: 17, a polypeptide encoding the elF3f protein represented by the sequence SEQ ID NO: 18, a polypeptide encoding the FERM protein represented by the sequence SEQ ID NO: 19, a polypeptide encoding the MDA-7 protein represented by the sequence SEQ ID NO: 20, a polypeptide encoding the truncated MDA-7 protein represented by the sequence SEQ ID NO: 21 and a polypeptide represented by a sequence having 80%, in particular 90%, particularly 95% sequence identity. with one of the sequences SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21., Claim 14. Combination or fusion protein or pharmaceutical composition for its use according to claim 12, wherein the molecule of interest comprises or consists of a polypeptide of interest selected from a polypeptide encoding the eGFP protein represented by the sequence SEQ ID NO: 17, a polypeptide encoding the elF3f protein represented by the sequence SEQ ID NO: 18, a polypeptide encoding the FERM protein represented by the sequence SEQ ID NO: 19, a polypeptide encoding the MDA-7 protein represented by the sequence SEQ ID NO: 20, a polypeptide encoding the truncated MDA-7 protein represented by the sequence SEQ ID NO: 21 and a polypeptide represented by a sequence having 80%, in particular 90%, particularly 95% sequence identity with one of the sequences SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO:

21. Claim 15. Fusion protein according to claim 7 comprising or consisting of an amino acid sequence selected from the sequences SEQ ID NO: 23 to SEQ ID NO: 29, SEQ ID NO: 31 to SEQ ID NO: 37, SEQ ID NO: 39 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 53 and SEQ ID NO: 55 to SEQ ID NO: 61.

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