The invention pertains to the field of molecular means capable of binding to, and preferably of chelating, cGMP, appropriate for use in vitro or in vivo and preferably capable of targeting specific cellular compartments.

cGMP is a second messenger involved in a wide range of signaling pathways and cellular processes including neurotransmission, calcium homeostasis, phototransduction, lipid metabolism and cation channel activity. The diversity of these processes suggest that cGMP signals are tightly controlled in space and time to achieve specific modulation of its downstream pathways. However, manipulating cGMP is mostly achieved using pharmacological approaches either altering the synthesis or degradation of this cyclic nucleotide, or manipulating downstream signaling pathways. This techniques lacks both cellular and subcellular specificity. There is currently no tools capable of binding specifically cGMP and which may be used to altering physiological variations of this second messenger with cellular and subcellular specificity in vitro and in vivo. Recently, optogenetic strategies have emerged to impose acute and extrinsic modulation of cGMP and investigate the dynamics of cGMP signaling. However, they are difficult to use in vivo or for chronic manipulations and are not able to modify endogenous cGMP signals.

The proteins from the cGMP-dependent Protein Kinase (PKG) family are able to bind cGMP, and their cGMP binding sites have been identified.

The proteins of the PKG family are serine/threonine kinases present in a variety of eukaryotes ranging from the unicellular organism Paramecium to humans. Two PKG genes, coding for PKG type I (PKG-I) and type II (PKG-II), have been identified in mammals. The N-terminus of PKG-I is encoded by two alternatively spliced exons that specify for the PKG-I and PKG-Iisoforms. PKG-Iis activated at about 10-fold higher cGMP concentrations than PKG-I. The PKG-I and PKG-II are homodimers of two identical subunits (about 75 kDa and about 85 kDa, respectively) and share common structural features.

Each subunit is composed of three functional domains:an N-terminal domain that mediates homodimerization, suppression of the kinase activity in the absence of cGMP, and interactions with other proteins including protein substrates;a regulatory domain that contains two non-identical cGMP-binding sites;a kinase domain that catalyzes the phosphate transfer from ATP to the hydroxyl group of a serine/threonine side chain of the target protein

In humans, two isoforms of PKG-I have been described: PKG-I and PKG-I. The regulatory domain and kinase domain are identical in all PKG-I isoforms, but their N-terminal domains differ.

It is difficult to envision how cGMP chelators could be derived from these proteins. On the one hand, PKG catalytic activity is likely to induce unwanted side effects both when used in vitro as biological tools or when used in a therapeutic treatment, thereby preventing the use of the whole protein. On the other hand, Dotsmann et al. (FEBS Lett.;2; 398(2-3):206-10, 1996) have shown, using deletion mutants of PKG-I, that the deletion of the catalytic domain has a deleterious effect on their affinity constant to cGMP, thus precluding the use of catalytic-deficient proteins derived from the PKG family.

There is thus a need for improved means to manipulate cGMP signaling, and in particular intracellular cGMP concentration.

The inventors have designed chimeric polypeptides derived from the isoforms PKG-I and PKG-I, devoid of any catalytic domain. The polypeptides of the invention comprise a chimeric construction derived from the N terminus part of PKG-I and PKG-I, and the two cGMP binding sites of the wild type PKG.

Surprisingly, despite the deletion of the catalytic domain, which had been shown to affect PKG-I affinity constant, the PKG-I/PKG-I-based chimeric polypeptide of the invention is able to efficiently bind and sequester cGMP. As shown in the experimental part, the polypeptides of the invention are capable of sequestering cGMP after nitric oxide induction, in presence of the FRET sensorThPDE5VV, thus suggesting its high affinity for cGMP. The polypeptide of the invention can easily be further functionalized using simple molecular tools, for instance by addition of fluorescent peptides. The polypeptide of the invention can therefore be utilized in a wide variety of applications, either in vitro and in vivo, for instance to prevent the activation of cGMP-dependent cellular processes. These properties render the polypeptide of the invention particularly useful for the prevention or treatment of pathologies associated with intracellular cGMP signaling dysfunction.

The invention pertains to a polypeptide comprising:a chimeric peptide derived from the sequence SEQ ID NO: 1 and from the sequence SEQ ID NO: 2;a cGMP binding domain comprising or consisting of the sequence SEQ ID NO: 6; and,wherein said polypeptide is devoid of any catalytic domain, and its functional variants.

The polypeptide of the invention is preferably an isolated, recombinant or synthetic polypeptide.

The sequence SEQ ID NO: 1 corresponds to residues 1 to 89 of bovine PKG-I, which is identical to residues 1 to 89 of human PKG-I. The sequence SEQ ID NO: 2 corresponds to residues 1 to 104 of bovine PKG-I, which is identical to residues 1 to 104 of bovine PKG-I.

In the context of the invention, the terms ‘chimeric peptide derived from the sequence SEQ ID NO: 1 and from the sequence SEQ ID NO: 2_ refer to a peptide comprising or consisting of at least a sequence corresponding to SEQ ID NO: 1 or a fragment thereof, and of at least a sequence corresponding to SEQ ID NO: 2 or a fragment thereof.

The inventors have in particular identified that the following chimeric peptides derived from the sequence SEQ ID NO: 1 and from the sequence SEQ ID NO: 2 are of particular interest when used in the polypeptide:the peptide of sequence SEQ ID NO: 3, which corresponds to residues 1-78 of SEQ ID NO: 1, fused to residues 94 to 104 of SEQ ID NO: 2;the peptide of sequence SEQ ID NO: 4, which corresponds to residues 1-65 of SEQ ID NO: 2, fused to residues 51-78 of SEQ ID NO: 1, fused to residues 94 to 104 of SEQ ID NO: 2; and,the peptide of sequence SEQ ID NO: 5 which corresponds to residues 1-50 of SEQ ID NO: 1, fused to residues 66-83 of SEQ ID NO: 2, fused to residues 69-78 of SEQ ID NO: 1, fused to residues 94 to 104 of SEQ ID NO: 2.

Advantageously, in the polypeptide of the invention, the chimeric peptide consists of or comprises the sequence SEQ ID NO: 3, 4 or 5, preferably the sequence SEQ ID NO: 3

Preferably, the chimeric peptide and the cGMP binding domain form a contiguous sequence, yet preferably the C-terminal end of the chimeric peptide is fused to the N-terminal end of the cGMP binding domain.

According to the invention, the cGMP binding domain comprises or consists of the sequence SEQ ID NO: 6. For the sake of clarity, the sequence SEQ ID NO: 6 corresponds to the consensus sequence for the minimal binding sequence of PKG-I and PKG-II, both of which are known to have the capacity for binding specifically cGMP.

Preferably, the cGMP binding domain comprises or consists of the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 8. For the sake of clarity, sequence SEQ ID NO: 7 and sequence SEQ ID NO: 8 correspond respectively to the minimal binding sequence of PKG-I and the minimal binding sequence of PKG-II.

Yet preferably, the cGMP binding domain comprises or consists of the sequence SEQ ID NO: 9 or the sequence SEQ ID NO: 10. For the sake of clarity, sequence SEQ ID NO: 9 and sequence SEQ ID NO: 10 correspond respectively to the cGMP binding sequence of human PKG-I and the cGMP binding sequence of bovine PKG-I.

In the context of the invention, the terms ‘devoid of any catalytic domain_ should be construed as indicating that the polypeptide of the invention does not contain any of the catalytic domains of either PKG-I or PKG-I, nor any other catalytic domain derived therefrom.

In the context of the invention, the terms ‘catalytic domain of PKG-I _ refer to the sequence SEQ ID NO: 11 and functional variants therefrom. The terms ‘catalytic domain of PKG-I_ refer to the sequence SEQ ID NO: 12 and functional variants therefrom.

As serine/threonine kinases, PKG-I and PKG-Icatalyze the transfer of phosphate from ATP, that is to say catalyze the phosphorylation, on serine or threonine residues in a large number of substrates, among which histone H2B.

In the context of the invention, the functional variants of SEQ ID NO: 11 encompass peptides which sequence derives from the sequence SEQ ID NO: 11 by insertions, substitutions, and/or deletion, of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to catalyze phosphorylate serine/threonine residues of PKG-I substrates, preferably of H2B. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 11 are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 11 is a peptide which sequence derives from the sequence SEQ ID NO: 11 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 11 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 11. In a preferred embodiment, a functional variant of SEQ ID NO: 11 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 11 and is a peptide which sequence derives from the sequence SEQ ID NO: 11 by conservative substitutions.

In the context of the invention, the functional variants of SEQ ID NO: 12 encompass peptides which sequence derives from the sequence SEQ ID NO: 12 by insertions, substitutions, and/or deletion, of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to catalyze phosphorylate serine/threonine residues of PKG-Isubstrates, preferably of H2B. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 12 are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 12 is a peptide which sequence derives from the sequence SEQ ID NO: 12 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 12 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 12. In a preferred embodiment, a functional variant of SEQ ID NO: 12 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 12 and is a peptide which sequence derives from the sequence SEQ ID NO: 12 by conservative substitutions.

The capacity to catalyze phosphorylate serine/threonine residues of PKG-I substrates, in particular of H2B, may easily be determined using standard methods known in the part, such as those disclosed by Glass and Krebs (J Biol Chem;254:9728-9738, 1979).

In the context of the invention, the terms ‘functional variants_ when referring to the polypeptide of the invention encompass polypeptides which sequence derives from the sequence of the polypeptide of the invention by insertions, substitutions, and/or deletion, of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to bind cGMP. The binding of cGMP to a given polypeptide may easily be determined by methods known in the art, such as detailed in Dotsmann et al.

Preferably, a functional variant of the polypeptide of the invention is a peptide which sequence derives from the sequence of the polypeptide of the invention by one or more conservative substitutions. Yet preferably, a functional variant of the polypeptide of the invention has a sequence which has at least 80, 85, 90 or 95% identity with the sequence of said polypeptide. In a preferred embodiment, a functional variant of the polypeptide of the invention has a sequence which has at least 80, 85, 90 or 95% identity with the sequence of said polypeptide and is a peptide which sequence derives from the sequence of the polypeptide of the invention by conservative substitutions.

In the sense of the present invention, the “percentage identity” or “% identity” between two sequences of nucleic acids or amino acids means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman, by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis., or by the comparison software BLAST NR or BLAST P). The percentage identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally-aligned sequences in which the nucleic acid or amino acid sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the amino acid, nucleotide or residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.

Preferably, the invention also pertains to modified polypeptides, which are derived from the polypeptide of the invention or its functional variants by introduction of chemical modification into one or more amino acid residues, peptide bonds, N- and/or C-terminal ends of the protein, aimed at increasing the stability, bioavailability or bioactivity of the protein, as long as the modified polypeptide remains functional.

The polypeptide of the invention may further be functionalized with peptide sequences having properties of interest, such as peptide signals, which are localization sequences that will enable targeting the polypeptide of the invention to specific subcellular compartment, or fluorescent peptides, that will be useful for in vitro detection of the polypeptide.

Peptides signals are peptides capable of targeting a polypeptide to a specific subcellular compartment, and are well known in the art. Of particular interest are the sequences capable of targeting peptides to the plasma membrane, and more particularly to lipid rafts. Lipid rafts are sphingolipid- and cholesterol-rich plasma membrane microdomains, which have been identified and proposed to function as platforms where signal transduction molecules may interact with receptors.

Advantageously, the polypeptide of the invention comprises a peptide signal. Preferably the peptide signal is capable of targeting the polypeptide of the invention to specific subcellular compartments, more preferably to the plasma membrane, yet preferably to lipid rafts.

Peptide signals capable of targeting a peptide to the plasma membrane are well known in the art. For instance, the N terminus domains of proteins from the Src family, and in particular the peptide of sequence SEQ ID NO: 13, which corresponds to the palmitoylation and myristoylation motifs of the N terminus part of the Lyn Kinase, have been shown to enable the localization of said proteins to lipid rafts. In contrast, the peptide of sequence SEQ ID NO: 14, which corresponds to the CaaX-polylysine motif derived from the protein K-Ras, is known to target proteins to the plasma membrane while restricting the localization to lipid rafts. These sequences may be used as peptide signal either alone or in tandem repeat.

Preferably, in the polypeptide of the invention, the peptide signal comprises or consists of the sequence SEQ ID NO: 13 or 14, more preferably the sequence SEQ ID NO: 13, or functional variants therefrom. Advantageously, in the polypeptide of the invention, the peptide signal comprises or consists of tandem repeats of the sequence SEQ ID NO: 13, or functional variants therefrom.

In the context of the invention, the functional variants of SEQ ID NO: 13 encompass peptides which sequence derives from the sequence SEQ ID NO: 13 by insertions, substitutions, and/or deletions of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to allow the localization of a peptide bound thereto or comprising thereof to lipid rafts. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 13 are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 13 is a peptide which sequence derives from the sequence SEQ ID NO: 13 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 13 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 13. In a preferred embodiment, a functional variant of SEQ ID NO: 13 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 13 and is a peptide which sequence derives from the sequence SEQ ID NO: 13 by conservative substitutions.

In the context of the invention, the functional variants of SEQ ID NO: 14 encompass peptides which sequence derives from the sequence SEQ ID NO: 14 by insertions, substitutions, and/or deletion, of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to allow the localization of a peptide bound thereto or comprising thereof to the cellular membrane, at the exclusion of lipid rafts. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 14 are substituted for other amino acid residues with similar chemical properties. (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 14 is a peptide which sequence derives from the sequence SEQ ID NO: 14 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 14 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 14. In a preferred embodiment, a functional variant of SEQ ID NO: 14 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 14 and is a peptide which sequence derives from the sequence SEQ ID NO: 14 by conservative substitutions.

The capacity of a peptide signal to allow the localization of a peptide bound thereto or comprising thereof to the cellular membrane, and in particular to lipid rafts or at the exclusion of lipid rafts may easily be determined using standard methods, in particular based on fluorescence microscopy and/or density gradients, such as detailed in the experimental part of the present application.

The peptide signal may be in C terminus or in N terminus of the polypeptide of the invention. Preferably, the peptide signal is in N terminus of the polypeptide of the invention.

The polypeptide of the invention may further be functionalized with fluorescent peptides, that will be useful for in vitro detection of the polypeptide.

Fluorescent peptides are well known in the art. Examples of fluorescent peptides are the green fluorescent protein (GFP), or GFP variants such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP) and their variants such as the optimized CFP Turquoise or the YFP variant Venus. Advantageously, the polypeptide of the invention further comprises a fluorescent peptide, preferably chosen in the list consisting of green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP), and their variants such as the optimized CFP Turquoise or the YFP variant Venus.

Another aspect of the invention relates to a polynucleotide encoding the polypeptide of the invention, and a recombinant vector comprising said polynucleotide.

In the context of the invention, the term ‘polynucleotide_ refers to a polydeoxyribonucleotides or polyribonucleotides, in the form of a separate fragment or a larger construct, and includes DNA, cDNA and RNA sequences which encode the polypeptide of the invention. Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term ‘isolated_ as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.

Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a eukaryotic or prokaryotic cell like a mammalian, bacterial or fungal cell. ‘Expression vectors_ are DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. Suitable selectable markers may for instance be chosen among the genes encoding neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 forS. cerevisiae. Some expression vectors do not contain an origin of replication for autonomous replication in host cells but rather depend on the ability of the vector to stably integrate (either randomly or by a homologous integration event) using a marker to select for integration/maintenance. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. The polynucleotide is inserted into the expression vector in proper orientation and correct reading frame for expression. Preferably, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Such vectors comprise YAC (yeast artificial chromosome), BAC (bacterial artificial), baculovirus vectors, phage, phagemid, cosmid, viral vector, plasmid, RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic DNA. Suitable vectors according to the present invention comprise, but are not limited to, YAC (yeast artificial chromosome), BAC (bacterial artificial), baculovirus vectors, phage, phagemid, cosmid, viral vector, plasmid, RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic DNA. Suitable plasmids to be used amammalian expression vectors include but are not limited to, pcDNA3, pcDNA3.1, pcDNAI, pcDNAIamp (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ≅ZD35 (ATCC 37565). Suitable plasmids to be used as bacterial expression vectors include but are not limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gtl 1 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia), and pQE vectors (Qiagen) including pQE70, pQE60, pQE-9. Suitable plasmids to be used as expression vectors in fungal cells include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen). Suitable plasmids to be used as expression vectors in insect cell include but are not limited to pBlueBacIII, pBlueBacHis2 (Invitrogen) and pFastBac1, pFastBacHT (Life Technologies). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, Dtype viruses, HTLV-BLV group, lentivirus, spumavirus. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.

Another aspect of the invention provides a host cell or a non-human organism transformed with said polynucleotide or recombinant vector. The non-human transgenic organism is obtained from a unicellular or pluricellular microorganism or a higher eukaryotic organism. The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to infection, transformation, transfection, lipofection, protoplast fusion, and electroporation. Modified host cells containing the expression vector may be clonally propagated and individually analyzed to determine whether they produce the polypeptide of the invention.

In a preferred embodiment said modified host cell is a eukaryotic cell such as a human cell. In another embodiment, said non-human transgenic organism is a transgenic plant, nematode, zebrafish or algae.

The polynucleotide, vector, host cell, and non-human transgenic organism of the invention are useful for the production of the polypeptide/chimeric protein of the invention using well-known recombinant DNA techniques.

Another aspect of the invention relates to the non-therapeutic use of the polypeptide, polynucleotide, vector of the invention, in vitro or in vivo, as cGMP chelating agent. Preferably the invention relates to the non-therapeutic use of the polypeptide, polynucleotide, vector of the invention, for stabilizing cGMP concentration, and/or for inhibiting cGMP signalization in vivo and/or in vitro. Yet preferably, the non-therapeutic use, when performed in vivo, is performed in a healthy subject.

The terms ‘stabilizing cGMP concentration_ should be construed as generally understood in the art, that is to say as the action of minimizing the variations of the concentration of free cGMP in a composition upon addition or induction of cGMP in said composition. In the context of the invention, the terms ‘concentration of free cGMP_ refer to the concentration of unbound cGMP. The variation, and in particular the increase, of cGMP concentration may easily be measured using for instance the biosensorThPDE5VV, by monitoring the FRET/CFP ratio, as disclosed in the experimental part. In the context of the invention, cGMP concentration can be considered as stabilized in a composition when the FRET/CFP ratio of the biosensorThPDE5VVvaries, preferably increases, of less than 10%, preferably less than 5% of the variation detected in the absence of the polypeptide, polynucleotide, vector of the invention, upon addition or induction of cGMP in said composition.

The terms ‘inhibiting cGMP signal_, ‘cGMP signal inhibition_ should be construed as generally understood in the art, that is to say as the action of inhibiting the downstream effectors known to be activated by cGMP as a second messenger, for instance cyclic nucleotide gated calcium channels (CNGs). The cyclic nucleotide-gated (CNG) channel of the rod photoreceptor cell plays a key role in phototransduction by controlling the flow of Na+and Ca2+into the rod outer segment (ROS) in response to light-induced changes in intracellular cGMP concentration. cGMP activates the opening of CNGs, leading to the influx of Na+and Ca2+ions through CNG channels in the cell. Methods for assessing the activation of photoreceptor channels by measuring the cGMP-activated currents at the cytoplasmic membrane using outer segment membrane patches have been thoroughly disclosed in Tanaka et al. (Biochemistry,28(7): 2776-84, 1989). The inhibition of the cGMP signal may thus easily be assessed by monitoring intracellular cGMP-activated currents at the cytoplasmic membrane, in the presence of the polypeptide, polynucleotide, vector of the invention, based on such methods.

It is well documented that a number of pathologies are associated with cGMP signaling dysfunction. The polypeptide, polynucleotide, vector, and host cell of the invention, may thus be particularly useful in preventing or treating such pathologies. In this context, the polypeptide, polynucleotide, vector, or host cell of the invention, may be formulated into a composition suitable for pharmaceutical use.

Another aspect of the invention relates to a pharmaceutical composition, comprising at least one polypeptide, polynucleotide, vector, and/or host cell of the invention, and, preferably, a pharmaceutically acceptable carrier. In the context of the invention, the terms “pharmaceutically acceptable” refer to carriers which can be used in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable, and which is acceptable for veterinary use as well as for human pharmaceutical use. Suitable vehicles or carriers include any pharmaceutically acceptable vehicle such as buffering agents, stabilizing agents, diluents, salts, preservatives, and emulsifying agents. Examples of suitable buffering agents include buffered solutes, such as phosphate buffered solution, chloride solutions or Ringers̆ solution. Examples of suitable stabilizing agents include human serum albumin (HSA), polyvinylpyrrolidone or hyaluronic acid.

The composition of the invention may be formulated according to the intended administration route. For instance, formulations suitable for oral administration include liquid or solid formulations such as tablets, pills, powders (hard or soft gelatine capsules) or granules. Typically, in such formulations, the polypeptide, polynucleotide, vector, and/or host cell of the invention is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica, in a stream of argon. These compositions may also comprise substances other than diluents, for example lubricants such as magnesium stearate or talc, coloring agent, coating (coated tablets) or varnish. Formulations suitable for injection, such as used in parenteral administration, are preferably sterile and fluid, and may preferably be aqueous or non-aqueous solutions, suspensions or emulsions.

Another aspect of the invention relates to the polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof, for use as a medicine, preferably for use in the prevention and/or treatment of pathologies associated with intracellular cGMP signaling dysfunction.

In the context of the invention, the terms ‘pathologies associated with intracellular cGMP signaling dysfunction_ should be construed according to their general meaning in the art. Preferably, said pathologies are associated with intracellular cGMP signaling dysfunction due to an abnormally high intracellular concentration of cGMP. In the context of the invention, ‘an abnormally high intracellular concentration of cGMP_ refer to an intracellular concentration of cGMP which is significantly higher than in cells from a healthy donor. Yet preferably, said pathologies are chosen in the list consisting of retinitis pigmentosa; cardiovascular diseases, preferably chosen in the list consisting of stroke, venous thrombosis or arterial thrombosis; schizophrenia; Huntington disease; achromatopia; cancer, preferably chosen in the list consisting of colorectal cancer, lung cancer, lung disease; erectile dysfunction and drug abuse.

In other terms, the invention pertains to a method for the prevention and/or treatment of pathologies associated with intracellular cGMP signaling dysfunction, wherein said pathologies are chosen in the list consisting of retinitis pigmentosa; cardiovascular diseases, preferably chosen in the list consisting of stroke, venous thrombosis or arterial thrombosis; schizophrenia; Huntington disease; achromatopia; cancer, preferably chosen in the list consisting of colorectal cancer, lung cancer, lung disease;

erectile dysfunction and drug abuse, comprising the step of administering the polypeptide, polynucleotide, vector, host cell of the invention, or a composition comprising thereof to a subject in need thereof, preferably in an effective amount. In the context of the invention, the terms ‘effective amount_ means that the amount of polypeptide, polynucleotide, vector, host cell of the invention, or of a composition comprising thereof is of sufficient quantity to obtain appropriate prevention and or regression of the symptoms of the above-recited pathologies. The effective amount and dosage regimen may be determined by the attending physician based on usual clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

The polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein can administered once or more than one time, in order to prevent or to treat a pathology as recited herein. Efficacy of the prevention and/or treatment can be monitored by periodic assessment.

The polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein can be administered topically or systemically, through the known routes of administration. Preferably, the polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein is administered through the enteric route, in particular by oral, sublingual, or rectal administration, or through the parenteral route, in particular by intracerebral, intramuscular, intradermal, transdermal, intraperitoneal or nasal administration.

In addition to the preceding features, the invention further comprises other features which will emerge from the following description, which refers to examples illustrating the present invention, as well as to the appended figures.

