Novel peptides and their use as transporters for the internalization of molecules of interest into target cells

Abstract

The invention relates to novel 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. Figure for the abstract: Fig. 1

Description

Title of the invention: Novel peptides and their use as carriers 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. More particularly, it relates to novel peptides or nucleic acids encoding such peptides, and their use 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, particularly polypeptides of interest, and the use of such a combination in the treatment, diagnosis, or prevention of diseases, particularly cancer.The invention further relates to a fusion protein comprising a novel peptide having cell-penetrating properties (referred to as a "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, and 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. Previous technique

[0002] Drugs derived from biotechnology, also called biopharmaceuticals or biological drugs, play an important role in the treatment of human diseases. They include, in particular, 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 (PNAs).

[0003] In some cases, it is necessary to use transporters for these biopharmaceuticals to be internalized into target cells. The internalization of therapeutic molecules into cells is the subject of much research aimed at finding new transporters, improving the efficiency of known transporters as well as the therapeutic molecules being transported, improving their targeting at the cellular level, and reducing the effective dose of certain treatments in order to decrease the risk of toxicity.

[0004] Several families of transporter peptides have already been identified. These natural or synthetic peptides are called PTDs (for "Protein Transduction Domain") in Anglo-Saxon terminology) or CPP (for "Cell-Penetrating Peptides" in Anglo-Saxon terminology) have the ability to transport and transfer molecules such as peptides or nucleic acids into cells according to different 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 the eGFP protein or α3-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 said protein interacts with the leucine zipper domain, leading to the formation of a hydrophobic pocket that stabilizes the ZEBRA protein / DNA complex.

[0007] To date, the internalization pathways used by transport peptides, known to those skilled in the art, such as endocytosis and macropinocytosis, required significant energy expenditure to achieve this intracellular penetration mechanism. Furthermore, the endocytosis mechanism leads to the internalization of molecules via endosomes, whose contents 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 intracellularly. Consequently, at an industrial production scale, to ensure efficient transduction of polypeptides of interest, it is necessary to produce a large quantity of transporter and polypeptides of interest. This sometimes requires a complex 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 administered quantity due to the risk of toxicity. It therefore appears necessary to use transport polypeptides with greater efficiency in cell penetration to mitigate 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 enabling the internalization of polypeptides of interest at low concentrations and preventing the degradation of these polypeptides of interest by a direct penetration mechanism in- dependent on endosomes.

[0009] The 2015 publication “MD 11 mediated delivery of recombinant eIF3f induces melanoma and colorectal carcinoma cell death” by R. Marchione et al. discloses the use of a complex formed from the eIF3f protein fused with a cell-penetrating peptide to increase the intracellular amount of available eIF3f protein in cancer cells and activate their apoptosis. The cell-penetrating peptide used is a fragment of the ZEBRA protein sequence (from amino acid position 178 to amino acid position 220) called MD11.

[0010] There remains today a need to find new carrier peptides with improved cell penetration potential and easier production, enabling the transport of molecules of interest into target cells at low concentrations, with high efficiency and improved affinity, while ensuring good stability and low toxicity within said cells. Presentation of the invention

[0011] The present invention aims to provide novel peptides as transporters for the internalization of molecules of interest into target cells. These novel peptides are mutants of a peptide fragment of the ZEBRA protein. This fragment, whose sequence is SEQ ID NO: 1, corresponds to the peptide sequence of the ZEBRA protein from amino acid position 178 to amino acid position 220. This fragment is known and will be referred to as MD11 in the remainder of this description. It is a cell-penetrating peptide (CPP), also called a transport peptide, transporter peptide, or simply a transporter or delivery system.

[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. Their advantage lies in their cellular penetration capacity, which is at least equal to or greater than that of the MD11 transport peptide. Furthermore, they are present in cells even when treated with low concentrations on the order of nanomolars, which supports direct and unmediated penetration into cells for the molecules they can transport. Finally, no toxicity of the peptides alone to the target cells has been demonstrated.

[0014] Cell penetration capacity refers to the efficiency of internalization of The percentage of molecules of interest is the percentage of treated cells containing these molecules in their cytoplasm. This internalization efficiency relies on detecting the molecules of interest in the transduced cells using, for example, microscopy or flow cytometry, or any other imaging techniques that allow visualization of internalization at the molecular level, such as bioluminescence resonance energy transfer (BRET), Förster resonance energy transfer (FRET), electron microscopy, atomic force microscopy (AFM), optogenetics, etc.

[0015] The term “carrier” refers to a molecule capable of transporting and transferring another different molecule across the plasma membrane to enable 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 into the inside of 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 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.

[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 the degeneracy of the genetic code known to a person 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 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. Such an expression vector may be of any type known in itself 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 genetic engineering elements, in particular replication origins and promoters, enabling autonomous control of vector replication in the host organism and specific expression of 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 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, or an expression vector comprising such a nucleic acid molecule. The host cells may be, for example, prokaryotic cells such as E. coli 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.Cô.

[0022] According to a fifth aspect, the present invention aims at the use of a 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 or of a nucleic acid molecule encoding 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 into target cells.

[0023] The cells that can be the target cells of an internalization process implemented by a transporter 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, and lymphoma cells. These human cells may also be normal cells, including fibroblasts, epithelial cells, lymphocytes, dendritic cells, and muscle cells such as myocytes, myoblasts, and myotubes, for example. These human cells may also be differentiated somatic cells that will undergo dedifferentiation to form induced pluripotent stem cells (iPSCs).To target certain cell lines, peptide sequences such as guidance peptides or targeting peptides (respectively "homing peptides" and "target peptides" in Anglo-Saxon terminology), nuclear localization signals or NLS (for "nuclear localization signal" in Anglo-Saxon 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 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.

[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 selected from polypeptides, DNA, RNA, oligonucleotides, siRNA, shRNA, miRNA, antisense RNA, or peptide nucleic acids (PNAs).

[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 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, and a molecule of interest which is a polypeptide of interest.

[0027] By “fusion protein” is meant 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 portion 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 encoding the fusion protein that 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 encoding the fusion protein 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 encoding the fusion protein according to the invention.

[0031] According to a particular embodiment of the present invention, the vector further comprises genetic engineering elements, in particular replication origins and promoters, enabling autonomous control of vector replication in the host organism and specific expression of 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 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. coli or Bacillus, or eukaryotic cells such as yeasts including Saccharomyces cerevisiae and Pichia pastoris, filamentous fungi including Trichoderma reesei and Aspergillus niger, insect cells using Baculovirus, or cell lines such as CHO, HEK 293, Cos, Per.C6.

[0033] In particular embodiments of the combination or fusion protein 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 fusion protein or combination of the subject of the present invention. Preferably, the composition comprises a pharmaceutically acceptable excipient and / or vehicle. The choice of a pharmaceutically acceptable excipient and / or 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 mentioned above, according to particular embodiments of the invention, 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 eIF3f 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 (full MDA-7 protein) and SEQ ID NO: 21 (truncated MDA-7 protein) and a polypeptide represented by a sequence having 80%, particularly 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 percentage of identity of peptide sequences is determined by direct comparison of two sequences of polypeptide molecules, by 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 of the two sequences, 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 peptides according to the principle of the degeneration of the genetic code known to a person skilled in the art. Brief description of the figures

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

[0040] [Fig.1] [Fig.1] illustrates a histogram showing the percentages of fluorescent HEK and B16-Ova cells after treatment with fluorescent fusion proteins of the present invention;

[0041] [Fig.2] [Fig.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 24 and 48 hours, allowing calculation of the IC50 of the potentially therapeutic fusion proteins that are the subject of the present invention. Description of embodiments

[0043] In the following 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 a person skilled in the art.

[0044] Production of MDmut and MD 11 plasmids

[0045] Starting from the original sequence of the cell penetration peptide (CPP) MD11, the inventors created 7 sequence modifications numbered from #2 to #8 according to their distance from the original sequence: - SEQ ID NO: 2 called MDmutlDelCys which corresponds to the sequence KRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLLK, - SEQ ID NO: 3 called MDmut2 which corresponds to the sequence RREKNR-VAARKCRAKFKNLLQHYREVAAAKSSENDRLRLLLK, - 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 RREKNR-VAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHKLEQL, - 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 the expression in bacteria of 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 KRYKNR-VASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQ (SEQ ID NO: 1) is also inserted into a pET_15b expression plasmid comprising the same organization of genetic engineering elements as the plasmids comprising the MDmut mutants.

[0048] The organization of the genetic engineering elements in each expression plasmid in the 5' to 3' direction is as follows:

[0049] Ndel Res Site - His Tag - TEV seq - MMP2 cleavage site - MD Seq - Xhol Res Site

[0050] in which:

[0051] Ndel Res Site = Ndel restriction site

[0052] His Tag = polyhistidine label

[0053] TEV seq = cleavage sequence of the Tev protease

[0054] MMP2 cleavage site = cleavage sequence by metalloproteinase 2 (tumor targeting)

[0055] MD Seq = the MD11 or MDmut sequence

[0056] Xhol Res Site = Xhol restriction site

[0057] All plasmids have been checked by two-way sequencing at the insertion sequence of the genetic elements encoding the fusion proteins of interest.

[0058] With this organization of the genetic engineering elements, the following cell penetration peptide (CPP) sequences are thus obtained: SEQ ID NO: 9 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQ for the CPP including the sequence MD11, SEQ ID NO: 10 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL K for the CPP including the sequence MDmutlDelCys, SEQ ID NO: 11 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LK for the CPP including the sequence Mdmut2, SEQ ID NO: 12 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL K for the CPP including the sequence Mdmut2DelCys, - SEQ ID NO: 13 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPKRYKNRVASRKCRAKFKQAETQK LISEIDLLRKQNEQLKHKLEQL for the CPP including the sequence Mdmut3, SEQ ID NO: 14 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQL for the CPP including the sequence Mdmut3DelCys, SEQ ID NO: 15 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQL for the CPP including the sequence Mdmut4, - SEQ ID NO: 16 which corresponds to the sequence MHHHHHHHHENLYFQSGALGLPRREKNRVAARKRAKFKNAETQKLI SEIDLLRKQNEQLKHKLEQL for the CPP including the sequence Mdmut4DelCys.

[0059] Production of plasmids of the polypeptides of interest

[0060] To obtain the sequences of the reporter molecules of interest, here polypeptides of the proteins eGFP (for "enhanced green fluorescent protein" in Anglo-Saxon terminology) for biotechnological use and eIF3f (for "eukaryotic Translation Initiation Factor 3 subunit F" in Anglo-Saxon terminology), FERM (corresponding to the FERM domain) and MDA-7 (for "Melanoma Differentiation Associated 7" in Anglo-Saxon terminology) whole or truncated, also known as IL-24, for therapeutic use, to be fused into our Mdmut CPP-mutants, 5 plasmids, called donors, containing the inserts of interest whose polypeptide sequences of interest are the following were used: - SEQ ID NO : 17 corresponding to the polypeptide MVSKGEELFTGVVPIL-VELDGDVNGHKFSVSGEGEGDATYGKLTLCFICTTGKLPVPWPTLVT TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADK QKNGIKVNFKIRHNIEDGSVQLADHYQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK of the eGFP protein, - SEQ ID NO : 18 corresponding to the polypeptide ATPAVPVSAPPATPTPV-PAAAPASVPAPTPAAAPVPAAAPASSSDPAAAAAATAAPGQTPAS AQAPAPAPALPGPALPLPGFPPGGRVVRLHPVILASIVDSYERRNEGA ARVIGTLLGTVDKHSVESTNCFSVPHNEVKFDMEVHAVNEGA KKVSPNELILGWYATGHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNG RMSIKAYVSTLMGVPGR™GVMFTPLTVKYAYYDTERIGVDLIMKTC FSPNRVIGLSSDDLQQVGGASARIQDALSTVLQYAEDVLSCGKVSADNTV GRFLMSLVNQVPKIVPDDFETMLNSNINDLLMVTYLANLTQSQIALNE KLVNL of the eIF3 protein, - SEQ ID NO : 19 correspondant au polypeptide MPKPINVRVTTMDAELE-FAIQPNTTGKQLFDQVVKTIGLREVWYFGLHYVDNKGFPTWLKLDKK VSAQEVRKENPLQFKFRAKFYPEDVAEELIQDITQKLFFLQVKEGILSD EIYCPPETAVLLGSYAVQAKFGDYNKEVHKSGYLSSERLIPQRVMDQ HKLTRDQWEDRIQVWHAEHRGMLKDNAMLEYLKIAQDLEMYGINY FEIKNKKGTDLWLGVDALGLNIYEKDDKLTPKIGFPWSEIRNISFNDK KFVIKPIDKKAPDFVFYAPRLRINKRILQLCMGNHELYMRRRKPDTIEV QQMKAQAREEKHQKQLER de la protéine FERM, - SEQ ID NO : 20 correspondent to the polypeptide NFQQRLQSLWTLARPFCP-PLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVV PQKLWEAFWAVKDTMQAQDNNTSCRLLQQEGLQNVSDAESCYLVH TLLEFYLKTVFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENE MFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYK L of the protein MDA-7, - SEQ ID NO : 21 correspondant au polypeptide GQEFHFGPCQVKGVVP-QKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTL LEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMF SIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL de la protéine MDA-7 tronquée.

[0061] The inventors have these sequences (SEQ ID NO: 17 to SEQ ID NO: 21) flanked by the restriction sites Xhol and BamHI in a pET15b plasmid. They were previously sequenced using the commercial primer pET_RP and their restriction profile was also analyzed by single and double digestions to ensure that they could be extracted without impacting the sequence to be translated.

[0062] Creation of the plasmid library

[0063] First, the 8 Mdmut plasmids and the 5 donor plasmids of the polypeptides of interest were transformed in E. coli XL 10 Gold bacteria. For this purpose, 25 µl of bacteria were transformed with 50 ng of plasmids by heat shock for 30 min on ice followed by 40 sec at 42°C before being transferred to ice. This step was followed by liquid culture in 100 µl of SOC culture medium at 37°C for 1 h, then plating onto a Petri dish containing LB (Lysogeny Broth) - Agar - Ampicillin medium and incubating at 37°C overnight. The following 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 25pL of water using the Macherey Nagel Miniprep kit according to the manufacturer's protocol.

[0064] The plasmids thus obtained were prepared for subcloning after double digestion with the restriction enzymes Xhol and BamHI. Two pg of plasmid were therefore digested with 5 units of each restriction enzyme for 3 h at 37 °C. Migration of the digestion products in a 0.6% agarose gel - 0.5X TAE - Gel Red IX for 1.5 h at 75 V 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.

[0065] The MDmut and MD11 plasmids, which can be described as recipients, gave their backbone containing the CPP-mutant or MD 11 sequences and the plasmids including the polypeptides of interest, described as donors, gave their insert containing the sequence of the polypeptides of interest.

[0066] The inserts corresponding to the polypeptides of interest were subcloned into each of the 8 MDmut plasmids, thus creating a new library of 40 recombinant plasmids enabling the synthesis of the fusion proteins. To this end, ligation was performed with 3 molar folds of each polypeptide insert 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 XL 10 Gold bacteria with half of the ligation product according to the same protocol as previously described.

[0067] Colonies transformed by recombinant plasmids were pre-selected by colony PCR. To perform this, a ready-to-use 2X commercial enzyme was used, with 10 pM of sense and antisense primers diluted halfway in sterile water. All colonies in the plate were pricked 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 - Red IX gel for 40 min at 100V was carried out.

[0068] After migration of the PCR products on agarose gel, the samples obtained from colony stings were compared to the positive control (donor plasmid) or negative control (Water) samples. One or two colonies that produced an amplicon of comparable size and appearance to the positive control were then cultured to amplify and purify the plasmids using a commercial "miniprep" kit before sending them for bidirectional sequencing (T7 and pET_RP primers) to verify the alignment of their sequence with the theoretical sequence.

[0069] New E. coli XL10 Gold bacteria were then retransformed from the plasmid samples validated by sequencing. This new transformation allowed the creation of a glycerol stock (freezing at -80°C of 750 µl of culture on the night of XL 10 bacteria with 25% sterile glycerol) and a stock of concentrated recombinant plasmids, purified using the Macherey Nagel “midiprep” kit according to the manufacturer’s protocol.

[0070] In each recombinant plasmid, the organization of the genetic engineering elements is as follows in the 5' to 3' direction:

[0071] Ndel Res Site - His Tag - TEV seq - MMP2 cleavage site - MD Seq - Xhol -PolyPep - BamHI

[0072] in which:

[0073] Ndel Res Site = Ndel restriction site

[0074] His Tag = polyhistidine label

[0075] TEV seq = cleavage sequence of the Tev protease

[0076] MMP2 cleavage site = cleavage sequence by metalloproteinase 2 (tumor targeting)

[0077] MD Seq = the MD11 sequence (SEQ ID NO: 1) or an MDmut sequence (SEQ ID NO: 2 to SEQ ID NO: 8)

[0078] XhoI = XhoI restriction site

[0079] PolyPep = the polypeptide sequence of interest (SEQ ID NO: 17 to SEQ ID NO: 21)

[0080] BamHI = BamHI restriction site

[0081] The following fusion protein sequences encoded by the recombinant plasmids thus created are obtained: SEQ ID NO: 22 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK for the CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence, fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 23 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL KLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG MDELYK for the CPP (SEQ ID NO: 10) including the engineering elements genetic and the MDmutlDelCys sequence fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 24 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LKLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK for the CPP (SEQ ID NO: 11) including the genetic engineering elements and the MDmut2 sequence fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 25 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL KLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG MDELYK for the CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 26 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHK LEQLLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSA MPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT LGMDELYK for the CPP (SEQ ID NO: 13) comprenant les Genetic elements and the MDmut3 sequence are combined with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 27 that corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQLLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK for CPP (SEQ ID NO: 14) including genetic engineering elements and the MDmut3DelCys sequence fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 28 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQLLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSA MPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT LGMDELYK for the CPP (SEQ ID NO: 15) comprising the genetic engineering elements and the MDmut4 sequence fused with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 29 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKL EQLLEMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK for the CPP (SEQ ID NO: 16) comprenant les Genetic elements and the sequence MDmut4DelCys by fusion with the eGFP protein (SEQ ID NO: 17). SEQ ID NO: 30 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPA SSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVV RLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHN ESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIH EYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTP LTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDA LSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNS NINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 9) including genetic engineering elements and the MD11 sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 31 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL KLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPASSS DPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVVRL HPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHNESE DEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIHEYY SREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTPLTV KYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDALST VLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNSNIN DLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutlDelCys sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 32 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LKLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPASS SDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVVR LHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHNES EDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIHEY YSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTPLT VKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDALS TVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNSNI NDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 11) including genetic engineering elements and the MDmut2 sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 33 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL KLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPASSS DPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVVRL HPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHNESE DEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIHEYY SREAPNPIHLTVDTSLQNGRMSIKAYVSTLGVPGRTMGVMFTPLTV KYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDALST VLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNSNIN DLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 34 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHK LEQLLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAP ASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRV VRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPH NESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLI HEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFT PLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQD ALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLN SNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 13) including genetic engineering elements and the MDmut3 sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 35 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQLLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPA SSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVV RLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHN ESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIH EYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTP LTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDA LSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNS NINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 14) including genetic engineering elements and the MDmut3DelCys sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 36 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQLLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAP ASSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRV VRLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPH NESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLI HEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFT PLTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQD ALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLN SNINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 15) to comprenant the genetic elements and the sequence MDmut4 with fusion with the protein eIF3f (SEQ ID NO: 18). SEQ ID NO: 37 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKL EQLLEATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPA SSSDPAAAAAATAAPGQTPASAQAPAQTPAPALPGPALPGPFPGGRVV RLHPVILASIVDSYERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHN ESEDEVAVDMEFAKNMYELHKKVSPNELILGWYATGHDITEHSVLIH EYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTP LTVKYAYYDTERIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDA LSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVPKIVPDDFETMLNS NINDLLMVTYLANLTQSQIALNEKLVNL for the CPP (SEQ ID NO: 16) including genetic engineering elements and the MDmut4DelCys sequence fused with the eIF3f protein (SEQ ID NO: 18). SEQ ID NO: 38 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWY FGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVA EELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNK EVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKD NAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDD KLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQL CMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 9) including the genetic engineering elements and the MD11 sequence fused with the FERM protein (SEQ ID NO: 19). SEQ ID NO: 39 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL KLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWYFG LHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVAEE LIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNKEV HKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDNA MLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDDKL TPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLC MGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutlDelCys sequence fused with the FERM protein (SEQ ID NO: 19). SEQ ID NO: 40 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LKLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWYF GLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVAE ELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNKE VHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDN AMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDDK LTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLC MGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 11) including genetic engineering elements and sequence MDmut2 fused with FERM protein (SEQ ID NO: 19). SEQ ID NO: 41 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL KLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWYFG LHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVAEE LIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNKEV HKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDNA MLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDDKL TPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLC MGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 12) comprising genetic engineering elements and the MDmut2DelCys sequence fused with the FERM protein (SEQ ID NO: 19). SEQ ID NO: 42 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHK LEQLLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVW YFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDV AEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYN KEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLK DNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKD DKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRIL QLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 13) to compare genetic elements and the MDmut3 sequence with fusion with the FERM protein (SEQ ID NO: 19). SEQ ID NO: 43 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQLLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWY FGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVA EELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNK EVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKD NAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDD KLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQL CMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 14) to compare the genetic elements and the sequence MDmut3DelCys with the protein fusion FERM (SEQ ID NO: 19). SEQ ID NO: 44 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQLLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVW YFGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDV AEELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYN KEVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLK DNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKD DKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRIL QLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER pour le CPP (SEQ ID NO :15) comprenant les éléments de génie génétique et la séquence MDmut4 en fusion avec la protéine FERM (SEQ ID NO : 19). SEQ ID NO: 45 which corresponds to the sequence MMHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKL EQLLEPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWY FGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVA EELIQDITQKLFFLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNK EVHKSGYLSSERLIPQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKD NAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWLGVDALGLNIYEKDD KLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQL CMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLER for the CPP (SEQ ID NO: 16) to compare the genetic elements and the sequence MDmut4DelCys with the protein fusion FERM (SEQ ID NO: 19). SEQ ID NO: 46 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLW SQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTS CRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLK SFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEA ALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 47 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL KLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQ VSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTSCR LLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSF STLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutlDelCys sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 48 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LKLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWS QVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTSC RLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKS FSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAA LTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 11) comprising genetic engineering elements and the MDmut2 sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 49 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL KLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQ VSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTSCR LLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSF STLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 50 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHK LEQLLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLL WSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNN TSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRT LKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDV EAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 13) comprising the genetic engineering elements and the MDmut3 sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 51 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQLLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLW SQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTS CRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLK SFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEA ALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 14) comprising the genetic engineering elements and the MDmut3DelCys sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 52 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQLLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLL WSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNN TSCRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRT LKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDV EAALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 15) including genetic engineering elements and the MDmut4 sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 53 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKL EQLLENFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLW SQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNNTS CRLLQQEGLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLK SFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEA ALTKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 16) comprising the genetic engineering elements and the MDmut4DelCys sequence fused with the MDA-7 protein (SEQ ID NO: 20). SEQ ID NO: 54 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLL LKQLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARL LQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFS TLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 9) comprising the genetic engineering elements and the MD11 sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 55 which corresponds to the sequence MMHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQLLQHYREVAAAKSSENDRLRLLL KLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQ QEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTL ANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTK ALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 10) comprising the genetic engineering elements and the MDmutlDelCys sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 56 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNLLQHYREVAAAKSSENDRLRLL LKLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLL QQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFST LANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALT KALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 11) comprising the genetic engineering elements and the MDmut2 sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 57 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNLLQHYREVAAAKSSENDRLRLLL KLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQ QEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTL ANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTK ALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 12) comprising the genetic engineering elements and the MDmut2DelCys sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 58 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKCRAKFKQAETQKLISEIDLLRKQNEQLKHK LEQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSAR LLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSF STLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 13) comprising the genetic engineering elements and the MDmut3 sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 59 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPKRYKNRVASRKRAKFKQAETQKLISEIDLLRKQNEQLKHKL EQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARL LQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFS TLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 14) comprising the genetic engineering elements and the MDmut3DelCys sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 60 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKCRAKFKNAETQKLISEIDLLRKQNEQLKHK LEQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSAR LLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSF STLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 15) comprising the genetic engineering elements and the MDmut4 sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21). SEQ ID NO: 61 which corresponds to the sequence MHHHHHHHHENLYFQS-GALGLPRREKNRVAARKRAKFKNAETQKLISEIDLLRKQNEQLKHKL EQLLEGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARL LQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFS TLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAAL TKALGEVDILLTWMQKFYKL for the CPP (SEQ ID NO: 16) comprising genetic engineering elements and the MDmut4DelCys sequence fused with the truncated MDA-7 protein (SEQ ID NO: 21).

[0082] Production and purification protocols for fusion proteins

[0083] 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 (addition of 1, 3 or 5% ethanol), optical density of production induction, concentration of production inducer (IPTG, isopropyl [3-Dl-thiogalactopyranoside); - Lysis: chemical and / or mechanical method, time, power, repetition; - Purification: solubilization or not, buffer, affinity, purity, column type, renaturation conditions, dialysis.

[0084] Production of fusion proteins comprising a CPP and the eGFP polypeptide

[0085] The optimal conditions for the 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) have been defined as described below.

[0086] 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 to 0.5 mM 0.26L

[0087] Bacterial lysis was optimal in two steps using Tris-NaCl-imidazole buffer. First, chemical lysis with lysozyme at 1 mg / ml for 30 min was performed on a gentle agitation wheel at 4°C to preserve protein integrity. This was followed by mechanical lysis using six 30-second sonication cycles: first three cycles at 30% power, then three cycles at 70% power. The bacterial lysates were placed on ice for at least 30 seconds between each sonication.

[0088] After centrifugation, the soluble fraction was passed through a Nickel column and the proteins were able to be eluted by competition with 500mM imidazole.

[0089] The purity, quality, and quantity of the proteins were analyzed by SDS-PAGE and Western blot. For this purpose, 15 µl of the sample were mixed with 5 µl of 4X reducing buffer and migrated into 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 stirring and then decolorized with water, or transferred onto a 0.2 µm nitrocellulose membrane using the Bio-Rad Mini-Protean system according to the manufacturer's protocol for the mixed molecular weight program. The membranes were then blocked with 5% skimmed milk - 0.1% TBS IX tween for 2 hours at room temperature with shaking, then incubated with an anti-His HRP antibody (for "HorseRadish Peroxidase" in Anglo-Saxon terminology) diluted in 1 / 50000 in the blocking solution. The anti-His antibody binds to the polyhistidine tag of fusion proteins and HRP allows its detection by the ChemiDoc Imaging System (BioRad) using chemiluminescence.

[0090] Coomassie blue staining of acrylamide gels allowed us to observe that the higher the production yield, the less efficient the protein solubilization. However, the quantities of solubilized protein remain very significant and sufficient for the purification of several milligrams of protein according to the protocol.

[0091] The western blot, for its part, indicated the presence of protein aggregates in the samples which could be eliminated by the addition of a very high speed centrifugation step.

[0092] Production of fusion proteins comprising a CCP and the eIF3f polypeptide

[0093] The optimal conditions for the production and purification of fusion proteins comprising a mutant CPP (MDmut) or MD11 and the eIF3f polypeptide (SEQ ID NO: 30 to SEQ ID NO: 37) are defined below.

[0094] The best production yield was observed after culture of E. coli BL21(DE3) bacteria transformed by recombinant plasmids encoding the sequences SEQ ID NO: 30 to SEQ ID NO: 37 overnight at 16°C after induction of production with 0.5 mM IPGT added to DO=1.

[0095] Bacterial lysis was optimal in two steps in MOPS-NaCl-imidazole buffer. First, chemical lysis with lysozyme at 1 mg / ml for 30 min on a gentle agitation wheel was performed at 4°C to preserve protein integrity. This was followed by mechanical lysis using five 30-second cycles of sonication at 70% power, with the bacterial lysates being placed on ice for at least 30 seconds between each sonication.

[0096] None of the tested protocols allowed obtaining a sufficient quantity of proteins in soluble form. After centrifugation, the soluble fraction was therefore removed and the inclusion bodies were washed in 3 different washing buffers (MOPS 20mM - NaCl 2M - Cysteine ​​15mM; MOPS 20mM - NaCl 250mM - Cysteine ​​15mM - Tritton X100 2%; MOPS 20mM - NaCl 250mM - Cysteine ​​15mM) then solubilized under denaturing conditions (MOPS 20mM - NaCl 250mM - Cysteine ​​15mM - Urea 8M).The proteins were purified on a Nickel column by imidazole competition (MOPS 20mM - NaCl 250mM - Cysteine ​​15mM - Urea 8M - Imidazole 500mM), the proteins were renatured by the drop-by-drop dilution method (Na Citrate 50mM - MgCl2 2mM - DTT 2mM - NaCl 50mM - L-Argine 500mM - Tween 20 0.5%) then dialyzed in 2 steps (Na Citrate 50mM - MgCl2 2mM - DTT 2mM - NaCl 50mM - L-Argine 50mM - N-Lauryl Sarcosine 0.01%; Na Citrate 50mM - NaCl 50mM - Glycerol 10%) to be in solution in a buffer. compatible with in cellulo and in vivo experiments.

[0097] The purity and quality of the proteins were analyzed by SDS-PAGE and Western blot using the same protocol as that used for the fusion proteins, including the eGFP protein, and described in the previous section. The treatment samples were quantified by the microBCA method according to the manufacturer's protocol.

[0098] In the elution, refolding, and dialysate samples, a single band, labeled with the anti-histidine antibody by Western blot, of 50 kDa was detected by Coomassie blue staining of the acrylamide gel. The treatment samples used for the in vitro experiments are therefore pure and of good quality. Indeed, the absence of any extra band by Western blot demonstrated the absence of degraded and / or aggregated proteins in the samples.

[0099] Production of fusion proteins comprising a CCP and the MDA-7 polypeptide or truncated MDA-7

[0100] The optimal conditions for the 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.

[0101] The best production yield was observed after culturing E. coli BL21(DE3) bacteria transformed with recombinant plasmids encoding sequences SEQ ID NO: 46 to SEQ ID NO: 61 overnight at 16°C after induction of production with IM of IPGT added to D026o>1-

[0102] Bacterial lysis was optimal in two steps using a 50 mM Tris-250 mM NaCl-5 mM imidazole buffer. First, chemical lysis with lysozyme at 1 mg / ml for 30 min on a gentle agitation wheel was performed at 4°C to preserve protein integrity. This was followed by mechanical lysis using five 30-second cycles of sonication at 70% power, with the bacterial lysates being placed on ice for at least 30 seconds between each sonication.

[0103] None of the tested protocols allowed obtaining a sufficient concentration of proteins in soluble form. After centrifugation, the soluble fraction was therefore removed and the inclusion bodies were washed in 3 different washing buffers (Tris 50 mM - [3-mercaptoethanol 20 mM - Tritton X100 2%; Tris 20 mM - [3-mercaptoethanol 20 mM; Tris 50 mM - DTT 20 mM - Guanidine Hydrochloride 6M) and then solubilized under Guanidine denaturing conditions. The proteins were purified on a nickel column by imidazole competition (Tris 50 mM - Guanidine Hydrochloride 6M - Imidazole 500 mM) and renatured by the drop-by-drop dilution method (Tris 50 mM - Guanidine Hydrochloride 1.4M - NaCl 50 mM - L-Argine 500 mM - Titton X100 0.5%) and then dialyzed in two steps to obtain a solution (Tris 50 mM - Guanidine Hydrochloride 0.75 M - NaCl 50 mM - L-Argine 50 mM - Tween20 0.5%; Tris 50mM - NaCl 50 mM - Glycerol 10%) in a buffer compatible with in cellulo and in vivo experiments.

[0104] The purity and quality of the proteins were analyzed by SDS-PAGE and Western blot, and then the proteins were quantified by the microBCA method. A single band, labeled with the anti-histidine antibody by Western blot, was detected by Coomassie blue staining of the acrylamide gel. The CPP fusion protein samples coupled to the whole or truncated MDA7 polypeptide are therefore pure and of good quality. Indeed, the absence of an extra band by Western blot demonstrated the absence of degraded and / or aggregated proteins in the samples.

[0105] Analysis of the cell penetration potential of CPPs

[0106] In order to demonstrate the high potential for intracellular penetration of MDmut mutant CPPs, flow cytometry and fluorescence microscopy analyses were performed.

[0107] 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 bound to a carrier, and to treatments with fusion proteins comprising either CPP MD11 (SEQ ID NO: 1) without the novel genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 17), or comprising CPP MD11 with the novel genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 22).

[0108] To count the number of fluorescent cells after treatment, HEK293 and B16-Ova cells were seeded and then treated to approximately 60% confluence with 200 nM of their respective fusion proteins in the absence of bovine serum for 4 hours. They were then supplemented with 5% bovine serum and incubated overnight. The following day, the cells were trypsinized, washed with PBS IX, and FACS-stained for analysis.

[0109] An untreated cell sample supplemented with Propridium iodide at 1 µg / ml was used to calibrate the blank and the live cell range. For the treated samples, a minimum of 25,000 live cells were analyzed for their green fluorescence.

[0110] The analysis results indicate the percentage of GFP-positive cells after treatment and are presented in [Fig. 1] in which: - MD 11-eGFP corresponds to the control fusion protein comprising the CPP MD11 sequence but without the genetic engineering elements (SEQ ID NO: 1) and the eGFP polypeptide (SEQ ID NO: 17); - #lA-eGFP corresponds to the fusion protein comprising the CPP MD11 sequence with genetic engineering elements and the eGFP polypeptide (SEQ ID NO: 22); - #2B-eGFP corresponds to the fusion protein comprising the CPP MDmutlDelCys sequence and the eGFP polypeptide (SEQ ID NO: 23); - #3C-eGFP corresponds to the fusion protein comprising the CPP MDmut2 sequence and the eGFP polypeptide (SEQ ID NO: 24); - #4D-eGFP corresponds to the fusion protein comprising the CPP sequence MDmut2DelCys and the eGFP polypeptide (SEQ ID NO: 25); - #5E-eGFP corresponds to the fusion protein comprising the CPP MDmut3 sequence and the eGFP polypeptide (SEQ ID NO: 26); - #6F-eGFP corresponds to the fusion protein comprising the CPP sequence MDmut3DelCys and the eGFP polypeptide (SEQ ID NO: 27); - #7G-eGFP corresponds to the fusion protein comprising the CPP MDmut4 sequence and the eGFP polypeptide (SEQ ID NO: 28); - #8H-eGFP corresponds to the fusion protein comprising the CPP sequence MDmut4DelCys and the eGFP polypeptide (SEQ ID NO: 29).

[0111] Flow cytometry showed that overall, under current treatment conditions with 3 to 5 replicas per condition, protein penetration is greater in HEK293 cells than in B16-Ova (B16) tumor cells. For the MD11-eGFP and #5E-eGFP fusion proteins, we are at the limit of significance (p=0.057). The same impaired penetration, compared to MD11-eGFP, is observed for the #4D-eGFP mutant in both cell lines.

[0112] 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 (MD 11-eGFP) with a rate of GFP-positive cells between 73% and 82% versus 57%.

[0113] In B16-Ova (B 16) cells, the penetration capacity of the #3C-eGFP, #5E-eGFP, #6F-eGFP, and #7G-eGFP mutants is equivalent to that of MD 11-eGFP. In contrast, the cell penetration capacity of the #2B-eGFP, #4D-eGFP, and #8H-eGFP mutants is significantly lower, between 15% and 20%, compared to over 41%.

[0114] Furthermore, it is agreed that the MD 11-eGFP or #lA-eGFP fusion proteins provide equivalent results in all respects despite the rearrangement of genetic engineering elements.

[0115] In parallel, the same cells (HEK293 and B 16-Ova) were also seeded onto glass slides equipped with culture chambers and then treated with the fusion proteins MD11-eGFP, #1A-eGFP, #3C-eGFP, #5E-eGFP, #7G-eGFP or buffer for 4 h in the absence of serum. The cells were then washed with PBS IX and then with heparin, fixed with paraformaldehyde, and observed by fluorescence microscopy. rescence. 5 successive focal planes spaced 2pm apart were captured in order to highlight the intracellular localization of proteins.

[0116] Representative images of cell fluorescence after treatment are given in [Fig.2].

[0117] 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.

[0118] Analysis of the antitumor therapeutic potential of the fusion protein comprising CPP and eIF3f polypeptide

[0119] In order to demonstrate the therapeutic potential of MDmut mutant CPPs fused to the translation regulation factor eIF3f (SEQ ID NO: 31 to SEQ ID NO: 37) on melanoma, the study continued with HEK293 and B16-Ova cells.

[0120] Viability was tested using Prestoblue®. For this purpose, cells were seeded in 96-well plates with black edges and transparent bottoms and then, the following day, treated at 60% confluence with 0.25X fetal bovine serum (FBS). In order to calculate the median inhibitory concentration (IC50) of each fusion protein, the proteins were diluted over a wide concentration range from 15 to 100 nM and incubated for 24 or 48 hours.

[0121] The viability curves of B16-Ova (B 16) and HEK293 cells after 24h and 48h of treatment with fusion proteins are illustrated in [Fig.3] in which: - 1E corresponds to the fusion protein comprising the CPP MD11 sequence and the eIF3f polypeptide (SEQ ID NO: 30); - 3E corresponds to the fusion protein comprising the CPP MDmut2 sequence and the eIF3f polypeptide (SEQ ID NO: 32); - 5E corresponds to the fusion protein comprising the CPP MDmut3 sequence and the eIF3f polypeptide (SEQ ID NO: 34); - 7E corresponds to the fusion protein comprising the CPP MDmut4 sequence and the eIF3f polypeptide (SEQ ID NO: 36).

[0122] These tests showed that the mutant CPP 3E 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 cells. The 48-hour IC50 was also not significantly different, ranging between 25 and 35 nM, an extremely low concentration that supports a direct, non-intermediate action of the fusion proteins in the cells.

[0123] More generally, it should be noted that the methods of implementation and realization of the invention considered above have been described as non-limiting examples and that other variants are therefore conceivable.

Claims

Demands

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

7.

2.

3. Nucleic acid molecule encoding a peptide according to claim 1. Expression vector comprising a nucleic acid molecule encoding a peptide according to claim 1.

4. Host cell comprising a nucleic acid molecule according to claim 2 or an expression vector according to claim 3.

5. Use of a peptide according to claim 1 or a nucleic acid molecule according to claim 2 or an expression vector according to claim 3 or a host cell according to claim 4, for obtaining a transporter for the internalization of a molecule of interest into target cells.

6. Combination comprising a carrier and a molecule of interest, said carrier being a peptide according to claim 1.

7. Fusion protein comprising a carrier which is a peptide according to claim 1, and a molecule of interest which is a polypeptide of interest.

8. Nucleic acid molecule encoding the fusion protein according to claim 7.

9. Expression vector comprising a nucleic acid molecule encoding the fusion protein according to claim 7.

10. Host cell comprising a nucleic acid molecule according to claim 8 or an expression vector according to claim 9.

11. Pharmaceutical composition comprising a combination according to claim 6 or a fusion protein according to claim 7 and a pharmaceutically acceptable excipient and / or vehicle.

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.

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, in which 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 eIF3f 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%, in particular 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.

14. A fusion combination or protein or pharmaceutical composition for 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 eIF3f 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.

15. Fusion protein according to claim 7 comprising or consisting of an amino acid sequence selected from the sequences SEQ ID NO: 24, 26, 27 and 28, SEQ ID NO: 32, 34, 35 and 36, SEQ ID NO: 40, 42, 43 and 44, SEQ ID NO: 48, 50, 51 and 52 and SEQ ID NO: 56, 58, 59 and 60.

Images