WO2005010038A2 - Peptide inhibiteur de la traduction des proteines et ses utilisations pour le controle de la traduction des proteines - Google Patents
Peptide inhibiteur de la traduction des proteines et ses utilisations pour le controle de la traduction des proteines Download PDFInfo
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- WO2005010038A2 WO2005010038A2 PCT/FR2004/050345 FR2004050345W WO2005010038A2 WO 2005010038 A2 WO2005010038 A2 WO 2005010038A2 FR 2004050345 W FR2004050345 W FR 2004050345W WO 2005010038 A2 WO2005010038 A2 WO 2005010038A2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K2319/00—Fusion polypeptide
- C07K2319/85—Fusion polypeptide containing an RNA binding domain
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
- C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
- C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
Definitions
- the present invention relates to the field of regulation of protein synthesis by controlling the post-transcriptional stages of translation of messenger RNA into proteins.
- the main industrial applications of the invention are the control of the production of proteins of interest in a bioreactor, the control of the production of proteins of interest in cell therapy (somatic gene therapy), or even the control of the production of proteins. as part of antiviral therapy.
- Proteins capable of acting, at the post-transcriptional level, on the level of translation of proteins by cells are known in the prior art.
- the post-transcriptional biological activity of certain proteins can consist of a modification of the metabolism of messenger RNAs, for example by modification of the stability and half-life of messenger RNAs, by activating the translation of messenger RNAs, or by a change in the transport or location of messenger RNAs.
- proteins originating from yeast such as Pablp, Publ p, She2p, She3p; originating from the xenopus such as Xp54 and PAP1; or mammalian proteins such as hUPF1, hUPF2, hUPF3a, hUPF3b, RNP S1, Y14, DEK, REF2, SRm160, elF-4E, elF-4G, REV, TAP1 and NXF3 were capable, after specific binding to RNA messenger, to modify the metabolism of messenger RNA to which these proteins bind, for example by stabilizing this messenger RNA, by stimulating the translation of messenger RNA, by stimulating the nucleus-cytoplasm export of messenger RNA or by stimulating polyadenylation of messenger RNA (COLLER et al., 2002).
- yeast such as Pablp, Publ p, She2p, She3p
- mammalian proteins such as hUPF1, hUPF2, hUPF3a, hUPF3
- RNA binding protein of known specificity, such as the protein MS2CP.
- the activity of the fusion protein is tested on a reporter DNA construct encoding a messenger RNA comprising (i) the target nucleotide motif of the RNA binding protein and (ii) an open reading frame encoding a reporter protein, such as luciferase or beta-globin (COLLER et al., 2002, PCT application No. WO 99 / 60.408).
- a fusion polypeptide comprising an RNA binding protein fused to a protein derived from the factor elF4G, in order to specifically activate the translation of proteins of interest (PCT request No. WO 00 / 53 779).
- the EDEN-BP protein of Xenopus laevis was the first factor acting in trans for which it has been directly demonstrated an essential role in the specificity of desadenylation of messenger RNAs (PAILLARD et al., 1998).
- a peptide inhibiting protein translation is provided, characterized in that its size is at most 250 amino acids and in that it comprises an amino acid sequence having at least 85 percent identity with the amino acid sequence SEQ ID No. 1.
- Another subject of the invention consists of a fusion polypeptide capable of specifically inhibiting the translation of a target polynucleotide of interest, characterized in that said polypeptide comprises an inhibitor peptide as defined above, said inhibitor peptide being fused with an RNA binding protein.
- the invention also provides nucleic acids comprising a polynucleotide encoding the inhibitor peptide or the fusion polypeptide defined above.
- the invention also relates to a system for controlling the translation of a polynucleotide of interest comprising: (a) a first nucleic acid consisting of a nucleic acid encoding a fusion peptide as defined above; (b) a second nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide encoded by the first nucleic acid as defined in (a) ; (ii) the polynucleotide of interest.
- the invention also relates to vectors into which the various nucleic acids included in the translation control system of a polynucleotide of interest defined above are inserted, as well as to methods for controlling the translation of a target polynucleotide of interest, said methods implementing the translation control system defined above.
- the invention also relates to kits or kits for controlling the translation of a target polynucleotide of interest.
- the invention also relates to the use of a control system or a kit as defined above for controlling the translation of a target polynucleotide of interest.
- the invention also relates to pharmaceutical compositions comprising a fusion polypeptide comprising a protein translation inhibitor peptide, said peptide being fused with an RNA-binding protein, as described above.
- FIG. 1 illustrates a scheme of action of a fusion polypeptide according to the invention comprising the inhibitory peptide Pep58X fused with the RNA binding protein MS2CP.
- the fusion polypeptide is annotated "MS2CP-Pep58X”.
- MS2CP-Pep58X At the bottom of the figure is represented a target messenger RNA comprising the target nucleotide motif of the RNA-binding protein MS2CP, which has been designated “MS2”, and which is represented in the figure by a rod-loop structure, and an expression cassette for the target protein of interest.
- FIG. 2 is a diagram showing the map of plasmid pMS2CP-Pep58X.
- "CV” indicates the promoter of the cauliflower mosaic virus, which controls the expression of the open reading frame encoding the fusion polypeptide between the binding protein and the RNA MS2CP and the inhibiting peptide Pep58X .
- the open reading frame codes for a fusion polypeptide in which the protein MS2CP and the inhibitor peptide Pep58X are separated by the peptide HA (sequence “YPYDVPDYA” [SEQ ID No. 11] ranging from amino acid 98 to amino acid 106 of the hemaglutinin protein HA1), which constitutes a label for detection and purification of the fusion polypeptide.
- T7 designates the promoter of phage T7, which allows RNA synthesis in vitro.
- the vector also comprises a neomycin resistance gene (“Neo r ”) placed under the control of the promoter of the SV40 virus.
- the vector also includes an open reading frame encoding an ampicillin resistance protein.
- the 3 'end of the open reading frame coding for the fusion polypeptide comprises a polyadenylation signal sequence derived from the cDNA coding for bovine growth hormone BGH (for "Bovine Growth Hormone”).
- Figure 3 is a diagram of the reporter vector pRLucLuc-CMVin + 3'UTRGb (MS2) n.
- the bidirectional CMV promoter controls the expression of two open reading frames, respectively (i) an open reading frame coding for luciferase R (“Luc R”) and comprising a 3'UTR region containing eight copies of the site nucleotide MS2, recognition site of the protein MS2CP and (ii) an open reading frame encoding the luciferase F protein (“LucF").
- FIG. 4 illustrates a diagram of the operation of an expression system I.
- FIG. 4A illustrates the synthesis of the fusion polypeptide Pep58X- RNA binding protein when the promoter containing the TetP sequence is activated by the activating protein tTA , which is itself expressed constitutively.
- FIG. 4B illustrates the absence of production of the fusion polypeptide Pep58X-RNA-binding protein in a situation in which the activating protein tTA is produced in the presence of tetracycline and does not activate the TetP sequence of the promoter controlling l of the Pep58X-RNA binding protein gene.
- FIG. 4A illustrates the synthesis of the fusion polypeptide Pep58X- RNA binding protein when the promoter containing the TetP sequence is activated by the activating protein tTA , which is itself expressed constitutively.
- FIG. 4B illustrates the absence of production of the fusion polypeptide Pep58X-RNA-binding protein in a situation in which the activ
- FIG. 5 illustrates the operating principle of the expression system II.
- FIG. 5A illustrates the repression of the expression of the fusion polypeptide Pep58X-RNA-binding protein under the effect of the inhibition of the promoter containing the sequence TetO by the repressor protein Tet (r), which is itself even expressed constitutively.
- FIG. 5B illustrates the activation of the synthesis of the fusion polypeptide Pep58X-RNA binding protein when the repressor protein Tet (r) is brought into contact with tetracycline, which deactivates it and prevents it from inhibiting the promoter containing the sequence TetO.
- FIG. 6 illustrates the results of inhibitory activity of an inhibitory peptide of the invention on the translation of the reporter protein CAT in Xenope.
- Fig. 6A expression of the CAT-B2 mRNA in the absence of peptide, or in the presence of the peptides Pep58X (“58”), Pep60X (“60”) and Pep61X (“61”).
- Fig. 6B expression of CAT-B1 (“B1”) and CAT-B2 mRNAs
- FIG. 7 illustrates the results of the translation of the marker protein luciferase by implementing a translation control system including the vectors pMS2CP-Pep58X, pMS2CP (used as control), and the reporter vector pRLuc Luc CMVin + 3'UTRgb
- This first peptide of 28 amino acids is designated “Pep58X” for the purposes of the present description.
- a peptide having a very high degree of amino acid identity with the inhibiting peptide Pep 58X defined above, said peptide being designated Pep58H also possessed the activity inhibiting translation protein that is wanted.
- the peptide Pep58H consists of a peptide of 28 amino acids in length having the amino acid sequence ranging from the amino acid at position 183 to the amino acid at position 210 of the human CUGBP protein. It has been shown in the examples that the level of inhibitory activity of Pep58X and Pep58H is very similar. It has also been shown that the level of inhibitory activity of Pep58H is identical to that observed for the complete CUGBP protein. Conversely, it has also been shown according to the invention that other peptides of 28 amino acids in length and derived from the region of 84 amino acids ranging from amino acid at position 155 to amino acid at position 238 of the EDEN-BP protein of sequence SEQ ID No. 9 had no inhibitory activity on the translation of proteins. he these are in particular the peptides designated respectively Pep ⁇ OX and Pep61X in the examples.
- the peptide of 28 amino acids derived from the protein EDEN-BP of Xenopus laevis designated Pep58X in the present description, and which has the amino acid sequence SEQ ID No. 1, is capable of inhibiting, in a non-specific manner, the translation of reporter messenger RNA coding for the enzyme CAT (chloramphenicol acetyl transferase).
- CAT chloramphenicol acetyl transferase
- the peptide Pep58X was co-injected with different reporter messenger RNAs in embryos of the Xenopus amphibian (Xenopus laevis). After expression of the reporter genes, protein extracts were prepared and the activity of the reporter gene was assayed.
- the peptide Pep58X inhibits the translation of reporter messenger RNA coding for the CAT enzyme, whether the construction of reporter messenger RNA also includes the EDEN-BP nucleotide site for binding to the EDEN-BP protein or that the reporter messenger RNA does not include any site EDEN nucleotide specific for the binding of RNA to the EDEN-BP protein. Consequently, it is shown according to the invention that a peptide comprising the amino acid sequence SEQ ID No. 1, as is the case with the peptide Pep58X, generally and nonspecifically inhibits the translation of cellular messenger RNAs. The general non-specific inhibitory activity of protein translation demonstrated with the peptides Pep58X of sequence SEQ ID No. 1 and Pep58H of sequence SEQ ID No.
- the size of the non-specific inhibitory peptides for the translation of messenger RNAs into proteins according to the invention, which all have a strong amino acid sequence identity with the peptide Pep58X, is at most 250 amino acids. Although peptides longer than 250 amino acids may also have inhibitory properties for translation of proteins, the applicant thinks, without wishing to be bound by any theory, that such large peptides are capable of possessing a reduced inhibitory activity, in particular due to the creation of conformation constraints of the peptide.
- the subject of the invention is therefore a peptide inhibitor of protein translation, characterized in that its size of at most 250 amino acids and in that it comprises an amino acid sequence having at least 85% identity with the amino acid sequence SEQ ID No. 1 of Pep58X.
- protein, polypeptide and peptide used in the present description are interchangeable and denote a linear chain of amino acid residues linked to each other by a peptide bond between the alpha-amino group and the carboxy group of two residues contiguous amino acids.
- polypeptides comprising at least one non-peptide bond such as a retro-inverso bond (NHCO), a carba bond (CH -CH 2 ) or even a ketomethylene bond (CO-CH 2 ).
- a protein translation inhibitor peptide according to the invention comprises an amino acid sequence preferably having at least 86%, 87%, 88%, 89%, 89.3%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of amino acid identity with the peptide inhibitor of sequence SEQ ID No. 1.
- the “percentage identity” between two amino acid sequences is determined by comparing the two optimally aligned sequences, through a comparison window.
- the part of the amino acid sequence in the comparison window can thus include additions or deletions (for example “gaps”) with respect to the reference sequence (which does not include these additions or deletions) so to obtain an optimal alignment between the two sequences.
- the percentage identity between the two amino acid sequences is calculated by determining the number of positions at which an identical amino acid residue is observed for the two sequences compared after alignment, and then dividing the number of positions to which there is identity between the two amino acid residues compared, by the total number of positions in the comparison window, then by multiplying the result by one hundred in order to obtain the percentage of identity between the two acid sequences amines.
- the optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
- the peptides belonging to the family of protein translation inhibitor peptides according to the invention therefore have, for some of them, an amino acid sequence comprising one or more substitutions, additions, or deletions of an amino acid, compared to the inhibitory peptides comprising the sequence SEQ ID No. 1.
- An illustrative example of such peptides is the sequence inhibitor peptide SEQ ID No. 2.
- Part of the invention are peptides whose amino acid sequence has one or more substitutions of an amino acid with an equivalent acid, compared to peptides which inhibit the translation of proteins as generally defined above.
- amino acids which have an inhibitory activity of the same order of magnitude as the inhibitory activity of the reference inhibitor peptide that is to say amino acids, which, when replace an amino acid present in the amino acid sequence of the peptide reference inhibitor, have an inhibitory activity having the same order of magnitude as that of the reference inhibitor peptide.
- conservative amino acid substitutions are the replacements of an amino acid by another amino acid belonging to the same class.
- the aliphatic amino acids Ala, Val, Leu and Ile are thus interchangeable; amino acids having a hydroxyl residue such as Ser and Thr; the amino acids acids Asp and Glu; amino acids having an amide function such as Asn and Gln; basic amino acids such as Lys and Arg; and aromatic amino acids such as Phe and Tyr.
- the level of inhibitory activity of an inhibitor peptide according to the invention can be easily determined by a person skilled in the art, for example by calculating, for each concentration of a series of increasing intracellular concentrations of inhibitor peptide, the percentage of expression of a marker protein encoded by a reporter mRNA, relative to the level of expression of said marker protein in the absence of the inhibitor peptide, then by calculating the slope of the straight line connecting the different values of percentage of expression of the marker protein, for the different increasing values of inhibitor peptide concentration, as detailed in the examples.
- - P is the value of the line of inhibition slope
- a determined inhibitory peptide has a level of activity inhibiting the translation of proteins of the "same order of magnitude" as the inhibitory activity level of a reference peptide when the P value of said determined peptide is understood. Between - 0.3 and -0.8, preferably between - 0.4 and - 0.7, the P value being preferably around - 0.6.
- EDEN-P protein has the amino acid sequence referenced in the present description as the sequence SEQ ID No. 9.
- the amino acid sequence 156-405 included in the sequence SEQ ID No. 9 of EDEN-BP consists of the amino acid sequence which is between the second and third RNA binding domains of the EDEN protein -BP.
- a protein translation inhibitor peptide as defined above advantageously has a size of at most 50 amino acids in length.
- a protein translation inhibitor peptide of the invention is characterized in that its size is at most 30 amino acids in length and is most preferably at most 28 amino acids in length.
- a protein translation inhibitor peptide which is characterized in that it comprises an amino acid sequence having at least 89%, preferably 89.3%, of identity with the acid sequence.
- amines SEQ ID N ° 1. Meets the definition of an inhibitory peptide above, a peptide characterized in that it comprises the sequence SEQ ID No. 1, in particular the peptide of sequence SEQ ID No. 1. Also included in the definition is a peptide above, a peptide characterized in that it comprises the sequence SEQ ID No.
- the invention also relates to a method for the production of a protein translation inhibitor peptide as defined above, said method comprising the steps of: (a) inserting a nucleic acid encoding said inhibitor peptide into a appropriate expression vector; (b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector obtained in step (a); (c) recovering the conditioned culture medium or lysing the host cell, for example by sonication or by osmotic shock; (d) separating and purifying, from said culture medium, or also from the cell lysates obtained in step (c), said inhibitor peptide; (e) if appropriate, characterize the recombinant inhibitor peptide produced.
- the inhibitor peptides according to the invention can be characterized by fixation on an immunoaffinity chromatography column on which antibodies directed against said peptide have been immobilized beforehand.
- an inhibitor peptide according to the invention can be purified by passage through an appropriate series of chromatography columns, according to methods known to those skilled in the art and described for example in AUSUBEL et al. (1989).
- An inhibitor peptide according to the invention can also be prepared by conventional techniques of chemical synthesis, either in homogeneous solution or in solid phase.
- an inhibitor peptide according to the invention may be prepared by the technique in homogeneous solution described by HOUBEN WEIL (1974) or also by the solid phase synthesis technique described by MERRIFIELD (1965a, 1965b).
- the invention also relates to a nucleic acid comprising a polynucleotide encoding a protein translation inhibitor peptide as defined above.
- said nucleic acid is characterized in that it comprises a regulatory polynucleotide under the control of which is placed the polynucleotide encoding said protein translation inhibitor peptide.
- the invention also relates to a nucleic acid as defined above, characterized in that it is inserted into a recombinant cloning or expression vector.
- the invention also relates to a recombinant cloning or expression vector comprising a nucleic acid as defined above.
- a recombinant cloning or expression vector comprising a nucleic acid as defined above.
- Any suitable vector known or defined in the present description can be used.
- the inhibitory peptides as defined above are used to non-specifically inhibit the translation of proteins, preferably in cellular systems, although the peptides inhibitors can also be used to inhibit the translation of proteins in acellular systems, for example from a cell lysate, for example by incubating an extract of a cellular messenger RNA in an acellular system consisting of a reticulocyte lysate rabbit according to the conventional technique which is described by UCHIDA et al. (2002).
- the inhibitory peptides as defined above are used in order to disturb the cellular metabolism to the point of causing the death of the cells in which these inhibitory peptides have penetrated.
- the protein translation inhibitor peptides as defined above can be used to eliminate certain categories of cells against which they can be targeted, such as tumor cells.
- the inhibitory peptides according to the invention can be fused with a peptide or a protein or else coupled to a polysaccharide, said peptide, said protein or said polysaccharide consisting of ligands recognized specifically by receptors expressed in target cells, by example on the membrane surface of target cells, said target cells then internalizing the inhibitory peptides which will cause their death by blocking the translation of cellular proteins.
- peptides or proteins which can be fused with an inhibiting peptide according to the invention are in particular antibodies or antibody fragments which specifically recognize antigens expressed specifically by certain cell categories, such as tumor cells.
- a fusion protein can be prepared between an inhibitor peptide according to the invention and an antibody or an antibody fragment, for example a Fab fragment or even an F (ab ′) 2 fragment specifically recognizing an antigen expressed selectively by the targeted tumor cells, such as for example the Tn tumor antigen well known in the state of the art.
- an antibody or an antibody fragment for example a Fab fragment or even an F (ab ′) 2 fragment specifically recognizing an antigen expressed selectively by the targeted tumor cells, such as for example the Tn tumor antigen well known in the state of the art.
- the tumor cells present in a population of cells taken from a patient can thus be eliminated by incubation of the cells originating from the patient with appropriate concentrations of the fusion protein.
- inhibitor-antibody peptide (or antibody fragment) followed by recovery of normal non-tumor cells.
- the peptide inhibitor-ligand fusion proteins of a cell receptor can also be used in vivo, for example in the context of anticancer therapies.
- Fusion polypeptides specific inhibitors of protein translation.
- the invention relates to fusion polypeptides capable of specifically inhibiting the translation of a target polynucleotide of interest into the corresponding protein, such a fusion polypeptide comprising a peptide inhibiting the translation of proteins such as defined previously in the description, said inhibitor peptide being fused with an RNA binding protein specifically recognizing a target nucleotide site of the messenger RNA which is targeted.
- the RNA binding protein MS2CP was capable of specifically inhibiting the translation of a messenger RNA comprising the target MS2 nucleotide site and an open reading frame encoding the luciferase marker protein, placed under the control of an appropriate promoter.
- the protein MS2CP contained in the fusion polypeptide selectively binds to its target nucleotide site MS2, which allows the inhibitor peptide Pep58X to specifically inhibit the expression of the messenger RNA or of the messenger RNA containing the target MS2 nucleotide site.
- the same results were reported by the applicant with a fusion polypeptide containing the inhibitory peptide Pep58H and the RNA-binding protein MS2CP.
- RNA-binding protein which is fused to the protein translation inhibitor peptide defined above, is chosen, preferably from MS2CP, N, IRP and U1A, listed in table 1 below.
- Table 1 Preferred mooring proteins
- the RNA binding protein chosen is located at the NH 2 -terminal end of the fusion polypeptide, although it can also be located at the COOH-terminal end of said fusion polypeptide.
- the RNA-binding protein can be directly fused to the inhibitor peptide, that is to say, depending on the case: - (i) when the RNA-binding protein is localized to the NH 2 -terminal end of the fusion polypeptide, the last amino acid in the COOH-terminal position of the RNA-binding protein is chemically linked, preferably by a normal peptide bond, to the amino acid located in position NH 2 -terminal of the inhibitory peptide; or - (ii) when the RNA binding protein is located in the COOH-terminal position of the fusion polypeptide, the amino acid located in the COOH-terminal position of the inhibitor peptide is directly linked, preferably by a normal peptide bond , to the amino acid located in the NH 2
- the RNA binding protein and the protein translation inhibitor peptide are not directly linked to each other, but are at otherwise separated from each other, in the fusion polypeptide, by a spacer amino acid sequence, preferably hydrophobic.
- the spacer amino acid sequence has a size sufficient to constitute a region of flexibility of the molecule protein allowing relative mobility of the RNA binding protein vis-à-vis the inhibitory peptide.
- the size of the spacer peptide is at least 3 amino acids in length and at most 50 amino acids in length.
- the size of the spacer peptide is between 5 and 30 amino acids in length, and most preferably between 5 and 20 amino acids in length.
- the spacer sequence when the spacer sequence, or spacer peptide, is hydrophobic, said spacer sequence facilitates the penetration of the fusion polypeptide through cell membranes.
- said spacer peptide mainly contains hydrophobic amino acids such as the amino acids valine, leucine or even isoleucine.
- the spacer peptide preferably comprises in its sequence at least 50% of hydrophobic amino acids, preferably at least 60% and very preferably at least 80% of hydrophobic amino acids.
- the spacer peptide consists of a chain of poly (Alanine) amino acids, comprising from 3 to 50, better from 5 to 30, advantageously from 5 to 20, and very preferably from 5 with 10 residues Alanine.
- the spacer amino acid sequence or spacer peptide constitutes a label allowing the detection or the purification of the fusion polypeptide present in a sample.
- the spacer peptide can consist of the “HA TAG” peptide, of sequence SEQ ID No. 11, as described in the examples.
- Specific and illustrative examples of specific inhibitory fusion polypeptides according to the invention consist of: the fusion polypeptide MS2CP-HA TAG-Pep58X of amino acid sequence SEQ ID No. 5, which is encoded by the nucleic acid of sequence SEQ ID N ° 7. the fusion polypeptide MS2CP-HA TAG-Pep58H of amino acid sequence SEQ ID No.
- the invention also relates to a nucleic acid comprising a polynucleotide encoding a fusion polypeptide as defined above.
- the preferred nucleic acids according to the invention are the following: the nucleic acid of sequence SEQ ID No. 7 encoding the fusion polypeptide MS2-HA TAG-Pep58X; the nucleic acid of sequence SEQ ID No. 8 encoding the fusion polypeptide MS2CP-HA TAG-Pep58H.
- the nucleic acid encoding a fusion polypeptide according to the invention is characterized in that it comprises a regulatory polynucleotide under the control of which is placed the polynucleotide encoding the fusion polypeptide.
- said nucleic acid is characterized in that the regulatory polynucleotide is an inducible regulatory polynucleotide.
- the invention also relates to a nucleic acid encoding a specific inhibitory fusion polypeptide as defined above, characterized in that it is inserted into a cloning or expression vector.
- the invention also relates to a recombinant cloning or expression vector, characterized in that it comprises a nucleic acid coding for a specific inhibitor peptide as defined above. Any cloning or expression vector known or described in the present invention can be used.
- the specific inhibitory fusion polypeptides as well as the nucleic acids encoding these fusion polypeptides of the invention have enabled applicants to achieve systems for controlling the translation of one or more target polynucleotides of interest, technical characteristics of these control systems being defined below.
- the invention relates to fusions between peptide and oligonucleotide capable of specifically inhibiting the translation of a target polynucleotide of interest into the corresponding protein.
- a fusion molecule comprising a protein translation inhibitor peptide as defined above in the description, said inhibitor peptide being fused with an oligonucleotide specifically recognizing a target nucleotide site of an mRNA which is targeted.
- An example of this type of oligonucleotide is the Aptastruc described in PCT application No. PCT / FR 95/01036.
- Fusion polypeptides specific for protein translation inhibitors can be integrated into different translation control systems for one or more target polynucleotides of interest.
- a first system for controlling the translation of one or more target polynucleotides of interest comprises a nucleic acid encoding an inhibitory peptide-RNA binding protein fusion polypeptide as defined above.
- a second control system of the invention consists of a control system which comprises an inhibitory peptide-RNA binding protein fusion polypeptide as defined above.
- said fusion polypeptide When the fusion polypeptide is expressed in cells thus transfected, said fusion polypeptide specifically binds to target messenger RNAs which comprise the target nucleotide site of the RNA-binding protein contained in said fusion polypeptide, whereby said fusion peptide inhibits the translation of proteins encoded by said target messenger RNAs.
- said fusion polypeptide when the specific inhibitory fusion polypeptide is brought into contact with the target cells, said fusion polypeptide is internalized in the cytoplasm of these cells and inhibits the translation of the proteins encoded by the target messenger RNAs containing, in their sequence, the target nucleotide site of the RNA binding protein contained in said fusion polypeptide.
- said control system comprises, on the one hand, a nucleic acid encoding the specific inhibitory fusion polypeptide and, on the other hand part, a second nucleic acid which constitutes the target nucleic acid of the fusion polypeptide and which codes for the protein of interest whose translation control is sought.
- the subject of the invention is also a system for controlling the translation of a target polynucleotide of interest comprising: (a) a first nucleic acid consisting of a nucleic acid comprising a polynucleotide encoding a specific inhibitory fusion polypeptide such as defined in this description; (b) a second nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide encoded by the first nucleic acid as defined in (a) ; and (ii) the polynucleotide of interest whose translation control is sought.
- the third control system is characterized in that the second nucleic acid, which codes for the protein of interest whose translation control is sought, comprises a regulatory polynucleotide under the control of which is placed the polynucleotide of interest encoding said protein of interest.
- a fourth translation control system consists of a translation control system for a target polynucleotide of interest which comprises: (a) an inhibitor peptide-RNA binding protein fusion polypeptide as defined previously in the description; (b) a nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide as defined in (a); and (ii) the polynucleotide of interest whose translation control is sought.
- the nucleic acid (b) of the protein translation control systems defined above comprises at least 2 copies, advantageously at least 3 or 4 copies of the target nucleotide sequence of the RNA-binding protein.
- the nucleic acid (b) comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 copies of said target nucleotide sequence. In certain cases, the nucleic acid (b) therefore contains up to 24 copies of said target nucleotide sequence.
- the invention also relates to a control system as defined above, characterized in that the nucleic acid or acids which are included therein are inserted into a recombinant expression vector.
- the invention also relates to a control system as defined above, characterized in that the nucleic acid (b) is inserted into the genome of a prokaryotic or eukaryotic host cell.
- the third and fourth translation control systems according to the invention make it possible to regulate in a controlled manner the expression of a gene previously inserted artificially into a cell, said gene preferably being a gene coding for a protein whose synthesis must be checked.
- a gene previously inserted artificially into a cell
- said gene preferably being a gene coding for a protein whose synthesis must be checked.
- the protein translation control system as defined above allows, due to the presence of the fusion polypeptide, the repression of the expression of a target polynucleotide of interest during the cell growth phase, then, if desired, the expression of the protein of interest, once the cell growth plateau has been reached within the bioreactor.
- control system Compared to other existing control systems, which act at the transcriptional level of RNA synthesis, and of which a high response inertia is observed as a result, such as for example the TeT on / Tet off system, the control system according to the invention allows a rapid repressive response or, on the contrary, activation of the synthesis of target proteins.
- the full potential of the protein translation control system according to the invention is reached when the expression of the inhibitory peptide-RNA binding protein polypeptide is inducible: thus, repression of the synthesis of said fusion polypeptide will allow translation of the target polynucleotide of interest into the corresponding protein, while activation of expression of the fusion polypeptide will inhibit translation of the target polynucleotide of interest into the corresponding protein.
- the fusion polypeptides are used in the context of a targeted antiviral treatment, using, as RNA binding protein, a protein which 'specifically binds to nucleotide sequences of Viral mRNAs.
- Inducible expression system for the fusion polypeptide constituting the protein translation control systems according to the invention.
- the invention therefore also relates to a nucleic acid comprising a polynucleotide coding for an inhibitory-RNA binding protein fusion polypeptide as defined above and which also comprises a direct action-sensitive regulatory polynucleotide or indirect, of an inducing signal, also designated as an inducible regulatory polynucleotide.
- Inducible regulation systems which can be used according to the invention are shown in FIGS. 4 and 5 respectively. Other inducible regulation systems, based on regulation principles identical to those represented in FIGS. 4 and 5 can also be used. Preferred are the inducible regulatory systems listed in Table 2.
- nucleic acid encoding a fusion polypeptide according to the invention as well as a nucleic acid comprising (i) at least one copy of a sequence target nucleotide of the RNA binding protein contained in said fusion polypeptide and (ii) the polynucleotide of interest whose translation control is sought, are inserted into a recombinant cloning or expression vector.
- vector within the meaning of the present invention is meant a circular or linear DNA or RNA molecule, which is either in the single strand or double strand form.
- a recombinant vector according to the invention is preferably an expression vector. It may especially be a vector of bacterial or viral origin.
- the nucleic acid coding for the inhibitor-RNA binding protein fusion polypeptide according to the invention is placed under the control of one or more sequences containing signals regulating its expression in the cells under consideration.
- a recombinant vector according to the invention advantageously comprises appropriate sequences for initiating and stopping transcription.
- the recombinant vectors according to the invention can include one or more origins of functional replication in the host cells in which their expression is sought, as well as, where appropriate, nucleotide selection marker sequences.
- the recombinant vectors according to the invention can also include one or more of the expression regulation signals as defined above in the description, including inducible regulatory polynucleotides.
- the preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC No. 37017) or also the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, Wl, United States) . Mention may also be made of other commercial vectors such as the vectors pQE70, qQE60, Pqe9 (QUIAGEN), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratag).
- an expression vector for a specific inhibitory fusion polypeptide as defined above is the vector pMS2CP-PEP58X deposited in the National Collection of Cultures of Microorganisms on July 8, 2003 under the access number I-3067.
- a preferred vector comprising the nucleic acid comprising (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the specific inhibitory fusion polypeptide and (ii) the polynucleotide of which translation control is sought is the vector pRLucLuc-CMVin + 3'UTRGb (MS2) n, which is described in the examples.
- the preferred eukaryotic expression vectors are those described in Makrides et al. (1999).
- a fusion protein specific inhibitor of protein translation is used to specifically inhibit the translation of one or more proteins of interest in cells.
- the present invention therefore also relates to a host cell transformed by a nucleic acid coding for a specific inhibitory fusion polypeptide according to the invention, or by a recombinant vector in which such a nucleic acid is inserted.
- the transformed host cell can be of bacterial, fungal or other eukaryotic cells. Most preferably, however, the host cell is a mammalian cell, including a human cell.
- prokaryotic or eukaryotic host cells comprising a protein translation control system as defined above, namely: - a nucleic acid encoding a fusion polypeptide specific inhibitor of protein translation according to the invention; - A nucleic acid comprising the polynucleotide of interest whose translation control is sought, and also comprising the target nucleotide site of the RNA binding protein contained in the fusion polypeptide.
- control system as defined above is characterized in that the nucleic acid (b) is inserted into the genome of the prokaryotic or eukaryotic host cell.
- the subject of the invention is also a method for controlling in vitro the translation of a target polynucleotide of interest, characterized in that it comprises the following steps: a) introducing into a prokaryotic or eukaryotic host cell a nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of an RNA-binding protein; (ii) the polynucleotide of interest; and (iii) a regulatory polynucleotide under whose control said polynucleotide of interest is placed.
- step b) cultivating the recombinant host cell obtained at the end of step a) in an appropriate culture medium, the recombinant host cell expressing said polynucleotide of interest; c) when desired, the expression of said polynucleotide of interest is inhibited by adding to the cell culture medium a specific inhibitory fusion polypeptide as defined in the present description.
- the subject of the invention is also a method for controlling in vitro the translation of a target polynucleotide of interest, characterized in that it comprises the following steps: a) a prokaryotic or eukaryotic host cell (1) is introduced into a nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of an RNA-binding protein; (ii) the polynucleotide of interest; and (iii) a regulatory polynucleotide under the control of which said polynucleotide of interest is placed, and (2) a nucleic acid comprising a polynucleotide encoding a specific inhibitory fusion polypeptide according to the invention placed under the control of an inducible regulatory polynucleotide ; b) cultivating the eukaryotic or prokaryotic host cell in an appropriate culture medium; c) when desired, an appropriate final concentration of an agent allowing activation or repression of the expression of the polynu
- the activation agent can consist of: - in tetracycline, when the inducible regulatory polynucleotide comprises the sequence TetO, - in tetracycline, when the inducible regulatory polynucleotide comprises the sequence Tet P.
- the invention also relates to a kit or kit for controlling the translation of a polynucleotide of interest, characterized in that it comprises a specific inhibitory fusion polypeptide as defined above.
- the invention also relates to a kit or kit for controlling the translation of a polynucleotide of interest, characterized in that it comprises: (a) a specific inhibitory fusion polypeptide as defined above; and (b) a recombinant vector into which a nucleic acid is inserted comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide as defined in (a ); (ii) the polynucleotide of interest
- the invention also relates to a kit or kit for controlling the translation of a polynucleotide of interest, characterized in that it comprises a recombinant vector into which is inserted a nucleic acid comprising a polynucleotide encoding a specific inhibitory fusion polypeptide, preferably placed under the control of a regulatory polynucleotide, most preferably under the control of an inducible regulatory polynucleotide.
- the invention also relates to a kit or kit for the control of interest, characterized in that it comprises: (a) a recombinant vector into which is inserted a nucleic acid comprising a polynucleotide encoding a specific inhibitory fusion polypeptide according to the invention, optionally placed under the control of a regulatory polynucleotide, most preferably placed under the control of an inducible regulatory polynucleotide; and (b) a recombinant vector in which a nucleic acid is inserted comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide as defined in (a) ; (ii) the polynucleotide of interest
- the kit or kit as defined above is characterized in that the recombinant vector (a) is the recombinant vector (a) is the vector Pms2cp-PEP58X deposited at the National
- the invention also relates to the use of a protein translation control system as defined in the present description, or of a kit as defined above for controlling the translation of a polynucleotide interest.
- a protein translation control system as defined in the present description
- a kit as defined above for controlling the translation of a polynucleotide interest.
- the above use is characterized in that the polynucleotide of interest is expressed in an acellular system.
- the above use is characterized in that the polynucleotide of interest is expressed in vitro by cells cultivated in a bioreactor.
- the subject of the invention is also a pharmaceutical composition comprising a fusion polypeptide specific for inhibiting protein translation as defined in the present description.
- the invention also relates to a pharmaceutical composition comprising: (a) a fusion polypeptide specific for the translation of proteins as defined in the present description; and (b) a recombinant vector into which is inserted a nucleic acid comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide as defined in (a); (ii) the polynucleotide of interest.
- the invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising a recombinant vector into which is inserted a nucleic acid comprising a polynucleotide encoding a fusion polypeptide specific inhibitor of protein translation according to the invention, optionally placed under the control of a polynucleotide regulator, preferably an inducible regulatory polynucleotide.
- the invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising: (a) a recombinant vector into which is inserted a nucleic acid encoding a fusion polypeptide specific inhibitor of protein translation as defined in the present description, optionally placed under the control a regulatory polynucleotide, preferably an inducible regulatory polynucleotide; and (b) a recombinant vector into which a nucleic acid is inserted comprising: (i) at least one copy of a target nucleotide sequence of the RNA-binding protein contained in the fusion polypeptide as defined in (a ); (ii) the polynucleotide of interest.
- the protein translation control systems according to the invention advantageously replace the regulator-inducer pair currently used by a single molecule, the specific inhibitory fusion polypeptide as defined above, which is capable of modulating directly and very specifically the expression of a gene coding for a protein of therapeutic interest.
- the regulation of the expression of the gene or genes of interest is carried out in a post-transcriptional manner, in the cytoplasm of the host cells, which allows rapid and reversible action of the inducer .
- the specific inhibitory fusion polypeptide will contain a protein that specifically binds to target cell messenger RNA.
- Cellular specificity of this type of treatment can also be obtained if the target messenger RNA is expressed only in a given cell type, and / or if the specific inhibitory fusion polypeptide used only interferes with specific regulators. of this cell type.
- the drugs currently used often have undesirable collateral effects linked to a lack of specificity of action.
- the targets of these drugs are cellular proteins such as polymerases.
- a specific inhibitory fusion polypeptide according to the invention is capable of acting directly and specifically on the messenger RNAs originating from the viral genome and not on the products coded by the genome of the infected cell.
- the RNA binding protein constituting the specific inhibitory fusion polypeptide of the invention is selected on the basis of its specificity for the viral messenger RNA which it is desired to neutralize. This strategy makes it possible to obtain very good specificity for targeting the specific inhibitory fusion polypeptide of the invention.
- a specific inhibitory fusion polypeptide according to the invention is particularly well suited to antiviral control.
- the recurring problem of the toxicity to cells of the protein of interest whose expression is sought can be overcome by repressing the expression of the gene of interest, thanks to the specific inhibitory fusion polypeptides of the invention, during the cell growth phase, then the gene coding for the protein of interest to be produced is activated during the subsequent phases, for example once the cells have reached a plateau growth phase.
- the concentration of the specific inhibitory fusion polypeptide present in the reaction medium it is probably possible to control very finely expression of the gene of interest during all stages of the bioreactor production process.
- the present invention is further illustrated, without however being limited, by the following examples.
- EXAMPLE 1 Protocol for the construction of the recombinant vectors MS2CP-Pep58X and MS2CP-Pep58H.
- the vector pMS2CP-HA was obtained by inserting the tag HA (TACCCATACGATGTTCCAGATTACGCT [SEQ ID No. 16]) in the vector pcNMS2 (Lykke Andersen, Cell (103) 1121-31).
- the digested plasmid pRL-Null (Promega) Xbal is linked to the hybrid oligos (and) to obtain the plasmid pRLuc-Xbl. Plasmid pGL3-
- the fragment comprising the 3 'untranslated b globin and 8 MS2 repeats obtained by NotI / Apal digestion of the plasmid pGB (8MS2) (Lykke Andersen, Cell (103) 1121-31) and inserted into pRLucLuc linearized by Pvull to obtain the plasmid pRLucLuc -CMVin + 3'UTRgb (MS2) 8 .
- the mRNAs used are prepared from the constructions described in Ezzedine et al., 2001.
- a femtomole of CAT-Eg2Delta2 mRNA, which does not contain an EDEN sequence in its 3 'untranslated, is co-injected with 200 ng of peptide in 2-cell stage Xenopus embryos. After 4.5 hours of incubation, five batches of three embryos are collected for each series of injection and protein extracts are prepared in order to measure the CAT activity.
- the cells are cotransfected with one of the vectors of the pMS2CP series and the reporter vector.
- the translation levels of Renillia luciferase (R) and Firefly luciferase (F) are determined by luminescence measurement (Promega's Dual Luciférase® Assay System).
- the quantity of fusion proteins produced is evaluated by Western blot (polyclonal anti HA antibody, Santa Cruz Biotechnology), and normalized in relation to the quantity of a ubiquitous cellular protein, the PCNA protein (monoclonal antibody Anti-Proliferating Cell Nuclear Antigen ( PCNA), Sigma Aldrich Company).
- the Luc F mRNA does not contain an MS2 site in its 3 'untranslated part and should not be affected by the expression of MS2CP-Pep58X.
- the expression of Luc F in the presence of MS2CP-Pep58X is analyzed by calculating d (LucF) / d (concentration of MS2CP-Pep58X). The results are shown in Fig 7A. The slope of the line obtained is close to a (0.998) verifying that the fusion protein produced does not affect the expression of luciferase F.
- the expression of Luc F was then used as an internal standard to estimate the effect of the fusion protein on the translation of the mRNA Luc R.
- the latter in fact comprises in its 3 'untranslated part the MS2 sites allowing the fixation of the protein MS2CP-Pep58X.
- the results are analyzed by calculating d (R / F) / d (MS2CP-Pep58X concentration) (Fig 7B).
- the experiment was carried out in parallel with MS2CP, the values obtained with MS2CP correspond to one hundred percent of Luc RB expression Results
- the results are presented in FIG.
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EP04767906A EP1658309B1 (fr) | 2003-07-21 | 2004-07-20 | Peptide inhibiteur de la traduction des proteines et ses utilisations pour le controle de la traduction des proteines |
DE602004003835T DE602004003835T2 (de) | 2003-07-21 | 2004-07-20 | Peptid-inhibitor der proteintranslation und dessen verwendung bei der steuerung der proteintranslation |
US10/565,438 US7863413B2 (en) | 2003-07-21 | 2004-07-20 | Peptide protein translation inhibitor and the use thereof for protein translation control |
CA2533479A CA2533479C (fr) | 2003-07-21 | 2004-07-20 | Peptide inhibiteur de la traduction des proteines et ses utilisations pour le controle de la traduction des proteines |
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Non-Patent Citations (6)
Title |
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CASTAGNETTI STEFANIA ET AL: "Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries" DEVELOPMENT (CAMBRIDGE), vol. 127, no. 5, mars 2000 (2000-03), pages 1063-1068, XP002262156 ISSN: 0950-1991 * |
COLLER JEFFERY ET AL: "Tethered function assays using 3' untranslated regions" METHODS (ORLANDO), vol. 26, no. 2, février 2002 (2002-02), pages 142-150, XP002262153 ISSN: 1046-2023 cité dans la demande * |
GOOD PETER J ET AL: "A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 37, 15 septembre 2000 (2000-09-15), pages 28583-28592, XP002262155 ISSN: 0021-9258 * |
MINSHALL NICOLA ET AL: "A conserved role of a DEAD box helicase in mRNA masking" RNA (NEW YORK), vol. 7, no. 12, décembre 2001 (2001-12), pages 1728-1742, XP001156011 ISSN: 1355-8382 * |
PAILLARD LUC ET AL: "East of EDEN was a poly(A) tail." BIOLOGY OF THE CELL (PARIS), vol. 95, no. 3-4, mai 2003 (2003-05), - juin 2003 (2003-06) pages 211-219, XP002262151 ISSN: 0248-4900 * |
TAKAHASHI NOBUHIRO ET AL: "Coexpression of the CUG-binding protein reduces DM protein kinase expression in COS cells" JOURNAL OF BIOCHEMISTRY (TOKYO), [Online] vol. 130, no. 5, novembre 2001 (2001-11), pages 581-587, XP002262154 ISSN: 0021-924X Extrait de l'Internet: URL:http://jb.bcasj.or.jp/130-5/5faawjtx.h tm> [extrait le 2003-11-20] * |
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WO2008009727A1 (fr) * | 2006-07-21 | 2008-01-24 | Centre National De La Recherche Scientifique (Cnrs) | Cytomoduline positive pour améliorer la productivité d'un bioréacteur |
US20130004991A1 (en) * | 2006-07-21 | 2013-01-03 | Institut National De La Recherche Agronomique | Positive cytomodulines to improve bioreactor productivity |
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EP1658309B1 (fr) | 2006-12-20 |
US7863413B2 (en) | 2011-01-04 |
CA2533479C (fr) | 2014-06-17 |
ATE348841T1 (de) | 2007-01-15 |
FR2857969A1 (fr) | 2005-01-28 |
EP1658309A2 (fr) | 2006-05-24 |
DE602004003835D1 (de) | 2007-02-01 |
WO2005010038A3 (fr) | 2006-02-09 |
US20070105764A1 (en) | 2007-05-10 |
CA2533479A1 (fr) | 2005-02-03 |
DE602004003835T2 (de) | 2007-08-09 |
FR2857969B1 (fr) | 2005-10-14 |
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