FR2843753A1 - Antisense nucleic molecule useful as inhibitor of angiogenesis in the treatment of angiogenic disorders, e.g., rheumatoid arthritis, atherosclerosis and endometriosis - Google Patents

Abstract

Inhibitor of angiogenesis (C) comprising an active substance chosen from at least one of a nucleic acid molecule, an antisense nucleic acid molecule, a polypeptide or an antibody, is new. Inhibitor of angiogenesis (C) comprising an active substance chosen from at least one of the following: (a) a gene comprising a nucleic acid molecule from an endothelial cell, whose expression is induced by an angiogenic factor and is inhibited by an angiostatic agent, chosen from any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 to SEQ ID NO. 15, SEQ ID NO. 17 to SEQ ID NO. 53, SEQ ID NO. 225 and SEQ ID NO.284 to SEQ ID NO. 290 and/or a sequence complementary to any of these and/or a fragment of any of these sequences; (b) a polypeptide sequence encoded by any of the above nucleic acid molecules, chosen from any of the sequences of SEQ ID NO. 54 to SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 to SEQ ID NO. 67, SEQ ID NO. 68 to SEQ ID NO. 102 and SEQ ID NO. 291 to SEQ ID NO. 297 and/or a fragment of any of these sequences; (c) an antisense nucleic acid molecule chosen from any of the sequences of SEQ ID NO. 6 , comprising at least 10 residues of a nucleic acid sequence chosen from SEQ ID NO. 108, SEQ ID NO. 110 and SEQ ID NO. 112; or (d) an antibody specific for a polypeptide encoded by the nucleic acid sequences chosen from SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 16; Independent claims are also included for: (1) antisense nucleic acid sequence (I) chosen from any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 to SEQ ID NO. 15, SEQ ID NO. 17 to SEQ ID NO. 53, SEQ ID NO. 225 and SEQ ID NO. 284 to SEQ ID NO. 290, comprising at least ten residues chosen from any of the sequences of SEQ ID NO. 103 to SEQ ID NO. 107, SEQ ID NO. 109, SEQ ID NO. 111 and SEQ ID NO. 113 to SEQ ID NO. 148 and/or a sequence having at least 85%, preferably 95% sequence identity with a sequence chosen from any of SEQ ID NO. 103 to SEQ ID NO. 107, SEQ ID NO. 109, SEQ ID NO. 111 and SEQ ID NO. 113 to SEQ ID NO. 148; (2) antibody (II) specific for any of the polypeptide sequences comprising any of the sequences of SEQ ID NO. 54 to SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 to SEQ ID NO. 67, SEQ ID NO. 68 to SEQ ID NO. 102 and SEQ ID NO. 291 to SEQ ID NO. 297; (3) preparation of the antibody (II) comprising in vivo or in vitro immunization of an immunocompetent animal cell, preferably of a vertebrate and most preferably of a mammal, with at least one of the polypeptide sequences chosen from any of SEQ ID NO. 54 to SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 to SEQ ID NO. 67, SEQ ID NO. 68 to SEQ ID NO. 102 and SEQ ID NO. 291 to SEQ ID NO. 297 (4) mammalian expression vector (III) comprising at least one antisense sequence chosen from above (1) and/or from SEQ ID NO. 149 to SEQ ID NO. 153, SEQ ID NO. 157, SEQ ID NO. 159 to SEQ ID NO. 163, SEQ ID NO. 164 to SEQ ID NO. 194; (5) preparation of a genetically modified cell, that over- or under-expresses a gene implicated in an angiogenic disorder, comprising inserting into a mammalian cell a vector (III); (6) genetically modified cell (IV) that over-expresses or under-expresses at least one gene involved in angiogenesis by a nucleic acid sequence chosen from any of SEQ ID NO. 1 to SEQ ID NO. 290 as in (1) or a fragment of any of these; (7) preparation of a cell line that stably expresses an expression vector (III) comprises: (a) introducing a gene offering resistance to at least an antibiotic into a genetically modified cell (IV); (b) cultivating the cells obtained in step (a) in the presence of the antibiotic to which the transformed cells are resistant; and (c) selecting viable cells. (8) therapeutic composition useful to treat or diagnose an angiogenic disorder in mammals, preferably in humans, comprising (IV) or a cell line prepared as above in (7); (9) preparation of a recombinant protein comprising: (a) construction of an expression vector (III)

Description

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 GENES INVOLVED IN THE REGULATION OF ANGIOGENESIS, PHARMACEUTICAL PREPARATIONS CONTAINING SAME AND THEIR USE.

 The present invention belongs to the field of pharmaceutical compositions useful for the treatment of pathologies resulting from a deregulation of the mechanism of angiogenesis.

 It relates to compositions comprising, on the one hand, sequences of new genes whose function was not identified to date and whose implication in the mechanism of angiogenesis has been demonstrated for the first time by the Applicant. and on the other hand gene sequences, at least one of whose functions has been previously identified, but whose implication, as genes constituting endothelial cells, in the mechanism of angiogenesis has been demonstrated for the first time during the work performed by the applicant in the context of the present invention. These genes are identified by their nucleotide sequences in the attached sequence listing. The present invention also relates to the polypeptide sequences of the factors encoded by said genes which find their application in the clinical study of the process of angiogenesis, the prognosis, the diagnosis and the treatment of pathologies related to this process as well as in the implementation of pharmacological, pharmacogenomic or pharmacosignalitic tests.

 Angiogenesis is a fundamental process by which new blood vessels are formed. This process is essential in many normal physiological phenomena such as reproduction, development or healing. In these normal biological phenomena, angiogenesis is under strict control, ie it is triggered for a short period, a few days, then

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 completely inhibited. However, several pathologies are related to invasive and uncontrolled angiogenesis.

Arthritis, for example, is a condition caused by damage to cartilage by invasive neovessels. In diabetic retinopathy, the invasion of the retina by the neovessels leads to the blindness of the patients; the neovascularization of the ocular apparatus presents the major cause of blindness and this neovascularization dominates about twenty diseases of the eye. Finally, tumor growth and metastasis are directly related to neovascularization and are dependent on angiogenesis. The tumor stimulates the growth of neovessels for its growth itself.

In addition, these neovessels present tumor escape routes to join the bloodstream and cause metastases in distant sites such as the liver, lung or bone.

 In other pathologies such as cardiovascular diseases, peripheral artery diseases, vascular or cerebral lesions, angiogenesis may present an important therapeutic basis. Indeed, the promotion of angiogenesis in damaged areas can lead to the formation of lateral and alternative blood vessels to damaged vessels thus providing blood and consequently oxygen and other nutritive and biological factors necessary for tissue survival. concerned.

 The formation of neovessels by endothelial cells involves the migration, growth, and differentiation of endothelial cells. The regulation of these biological phenomena is directly linked to gene expression. In terms of angiogenesis, an ever increasing number of studies show that the regulation of angiogenesis is through an equilibrium between factors acting directly on the endothelial cell.

These factors can be stimulating, on the one hand, such as,

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 among others, VEGF, FGFs, IL-8, HGF / SF, PDGF.

They can also be inhibiting, such as, among others, IL-10, IL-12, gro-a and ss, platelet factor 4, angiostatin, the human chondrocyte-derived inhibitor, thrombospondin, leukemia inhibitory factor (Jensen, Neural Surg., 1998, 49, 189-195, Tamatani et al., Carcinogenesis, 1999, 20, 957-962, Tanaka et al., Cancer Res., 1998,58). , 3362-3369, Ghe et al., Cancer Res., 1997, 577, 3733-3740, Kawahara et al., Hepatology, 1998, 28, 1512-1517, Chandhuni et al., Cancer Res., 1997, 57, 1814-1819, Jendraschak and Sage, Semin.

Cancer Biol., 1996, 7, 139-146; Majewski et al., J.

Invest. Dermatol., 1996, 106, 1114-1119).

 The control of angiogenesis represents a strategic axis, at the same time of fundamental research, in order to improve our understanding of the numerous pathological phenomena related to the angiogenesis, but also a base for the elaboration of the new therapies intended to treat the pathologies related to angiogenesis.

 In order to control angiogenesis, several pharmaceutical groups have developed therapeutic strategies based directly on the use of paracrine signals, stimulatory and inhibitory factors, as agents to promote or inhibit angiogenesis. These strategies are based essentially on the use of these factors in their polypeptide form as stimulating agents or inhibitors of angiogenesis, or even more recently in the form of expression vectors coding for the chosen factors.

 A method for identifying novel genes involved in the regulation of angiogenesis has been developed. It has been the subject of a French patent application published under No. 2798674 and a PCT international patent application published under the

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WO 01/218312. This method has the particularity of faithfully translating the intimate mechanism regulating angiogenesis by taking into account all the extracellular factors described as angiogenesis regulating agents, that is to say the angiogenic factors, the angiostatic factors, as well as the angiogenesis factors. different components of the extracellular matrix. This methodology consists of implementing these different extracellular factors through four well-defined experimental conditions. The endothelial cells are cultured on a component and / or a well-defined mixture of several components of the extracellular matrix and placed under the four experimental conditions, namely:
A control condition where the endothelial cells are not stimulated.

 An angiogenic condition where the endothelial cells are stimulated by one or more angiogenic factors.

 A condition of inhibition of angiogenesis in which the endothelial cells are stimulated by one or more angiogenic factors and placed in the presence of one or more angiostatic factors.

 Another control condition where the endothelial cells are stimulated by one or more angiostatic factors.

 These four conditions make it possible to obtain angiogenesis-specific mRNA preparations, namely the angiogenic state and / or the inhibition of angiogenesis, and make it possible to detect the genes coding for the cellular constituents involved in the regulation of angiogenesis. angiogenesis, including positive regulators and

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 negative regulators. Thus, the method described above allows the systematic screening of all the angiogenic and angiostatic factors as well as the different components of the extracellular matrix in order to identify and identify the genes encoding the cellular constituents involved in the regulation of the cell. angiogenesis. In addition, since gene expression can be analyzed throughout the kinetics of neovessel formation by endothelial cells, this approach provides an in vitro methodology for linking gene expression to biological functional parameters of the cell. angiogenesis.

 The identification of the fifty-four genes reported below was performed according to the methodology described above, using the angiogenic and angiostatic factors, as well as type I collagen as a component of the extracellular matrix to reproduce the four experimental conditions.

 The Applicant has proved the involvement of these fifty-four new genes identified by the sequences SEQ ID No. 1 to SEQ ID No. 53 and SEQ ID No.

225 in the list of sequences in the appendix, in the mechanism of regulation of angiogenesis.

 More particularly, the invention relates to a pharmaceutical composition active on angiogenesis phenomena comprising as active agent at least one substance chosen from: (i) a nucleic acid molecule of a gene of an endothelial cell, of which the expression is induced by an angiogenic factor and inhibited by an angiostatic agent, a complementary sequence or a fragment thereof, (ii) a polypeptide sequence encoded by said acid molecule

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 nucleic acid, or a fragment thereof (iii) a molecule capable of inhibiting the expression of a nucleic acid molecule according to (i) or of binding to a polypeptide sequence according to (ii).

 According to a particular mode of implementation, the pharmaceutical composition of the invention comprises, as active compound, at least one nucleotide sequence selected from the set of nucleotide sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. : 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing, their complementary sequences and corresponding antisense sequences, or a fragment thereof.

 For the purposes of the present invention, nucleotide sequences having minor structural modifications, which do not alter their function, such as deletions, mutations or additions of bases, whose identity is at least 90% with the nucleotide sequences identified as SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing.

 According to another particular mode of implementation, the pharmaceutical composition, which regulates angiogenesis, comprises at least one angiogenesis inhibiting sequence.

 According to a particular mode of implementation, the pharmaceutical composition regulating angiogenesis comprises at least one angiogenesis stimulating sequence.

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 According to a particular mode of implementation, the pharmaceutical composition of the invention comprises one or more angiogenesis inhibiting sequences comprising an antisense sequence of at least one sequence chosen from SEQ ID N 1 to SEQ ID N 5, SEQ ID No. 7 to SEQ ID No. 9, SEQ ID No. 11 to SEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. 53, SEQ ID No. 225, and SEQ ID No. 284 to SEQ ID No. 290 in the list. sequences attached.

 Preferably, the pharmaceutical composition of the invention comprises one or more antisense sequences chosen from SEQ ID N 103 to SEQ ID SEQ ID No. 107, SEQ ID No. 109, SEQ ID No. 111 and SEQ ID No. 113 to SEQ ID No. 148 in the attached sequence listing.

 According to a second embodiment, the pharmaceutical composition of the invention comprises one or more angiogenesis stimulating sequences comprising an antisense sequence of at least one sequence chosen from the sequences SEQ ID N 6, SEQ ID N 8, SEQ ID N 10 and SEQ ID N 16 in the attached sequence listing.

 Preferably, the pharmaceutical composition of the invention comprises antisense sequences chosen from the sequences SEQ ID No. 108, SEQ ID No. 110 and SEQ ID No.

112 in the attached sequence listing.

 The subject of the invention is also a pharmaceutical composition intended for the diagnosis and / or treatment of pathologies related to angiogenesis, characterized in that it comprises at least one polypeptide sequence, selected from polypeptide sequences identified under the numbers SEQ ID No. : 54 to SEQ ID No. 102 or from polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID No.

: 297 in the attached sequence listing.

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 For the purposes of the present invention, polypeptide sequences having minor structural modifications, which do not alter their function, such as deletions, mutations or additions of amino acid residues, the identity of which is identical, should be considered as equivalent sequences. at least 85%, preferably at least 90% with the polypeptide sequences identified as SEQ ID NO: 54 to SEQ ID No. 102 or with the polypeptide sequences identified as SEQ ID No. : 291 to SEQ ID No.

 297 in the attached sequence listing.

 The subject of the invention is also a pharmaceutical composition intended for the diagnosis and / or treatment of pathologies related to angiogenesis comprising at least one antagonist of one or more polypeptide sequences mentioned above.

 An antagonist is any compound that inhibits the biological activity of said polypeptide sequences in the mechanism of angiogenesis.

 By way of example of such antagonists, there may be mentioned an antibody having an affinity for said polypeptide sequence.

 The invention also relates to antibodies having an affinity for each of the polypeptide sequences identified as SEQ ID NO: 54 to SEQ ID No. 102 or to the polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID Nos. No.: 297 in the attached sequence listing, as well as therapeutic compositions comprising them.

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 Said antibodies can be obtained by any method of immunization in vivo or in vitro, of an animal, in particular a vertebrate and preferably a mammal with any of the polypeptide sequences identified under the numbers SEQ ID No. : 54 to SEQ ID No. 102 or the polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID NO: 297 in the attached sequence listing, or one of their fragments retaining immunogenicity of the total protein.

 The antibodies may be polyclonal or monoclonal antibodies. (Kohler G. and Milstein C.

Nature. 1975 Aug 7; 256 (5517): 495-7.)
The invention also relates to a therapeutic or diagnostic composition comprising one or more antibodies having an affinity with one or more of the polypeptide sequences identified as SEQ ID NO: 54 to SEQ ID No. 102 or with the polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID NO: 297 or by one of their fragments retaining this affinity, or prepared as indicated above.

 Another subject of the invention relates to the antisense nucleotide sequences of the nucleotide sequences identified under the numbers SEQ ID No: 1 to SEQ ID No: 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the list of sequences in annex.

 In the context of the present invention, an antisense sequence is understood to mean: DNA sequence, of at least 10 months, complementary to an mRNA which, by hybridizing with a portion of the initial mRNA sequence, inhibits its expression, that is, its translation into a protein.

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 Most preferably, the subject of the invention is the antisense sequences having an identity of at least 85%, preferably of 95% with a sequence chosen from the sequences identified under the numbers SEQ ID N 103 to SEQ ID N 148 in the list of sequences in annex.

 The subject of the invention is also a mammalian expression vector comprising at least one antisense sequence defined above.

 More particularly, said vector is chosen from the group of vectors GS-VI to GS-V46, identified by their sequence, bearing the numbers SEQ ID N 149 to SEQ ID N 194 in the sequence listing in the appendix.

 The introduction of said sequences SEQ ID No. 103 to SEQ ID No. 148 or one of their homologues into mammalian expression vectors, and the subsequent insertion of said vectors into mammalian cells makes it possible to obtain cell lines under-expressing genes involved in the mechanism of angiogenesis.

 These particularly preferred primers are shown in Table I below and identified with sequence numbers SEQ ID No. 195 to SEQ ID No. 222, SEQ ID No. 226 to SEQ ID No. 283 and SEQ ID No. .: 298 to SEQ ID No. 299 in the attached sequence listing.

 The subject of the invention is also a mammalian expression vector, said vector comprising at least one antisense sequence of at least one of the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ. ID No 284 to SEQ ID No. 290 in the

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 list of sequences in the appendix, as well as a promoter which allows the expression of said antisense DNA.

 For the construction of these vectors, specific primers for each of the identified sequences are drawn.

 Advantageously, the amplification of the fragment of the bacterial plasmid comprising the cloned gene is carried out by means of primers hybridizing to the regions of the plasmid flanking the cloned gene. They also include in their ends the restriction sites not contained in the cloned fragment and present in the multisite region of the expression vector.

 For example, in the context of the cloned fragments used in the present invention, the restriction sites used, with the pCI expression vector, are the salI and MluI sites. These two restriction sites can be interchanged depending on whether the fragment has been cloned into the bacterial plasmid in its sense or antisense orientation.

By way of example, primers GS-PGS-F (SEQ ID No. 223) and GS-PGM-R (SEQ ID No. 224) are used for fragments cloned in sense orientation in the bacterial plasmid. N15)
These particular primers can be used universally to transfer all cloned fragments in sense orientation into a bacterial vector to integrate the expression vector into the antisense orientation.

 Primers GS-PGM-F (SEQ ID No. 300) and GSPGS-R (SEQ ID No. 301) are used for fragments cloned in antisense orientation in the bacterial plasmid (GS-N46).

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 These particular primers can be used universally to transfer all cloned fragments in antisense orientation into a bacterial vector to integrate the expression vector into the antisense orientation.

 The subject of the invention is also a mammalian expression vector comprising at least one nucleotide sequence chosen from the set of sequences identified under the numbers SEQ ID. N 1 to SEQ ID. N 53, SEQ ID No. 225 and SEQ ID. N 284 to SEQ ID. N 290 in the attached sequence listing or one of their fragments.

 These constructs are useful on the one hand for preparing therapeutic compositions for the treatment, by cell therapy, of angiogenic disorders and, on the other hand, for verifying the efficacy of a treatment of an angiogenic disorder in a mammal, in particular in a patient. to be human, or else to verify the functionality of the genes possibly involved in the mechanism of angiogenesis, in said mammal.

 Thus the subject of the invention is also a genetically modified cell comprising at least one of the vectors comprising antisense sequences, under-expressing at least one nucleotide sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing.

 Another object of the invention is a method for preparing a genetically modified cell line stably expressing a

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 expression vector, said vector comprising at least one antisense sequence of at least one of the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the list of sequences in the appendix, as well as a promoter which allows the expression of said antisense DNA comprising the following steps: (a) introducing a resistance gene to at least one antibiotic into said genetically modified cell, b) culturing the cells obtained in step (a) in the presence of said antibiotic, c) the viable cells are selected.

 The invention also relates to a pharmaceutical composition for the diagnosis and / or treatment of angiogenesis-related pathologies comprising, as active principle, said genetically modified cell.

 On the other hand, the invention also relates to a genetically modified cell comprising at least one vector comprising a nucleotide sequence chosen from the set of sequences identified under the numbers SEQ ID. N 1 to SEQ ID. N 53, SEQ ID No. 225 and SEQ ID. N 284 to SEQ ID. N 290 in the attached sequence listing or a fragment thereof, overexpressing said sequence.

 The invention also relates to a method for preparing a genetically modified cell line stably expressing an expression vector, said vector comprising at least one of the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing or a fragment thereof, as well as a promoter that allows the expression of said antisense DNA, comprising the following steps:

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 (a) introducing a resistance gene for at least one antibiotic into said genetically modified cell, b) culturing the cells obtained in step (a) in the presence of said antibiotic, c) selecting the viable cells.

 It can therefore be envisaged to isolate human cells and to transfect them in vitro with at least one of the above-defined vectors, coding for at least one of the genes whose sequences are identified under the numbers SEQ ID No.: 1 to SEQ ID No. 53, SEQ ID No; and SEQ ID No. 284 to SEQ ID No. 290 or one of their fragments. These genetically modified cells can then be administered to a mammal, preferably a human mammal.

 Therapeutic compositions containing such cells may be in the form of simple cell suspensions, but they may also be encapsulated in a suitable device, for example using semipermeable membranes.

 Another subject of the invention is a method for preparing a protein encoded by at least one of the genes whose sequences are identified under the numbers SEQ ID NO: 1 to SEQ ID No. 53, SEQ ID No: 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing or a fragment thereof.

 These proteins identified by the sequences SEQ ID No. 54 to SEQ ID No. 102 and SEQ ID No. 291 to SEQ ID No.

297 in the attached sequence listing, or fragments thereof can be produced as recombinant proteins in vitro, by introducing into a suitable host an appropriate corresponding expression vector, then purified and subsequently used as a therapeutic agent.

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 Such a method of preparing a recombinant protein comprises the steps of: a) constructing an expression vector comprising at least one of those identified as SEQ ID NO: 1 to SEQ ID NO: 53 , SEQ ID No: 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing or a fragment thereof. b) introducing said vector into a cellular host, c) culturing said cells in a suitable medium, d) purifying the expressed proteins or a fragment thereof.

 The invention also relates to a recombinant protein obtained by the above method.

 By way of example, recombinant protein expression systems in bacteria such as E.

Coli can be used to express non-glycosylated proteins or polypeptides.

 The coding sequence or a partial sequence of the gene of interest is amplified by PCR using primers specific for this gene with at the ends of the preferably different restriction enzyme sites to allow the orientation of the gene in the expression vector. . The amplified DNA is purified and then digested with the restriction enzymes then inserted by ligation to the expression vector previously digested with these same restriction enzymes. A large number of vectors can be used as the vector pBR322 (Bolivar et al., Gene 2 (1977) 95-113) or its derivatives, containing, for example, the bacteriophage T7 RNA polymerase promoter for a high level of control. expression, such as plasmid pET3a (Studier and Moffatt, 1986, J Mol Biol, 189 (1): 113-30), preferably containing sequences that encode selection markers (antibiotic resistance), a

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 multiple cloning site containing restriction enzyme sites suitable for DNA insertion, the cell / host system is preferably an inducible system such as that used for in vivo radiolabeling of FGF2 growth factor (Colin et al. , 1997, Eur.J.Biochem., 249, 473-480) and already described by Patry et al (1994, FEBS Lett, 349 (1): 23-8), it may also contain a region coding for a poly tail. histidine at the end of the polypeptide of interest to facilitate purification.

 The amplified DNA is ligated into the plasmid which is transformed into the bacterium according to the method described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.). The transformed cells are spread on LB agar medium containing the antibiotics, the antibiotic resistant colonies are then controlled by PCR and gel analysis. Plasmid DNA can be isolated and sequenced to confirm the construct. The production and purification of the recombinant protein can be carried out as described (Patry et al., 1994, FEBS Lett, 349 (1): 23-8), 473-480. Briefly, an isolated colony is inoculated into the medium. liquid culture such as LB broth medium supplemented with antibiotics. After overnight incubation, the preculture can be used to seed a culture of greater volume. The expression of the polypeptide is then induced, the cells develop for a few hours and then are harvested by centrifugation. The cell pellet may be lysed by chemical agents known in the art or mechanically by sonication for example. The protein can be purified by virtue of its physicochemical properties as described for the purification of recombinant FGF2 (Colin et al., 1997, Eur.J.Biochem., 249, 473-480) or if the protein is labeled with a poly histidine tail, can be purified via this tail by immobilization on a metal ion chelating support

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 as described (Tang et al., Protein Expr Purif 1997 Dec; 11 (3): 279-83).

 By way of example, recombinant protein expression systems for expressing polypeptides having post translational modifications such as glycosylation, such as eukaryotic systems (yeasts, plants, insects) can be used.

 Thus, the recombinant protein can be produced for example in Pichia Pastoris yeast as described by Sreekrishna et al (1988, Basic Microbiol, 28 (4): 265-78). The amplified DNA will be introduced in the same manner after digestion and ligation in a Pichia Pastoris expression vector, preferably containing a sequence encoding a selection marker (Scorer et al, Biotechnology (NY), 1994 Feb; 2): 181-4). The protein may be either intracellular or secreted if the vector contains at the end of the introduced gene a coding sequence for a secretory signal sequence such as, for example, the prepropeptide factor of Saccharomyces cerevisiae (Cregg et al., 1993, Scorer et al. 1993). A Histidine tail may also be added to one end of the recombinant protein to facilitate purification (Mozley et al., 1997, Photochem Photobiol, 66 (5): 710-5).

 Preferably, said host is chosen from: a bacterium, a yeast, an insect cell and a mammalian cell, a plant cell.

 Administration of therapeutic compositions comprising such proteins may be, for example, topically, orally, intradermally, transdermally, intravenously.

 The fragments of said proteins can be used as antagonists of the protein of which they

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 are from. Thus, the appropriate administration to an animal of a therapeutic composition comprising such fragments is recommended to induce a decrease in the activity of said protein in the mechanism of angiogenesis in a given pathology.

 The present invention also relates to a method for diagnosing an angiogenic pathology in a mammal, in particular in a human being, of detecting in the cells of said mammal the overexpression or the under-expression of one or more nucleotide sequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing.

 Such a diagnostic method comprises the following steps: the detection of the expression of one or more of said nucleotide sequences SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290; by a cell population of a mammal, - the detection of the expression of the same sequence (s) by a reference cell population whose angiogenic state is known, - the identification of any differences the level of expression of the same sequence (s) by the two cell populations.

 The present invention also relates to a method for diagnosis and prognosis of an angiogenic pathology in a mammal, in particular in a human being, of detecting in the cells of said mammal the overexpression or the under-expression of one or more identified polypeptide sequences. by the

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 SEQ ID NO: 54 to SEQ ID No. 102 or the polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID NO: 297 in the attached sequence listing.

 According to a preferred embodiment, said method comprises the following steps: a) detecting the expression of one or more of said polypeptide sequences SEQ ID No. 54 to SEQ ID No. 102 or with the sequences polypeptides identified by SEQ ID NO: 291 to SEQ ID No.

 297 by a cell population of a mammal, b) the detection of the expression of the same polypeptide sequence (s) by a reference cell population whose angiogenic state is known, ) identification of any differences in the level of expression of the same polypeptide sequence (s) by the two cell populations.

 According to a particular mode of implementation, in the diagnostic method of the invention the detection of the expression of the sequences is carried out after putting the endothelial cells in the presence of a biological fluid from a patient.

 The present invention also relates to a method for verifying the therapeutic efficacy of an angiogenic treatment in a mammal, in particular in a human being, by the identification of a cell population in said mammal capable of overexpressing or under-expressing one or more a plurality of nucleotide sequences identified by the numbers SEQ ID N 1 to SEQ ID N 53, SEQ ID No. 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing.

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 Such a method for verifying the therapeutic efficacy comprises the following steps: the detection of the expression of one or more of said nucleotide sequences SEQ ID No. 1 to SEQ ID No. 53 and SEQ ID No. 225 by a cell population of a mammal to which a therapeutic composition for treating an angiogenic disorder is administered, - the detection of the expression of the same nucleotide sequence (s) by a reference cell population whose angiogenic state is known identifying any differences in the level of expression of the same nucleotide sequence (s) by the two cell populations.

According to preferred embodiments, the verification method is carried out on a cell population of a mammal in vivo, ex-vivo, or on an isolated cell population of said mammal in vitro.
According to a particular mode of implementation, in the verification method of the invention the detection of the expression of the sequences is carried out after putting the endothelial cells in the presence of a biological fluid from a patient.

 The present invention also relates to a method for screening compounds useful for the angiogenic treatment of a mammal, in particular a human being.

 According to a preferred embodiment, such a screening method comprises the following steps:

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 detecting the expression of one or more of said nucleotide sequences SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 by a cell population placed in the presence of a compound likely to have a therapeutic effect on an angiogenic disorder, - the detection of the expression that (s) same (s) sequence (s) nucleotide (s) by a reference cell population whose angiogenic state is known, identification of the possible differences in the level of expression of the same nucleotide sequence (s) by the two cell populations.

 According to another preferred embodiment, such a screening method also comprises the following steps: the detection of the expression of one or more of said polypeptide sequences identified by the numbers SEQ ID No. 54 to SEQ ID No.: 102 or with the polypeptide sequences identified as SEQ ID NO: 291 to SEQ ID NO: 297 in the attached sequence listing by a cell population placed in the presence of a compound likely to have an effect. therapy on an angiogenic disorder, - the detection of the expression of the same polypeptide sequence (s) by a reference cell population whose angiogenic state is known, - the identification of any differences in the level of expression of that (s). ) same polypeptide sequence (s) by the two cell populations.

 According to a particular mode of implementation, in the screening method of the invention, the detection of the expression of the sequences is carried out after having set

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 endothelial cells in the presence of a biological fluid from a patient.

 Among the angiogenic disorders that can be diagnosed or treated with the pharmaceutical compositions of the invention, mention may be made of: vascularization of tumors, retinopathies, rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of ovarian, psoriasis, endometrium associated with neovascularization, restenosis due to balloon angioplasty, tissue overproduction due to scarring, peripheral vascular disease, hypertension, vascular inflammation, disease, and Raynaud's phenomenon, aneurysm, arterial restenosis, thrombophlebitis, lymphagyte, lymphodema, scarring and tissue repair, ischemia, angina, myocardial infarction, chronic heart disease , heart failure such as congestive heart failure, age-related macular degeneration, and osteoporosis.

 The invention also relates to a device comprising a support comprising one or more probes specific for one or more nucleotide sequences identified under the numbers SEQ ID No: 1 to SEQ ID No: SEQ ID No. 225 and SEQ ID No. 284 SEQ ID No. 290, in the attached sequence listing for the implementation of the screening method of the invention.

 In the context of the present invention, the term "probe" is understood to mean any single-stranded DNA fragment whose sequence is complementary to a desired sequence: this can thus be detected by hybridization with the labeled probe (by incorporation of radioactive atoms

<Desc / Clms Page number 23>

 or fluorescent groups), which acts as a molecular "hook".

 According to preferred embodiments, the support of said device is selected from a glass membrane, a metal membrane, a polymer membrane, a silica membrane.

 Such devices are, for example, DNA chips comprising one or more nucleotide sequences identified under the numbers SEQ ID No: 1 to SEQ ID No: SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290, in the list of sequences in annex.

 The subject of the invention is also a kit intended for measuring the differential display of genes involved in angiogenic disorders comprising a device as previously described, specific primers and the accessories necessary for the amplification of the sequences extracted from a sample, their hybridization with the probes of the device and the realization of the differential display measurements.

The invention also relates to a kit for the measurement of the differential display of genes involved in angiogenic disorders comprising a genetically modified cell line stably expressing the vector expressing at least one of the nucleotide sequences identified under the numbers SEQ. ID No: 1 to SEQ ID No: SEQ ID No. 225 and SEQ ID No.
284 to SEQ ID No. 290, in the attached sequence listing, or a fragment thereof as the reference cell population and the means necessary for measuring said differential display.

 The invention also relates to a kit intended for measuring the differential display of genes

<Desc / Clms Page number 24>

 involved in angiogenic disorders comprising a genetically modified cell line stably expressing the vector expressing at least one antisense sequence of one of the nucleotide sequences identified as SEQ ID No: 1 to SEQ ID No: SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290, in the attached sequence listing, or fragment thereof, as the reference cell population and the means necessary for measuring said differential display.

 Verification of the involvement of the fifty-four identified genes and their homologs in the mechanism of angiogenesis was carried out according to the methodology described in the Material and Methods section.

This involvement is illustrated by means of Figures 1 to 11 in the appendix, in which:
Figure 1 shows that the expression of GS-V1, GS-V2, GS-V4, GS-V5, GS-V15 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 1A) GS-V1 coding for the specific antisense transcript of GS-N1; 1B) GS-V2 encoding the antisense transcript specific for GS-N2; 1C) GS-V4 encoding the GS-N4 specific antisense transcript; 1D) GS-V5 encoding the specific antisense transcript of GS-N5; 1E) GS-V15 encoding the antisense transcript specific for GS-N15; 2F) the empty vector (Control).

 Figure 2 shows that expression of GS-V3, GS-V14 in human endothelial cells inhibits formation of capillary tubes: endothelial cells transfected with 2A) GS-V3 encoding GS-N3 specific antisense transcript; 2B) GS-V13 encoding the antisense transcript specific for GS-N13; 2C) the empty vector (Control).

<Desc / Clms Page number 25>

 Figure 3 shows that the expression of GS-V6, GS-V8, GS-V10 in human endothelial cells induces the formation of capillary tubes: transfected endothelial cells where 3A) GS-V6 encoding GS-V specific antisense transcript N6; GS-V8 encoding the specific antisense transcript of GS-N8; 3C) GS-V10 encoding the antisense transcript specific for GS-N10 and its counterpart GS-N54; 3D) the empty vector (Control).

 Figure 4 shows that the expression of GS-V7, GS-V9, GS-V11, GS-V12, GS-V14 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 4A) GS-V7 coding for the specific antisense transcript of GS-N7; 4B) GS-V9 encoding the antisense transcript specific for GS-N9; 4C) GS-V11 encoding the specific antisense transcript of GS-N11; 4D) GS-V12 encoding the antisense transcript specific for GS-N12; 4E) GS-V14 encoding the specific antisense transcript of GS-N14 and 4F) the empty vector (Control).

 Figure 5 shows that the expression of GS-V16, GS-V17, GS-V18, GS-V19, GS-V21 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 5A) GS-V16 coding for the specific antisense transcript of GS-N16; 5B) GS-V17 encoding the antisense transcript specific for GS-N17; 5C) GS-V18 encoding the antisense transcript specific for GS-N18; 5D) GS-V19 encoding the antisense transcript specific for GS-N19; 5E) GS-V21 encoding the specific antisense transcript of GS-N21; 5F) the empty vector (Control).

 Figure 6 shows that expression of GS-V22, GS-V24, GS-V25, GS-V26, GS-V27 in human endothelial cells inhibits tube formation

<Desc / Clms Page number 26>

 capillaries: endothelial cells transfected with 6A) GS-V22 encoding the antisense transcript specific for GS-N22; 6B) GS-V24 encoding the antisense transcript specific for GS-N24 and its counterpart GS-N49; encoding the antisense transcript specific for GS-N25 and its counterpart GS-N50; 6D) GS-V26 encoding the GS-N26 specific antisense transcript; encoding the specific antisense transcript of GS-N27 and its counterpart GS-N51; 6F) the empty vector (Control).

 Figure 7 shows that the expression of GS-V28, GS-V29, GS-V30, GS-V31, GS-V32 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 7A) GS-V28 coding for the antisense transcript specific for GS-N28; 7B) GS-V29 encoding the specific antisense transcript of GS-N29 and its counterpart GS-N52; 7C) GS-V30 encoding the antisense transcript specific for GS-N30; 7D) GS-V31 encoding the antisense transcript specific for GS-N31 and its counterpart GS-N53; 7E) GS-V32 encoding the antisense transcript specific for GS-N32; 7F) the empty vector (Control).

 Figure 8 shows that the expression of GS-V33, GS-V34, GS-V35, GS-V37, GS-V38 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 8A) GS-V33 coding for the specific antisense transcript of GS-N33; 8B) GS-V34 encoding the antisense transcript specific for GS-N34; 8C) GS-V35 encoding the specific antisense transcript of GS-N35; 8D) GS-V37 encoding the GS-N37 specific antisense transcript; encoding the specific antisense transcript of GS-N38; 8F) the empty vector (Control).

<Desc / Clms Page number 27>

 Figure 9 shows that the expression of GS-V40, GS-V42, GS-V43, GS-V44, GS-V45 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 9A) GS-V40 coding for the specific antisense transcript of GS-N40; 9B) GS-V42 encoding the specific antisense transcript of GS-N42; encoding the specific antisense transcript of GS-N43; 9D) GS-V44 encoding the specific antisense transcript of GS-N44; encoding the specific antisense transcript of GS-N45; F) the empty vector (Control).

 Figure 10 shows that the expression of GS-V20, GS-V23, GS-V36 GS-V39 GS-V41 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 10A) GS-V20 encoding the antisense transcript specific for GS-N20; 10B) GS-V23 encoding the antisense transcript specific for GS-N23 and its counterparts GS-N47 and GS-N48; 10C) GS-V36 encoding the GS-N36 specific antisense transcript; 10D) GS-V39 encoding the specific antisense transcript of GS-N39; encoding the specific antisense transcript of GS-N41; 10F) the empty vector (Control).

 Figure 11 shows that the expression of GS-V46 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 11A) GS-V46 encoding the antisense transcript specific for GS-N46; 11B) the empty vector (Control).

MATERIAL AND METHODS
1.Culture of cells and angiogenesis test

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Umbilical vein endothelial cells (HUVECs) under said four culture conditions are then used to identify genes encoding the cellular components involved in the regulation of angiogenesis.

 The endothelial cells are maintained in complete medium (EGM-2 from Clonetics).

 For the identification of genes involved in angiogenesis in in vitro angiogenesis assays according to the model of Montesano et al (1986, Proc Natl Acad Sci U S A, 83 (19): 7297-301). The cells are first seeded on a type I collagen gel in complete medium until confluence. Then, the reference HUVEC cells are cultured in serum-depleted medium and without growth factors: EBM-2 + 2% serum and various factors are added under test conditions.

 FGF2: at concentrations of between 5 ng / ml and 60 ng / ml, preferably between 10 and 40 ng / ml; VEGF: at concentrations of between 10 ng / ml and 60 ng / ml, preferably between 30 ng / ml and 50 ng / ml; PF4: at concentrations of between 0.1 and 5 jug / ml, preferably between 0.5 g / ml and 1 g / ml; TNF-α at concentrations of between 20 ng / ml and 100 ng / ml, preferably between 30 ng / ml and 60 ng / ml; IFN-γ: at concentrations of between 50 ng / ml and 200 ng / ml, preferably between 80 ng / ml and 120 ng / ml.

 Human endothelial cells placed under the four aforementioned culture conditions are then used to identify genes encoding the cellular constituents involved in the regulation of angiogenesis.

 2. Angiogenic and angiostatic factors.

 Angiogenic and angiostatic factors, affecting the expression of genes identified in

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 correlation with the formation of neovessels or the inhibition of neovessels respectively, used, for example, in the context of the present invention, are illustrated below.

 VEGF = vascular endothelial growth factor.

 FGF2 = Basic growth factor of the fibroblast.

 HGF = Growth factor of the hepatocyte.

 PF4 = Platelet factor 4.

 IFN-y = interferon gamma.

TNF-a = tumor necrosis factor alpha
TNF-which is a regulator of angiogenesis, it can induce angiogenesis in vivo but also inhibit the formation of vessels in vitro (Frater-Schroder et al., 1987, Proc Natl Acad Sci USA, 84 (15): 5277-81, Fajardo et al., 1992, Am J Pathol Mar, 140 (3): 539-44, Niida et al., 1995, Neurol Med Chir (Tokyo), 35 (4): 209-14. In vitro angiogenesis, TNF-α is used under conditions of inhibition of angiogenesis.

 3. Comparison of gene expressions.

 The gene expressions can then be compared using DNA chips, SAGE, a quantitative PCR amplification reaction, viral vectors to construct subtractive libraries, or differential display analysis.

 In the context of the experimental work leading to the present invention, the Applicant has preferentially used the differential display technique for the identification of said genes.

Differential display
Total RNAs are prepared from HUVEC cells grown on a collagen gel in the presence of the various factors used, according to the method

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 RNeasy Mini kit (Qiagen) by integrating a digestion step with DNase I according to the protocol recommended by the manufacturer.

 The differential display from the total RNAs is carried out according to the method described by Liang and Pardee (1992, Science, 14; 257 (5072): 967-7) using aP33-ATP in isotopic dilution during the PCR amplification for band visualization by autoradiography of electrophoresis gels.

 Thus, the DNA fragments differentially present on the gel as a function of the culture conditions analyzed are cut, reamplified, cloned in a PGEM easy vector plasmid, Promega), sequenced and identified by questioning the BLAST library.

 4. Verification of the implication of the identified genes in the mechanism of angiogenesis.

Gene functionality test
In a second step, the functionality of each identified sequence is tested on the in vitro angiogenesis model with the endothelial cells transfected with an expression vector comprising an antisense oligonucleotide of said sequence.

 For the construction of these vectors, specific primers for each of the identified sequences are drawn. These primers are shown in Table I below and identified with sequence numbers SEQ ID No. 195 to SEQ ID No. 222, SEQ ID No. 226 to SEQ ID No. 283 and SEQ ID No. 298 to SEQ ID No. 299 in the attached sequence listing.

Table I

<Tb>
<tb> SEQ.ID <SEP> Name
<tb> ~~~~~~~~~~~~~~~~~~~~ primer
<Tb>

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SÉO ID # SÉQ.ID amorce amorce GVl-1

SEO ID # SEQ.ID primer primer GVl-1

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 1 <SEP> (GS-N1) <SEP> GV1-2
<tb> GV2-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 2 <SEP> (GS-N2) <SEP> GV2-2
<tb> GV3-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 3 <SEP> (GS-N3) <SEP> GV3-2
<tb> GV4-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 4 <SEP> (GS-N4) <SEP> GV4-2
<Tb>

GV5-1

GV5-1

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 5 <SEP> (GS-N5) <SEP> GV5-2
<tb> GV6-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 6 <SEP> (GS-N6) <SEP> GV6-2
<tb> GV7-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 7 <SEP> (GS-N7) <SEP> GV7-2
<tb> GV8-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 8 <SEP> (GS-N8) <SEP> GV8-2
<tb> GV8-2
<tb> GV9-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 9 <SEP> (GS-N9) <SEP> GV9-2
<Tb>

GV9-2 GV10-1

GV9-2 GV10-1

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 10 <SEP> (GS-N10) <SEP> GV10-2
<tb> GV10-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 11 <SEP> (GS-N11) <SEP> GV11-2
<tb> GV11-2
<tb> GV12-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 12 <SEP> (GS-N12) <SEP> GV12-2
<tb> GV13-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 13 <SEP> (GS-N13) <SEP> GV13-2
<tb> GV14-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 14 <SEP> (GS-N14) <SEP> GV14-2
<Tb>

GV14-2 GS-PGS#F

GV14-2 GS-PGS # F

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 15 <SEP> (GS-N15) <SEP> GS-PGM-R
<Tb>

GS-PGM-R GV10-1

GS-PGM-R GV10-1

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 16 <SEP> (GS-N54) <SEP> GV10-2
<tb> GV16-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 17 <SEP> (GS-N16) <SEP> GV16-2
<tb> GV16-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 18 <SEP> (GS-N17) <SEP> GV17-1
<tb> GV18-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 19 <SEP> (GS-N18) <SEP> GV18-2
<Tb>

GV18-2 ~~ G V 19 #

GV18-2 ~~ GV 19 #

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 20 <SEP> (GS-N19) <SEP> GV19-2
<tb> GV19-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 21 <SEP> (GS-N20) <SEP> GV20-2
<tb> GV20-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 22 <SEP> (GS-N21) <SEP> GV21-1
<tb> GV22-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 23 <SEP> (GS-N22) <SEP> GV22-2
<tb> GV22-2
<tb> GV23-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 24 <SEP> (GS-N23) <SEP> GV23-2
<tb> GV24-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 25 <SEP> (GS-N24) <SEP> GV24-2
<tb> GV25-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 26 <SEP> (GS-N25) <SEP> GV25-2
<Tb>

<Desc / Clms Page number 32>

<Tb>
<tb> SEQ.ID <SEP> Name
<tb> primer
<tb> GV26-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 27 <SEP> (GS-N26) <SEP> GV26-2
<tb> GV27-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 28 <SEP> (GS-N27) <SEP> GV27-2
<Tb>

GV27-2 GV2 8 #

GV27-2 GV2 8 #

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 29 <SEP> (GS-N28) <SEP> GV28-2
<tb> GV29-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 30 <SEP> (GS-N29) <SEP> GV29-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 31 <SEP> (GS-N30) <SEP> GV30-2
<tb> GV30-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 32 <SEP> (GS-N31) <SEP> GV31-2
<tb> GV32-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 33 <SEP> (GS-N32) <SEP> GV32-2
<tb> GV32-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 34 <SEP> (GS-N33) <SEP> GV33-2
<tb> GV33-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 35 <SEP> (GS-N34) <SEP> GV34-2
<tb> GV34-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 36 <SEP> GS-N35) <SEP> GV35-1
<tb> GV35-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 37 <SEP> (GS-N36) <SEP> GV36-2
<tb> GV36-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 38 <SEP> (GS-N37) <SEP> GV37-2
<tb> GV37-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 39 <SEP> (GS-N38) <SEP> GV38-2
<Tb>

GV39-1

GV39-1

<Tb>
<tb> SEQ <SEP> ID <SEP> N 40 <SEP> (GS-N39) <SEP> GV39-2
<tb> GV40-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 41 <SEP> (GS-N40) <SEP> GV40-2
<tb> GV40-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 42 <SEP> (GS-N41) <SEP> GV41-2
<tb> GV42-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 43 <SEP> (GS-N42) <SEP> GV42-2
<tb> GV43-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 44 <SEP> (GS-N43) <SEP> GV43-2
<tb> GV44-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 45 <SEP> (GS-N44) <SEP> GV44-2
<tb> GV45-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 46 <SEP> (GS-N45) <SEP> GV45-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 47 <SEP> (GS-N46) <SEP> GS-PGM-F
<Tb>

GS-PGS-R GV23-1

GS-PGS-R GV23-1

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 48 <SEP> (GS-N47) <SEP> GV23-2
<tb> GV23-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 49 <SEP> (GS-N48) <SEP> GV23-2
<tb> GV24-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 50 <SEP> (GS-N49) <SEP> GV24-2
<tb> GV24-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 51 <SEP> (GS-N51) <SEP> GV27-2
<tb> GV27-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 52 <SEP> (GS-N52) <SEP> GV29-1
<Tb>

<Desc / Clms Page number 33>

<Tb>
<tb> SEQ.ID <SEP> Name
<tb> primer
<tb> SEQ <SEP> ID <SEP> No <SEP> 53 <SEP> (GS-N53) <SEP> GV31-1
<tb> GV31-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 225 <SEP> (GS-N50) <SEP> GV50-1
<Tb>

These primers contain at each of their ends a site of a different restriction enzyme (SalI, GTCGAC or MluI: ACGCGT).

 Amplified fragments of each gene are obtained by PCR from the bacterial plasmids containing the identified gene fragment using said primers.

 These fragments are purified, digested with SalI and MluI restriction enzymes and inserted into an expression vector in mammals of the pCi-neo vector type (Promega), itself digested with these two restriction enzymes.

 Each fragment is introduced in the antisense orientation.

 In the particular case of the GS-N15 and GS-N46 sequences, the amplification of the fragment cloned into the bacterial plasmid is carried out using particular primers, chosen from the GS-PGS-F, GS-PGM-R or GS sequences. -PGM-F and GS-PGS-R hybridizing to the regions of the plasmid flanking the cloned gene and also comprising in their ends the restriction sites (SalI and MluI sites), not contained in the cloned fragment, and present in the region multisite of the expression vector.

 These two restriction sites can be interchanged depending on whether the fragment has been cloned into the bacterial plasmid in its sense or antisense orientation.

 Controls performed with these primers, which can be considered as universal primers, in

<Desc / Clms Page number 34>

 absence of the cloned gene, (empty plasmid) showed that the amplified fragment of the plasmid (40 bp) when integrated into the expression vector does not alter the formation of neovessels in the in vitro functionality test. The results obtained with this vector thus constructed are identical to those obtained with the empty vector (results not shown) and show that these additional base pairs do not alter the effect of the specific antisense fragments of the identified sequence.

 In general, the vectors that can be used for demonstrating the functionality of the genes identified in the present invention in the mechanism of angiogenesis include any mammalian expression vector system comprising a promoter which allows the expression of a cloned gene, by way of example, mention may be made of the strong promoter of human cytomegalovirus (CMV).

Other constitutive or inducible expression vectors that can also be used are indicated in the non-exhaustive list given below:
Vectors marketed by the Promega Company; vectors with a strong promoter for a high level of constitutive expression of the genes in mammalian cells (pCI Mammalian Expression Vector, Vector Expression Vector Cloning Vector pALTER (R) * - MAX), vectors marketed by the company Invitrogen: ( pcDNA3 .1, - / hygro, - / Zeo, pcDNA4 / HisMAx, -E, pBudCE4, pRcRSV, pRcCMV2, pSecTag2, - / hygro secretion vectors, the vectors pEBVHis A, B and C), expression vectors in the mammals marketed by Clontech (pIRES, pIRES-EYFP pIRES2-EGFP, pCMV-Myc and pCMV-HA), EpitopeTagged pTRE, VP16 Minimal Domain vectors (ptTA 2, ptTA 3 and ptTA 4), Tet expression vectors bidirectional (pBI, pBI-EGFP, pBI-G, pBI-L), pRevTRE, pTRE2, pLEGFP-N1 retroviral vector pLEGFP-C1, adeno-viral expression systems Adeno-X, pCMS-EGFP, pdlEGFP-N1, pd2ECFP- nl,

<Desc / Clms Page number 35>

 pd2EYFP-N1, pEGFP (-Cl, -C2, -C3, -Ni, -N2, -N3), pEYFP-C1, -N1.

 Each vector comprising said antisense fragment is then produced in E. coli, extracted, purified and quantified. One g of each vector is incubated in the presence of a transfecting agent (effectene, Qiagen) following the protocol recommended by the manufacturer with the endothelial cells. Twenty-four hours after transfection, the endothelial cells are trypsinized and plated on the extracellular matrix containing the angiogenic factors in this case the matrigel according to the model described by Grant et al. (1989, Cell, 58 (5): 933- 43). After 24h incubation, vessel formation is observed and compared to control cells transfected with the empty mammalian expression vector.

 5. Establishment of the library of stable lines expressing the expression vectors containing the gene sequences or their fragments or the antisense sequences.

 Expression systems may include an antibiotic selection marker (an antibiotic resistance gene) for selecting transfected cells stably expressing the vector comprising the nucleic acid cloned into said vector and either same vector, either in a second cotransfected vector.

 This expression vector may be a constitutive or inducible expression system.

 In the particular example described below, the stable lines for the expression of the antisense oligonucleotide corresponding to each identified gene were obtained with a constitutive expression vector and after selection in the presence of antibiotic.

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 For this purpose, 24 hours after the transfection performed under the conditions described above, the BAEC endothelial cells are trypsinized and seeded at a rate of 80,000 cells / well in a six-well plate in the presence of 700 g / ml of the antibiotic G418. (Promega). A control well is inoculated with untransfected cells. The medium is changed every three days with an antibiotic refill. The control cells are removed after 8 to 10 days, the antibiotic resistant cells are harvested at confluence (after 2 to 3 weeks) and then transferred into culture flasks always in the presence of the antibiotic. Stable lines are then tested for their ability to form or not form vessels in the in vitro angiogenesis assay.

6. Results
6. 1 Identification of genes.

 The nucleic acid sequences named GS-N1, GS-N2, GS-N3, GS-N4, GS-N5, GS-N6, GS-N7, GS-N8, GS-N9, GSN10 (or GS-N54) , GS-N11, GS-N12, GS-N13, GN-14 and GS-N15, and respectively the proteins encoded by said GS-N1 nucleic acids at GS-N13 and GS-N15 named: angioinducine, angiodockine, angioblastine, angioreceptin , angiodensin, angiopartnerine, vassoserpentine, angiosulfatin, vassoreceptin, angiokinasine, vassosubstratine, angiosignalin, angiofoculin, angiohelicin, angioacyline, have not been previously identified as having any biological role, let alone one in the process of angiogenesis or differentiation of endothelial cells into capillary tubes. These proteins are described below.

 The differential display method described above allowed the identification of the following mRNAs:

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 GS-N1: a 1683bp mRNA identified by the sequence SEQ ID No: 1 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number BC008502.

 The sequence of this mRNA has a coding sequence from nucleotide 159 to nucleotide 458. We therefore identified a GS-P1 protein, resulting from the translation of this mRNA. This protein is composed of 99aa, identified as SEQ ID No. 54 in the attached sequence listing, called Angioinducine-GS-N2; a mRNA of 1649 bp, identified by the sequence SEQ ID No: 2 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number NM 022823.

 The sequence of this mRNA has a coding sequence from nucleotide 367 to nucleotide 1071. We therefore identified a GS-P2 protein, resulting from the translation of this mRNA. This protein is composed of 234 aa, identified as SEQ ID No 55 in the attached sequence listing, called Angiodockine.

 GS-N3: a mRNA of 5766 bp, identified by the sequence SEQ ID No: 3 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AB007963.

 The sequence of this mRNA has a coding sequence from nucleotide 978 to nucleotide 2465. We therefore identified a GS-P3 protein, resulting from the translation of this mRNA. This protein is composed of 495 aa, identified as SEQ ID No. 56 in the attached sequence listing, called Angioblastine.

 GS-N4: a 5242 bp mRNA identified by the sequence SEQ ID No: 4 in the attached sequence listing. BLAST search based on sequences

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 GENBANK allows to identify it under accession number AB037835.

 The sequence of this mRNA has a partial coding sequence from nucleotide 1 to nucleotide 4762. We therefore identified a GS-P4 protein, resulting from the translation of this mRNA. This protein is composed of 1586 aa, identified as SEQ ID No. 57 in the attached sequence listing, called Angioreceptin-GS-N5: a mRNA of 2153 bp identified by the sequence SEQ ID No: 5 in the sequence listing in annex. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AK025682.

 The sequence of this mRNA has a coding sequence from nucleotide 38 to nucleotide 691. We therefore identified a GS-P5 protein, resulting from the translation of this mRNA. This protein is composed of 217 aa, identified as SEQ ID No. 58 in the attached sequence listing, called Angiodensin.

 GS-N6: a 3005 bp mRNA identified by the sequence SEQ ID No: 6 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AK023284.

 The sequence of this mRNA has a coding sequence from nucleotide 90 to nucleotide 773. We therefore identified a GS-P6 protein, resulting from the translation of this mRNA. This protein is composed of 227 aa, identified as SEQ ID No 59 in the attached sequence listing, called Vasoserpentine.

 GS-N7: an ARN of 4397 identified by the sequence SEQ ID No: 7 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AB033073.

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 The sequence of this mRNA has a partial coding sequence from nucleotide 286 to nucleotide 2943. We therefore identified a GS-P7 protein, resulting from the translation of this mRNA. This protein is composed of 885 aa, identified as SEQ ID No 60 in the attached sequence listing called Angiosulfatin.

 GS-N8: a mRNA of 5844 bp identified by the sequence SEQ ID No: 8 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number AB023187 :.

 The sequence of this mRNA has a partial coding sequence from nucleotide 1 to nucleotide 3456. We therefore identified a GS-P8 protein, resulting from the translation of this mRNA. This protein is composed of 1151 aa, identified as SEQ ID No 61 in the attached sequence listing called Vassoreceptin.

 GS-N9: a 4266 bp mRNA identified by the sequence SEQ ID No: 9 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AB014587.

 The sequence of this mRNA has a partial coding sequence from nucleotide 1 to nucleotide 3528. We therefore identified a GS-P9 protein, resulting from the translation of this mRNA. This protein is composed of 1175 aa, identified as SEQ ID No. 62 in the attached sequence listing called Angiokinasine.

 Angiokinasine is homologous with MAP4K4 (SEQ ID No. 224 in the attached sequence listing), n accession: XM-038751 (Nucleic sequence: 4197 bp, protein sequence: 1141aa).

 One of the mechanisms by which cells respond to external stimuli is the recruitment of chains consisting of a set of proteins that relay the external signal to the inside of the cells. In

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 providing signal transduction, this chain changes the intracellular environment thus controlling gene transcription (reviewed: Avruch, 1998, Mol Cell.

Biochem., 182, 31-48; Karin, 1998, Ann. N. Y. Acad. Sci.

851.139-146). A significant number of these chains and therefore the signaling pathways that are highly conserved through evolution are collectively referred to as the mitogen-activated protein kinase (MAPK) voices (Gupta et al, 1996, EMBO J., , 2760-2770, Madhani and Fink, 1998, Trends Genet 14, 151-155). The classical MAPK voice is triggered by the binding of growth factors to their cell surface receptor leading to the activation of the Ras protein, GTPase. This voice results in the activation of protein kinases regulated by extracellular signals (ERKs) leading to gene transcription and cell proliferation. A parallel voice of MAPK is stimulated by stress factors such as osmotic shock, cytotoxic products, UV radiation or inflammatory cytokines. This voice results in the activation of stress-activated protein kinases, known as c-Jun N-terminal kinases (SAPK / JNKs) (Karin, 1998, Ann.No. Acad Acad Sci. 146). A second voice activated by stress leads to the activation of the MAPK p38. The effect of activation by stress covers proliferation, differentiation or gene transcription leading to cell cycle arrest and / or apoptosis depending on cell type and stimulus (Karin, 1998, Ann. NY Acad.

Sci. 851, 139-146).

 Several studies have reported a role for MAPKs 1 and 2 as well as p38 MAPK in signal transduction voices elicited by angiogenic or antiangiogenic factors during angiogenesis, however no role has been reported for MAKP4 in this process (Tanaka et al, 1999, Jpn J Cancer Res, 90: 654 et al, 2000, Cancer Res, 60: 712-721, Gupta et al, 1999, Exp Cell Res, 247: 495-504; ,

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 1998, Nature, 391: 24-25; et al., Oncogene, 1997, 15: 2169-2177; Shore et al., 1997, Placenta, 18: 657-665).

 MAPK 4 is a member of the MAPK family. This kinase directly phosphorylates and therefore activates kinases acting directly on the c-Jun N-terminal (JNK), in response to inflammatory stress and / or cytokines. MAPK 4 is expressed in different tissues, however there is an abundance of expression of this kinase in skeletal muscle and brain. The deficient mice of the MAPK 4 gene develop abnormal hepatogenesis and die in the embryogenic state on the fourteenth day. However, cell lines deficient in MAPK 4 have been obtained. These lines are characterized by the absence of JNK-dependent gene transcription and the AP-1 transcription factor. In addition, T cells deficient in MAPK 4 show a decoupling of IL-2 production following activation of T cell receptors suggesting a key role for MAPK4 / JNK in the inflammatory process. The mutation of MAPK4 in some carcinomas indicates that it can play a role in suppressing tumors. Although the expression control and activity of MAPK4 are currently under intense analysis and studies, they are in an approach to develop anti-inflammatory and anticancer therapy. However, the role of MAPK4 in angiogenesis has not been demonstrated to date.

 Pedram et al (Endocrinology, 2001,142: 1578-86) have shown that the natriuretic peptide suppresses or inhibits VEGF-induced angiogenesis, and they have also shown that activation of kinases acting directly on the N-terminal of c-Jun is an important step in the induction of angiogenesis by VEGF. In contrast, Jimenez et al, Oncogene, 2001,20: 3443-3448) reported that activation of kinases acting on the N-terminal of c-Jun is necessary for inhibition of neovascularization by thrombospondin 1 .

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 In both cases, these two recent studies do not relate to a specific role of MAP4K4 in the regulation of angiogenesis.

 GS-N10: a mRNA of 2034 bp identified by the sequence SEQ ID No: 10 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number XM 035658.

 This sequence is homologous to the GSN54 sequence.

 GS-N54: a mRNA of 4749 bp identified by the sequence SEQ ID No: 16 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AK024248. This mRNA does not have a coding sequence.

 The GS-N10 mRNA sequence has a coding sequence from nucleotide 618 to nucleotide 1787. We therefore identified a GS-P10 protein, resulting from the translation of this mRNA. This protein is composed of 389 aa, identified as SEQ ID No 63 in the attached sequence listing called Vassosubstratine.

 GS-N11: a called 1817bp mRNA identified by the sequence SEQ ID No: 11 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number NM 032181.

 The sequence of this mRNA has a coding sequence from nucleotide 439 to nucleotide 897. We therefore identified a GS-P11 protein, resulting from the translation of this mRNA. This protein is composed of 152 aa, identified as SEQ ID No 64 in the attached sequence listing, called Angiosignaline.

 GS-N12: a 4131 bp mRNA identified by the sequence SEQ ID No: 12 in the attached sequence listing. BLAST search based on sequences

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 GENBANK allows to identify it under the accession number AB023233.

 The sequence of this mRNA has a partial coding sequence from nucleotide 1 to nucleotide 2384. We therefore identified a GS-P12 protein, resulting from the translation of this mRNA. This protein is composed of 793 aa, identified as SEQ ID No 65 in the attached sequence listing, called Angiofoculin.

 GS-N13: a 2566 bp mRNA identified by the sequence SEQ ID No: 13 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under number XM ~ 018273.

 The sequence of this mRNA has a coding sequence from nucleotide 426 to nucleotide 2345. We therefore identified a GS-P13 protein, resulting from the translation of this mRNA. This protein is composed of 639 aa, identified as SEQ ID No. 66 in the attached sequence listing called Angiohelicin.

 GS-N14: an 1830bp mRNA identified by the sequence SEQ ID No: 14 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AK022109.

 This sequence does not contain a coding sequence.

 GS-N15: a mRNA of 6253 bp identified by the sequence SEQ ID No: 15 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number NM 014873.

 The sequence of this mRNA has a coding sequence from nucleotide 228 to nucleotide 1340. Thus, a GS-P15 protein resulting from the translation of this mRNA has been identified. This protein is composed of 370 aa, identified as SEQ ID No 67 in the attached sequence listing, called angioacyline.

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 The nucleic acid sequences designated GS-N16 to GS-N53 identified by the numbers SEQ ID NO: 17 to SEQ ID NO: 53, and SEQ ID No.: in the attached sequence listing and respectively the proteins encoded by said nucleic acids, identified as SEQ ID NO: SEQ ID NO: in the attached sequence listing have not previously been identified as having a biological role in the process of angiogenesis or differentiation of endothelial cells in capillary tubes. These sequences are described below.

 -GS-N16: a mRNA of 3139 bp identified by sequence number SEQ ID No: 17 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number XM ~ 011833 (Homo sapiens phosducin-like (PDCL) in the GENBANK sequence database.

 The sequence of this mRNA has a coding region from nucleotide 167 to nucleotide 1072. A GS-P16 protein resulting from the translation of this mRNA has therefore been identified.

 This protein whose sequence is identified under the number SEQ ID No: 68 in the attached sequence listing is a phosducin analogue called PDCL, composed of 301 aa.

 G proteins (guanine binding proteins) play a major role in transmembrane signaling pathways by transmitting extracellular signals through transmembrane receptors to their appropriate intracellular effectors (Gilman, 1987, Annu Rev. Biochem., 56, 615-649; Simon et al., 1991, Science, 252, 802-808). After binding of the ligand, the receptor catalyzes the exchange of GDP for a GTP on the alpha subunit of the heterotrimeric G protein which causes

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 its activation and the dissociation of the alphaGTP subunit from beta and gamma subunits (Gilman, 1987, Annu Rev.

Biochem.,. 56, 615-649). The G protein-dependent signaling pathways are designed to amplify and integrate a multiplicity of both stimulatory and inhibitory responses and their importance in cellular function is such that they are tightly regulated. PhLP (phosducin-like protein) is one of these regulatory elements, it belongs to the phosducin family and its isoforms, proteins that bind the beta / gamma subunits of the G protein, thus blocking their function (Lee et al, 1987 Biochemistry 26, 933-3990, Miles et al., 1993, Proc Natl Acad Sci USA, 90, 10831-10835, Craft et al., 1998, Biochemistry 37, 15758-15772). Phosducin, strongly expressed in photoreceptor cells of the retina (Lee et al., 1987, Biochemistry 26, 393-3990, Wilkins et al, 1996, J. Biol.

Chem., 271, 19232-19237) has been proposed to intervene in the adaptation to light (Willardson et al, 1996, Proc.

Natl. Acad. Sci. U.S.A., 93, 1475-1479). On the other hand, the function of PhLP is less known, this protein is more widely expressed (Miles et al., 1993, Proc Natl Acad.

Sci. USA 90, 10831-10835) and also binds the beta / gamma subunits of the G protein with high affinity (Schröder and Lohse, 1996, Proc Natl Acad Sci USA, 93, 2100-2104, Thibault et al. , 1997, J. Biol Chem., 272, 12253-12256). This protein is proposed to represent a phosducin homolog that regulates a number of G protein-dependent pathways in many cell types (Savage et al., 2000, J. Biol Chem., Vol 275, 39, 30399-30407. ).

 But to date, no role of PhLP has been described in the regulation of angiogenesis.

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 GS-N17: a mRNA of 2326 bp identified by the sequence number SEQ ID No. 18 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number BC011860 (Ribosomal Protein L3 (RPL3)), in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 1030 to nucleotide 2241, identified as SEQ ID No. 69 in the attached sequence listing.

This polypeptide sequence of 403 aa is identical (100% identity) to the human L3 ribosomal protein (RPL3) whose nucleic sequence is shorter, no accession BC006483 (SEQ ID N 285), BC012786 (SEQ ID N 286).

 L3 ribosome protein (RPL3) is a highly conserved protein (Herwig et al., 1992, Eur J Biochem, 207 (3): 877-85, Van Raay et al, 1996, Genomics: 37 (2): 172-6). located in the large ribosomal subunit. In E. coli, this protein is known to bind 23S ribosomal RNA and participate in the formation of the ribosomal center peptidyltransferase (Noller, 1993, Bacteriol.

175: 5297-530039; Noller, 1997, Annu. Rev. Biochem. 66: 679-716).

 Although the differential expression of L3 ribosomal protein has been demonstrated in several studies: in skeletal muscle in a mouse obesity study model (Vicent et al, 1998, Diabetes 47: 1451-8), in the hypothalamus and brown adipose tissue in mice involving RLP3 in the regulation of energy balance (Allan et al, 2000, Physiological Genomics, 3: 149-156), differential expression was not described during angiogenesis nor in the regulation of angiogenesis.

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 GS-N18: a mRNA of 3937 bp identified under the sequence number SEQ ID No. 19 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number XM ~ 042798 (Protein 20 RING finger protein 20 (RNF20)) in the base of GENBANK sequences.

 This mRNA has a coding sequence from nucleotide 89 to nucleotide 3016, its direct translation makes it possible to identify a protein homologous to the protein RNF20 composed of 975 aa, (GS-P18), identified under the number SEQ ID No. 70 in the list of sequences in annex.

 This GS-P18 sequence has a total identity with the protein sequence deduced from the nucleotide sequence identified under the accession number AF265230 in the GENBANK database and under the number SEQ ID No.

287 in the attached sequence listing. The nucleic sequences corresponding to the two proteins have a homology of 99% between them. the RNF20 protein is still little known, but it is characterized by the presence of the RING FINGER domain. Ring finger proteins are likely to play a role in the formation and architecture of large protein complexes that contribute to various cellular processes such as signal transduction, oncogenesis, apoptosis, development, differentiation, regulation genes, ubiquination (Saurin et al., 1996, Trends Biochem Sci 21,208-214, Borden, 2000, J. Mol Biol., 295, 1103-1112, Topcu et al., 1999, Oncogene 18,7091- 7100).

 To date, it has not been described that the RNF20 protein is involved in the regulation of angiogenesis.

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 GS-N19: a mRNA of 2167 bp identified under the number SEQ ID No. 20 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number BC002781 (Protein analog of the splice factor, arginine / serine-rich 4), in the GENBANK sequence database.

 The sequence of this mRNA has a coding region, from nucleotide 107 to nucleotide 1591. A GS-P19 protein resulting from the translation of this mRNA, presented under the number SEQ ID No. 71 in the attached sequence listing has therefore been identified. This protein composed of 494aa is homologous to the splice factor, arginine / serinerich 4 (SFRS4) also called SRp75 and has the same characteristic domains.

 SRp75 belongs to the SR protein family because it contains an N-terminal conserved domain, RRM (RNA recognition motif), a glycine-rich region, an RRM homologous inner region, and a long ( 315 aa) C-terminal domain consisting essentially of alternating serine and arginine residues (RS domain) (Zahler et al., Mol Cell Biol 1993 Jul; 13 (7): 4023-8). SR proteins are a family of nuclear phosphoproteins that are necessary for constitutive splicing but also influence the regulation of alternative splicing. SR proteins have a module structure (one or two RRM domains and one RS domain). Each domain in SR proteins is a functional module. The coordinated action of the RRM domains determines their specificity of binding to the RNAs, whereas the RS domains function as splice enhancers (Caceres et al., 1997, J. Cell Biol., 138,225-238; Chandler et al., 1997, Proc Natl Acad Sci USA, 94, 3596-3601, Mayeda et al., 1999, Mol Cell Biol, 19, 1853-1863, Graveley and Maniatis, 1998, Mol Cell, 1,765,

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 771). Various studies have suggested unique functions in the alternative splicing of pre-mRNAs for particular SR proteins, especially since they are expressed differentially in a variety of tissues.

These SR proteins are thus presented as crucial in the regulation of splicing during cell development and differentiation (Zahler et al., Science 1993 Apr 9; 260 (5105): 219-222; Fu, 1993, Nature, 365 (6441): 82-8, Caceres and Krainer, 1997, (Krainer ed.), Oxford University Press, Oxford, UK, pp. 174-212, Valcarcel and Green, 1996, Trends Biochem Sci, 21 (8): 296-301). A recent study shows a variable level of expression of SRp75 in different lymphoid cell lines (Dam et al., 1999, Biochim Biophys Acta; 1446 (3): 317-33.

 To date, no role in the regulation of angiogenesis is described for SFRS4 or SRp75 nor for the homologous protein of this factor.

 GS-N20: a 5801 bpARNA identified under the number SEQ ID No. 21 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number U65090 (Carboxypeptidase D) in the GENBANK sequence database
The sequence of this mRNA has a coding region of nucleotide 36 to nucleotide 4169. A GS-P21 protein, resulting from the direct translation of this mRNA, presented under the number SEQ ID No. 72 in the attached sequence listing has therefore been identified. . This protein called Carboxypeptidase D is composed of 1377 aa.

 Carboxypeptidase D (CPD of the S10 family of serine peptidases) is a transmembrane protein (180 kDa) that transects the proteins in the transpor-

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 Golgi and especially proteins secreted via the constitutive pathway such as growth factors and their receptors: insulin receptor, insulin receptor of growth factors (Reznik et al, 1998, J. Histochem Cytochem., 46, 1359). It is a carboxypeptidase with activity identical to carboxypeptidase E (CPElike) which is widely distributed in tissues. Carboxypeptidases mediate the removal of basic amino acids from the C-terminus of the peptide to generate either the bioactive product or the precursor for the formation of the C-terminal amide group (Fricker, 1988, Annu Rev. Physiol., 50, 309-321 Fricker, 1991, (ed) Peptide Biosynthesis and Processing, pp. 199-230, CRC Press, Boca Raton, FL).

 CPD consists in man of three carboxypoeptidase-like domains, a transmembrane domain and a small cytosolic tail of 58 residues (Novikova et al., 1999, J. Biol Chem., 274, 2887). capable of binding Protein Phosphatase A (PP2A) (Varlamov et al., 2001, J. Cell Science, 114, 311). It is a highly conserved protein between species with similar enzymatic properties.

 CPD is highly expressed in the human placenta. It is found especially in endothelial cells, trophoblasts, amniotic epithelial cells, chorionic endothelial villi cells or vascular smooth muscle cells of umbilical cords (Reznik et al., 1998, J. Cytochem., 46, 1359). CPE and CPD are also involved in the development of the precursor of endothelin 1 (ET-1).

This suggests that CPE and CPD are involved in the development of some umbilical peptides and placenta with autocrine and / or paracrine functions.

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 To date, no regulatory role of the CPD protein in angiogenesis has been described.

 GS-N21: an 8171 bpARNA identified under the number SEQ ID No. 22 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number NM ~ 004652 (Ubiquitin-specific Protease 9) in the GENBANK sequence database
The sequence of this mRNA has a coding sequence from nucleotide 60 to nucleotide 7751. It has thus been identified a protein resulting from the translation of this mRNA. This protein called GS-P21, ubiquitin-specific Protease 9, is composed of 2563 aa. It is identified as SEQ ID No 73 in the attached sequence listing
The USP9X protein belongs to the family of UBPs (Ubiquitin proteases), a group of enzymes whose function is to reverse the ubiquitination reaction by removing the ubiquitin residue from many substrates involved in cell division, growth, differentiation, signaling or transcription activation (Liu et al., 1999, Mol Cell Biol, 4, 3029-38, Zhu et al., 1996, Proc Natl Acad Sci USA 93: 3275-3279, Verma et al. , 1995, Genes Dev. 9: 2723-2735) Protein ubiquitination is an important regulatory phenomenon of biological pathways such as cytokine-activated transduction (Baek et al, 2001, Blood, 98, 636-642). UBPs are characterized by a conserved domain with surrounding divergent sequences and more particularly at the Nterminal portion allowing substrate specificity. These N-terminal discrepancies can alter the localization

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 and confer multiple functions to the different members of the larger family of UBPs (Lin et al., 2001, J Biol Chem, 276 (23): 20357-63). Some ubiquitin-specific proteases have already been described or suggested involved in some biological processes, such as UBP109 in embryonic development (Parket al, 2000, Biochem J, 349, 443-53), UBP43 in the differentiation of hematopoietic cells (Liu et al, 1999, Mol Cell Biol, 4, 3029-38). The ubiquitination pathway is involved in angiogenesis (Ravi et al., 2000, 14, 34-44, Sutter et al., Proc Natl Acad Sci USA, 97, Issue 9.4748-4753). USP9X has never been described to date as a regulator of angiogenesis.

* - GS-N22: a 3851 bpRNA identified under the number SEQ ID No. 23 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number NM-002525 (Nardilysin (N-arginine dibasic convertase) (NRD1)) in the base of GENBANK sequences.

 The sequence of this mRNA has a coding sequence from nucleotide 136 to nucleotide 3795. Thus, a GS-P22 protein resulting from the translation of this mRNA has been identified. This Nardilysin protein is composed of 1219aa. It is identified by the number SEQ ID No 74 in the attached sequence listing.

 N-arginine dibasic convertase (NRD convertase, nardilysin, EC 3. 4.24.61) is a metalloendopeptidase of the family of insulinases which specifically cleaves peptides (particularly neuroendocrine peptides such as somatosatin-28, dynorphin-A, natriuric atrial factor) on the N-terminal side of an Arginine residue at dibasic sites in vitro (Cohen et al, 1995, Methods Enzymol.,

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 248.703 to 716; Hospital et al., 1997, Biochem. J., 327, 733-779). In vivo, its exact function remains poorly known but many enzymes of the same family are involved in the maturation of prohormones and proproteins (Winter et al., 2000, Biochem J., 351, 755-764). The NRD convertase activity is present mainly in the endocrine tissues and mainly in the testes (Chesneau, et al., 1994, J. Biol Chem., 269, 2056-2061). It can be localized both in the cytoplasm and at the cell surface (Hospital et al, 2000, Biochem J., 349, 587-597).

 Currently little is known about the regulation of the expression and activity of NRD convertase. The activity appears to be regulated by the amines that bind the acid (stretch) domain of the enzyme (Csuhai et al., 1998, Biochemistry, 37 (11): 3787-94). Retinoic acid has also been shown to modulate the expression of the enzyme in human neuroblastic lineages (Draoui et al, 1997, J. Neurooncol., 31, 99-106).

 In the adult rat, the regulated expression of NRD convertase during spermatogenesis and its concentration in the flagellum suggests a role of this enzyme in the differentiation of male germ cells (Chesneau, et al., 1996, J. Cell Sci. 2737-2745). This enzyme is also proposed to play a specific role in neuronal development (Fumagalli et al., 1998, Genomics 47, 238-245). Recently, NRD convertase has been described as a new specific heparin-binding EGF-like growth factor receptor (HB-EGF) that controls cell migration (Nishi et al, 2001, EMBO J, 20 (13): 3342- 50).

 On the other hand, no role has been discovered to date, in the regulation of angiogenesis

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 GS-N23: a 13107 bpARNA identified under the number SEQ ID No. 24 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number XM ~ 016303 (myeloid / lymphoid or mixedlineage leukemia (MLL) mRNA) in the base of GENBANK sequences.

 The mRNA sequence identified by the number SEQ ID No. 24 has a coding sequence of the nucleotide 1872 to the nucleotide 10001. It was thus identified a GSP23 protein resulting from the translation of this mRNA. This protein called MLL is composed of 2709aa. It is identified by the number SEQ ID No 75 in the attached sequence listing.

 This GS-N23 sequence has at least 86% homology with the following sequences: GS-N47: a 14255 bpRNA identified under the number SEQ ID No. 48 in the attached sequence listing.

A BLAST search makes it possible to identify it under the accession number L04731 in the GENBANK sequence database.

 This mRNA sequence has no coding sequence. It includes the sequence GS-N23, with a homology of 90%.

 - GS-N48: a ARN of 11910 bp identified by the number SEQ ID No. 49 in the attached sequence listing.

A BLAST search makes it possible to identify it under the accession number NM ~ 005933 in the GENBANK sequence database.

 The mRNA sequence identified by the number SEQ ID No. 48 has a coding sequence from nucleotide 1 to nucleotide 11910. It has thus been identified a GSP48 protein resulting from the translation of this mRNA. This protein called MLL is composed of 3969aa. It is identified by the number SEQ ID No 99 in the attached sequence listing.

 The acute lymphoblastic leukemia-1 (ALL) -l or Myeloid-lymphoid or Mixed-lineage Leukemia (MLL) gene

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 or called human tri-thorax (HRX) on the human chromosome, band q23 is the site of many locally grouped chromosomal alterations (deletions, partial duplications, translocations) associated with several types of leukemias. Structurally variant proteins derived from the altered gene are presumed to cause malignant transformation of hematopoietic progenitor cells (Nilson, Br J Haematol, 1996, 93 (4): 966-72, Kobayashi et al, 1995, Leuk Lymphoma, 17 (5-6). ): 391-9).

 The MLL protein is a large nuclear protein with zinc finger motifs and SET domain, highly conserved 200aa located in its C-terminal portion. This protein is widely expressed during embryogenesis; studies have shown that this protein is a positive regulator of the Hox homeobox genes (Yu, et al., 1995, Nature (London) 378, 505-508). The MLL protein is described as being involved in transcriptional maintenance in development that functions in multiple morphogenetic processes (Yu et al, 1998, Vol 95, Issue 18,10632-10636). The MLL protein is suggested to play a role in the regulation of both cell proliferation and survival in the developing embryo (Hanson et al., 1999, Proc Natl Acad Sci.

USA, 96, Issue 25, 14372-14377).

 To date, no role has been described for the MLL protein in the regulation of angiogenesis.

 GS-N24: an ARN of 10330 identified as SEQ ID No 25 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number U72937 (Helicase and ATPase dependent on DNA (ATRX), product 2 of alternative splicing) in the base of GENBANK sequences.

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 This sequence is homolocal, with at least 95% identity of the sequence: GS-N49: a 10452 bpARNA identified under the number SEQ ID No. 50 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number U72936 in the GENBANK sequence database.

 The mRNA sequence identified under the number SEQ ID No. 25 has a coding sequence of nucleotide 216 to nucleotide 7694. It has thus been identified a GSP24 protein resulting from the translation of this mRNA. This protein called ATRX product 2 is composed of 2492 aa. It is identified as SEQ ID No 76 in the attached sequence listing.

 The mRNA sequence identified by the number SEQ ID No. 50 has a coding sequence from nucleotide 950 to nucleotide 7816. Thus, a GSP49 protein resulting from the translation of this mRNA was identified. This protein called ATRX product 1 is composed of 2288aa. It is identified by the number SEQ ID No 100 in the attached sequence listing.

 The proteins identified by the numbers SEQ ID No. 76 and 100 are 90% homologous.

 Since its identification in 1995, the ATRX gene (synonyms XNP, XH2) has been shown to be the gene for many forms of disease, various X-linked mental retardation syndromes are linked to mutations in this gene. (Review: Gibbons et al. Higgs, 2000, Am J Med Genet, 97 (3): 204-12). This gene encodes a protein of the SNF2 subgroup of the helicase superfamily and ATPases (Picketts et al., 1996, Hum Mol Genet (12): 1899-907), these domains suggest that the ATRX protein has a role in the transcriptional regulation via an effect on the structure and / or function of chromatin, but its role

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 exact is not yet known. No role in the regulation of angiogenesis has been described to date.

 GS-N25: a 1777 bp ARNOM identified as SEQ ID No. 26 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number NM ~ 006476 (Carrier of sialic acid CMP, member 1 (SLC35Al)) in the base of GENBANK sequences.

 The sequence of this mRNA has a coding sequence from nucleotide 28 to nucleotide 1041. Thus, a GS-P25 protein resulting from the translation of this mRNA has been identified. This protein called sialic acid transporter-CMP is composed of 337a. It is identified by the number SEQ ID No 77 in the attached sequence listing.

 GS-N50: a 1874 bpRNA identified as SEQ ID No 225 in the attached sequence listing.

A BLAST search can identify it under accession number BC008372 in the GENBANK sequence database. The mRNA sequence identified by the number SEQ ID No. 225 has no coding sequence.

 The CMP-sialic acid transporter is involved in the process of maturation of glycosylation and more particularly sialylation; it allows the translocation of cytosolic CMP-sialic acid through the membrane of the Golgi apparatus necessary for the sialylation of membrane or secreted proteins as well as lipids in this compartment.

(Hirschberg and Snider, 1987, Annu Rev. Biochem., 56, 63-88, Hirschberg, 1996, in Organellar Ion Channels and Transporters Society of General Physiologist, 49th Annual Symposium (Clapham, DE, and Ehrlich, BE, eds) , pp.

105-120, Rockefeller University Press, New York). The

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 CMP-sialic acid transport regulation is still poorly known except that an increase in sialylation has been observed on the surface of tumor cells (Santer et al., 1989, Eur J. Biochem., 181, 249-260; Saitoh et al., 1992, J. Biol Chem., 267, 5700-5711, Bresalier et al., 1996, Gastroenterology, 110, 1354-1367, Gorelik et al, 1997, Cancer Res 57,332-336) and that Inhibition of the CMP-sialic acid transporter reduces the growth and metastasis of tumor cells (Harvey, BE, and Thomas, P. (1993) Biochem Biophys Res 190.571-575). But to date, no involvement of this transporter has been reported in the regulation of angiogenesis.

 - GS-N26: a ARN of 3982 bp identified as SEQ ID No 27 in the attached sequence listing.

A BLAST search can identify it under accession number U26710 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence of nucleotide 323 to nucleotide 3271. It has thus been identified a GS-P26 protein resulting from the translation of this mRNA. This protein called cbl-b is composed of 982aa.

 It is identified by the number SEQ ID No 78 in the attached sequence listing.

 Cbl-b belongs to the Cbl family, highly conserved across species. The Cbl proteins are characterized in their N-terminal part by a putative phosphotyrosine-binding domain and a RING finger motif in the C-terminal part, the mammalian Cbl proteins contain a proline-rich region, conserved tyrosine residues and a motif. leucine Zipper. Cbl proteins participate in protein signaling

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 tyrosine kinase receptors, as well as cytokine antigens and receptors by associating with their cytoplasmic tail ensuring signal continuity of these receptors. The Cbl protein is recruited by the tyrosine kinase module of these receptors and the tyrosines phosphorylated after cellular activation. Cbl functions as a docking protein and associates with molecules containing the SH2 and SH3 domains, including the family of Crk and Vav adapters. Cbl proteins are proposed both as negative regulators of tyrosine kinase receptor signaling (Smit et al., Crit Rev Oncog 1997; 8: 359-79) and as positive modulators of receptor signaling such as the receptor superfamily. TNF (Arron et al., J Biol Chem 2001 Aug 10; 276: 30011-7).

 The Cbl-b protein, widely expressed in many tissues and cells including hematopoietic cells (Keane et al., Oncogene 1995 Jun 15; 10 (12): 2367-77), is involved in setting up the activation threshold. lymphocytes (Bachmaier et al., Nature 2000, 403: 211-6, Fang et al., J Biol Chem 2001, 276: 4872-8). The P85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) has been identified as its substrate. Cbl-b, by its ligase activity of the ubiquitin E3 protein, negatively regulates this P85 regulatory subunit (Fang D, Nat Immunol 2001 Sep; 2 (9): 870-5).

Cbl-b is also a negative regulator of epidermal growth factor receptor signaling, EGFR (Ettenberg et al., Oncogene 1999; 18: 1855-66; Ettenberg et al., J Biol Chem 2001; 276: 27677-84).

 However, to date, it has not been described that Cbl-b plays a role in the regulation of angiogenesis.

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 GS-N27: a mRNA of 3385 bp identified under the number SEQ ID No. 28 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number XM ~ 39529 in the GENBANK sequence database.

 This sequence has 86% homology with the following sequence: a 4461 bp cDNA (DKFZp564D173) identified under the number SEQ ID No. 51 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AL110212 in the GENBANK sequence database. There is no corresponding coding sequence.

 The mRNA sequence identified as SEQ ID No. 27 has a coding sequence from nucleotide 107 to nucleotide 451. Thus, a GSP27 protein resulting from the translation of this mRNA was identified. This protein called Histone H2A.F / Z variant (H2AV) is composed of 114aa. It is identified as SEQ ID No 79 in the attached sequence listing.

 The basic unit of chromatin in eukaryotes is the nucleosome. A nucleosome consists of a 146bp DNA sequence wrapped around an octamer of proteins, histones H2A, H2B, H3, H4 (Lugeret et al., 1997, Nature, 389, 251-260). Heterogeneity in nucleosome structure may be a transcriptional regulatory mechanism. This heterogeneity is created either by post-translational modifications of histones such as acetylation, phosphorylation, methylation, ubiquitination (Mizzen et al., Cold Spring Harb Symp Quant Biol 1998; 63: 469-81; Workman and Kingston, 1998, Annu Rev. Biochem., 67: 545-579) or the incorporation of histone variants into the nucleosome. The different histone variants allow the specialization of the

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 nucleosome structure for specific purposes; Sperm-specific histone variants, for example, facilitate the dramatic compaction of DNA that occurs during spermatogenesis (Wolffe, 1998, Chromatin: Structure and Function, 3rd Ed., Academic Press, San Diego, Doenecke and al, 1997, Adv Exp Med Med Biol., 424, 37-48).

 Histone H2A.F / Z is a family of histone H2A variants that is highly conserved across species and substantially divergent from histone H2A of phase S in given species (Jackson et al, 1996, Trends Biochem Sci., 221, 466-467, Jiang et al, 1998, Biochem Biophys Res Commun, 245, 613-617). The exact function of H2A.F / Z is not yet known, but this histone may play a role in transcriptional regulation because in Tetrahymena, its expression is associated with the transcriptionally active macronucleus and in Drosophila its incorporation into the chromatin during the development coincides with the onset of zygotic gene expression (Clarkson et al., 1999, DNA Cell Biol., 18, 457-462, Stargell et al, 1993, Genes Dev., 7, 2641-2651).

 On the other hand, to date, no role is known for histone H2A.F / Z in the regulation of angiogenesis.

 GS-N28: a 1128-bpARNA identified as SEQ ID No. 29 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number NM ~ 001320 in the GENBANK sequence database.

 This sequence has 77% homology with the sequence identified under the accession number M30448 in the GENBANK database and identified under the number SEQ ID No. 289 in the attached sequence listing.

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 The sequence of this mRNA has a coding sequence from nucleotide 97 to nucleotide 744. It has thus been identified a GS-P28 protein resulting from the translation of this mRNA. This protein called Casein kinase II, subunit Beta is composed of 215 aa. It is identified as SEQ ID No 80 in the attached sequence listing.

 Casein kinase II (CKII) is a ubiquitous serine / threonine kinase that is located both in the cytosol and in the nucleus of eukaryotic cells. CKII phosphorylates more than one hundred substrates, many of which are involved in the control of cell division and signal transduction (reviewed: Allende and Allende, 1995, The FASEB Journal, Vol 9,313-323). CKII exists as a tetramer composed of two alpha and / or alpha 'catalytic subunits and two regulatory subunits (beta). The beta subunit seems to act to stabilize the alpha and / or alpha subunits and also to influence the specificity of the substrate and the kinetic properties of the enzyme (Dobrowolska et al, 1999, Mol Cell Biochem, 191 (1-2) : 3-12). Some studies have shown that casein kinase activity is stimulated by hormones or growth factors such as insulin, IGF-I, EGF (Sommercorn et al., 1987, Proc Natl Acad Sci USA, 84,8834-8838, Klarlund et al., 1988, J. Biol., Chem., 263, 1872-15875, Ackerman and Osheroff, 1989, J. Biol Chem., 264, 11958-11965), this activation resulting from a increased phosphorylation of the beta subunit of casein kinase (Ackerman et al, 1990, Proc Natl Acad Sci USA, 87, 821-825). Different studies have shown a dysregulated expression of CKII in tumors. Recent studies have shown that overexpression of CKII in tumor cells is not only a reflection of cell proliferation.

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 tumors, but it can also reflect the pathobiological characteristics of the tumor. Deregulation of CKII could influence the apoptotic activity of these cells (reviewed: Tawfic et al., 2001, Histol Histopathol, 16 (2): 573-82). This enzyme is described as having a possible role in oncogenesis (Yu et al., J Cell Biochem, 1999, 74 (1): 127-34).

 On the other hand, no differential expression of CKII or of its beta subunit during angiogenesis has been described or a role in the regulation of angiogenesis has been demonstrated.

 GS-N29: an ARN of 18207 bp identified as SEQ ID No 30 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AF156100 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 230 to nucleotide 17140. Thus, a GS-P29 protein resulting from the translation of this mRNA was identified. This protein called Hemicentine is composed of 5636aa. It is identified as SEQ ID No 81 in the attached sequence listing.

 This GS-29 sequence comprises the sequence GSN52 below, having a 99% homology with it.

 GS-N52: an 8546 bp mRNA identified as SEQ ID No. 52 in the attached sequence listing. A BLAST search can identify it under the accession number AJ306906 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence for a GS-P52 protein of 2673aa identified as SEQ ID No. 101 in the attached sequence listing.

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 Recently described, hemicenin is an extracellular matrix protein of the immunoglobulin superfamily, which is involved in attachment and cell migration on the basement membrane (Vogel and Hedgecock, 2001, Development, 128 (6): 883- 94). Its role in the regulation of angiogenesis is not described at present. This protein contains the sequence of fibulin-6 which belongs to the family of fibulins, extracellular matrix proteins and blood whose two most studied members are fibulin 1 and fibulin 2 (Alexande and Detwiler, 1984, Biochem. J., 217, 67-71, Argraves et al., 1990, J. Cell Biol., 111, 355-3164, Kluge et al., 1990, Eur J. Biochem., 193, 651-659 et al. J. Cell Biol., 123, 1269-1277). They interact with proteins involved in cell adhesion such as fibronectin, laminin, fibrinogen (Brown et al., 1994, Cell Sci, 107 (Pt 1), 329-38, Tran et al, 1995, J Biol Chem. 270 (33): 19458-64, Godyna et al., 1995, Matrix Biol, 14 (6): 467-77) or endostatin which is an angiogenesis inhibitor (Sasaki et al, 1998, EMBO J. , 17 (15): 4249-56) which gives them a regulatory function in various biological processes. Fibulin 1, for example, has been described which may play a role in the regulation of the neurotrophic activity of amyloid beta precursor protein (Ohsawa et al., 2001, J. Neurochem; 76 (5): 1411-20), in homeostasis and thrombosis (Tran et al., 1995, J Biol Chem, 270 (33): 19458-64).

 By cons, the function of fibulin 6 is not yet known, and in particular, no role of this protein has been described until today in the regulation of angiogenesis.

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 GS-N30: a 4325 bp RNA identified under the number SEQ ID No. 31 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number NM ~ 015180 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 123 to nucleotide 3041. Thus, a GS-P30 protein resulting from the translation of this mRNA has been identified. This protein called SYNE-2 is composed of 1092aa.It is identified by the number SEQ ID No. 82 in the attached sequence listing.

 The Syne-2 protein is poorly known, it has recently been described with a homologous protein, Syne-1 protein (synaptic nuclear envelope-1). Syne-1 protein is associated with nuclear envelopes in smooth muscle, cardiac and skeletal cells. Syn-1 is described as the first protein found to be selectively associated with the synaptic nucleus and is suggested to be involved in the formation or maintenance of nuclear aggregates in the muscle junction (Appel et al., 2000, J. Biol Chem., Vol. 275, Issue 41, 3186-31995). Syn-2 differs from Syn-1 in the distribution and by its lower level of expression. Both homologous proteins show repetitive domains of spectrin that are present in different proteins involved in the structure of the cytoskeleton (Yan et al., Science 1993 Dec 24; 262 (5142): 2027-30).

 To date, no role of Syn-2 is described in the regulation of angiogenesis.

 GS-N31: a 4248 bpARNA identified as SEQ ID No. 32 in the attached sequence listing.

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A BLAST search makes it possible to identify it under the accession number AF261758 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 100 to nucleotide 1650. Thus, a GS-P31 protein resulting from the translation of this mRNA was identified. This protein called Seladine-1 is composed of 516aa. It is identified by the number SEQ ID No 83 in the attached sequence listing.

 This mRNA sequence identified as SEQ ID No. 32 in the attached sequence listing is homologous to a 4187 bp sequence. A BLAST search makes it possible to identify it under accession number D13643 in the GENBANK sequence database. It is identified by the number SEQ ID No. 53 in the attached sequence listing.

 This mRNA has a partial coding sequence. A GS-P53 protein of 528aa resulting from the translation of this mRNA is thus identified. It is identified as SEQ ID No 102 in the attached sequence listing.

 The gene seladin-1 has recently been identified in the human brain. Seladin-1 appears to be an important factor for cell protection against beta-amyloid peptide toxicity and oxidative stress (Greeve et al, 2000, J Neuroscience, (19): 7345-7352).

These authors suggest that seladin-1 may be involved in the regulation of survival and cell death and that decreased expression of this protein in specific neurons may be a cause for selective vulnerability in Alzheimer's disease.

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 On the other hand, to date, no role in the regulation of angiogenesis has been described for seladin-1.

 GS-N32: a mRNA of 7764 bp identified under the number SEQ ID No. 33 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number NM ~ 001271 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 708 to nucleotide 5927. Thus, a GS-P32 protein resulting from the translation of this mRNA was identified. This protein called CHD2 is composed of 1739aa. It is identified by the number SEQ ID No. 84 in the attached sequence listing.

 The CHD2 protein belongs to the family of CHD proteins characterized by a chromodomain. The "chromo" (CHRromatin Organization Modifier) domain is a conserved region of about 60 amino acids found in a variety of proteins including Drosophila melanogaster HP1 proteins, which binds to heterochromatin; 4 genes of this family have been identified in the human genome: CHD1, CHD2, CHD3 and CHD4 (Woodage et al., 1997, 94, 11472-11477). Chromodomain confers a role on the protein in chromatin compaction (Paro 1990, Trends Genet 6: 416-421, Singh et al., 1991, Nucleic Acids Res 19: 789-794, Aasland and Stewart 1995). Nucleic Acids Res 23: 3168-3173, et al, 1995, Nucleic Acids Res.

23: 4229-4233, Messner et al, 1992, Genes Dev., 6, 1241-1254; James and Elgin, 1986, Mol. Cell.Biol, from 6.3862 to 3872). CHDs contain a second conserved domain, the Myb domain that is involved in DNA binding (Klempnauer and Sippel, 1987, EMBO J., 6: 2719-2725). In addition to these domains, CHD2 contains the SNF2 domain, found in

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 proteins involved in a variety of processes such as transcriptional regulation (eg SNF2, STH1, brahma, MOT1), DNA repair (eg ERCC6, RAD16, RAD5), DNA recombination (eg, RAD54 Eisen et al, 1995, Nucleic Acids Res, 23 (14): 2715-23) and finally a conserved domain of the helicase superfamily, the DEAH box helicases. Helicases are involved in the unwinding of nucleic acids (Matson and Kaiser Roger, 1990, Annu Rev. Biochem 59,289-329). DEAH box helicases have been proposed to be involved in mRNA splicing and cell cycle progression (Ludgren et al, 1996, Mol Biol Cell 7, 1083-1094, Imamura et al, 1998, Nucleic Acids Res , 26 (9): 2063-8).

CHD can play an important role in regulating gene transcription. (Delmas et al., 1993, Proc Natl Acad Sci, 90, 2414-2418, Woodage et al., 1997, Proc Natl Acad Sci USA, 94, 11472-11477, Tran et al, 2000, The EMBO Journal, 19,2323-2331)
A recent study has shown that CHD4 is induced when endothelial cells are stimulated by TNF-alpha (Murakami et al, 2000, J Atheroscler Thromb, 7 (1): 39-44).

 No study shows an involvement of CHD2 in the regulation of angiogenesis.

 GS-N33: a 4693 bp mRNA identified under the number SEQ ID No. 34 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number XM ~ 042555 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 1702 to nucleotide 4107. Thus, a GS-P33 protein resulting from the translation of this mRNA was identified. This protein called BRD2 is composed of

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 801aa. It is identified as SEQ ID No 85 in the attached sequence listing.

 The role of the BRD2 protein is not known.

This protein is characterized by two bromodomains. The bromodomain is a conserved region, first identified as a signature of 61-63 amino acids, its function not being known (Haynes et al., Nucl Acids Res., 1992, 20, 2603). Subsequently, this domain has been identified in transcription factors, coactivators and other proteins are involved in the transcription or remodeling of chromatin and its bounds have been extended up to 110 amino acids. The growing number of proteins containing this domain is more than forty (Jeanmougin et al, 1997, Trends Biochem Sci 22, 151-153, Winston and Allis, 1999, Nature Struct Biol 6.601-1604). Some proteins have only one copy of the domain others present two copies of the pattern. The BRD2 protein is characterized by two bromodomains, as is the case of the BDF1 transcription factor (Tamkun, 1995, Curr, Opin, Genet, Dev, 5: 473-477), or the TAFII250 protein, the largest subunit. multiprotein complex, TFIID involved in transcription initiation (Jacobson et al, 2000, Science, 288 (5470): 1422-5). The exact function of this domain is not yet known but it is thought to be involved in protein-protein interactions and may be important for the assembly or activity of the multiple complex components involved in chromatin modification and control. of a wide variety of eukaryotic genes including those that control growth. A wide variety of chromatin-directed functions, including but not limited to phosphorylation, acetylation, methylation, co-activation, or

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 Transcriptional recruitment characterizes complexes that contain bromodomain motifs. Their versatility and ubiquity ensure diverse, fast and flexible transcriptional responses (reviewed: Denis, 2001, Frontiers in Bioscience 6, d849-852).

 The differential expression of bromodomain-containing proteins has already been shown in particular that of a RING3 kinase homologous protein whose expression is induced by VEGF or bFGF in endothelial cells, this protein is suggested as a new target of the VEGF and bFGF activated signaling voice which allows endothelial cells to enter the proliferative phase of the angiogenesis process (BelAiba et al, 2001, Eur J Biochem, 268 (16): 4398-407).

 On the other hand, the BRD2 protein has never been described involved in the regulation of angiogenesis.

 GS-N34: a 2983 bpRNA identified under the number SEQ ID No. 35 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number BC007429 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 200 to nucleotide 1069. It has thus been identified a GS-P34 protein resulting from the translation of this mRNA. This protein called Syntaxin 3A is composed of 289aa. It is identified as SEQ ID No 86 in the attached sequence listing.

 Syntaxin 3A belongs to the family of syntaxins / epimorphins which is characterized by a size between 30 and 40 kDa, a highly hydrophobic C-terminus that is probably involved in the anchorage of

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 the protein in the membrane and a well-conserved central region that appears to be in a "coiledcoil" conformation. The specific profile of this family is based on the most conserved region of the "coiled-coil" domain. Syntaxins are involved in the intracellular transport of vesicles and their attachment to the plasma membrane. Recent work suggests that different syntaxin isoforms may interact with a defined group of membrane transport proteins and thus regulate their transport activity (reviewed: Saxena et al., 2000, Curr Opin Nephrol Hypertens, 9 (5): 523-7). .

 Syntaxin 3A is one of two isoforms (3A and 3B) identified in humans, derived from an alternative splicing of the same gene. The increase in syntaxin 3A expression has already been demonstrated during various biological processes such as neutrophil differentiation of HL-60 cells or in granule cells of the dentate (dentate granule cells) during propagation. synaptic plasticity in the nervous system (Rodger et al, 1998, J Neurochem, 71 (2): 666-675, Martin-Martin et al, 1999, J Leukoc Biol, 65 (3): 397-406).

 On the other hand, to date, no increased expression of syntaxin 3A has been described during angiogenesis and therefore no implication in the regulation of angiogenesis has been reported.

 - GS-N35: a 12227pb ARN identified with the number SEQ ID No. 36 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number NM ~ 015001 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 205 to nucleotide 11199. Thus, a GS-P35 protein resulting from translation has been identified.

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 of this mRNA. This protein called SHARP is composed of 3664aa. It is identified as SEQ ID No 87 in the attached sequence listing.

 Sharp (SMART / HDAC1 Associated Repressor Protein) is a recently isolated gene (Nagase et al., 1999, DNA Res, 6 (1): 63-70). SHARP is a potential repressor for transcription whose repression domain (RD) directly interacts with SMRT and at least 5 members of the NuRD complex (nucleosome remodeling and histone deacetylase activities) including histone deacetylases HDAC1 and HDAC2. In addition, SHARP binds to the ARS coactivator of the steroid receptor RNA by an intrinsic RNA-binding domain and suppresses steroid receptor transcriptional activity. In this way, SHARP has the ability to modulate both leaved and unligated nuclear receptors. The SHARP expression is itself inducible by steroids suggesting a simple feedback mechanism for attenuation of the hormonal response (Shi et al, Genes Dev 2001 May 1; 15 (9): 1140-51) . Histone deacetylases (HDAC) have been shown to be involved in the induction of angiogenesis by suppressing the expression of tumor suppressor genes (Kim et al, 2001, Nat Med, 7 (4): 437-43).

 However, no role of the SHARP protein has been described in regulation in angiogenesis.

 GS-N36: a 5376 bp mRNA identified under the number SEQ ID No. 37 in the attached sequence listing.

A BLAST search makes it possible to identify it under the accession number AF352051 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 92 to nucleotide 4962. It has thus been identified a GS-P36 protein resulting from translation.

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 of this mRNA. This protein called Protein linked to the proliferation potential is composed of 1616aa. It is identified by the number SEQ ID No 88 in the attached sequence listing.

 This sequence identified under the number SEQ ID No. 37 has 92% homology with the sequence coding for the protein RPBB6 (retinoblastoma-binding protein 6) of 948aa identified under the number SEQ ID No. 288 in the attached sequence listing. The nucleotide sequence corresponding to this protein comprises 2994bp and accession number NM ~ 006910 on the basis of GENBANK sequences.

 The exact role of Protein related to the identified potential proliferation potential (proliferation potential-related protein) is not yet known. Like its homologous proteins, it presents finger domains of Zinc known to be involved in the protein-protein interaction.

This protein is homologous to a member of the family of retinoblastoma protein binding proteins (pRb), retinoblastoma-binding protein 6 (RPBB6), also called RBQ-1 (Sakai et al, 1995, Genomics, 30 (1): 98 -101).

The pRb protein (tumor suppressor called retinoblastoma) acts to control cell proliferation, inhibit apoptosis and induce cell differentiation by associating with a large number of proteins (reviewed: Morris and Dyson, 2001, Adv Cancer Res; 82: 1-54).

 To date, the protein linked to the proliferation potential has never been described as being involved in the regulation of angiogenesis.

 GS-N37: a 6626 bp RNA identified as SEQ ID No. 38 in the attached sequence listing.

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A BLAST search makes it possible to identify it under accession number XM ~ 005338 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 245 to nucleotide 2989. It has thus been identified a GS-P37 protein resulting from the translation of this mRNA. This protein called Protein HIP1 is composed of 914aa. It is identified by the number SEQ ID No. 89 in the attached sequence listing.

HIP1 (Huntingtin interacting protein 1) protein is a 116 kDa protein that binds protein
Huntingtin, mutated gene product in Huntington's disease (Kalchman et al, 1997, Nat.

Genet., 16, 44-53; The Huntington's Disease Collaborative Research Group, 1993, Cell 72, 971-983). HIP1 is predominantly expressed in the brain, but is also detected in other tissues (Wanker et al, 1997, Human Molecular Genetics, Vol 6,487-495). The function of HIP1 is not yet well known, but it shares biochemical characteristics and conserved domains with Sla2p, a protein essential for cytoskeletal function in Saccharomyces cerevisiae (Kalchman et al., 1997, Nat Genet 16,44-53). Holtzman et al., 1993, J. Cell Biol 122, 635-644). HIP1 also contains a leucine Zipper domain and is homologous in its Talaline Cterminal Part, a cytoskeletal protein involved in cell-substratum and cell cell interactions (Rees et al, 1990, Nature, 347 (6294): 685-9). the HIP1 protein has been shown to be involved in apoptosis (Hackam et al., 2000, J. Biol.

Chem., Vol. 275, Issue 52.41299-41308).

 To date, no role has been described for HIP1 in angiogenesis.

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 GS-N38: a mRNA of 2366 bp identified under the number SEQ ID No. 39 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number BC000335 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 12 to nucleotide 2237. Thus, a GS-P38 protein resulting from the translation of this mRNA has been identified. This protein called Nucleoporine 88kDa is composed of 741aa. It is identified by the number SEQ ID No. 90 in the attached sequence listing.

 Nucleoporin 88 (Nup88) is a nuclear membrane protein likely to be involved in nucleocytoplasmic transport (Fornerod et al., 1997, EMBO J, 16: 807-816, et al., Oncogene 1996, 13: 1801-1808). Nup88 was found to be associated with the central CAN / Nup214 domain, a component of a nuclear pore complex likely involved in nuclear protein import, nuclear mRNA export, and cell cycle regulation (vanilla). Deursen et al., 1996, EMBO J., 15: 5574-5583). Nup 88 has been shown to be widely distributed and overexpressed in cancer cells and tissues (Martinez et al., 1999, Cancer Research 59,5408-5411, Gould et al., 2000, American Journal of Pathology, 157: 1605). 1613).

 To date, Nup88 has never been described as being involved in the regulation of angiogenesis.

 GS-N39: a mRNA of 1543 bp identified under the number SEQ ID No. 40 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number XM ~ 049486 in the GENBANK sequence database.

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 The sequence of this mRNA has a coding sequence from nucleotide 86 to nucleotide 412. Thus, a GS-P39 protein resulting from the translation of this mRNA has been identified. This protein called Protein 1A binding FK506 is composed of 108aa. It is identified by the number SEQ ID No 91 in the attached sequence listing.

 FKBPs (FK506 Binding Protein) are major binding proteins with high affinity in vertebrates for the immunosuppressive drug FK506 (1990, Tropschug et al, Nature 346: 674-677, Stein, 1991, Curr.

Biol. 1: 234-236; Siekierka et al., 1990, J. Biol. Chem. 265: 21011-21015). Several members of the FKBP family have been identified in many tissues and in various cellular compartments, the most well known being FKBP12, a cytoplasmic isoform (Galat and Metcalf, 1995, Prog.

Biophys. Mol. Biol. 63.67 to 118; Kay, 1996, Biochem. J. 314, 361-385). FKBPs belong to a large family of cis-trans peptidyl propyl isomerases, (PPIase or rotamase). PPIase is an enzyme that accelerates protein folding by catalyzing cis-trans isomerization of peptide bonds involving the proline residue (Fischer and Schmid, 1990, Biochemistry 29: 2205-2212). FKBPs are known to be involved in many cellular processes, such as cell signaling, protein transport and transcription. Inhibition studies of the gene encoding FKBPs in plants and animals have emphasized the importance of this family of proteins in the regulation of cell division and differentiation (reviewed: Harrar et al., 2001 Trends Plant Sci; : 426-31). However, although they bind surface receptors and despite their PPiase activity, their physiological function has yet to be defined. Recently, the PPiases have been proposed to play a

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 role in the functional rearrangement of components in receptor heterocomplexes (Schiene-Fischer and Yu, 2001, FEBS Lett, 495 (1-2): 1-6).

 To date, it has not been found that FKBP proteins are involved in the regulation of angiogenesis.

 GS-N40: a mRNA of 3824 bp identified under the number SEQ ID No. 41 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number XM ~ 042827 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 115 to nucleotide 3663. It has thus been identified a GS-P40 protein resulting from the translation of this mRNA. This protein called Protein SALF is composed of 1182 aa. It is identified by the number SEQ ID No. 92 in the attached sequence listing.

 Complementary DNA (cDNA) of the stoned B / TFIIA-alpha / beta-like factor (SALF) has recently been identified from a human placenta cDNA library (Ashok et al., 1999, J. Biol. Chem, Vol 274, Issue 25, 18040-18048). The role of SALF is not yet identified, however, this cDNA, described as rare, is identical to the sequence of the ALF protein with a larger N-terminal sequence containing a domain homologous to the Drosophila Stone B protein (Andrewe et al. , 1996, Genetics 143, 1699-1711) and the adapter proteins of Clathrin, 1 (AP47) and 2 (AP50) (Thurieau et al, 1988, DNA (NY) 7.663-669, Nakayama et al, 1991, Eur J. Biochem 202,569-574). The ALF protein (TFIIA-alpha / beta-like factor) is also a new factor, which is a functional homolog of the transcription factor TFIIA alpha / beta which, together with the factor TFIIA

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 gamma, can stabilize the interactions of TBP (TATA-binding protein) -TATA element and maintain in vitro transcription dependent on RNA polymerase II. ALF protein is described as a general transcription factor specific to testes (Ashok et al., 1999, J Biol Chem, Vol 274, Issue 25, 18040-18048).

 The "Stoned B / clathrin APlike" homology domain of SALF also reveals a potential function in Clathrin-dependent transport of membrane proteins.

 Chen et al (2001, Biochem Biophys Res Commun 23; 281 (2): 475-82) have shown that the SALF factor gene is induced by retinoic acid (or retinoids) in cultured smooth muscle cells.

 On the other hand, no involvement of SALF in the regulation of angiogenesis has been described to date.

 GS-N41: a 1365 bpARNA identified with the number SEQ ID No. 42 in the attached sequence listing. A BLAST search can identify it under accession number BC010862 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 27 to nucleotide 758. It has thus been identified a GS-P41 protein resulting from the translation of this mRNA. This protein is composed of 243 aa and is identified by the number SEQ ID No. 93 in the attached sequence listing.

 The P29 protein, recently described, is a protein that interacts with the GCIP protein (Grap2 cyclin-D interacting protein), itself interacting with cyclin D and Grap2 protein. (Chang et al., Biochem Biophys Res Commun 2000 Dec 20; 279 (2): 732-7). Grap2 is a

<Desc / Clms Page number 79>

 adapter protein, specific for leukocytes of the immune tissues (Qiu et al, 1998, Biochem Biophys Res Commun.

253.443-447). The adapter proteins play an essential role in the formation of intracellular signaling complexes relaying the extracellular signals of the plasma membrane to the nucleus of the cell. Grap2 is a central binding protein in the signaling and activation of immune cells. D-type cyclins respond to extracellular mitogenic signals (Sherr, 1993, Cell 73, 1059-1065). The GCIP protein, which is ubiquitously expressed in all human tissues, interacts with Grap2 and cyclins D, its expression regulates the phosphorylation of retinoblastoma protein (Rb) and consequently the factor-mediated transcription pathway. E2F1 transcription, belonging to a family of factors that plays a major role in proliferation, differentiation, apoptosis and cell cycle progression (Nevins, 1998, Cell Growth Differ 9.585-593, Chellappan et al, 1998, Curr Top Microbiol Immunol 227, 57-103, Dyson, 1998, Genes Dev 12,2245-2262). Thus, the GCIP is suggested to play a role in the control of cell proliferation and differentiation across signaling pathways controlled by Grap2 and cyclin D proteins (Xia et al, 2000, J Biol Chem, 275 (27): 20942 -8). The protein, P29 which could be localized in the nucleus and present in multiple tissues, was found to be associated with the GCIP and therefore, may be involved in the functional regulation of GCIP (Chang et al., Biochem Biophys Res Commun 2000 Dec 20; 279 (2): 732-7).

 The P29 protein therefore seems to play a role in signaling pathways involved in cell proliferation and differentiation. However no role in

<Desc / Clms Page number 80>

 the regulation of angiogenesis has not been demonstrated to date either for the P29 protein or for the homologous protein identified.

 GS-N42: a 6147 bp RNA identified as SEQ ID No. 43 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number NM ~ 013390 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 149 to nucleotide 4300. It has thus been identified a GS-P42 protein resulting from the translation of this mRNA. This protein called TMEM2 is composed of 1383aa. It is identified by the number SEQ ID No 94 in the attached sequence listing.

 The TMEM2 gene has been recently described (Scott et al., Gene 2000; 246 (1-2): 265-74); because of the structure, the encoded protein TMEM2 has been suggested to be transmembrane. The presence of the RGD motif suggesting a role in cell adhesion is an implication in cellular signaling pathways.

 At present no role for this protein has been described and in particular no role in the regulation of angiogenesis has been demonstrated.

 GS-N43: a mRNA of 4357 bp identified under the number SEQ ID No. 44 in the attached sequence listing.

A BLAST search makes it possible to identify it under the accession number AB029316 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 318 to nucleotide 2834. Thus, a GS-P43 protein resulting from the translation of this mRNA was identified. This protein called Dorfine is composed

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 from 838aa. It is identified by the number SEQ ID No 95 in the attached sequence listing.

 The protein called Dorfine is still very little studied, its gene has been recently identified. This protein is ubiquitously expressed in many organs. It binds specifically to ubiquitin-conjugating enzymes, UbcH7 and UbcH8 across its RINGfinger / IBR domain. Dorfine is proposed as a new member of ubiquitin ligases such as RING Finger - This protein is localized in the centrosome and probably acts in centers of microtubule organization (Niwa et al, 2001, Biochem Biophys Res Commun, 281 (3)). : 706-13). To date, no differential expression of Dorfin has been described nor a role in the regulation of angiogenesis.

 GS-N44: a mRNA of 1801bp identified under the number SEQ ID No. 45 in the attached sequence listing.

A BLAST search makes it possible to identify it under accession number XM ~ 032382 (member 2 of the transmembrane superfamily 4) in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 81 to nucleotide 815. Thus, a GS-P44 protein resulting from the translation of this mRNA has been identified. This protein called TM4SF2 is composed of 244aa. It is identified as SEQ ID No 96 in the attached sequence listing.

 The TM4SF2 protein belongs to the transmembrane superfamily type 4, some of whose members are known to be involved in angiogenesis such as CD9 or PETA-3 / CD151 (Klein-Soyer et al., 2000, Arterioscler Thromb Vasc Biol., 20: 360 Sincock et al., 1999, J.Cell.

Science, 112, 833-844). On the other hand, the function of TM4SF2

<Desc / Clms Page number 82>

 is not yet known. This protein is homologous to the TALLA-1 protein, proposed as a specific surface marker of neuroblastomas and neuroblastic leukemia (Takagi et al, Int J Cancer, 1995, 61 (5): 706-15).

 No role for TM4SF2 protein in angiogenesis has been described to date.

 - GS-N45: An ARN of 2081 bp identified as SEQ ID No 46 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number HSA133133 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 184 to nucleotide 1737. Thus, a GS-P45 protein resulting from the translation of this mRNA has been identified. This protein called Ecto-ATP diphosphohydrolase I is composed of 517aa.

 It is identified as SEQ ID No 97 in the attached sequence listing.

 Ecto-ATPases (cell surface ATPases) are ubiquitous enzymes in cells that hydrolyze extracellular ATP and ADP to AMP (reviewed: Plesner, 1995, Int Rev Cytol, 158: 141-214). The presence of ATPDases has been demonstrated on aortic endothelial cells and smooth muscle cells and described as being able to play a regulatory role in homeostasis and platelet reactivity by hydrolyzing ATP and ADP (Robson et al, 1997, J Exp Med, 185 (1): 153-63). The vascular ATPDase is closely homologous to the CD39 glycoprotein whose accession number in the GENBANK base is S73813 and is identified under the number SEQ ID No. 290 in the attached sequence listing, antigen of lymphoid cell activation. , also expressed by human endothelial cells (Kaczmarek et al,

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 1996, J. Biol.Chem, 271.51, 33116-33122). Recently, Goepfert et al., (2001, Circulation, n104 (25): 3109-3115) implanting bodies containing Matrigel in mutant mice characterized by the deficiency of CD39 expression have shown the absence of neovascular formation around implanted bodies. These observations led these authors to suspect a role for CD39 in the phenomenon of angiogenesis. However, the described experiment argues rather for an ill-defined role of CD39 in angiogenesis than for a direct and incontestable role. Indeed, since CD39-deficient mutant mice exist, it is possible that embryos of CD39-deficient mice may develop. However, without angiogenesis, there is no possible embryonic development. Therefore, either CD39 has a role in angiogenesis and therefore there will be no mutant CD39 deficient mutant mice, ie CD39 has no role in angiogenesis. In another article, Goepfert et al., (2001, Circulation, 104 (25): 3109-15) did not show the absence of CD39 expression in the endothelial cells of the mutant mice used.

 In sum, the study by Goepfert et al., (2001, Circulation, 104 (25): 3109-15) does not provide evidence for any role of CD39 in angiogenesis.

 CD39 and ecto-ATPDase I can be two forms resulting from an alternative splicing of the same gene.

The vector coding for the antisense oligonucleotide used in our study can both inhibit the expression of CD39 and ecto-ATPDase I.

 The results obtained with the vector GS-V45, in the context of the present invention, prove however that CD39 plays a direct role in angiogenesis.

<Desc / Clms Page number 84>

 It should also be noted that no role has been described to date for ecto-ATPDase I in the regulation of angiogenesis.

 GS-N46: A mRNA of 4332 bp identified under the number SEQ ID No. 47 in the attached sequence listing.

A BLAST search can identify it under the accession number AJ306399 in the GENBANK sequence database.

 The sequence of this mRNA has a coding sequence from nucleotide 56 to nucleotide 1828. Thus, a GS-P46 protein resulting from the translation of this mRNA was identified. This protein is composed of 590aa. It is identified by the number SEQ ID No 98 in the attached sequence listing.

 The expression of the mRNAs identified above is observed in human endothelial cells which form capillary tubes. The Applicant has thus demonstrated that the differential expression of the gene corresponding to each of these mRNAs accompanies the formation of neovessels by the endothelial cells.

 In addition, it is shown that the induction of the expression of said gene during angiogenesis is sensitive to the presence of different inhibitors. Indeed, when the human endothelial cells forming neovessels are stimulated by an angiogenic factor (indicated in column 2 of Table II), a high expression of this mRNA is observed, whereas when the same human endothelial cells are stimulated by the same angiogenic factor and placed in the presence of an anti-angiogenic factor (indicated in column 3 of Table II) (where angiogenesis is inhibited) it is observed that the expression of this gene is also inhibited.

 Table II

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<Tb>
<tb> Inductors <SEP> Inhibitors
<tb> SEQ.ID <SEP> of <SEP> from
<tb> the expression <SEP> the expression
<tb> SEQ <SEP> ID <SEP> No <SEP> 1 <SEP> (GS-N1) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 2 <SEP> (GS-N2) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 3 <SEP> (GS-N3) <SEP> FGF2 <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 4 <SEP> (GS-N4) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 5 <SEP> (GS-N5) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 6 <SEP> (GS-N6) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 7 <SEP> (GS-N7) <SEP> VEGF <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 8 <SEP> (GS-N8) <SEP> VEGF <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 9 <SEP> (GS-N9) <SEP> VEGF <SEP> IFN-y <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 10 <SEP> (GS-N10) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 11 <SEP> (GS-N11) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 12 <SEP> (GS-N12) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 13 <SEP> (GS-N13) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 14 <SEP> (GS-N14) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 15 <SEP> (GS-N15) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 17 <SEP> (GS-N16) <SEP> VEGF <SEP> PF4
<tb> SEQ <SEP> ID <SEP> No <SEP> 18 <SEP> (GS-N17) <SEP> VEGF <SEP> TSP-1
<tb> SEQ <SEP> ID <SEP> No <SEP> 19 <SEP> (GS-N18) <SEP> VEGF <SEP> PF4
<tb> SEQ <SEP> ID <SEP> No <SEP> 20 <SEP> (GS-N19) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 21 <SEP> (GS-N20) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 22 <SEP> (GS-N21) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 23 <SEP> (GS-N22) <SEP> VEGF <SEP> PF4 <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 24 <SEP> (GS-N23) <SEP> FGF2 <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 25 <SEP> (GS-N24) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 26 <SEP> (GS-N25) <SEP> VEGF <SEP> IFN-y <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 27 <SEP> (GS-N26) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 28 <SEP> (GS-N27) <SEP> VEGF <SEP> IFN-y <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 29 <SEP> (GS-N28) <SEP> VEGF <SEP> IFN-y <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 30 <SEP> (GS-N29) <SEP> FGF2 <SEP> Ang-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 31 <SEP> (GS-N30) <SEP> VEGF <SEP> TNF-a <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 32 <SEP> (GS-N31) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 33 <SEP> (GS-N32) <SEP> VEGF <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 34 <SEP> (GS-N33) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 35 <SEP> (GS-N34) <SEP> VEGF <SEP> TNF-a <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 36 <SEP> GS-N35) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 37 <SEP> (GS-N36) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 38 <SEP> (GS-N37) <SEP> FGF2 <SEP> Ang-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 39 <SEP> (GS-N38) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> N 40 <SEP> (GS-N39) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 41 <SEP> (GS-N40) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 42 <SEP> (GS-N41) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 43 <SEP> (GS-N42) <SEP> VEGF <SEP> Ang-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 44 <SEP> (GS-N43) <SEP> FGF2 <SEP> PF4
<tb> SEQ <SEP> ID <SEP> No <SEP> 45 <SEP> (GS-N44) <SEP> FGF2 <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 46 <SEP> (GS-N45) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 47 <SEP> (GS-N46) <SEP> FGF2 <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 48 <SEP> (GS-N47) <SEP> FGF2 <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 49 <SEP> (GS-N48) <SEP> FGF2 <SEP> TNF-a
<tb> SEQ <SEP> ID <SEP> No <SEP> 50 <SEP> (GS-N49) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 51 <SEP> (GS-N51) <SEP> VEGF <SEP> IFN-y
<tb> SEQ <SEP> ID <SEP> No <SEP> 52 <SEP> (GS-N52) <SEP> FGF2 <SEP> Ang-2
<tb> SEQ <SEP> ID <SEP> No <SEP> 53 <SEP> (GS-N53) <SEP> VEGF <SEP> TNF-a
<Tb>

<Desc / Clms Page number 86>

<Tb>
<tb> Inductors <SEP> Inhibitors
<tb> SEQ.ID <SEP> of
<tb> the expression <SEP> the expression
<tb> SEQ <SEP> ID <SEP> No <SEP> 225 <SEP> (GS- <SEP> VEGF <SEP> IFN- [gamma]
<tb> SEQ <SEP> ID <SEP> No <SEP> 16 <SEP> (GS-N54) <SEP> VEGF <SEP> IFN-y
<Tb>

It thus appears that there is a direct correlation between the expression of each GS-N1 GSN54 gene and the angiogenic state of human endothelial cells.

 6.2 Verification of the role of identified genes in the regulation of angiogenesis.

 In addition, the functional role of these genes in the formation of neovessels by human endothelial cells has also been shown.

 Indeed, an oligonucleotide specific for each of the identified genes, chosen from the oligonucleotides identified by the sequences SEQ ID No. 103 to SEQ ID. : 148 in the attached sequence listing was introduced into the vector pCI-neo expression vector in antisense orientation.

 The resulting vectors designated GS-V1 to GS-V46 identified by their sequence SEQ ID No: 149 to SEQ ID No: 194 in the attached sequence listing were used to repress expression of the gene encoding this mRNA in cells. endothelial cells following transfection of the latter by this vector.

 Human endothelial cells were then stimulated by angiogenic factors. The results obtained, for each of the sequences illustrated below, using the antisense sequences and the corresponding vectors, indicated in Table III, show that:

<Desc / Clms Page number 87>

 repression of the expression of the genes SEQ ID No. 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 to SEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. 53 and SEQ ID No. 225 inhibits the formation of neovessels by human endothelial cells and that - repression of genes SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. It stimulates the formation of neovessels by human endothelial cells despite the presence of various angiogenic factors.

 These results are illustrated in the corresponding figures 1 to 11 in the appendix.

TABLE III

<Tb>
<tb> MM <SEP> Genes <SEP> Protein <SEP> Sequences <SEP> Vector <SEP> with <SEP> Fig
<tb> NAME <SEP> SEQ.ID <SEP> SEQ. <SEP> ID <SEP> antisense <SEP> antisense <SEP> Fig <SEP> control
<tb> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ insert ~~~~ ~~~~~~~~~~~~
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 54 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP > No <SEP> 149 <SEP>
<tb> (GS-N1) <SEP> (GS-P1) <SEP> 103 <SEP> (GS-V1) <SEP> l <SEP> 1F
<tb> Angioinducine <SEP> (257bp) <SEP> (GS-V1
SEQ <SEP> ID <SEP> No <SEP > No <SEP> 150 <SEP>
<tb> (GS-N2) <SEP> (GS-P2) <SEP> 104 <SEP> (GS-V2) <SEP> 1B <SEP> 1F
<tb> Angiodockine <SEP> (202bp)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 56 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP > No <SEP> 151
<tb> 3 <SEP> (GS-N3) <SEP> (GS-P3) <SEP> 105 <SEP> (GS-V3) <SEP> 151 <SEP> 2A <SEP> 2C
<tb> (GS-N3) <SEP> Angioblastine <SEP> (242 <SEP> pb) <SEP> (GS-V3)
<tb> SEQ <SEP> ID <SEP> No <SEP> 4 <SEP> SEQ <SEP> ID <SEP> No <SEP> 57 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No
<tb> 4 <SEP> (GS-N4) <SEP> (GS-P4) <SEP> 106 <SEP> 152 <SEP> 1C <SEP> 1F
<tb> (GS-N4) <SEP> Angioreceptin <SEP> (211 <SEP> pb) <SEP> (GS-V4)
<tb> SEQ <SEP> ID <SEP> No <SEP> 5 <SEP> SEQ <SEP> ID <SEP> No <SEP> 58 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 153
<tb> 5 <SEP> (GS-N5) <SEP> (GS-P5) <SEP> 107 <SEP> (GS-V5) <SEP> 1D <SEP> 1F
<tb> Angiodensin <SEP> (191 <SEP> bp)
<tb> SEQ <SEP> ID <SEP> No <SEP> 6 <SEP> SEQ <SEP> ID <SEP> No <SEP> 59 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 154
<tb> 6 <SEP> (GS-N6) <SEP> (GS-P6) <SEP> 108 <SEP> (GS-V6) <SEP> 3A <SEP> 3D
<tb> Vassoserpentine <SEP> (238 <SEP> pb) <SEP>'~~~~' ~~~~~~~~~~~~
<tb> SEQ <SEP> ID <SEP> No <SEP> 7 <SEP> SEQ <SEP> ID <SEP> No <SEP> 60 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 155
<tb> 7 <SEP> (GS-N7) <SEP> (GS-P7) <SEP> 109 <SEP> (GS-V7) <SEP> 4A <SEP> 4F
<tb> ~~~~ <SEP> (GS-N7) <SEP> Angiosulfatin <SEP> (205 <SEP> pb) <SEP> (GS-V7) ~~~~~~~~~~~~
<tb> SEQ <SEP> ID <SEP> No <SEP> 8 <SEP> SEQ <SEP> ID <SEP> No <SEP> 61 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 156 <SEP>
<tb> 8 <SEP> (GS-N8) <SEP> (GS-P8) <SEP> 110 <SEP> (GS-V8) <SEP> 3B <SEP> 3D
<tb> Vassoreceptin <SEP> (186 <SEP> pb)
<tb> SEQ <SEP> ID <SEP> No <SEP> 9 <SEP> SEQ <SEP> ID <SEP> No <SEP> 62 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 157
<tb> (GS-N9) <SEP> (GS-P9) <SEP> 111 <SEP> (GS-V9) <SEP> 4B <SEP> 4F
<tb> Angiokinasine <SEP> (223bp)
<tb> SEQ <SEP> ID <SEP> No <SEP> 10 <SEP> SEQ <SEP> ID <SEP> No <SEP> 63 <SEP> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 158
<tb> (GS-N10) <SEP> (GS-P10) <SEP> 112 <SEP> (GS-V10) <SEP>
<Tb>

<Desc / Clms Page number 88>

...., Vecteur avec .ï. Gènes Protéine Séquences vecteur avec -:# Fig

...., Vector with .ï. Protein Genes Sequences vector with -: # Fig

<Tb>
<tb> NAME <SEP> SEQ.ID <SEP> SEQ. <SEP> ID <SEP> antisense <SEP> antisense <SEP> Fig <SEP> control
<tb> insere
<tb> Vassosubstratin <SEP> (247bp)
<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 11 <SEP> SEQ <SEP> ID <SEP> No <SEP> 64 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > 159
<Tb>

11 SEQ ID No (GS-Pll) 113 SEQ ID No 159 4C 4F (GS-N11) Angiosignaline (162pb) (GS-V11)

11 SEQ ID No (GS-Pll) 113 SEQ ID No. 159 4C 4F (GS-N11) Angiosignaline (162bp) (GS-V11)

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 12 <SEP> SEQ <SEP> ID <SEP> No <SEP> 65 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 160
<tb> 12 <SEP> (GS-N12) <SEP> (GS-P12) <SEP> 114 <SEP> (GS-V12) <SEP> 4D <SEP> 4F <SEP>
<tb> Angiofoculin <SEP> (166 <SEP> pb)
<tb> SEQ <SEP> ID <SEP> No <SEP> 13 <SEP> SEQ <SEP> ID <SEP> No <SEP> 66 <SEP> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 161
<tb> 13 <SEP> (GS-N13) <SEP> (GS-P13) <SEP> 115 <SEP> (GS-V13) <SEP> 2B <SEP> 2C
<tb> Angiohelicin <SEP> (135 <SEP> bp)
<tb> SEQ <SEP> ID <SEP> No
<tb> SEQ <SEP> ID <SEP> No <SEP> 14 <SEP> 116 <SEP> SEQ <SEP> ID <SEP> No <SEP> 16 <SEP> 2
<tb> (GS-N14) <SEP> (136) <SEP> (GS-V14)
<Tb>

SEQ ID No 15 SEQ ID No 67 SEQ ID No SEQ ID No 163 15 (GS-N15) (GS-Pl5) 117 (GS-V15) lA 1F

SEQ ID No. 15 SEQ ID No. 67 SEQ ID No. SEQ ID No. 163 15 (GS-N15) (GS-Pl5) 117 (GS-V15) 1F

<Tb>
<tb> (GS <SEP> N15) <SEP> Angioacyline <SEP> (152) <SEP> @
<tb> SEQ <SEP> ID <SEP> No <SEP> 16 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 158
<Tb>

16 (GS-N54) (247 pb) (GS-V10)

16 (GS-N54) (247bp) (GS-V10)

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 17 <SEP> SEQ <SEP> ID <SEP> No <SEP> 68 <SEP> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 164
<tb> 17 <SEP> (GS-N16) <SEP> (GS-P16) <SEP> 118 <SEP> (GS-V16) <SEP> 5A <SEP> 5F
<tb> PDCL <SEP> (417 <SEP> pb)
<tb> SEQ <SEP> ID <SEP> No <SEP> 18 <SEP> SEQ <SEP> ID <SEP> No <SEP> 69 <SEP> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 165
<tb> 18 <SEP> (GS-N17) <SEP> (GS-P17) <SEP> 119 <SEP> (GS-V17) <SEP> 5B <SEP> 5F
<Tb>

RPL3 (244 SEQ SEQ ID No 70 SEQ ID No SEQ 19 SEQIDNO 19 SE?Gfp; 7 SEQ12X;NO SEQIDNO 166 5C (GS-N18) (GS-P18) 120 (GS-V18) (GS-N18) homol.RNF20 (311 pb) (GS-V18) SEQ SEQ ID No 71 SEQ ID No SEQ 2Q SEQIDNO 20 (GS-P19) 121 SEQIDNo 167 5D (GS-N19) (GS-P19) 121 (GS-V19) (GS-N19) homol.SFRS4 (246 pb) (GS-V19) SEQ SEQ ID No 72 SEQ ID No SEQ SEQ ID No 21 (GS-P20) 122 SEQ ID No 168 10A (GS-N20) (GS-P20) 122 pb) (GS-V20) 10A (GS-N20) CPD (203 pb) (GS-V20) SEQ SEQ ID No 73 SEQ ID No SEQ 22 SEQ ID No 22 (GS-P21) 123 SEQ ID No 169 5E (GS-N21) (GS-P21) 123 (GS-V21) (GS-N21) USP9X (253 pb) (GS-V21)

RPL3 (244 SEQ SEQ ID NO: 70 SEQ ID NO SEQ 19 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 166 5C (GS-N18) (GS-P18) 120 (GS-V18) (GS-N18) homol.RNF20 (311bp) (GS-V18) SEQ SEQ ID No. 71 SEQ ID No SEQ 2Q SEQ ID NO: 20 (GS-P19) 121 SEQ ID NO 167 5D (GS-N19) (GS-P19) 121 (GS-V19) (GS-N19 homol.SFRS4 (246 bp) (GS-V19) SEQ SEQ ID No. 72 SEQ ID No. SEQ SEQ ID No. 21 (GS-P20) 122 SEQ ID No. 168 10A (GS-N20) (GS-P20) 122 bp) (GS-V20) 10A (GS-N20) CPD (203bp) (GS-V20) SEQ SEQ ID No. 73 SEQ ID No. SEQ 22 SEQ ID No. 22 (GS-P21) 123 SEQ ID No. 169 5E (GS-N21 ) (GS-P21) 123 (GS-V21) (GS-N21) USP9X (253 bp) (GS-V21)

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 23 <SEP> SEQ <SEP> ID <SEP> No <SEP> 74 <SEP> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No
<tb> 23 <SEP> (GS-N22) <SEP> (GS-P22) <SEP> 124 <SEP> 170 <SEP> 6A <SEP> 6F
<tb> (GS <SEP> NRD1 <SEP> (173 <SEP> pb) <SEP> (GS-V22)
<tb> SEQ <SEP> ID <SEP> No <SEP> 75 <SEP> SEQ <SEP> ID <SEP> No <SEP>
<tb> SEQ <SEP> ID <SEP> No <SEP> 24 <SEP> (GS-P23) <SEQ> SEQ <SEP> ID <SEQ> SEQ <SEP> ID <SEP> No <SEP> 171 <SEP > 10B <SEP> 10F
<tb> (GS-N23) <SEP> Homol. <SEP> HRX <SEP>, <SEP> (228 <SEP> 5 <SEP> (GS-V23) <SEP> lOB
<tb> ALL-1, <SEP> MLL <SEP>'~~~~~~~~~~~~~~~~~~~~~~~~~
<Tb>

SEQ SEQ ID No 76 SEQ ID No SEQ 25 SEQIDNO 25 (GS-P24) 1 SEQIDNO 172 6B 6F (GS-N24) (GS-P24) (381 pb) (GS-V24)

SEQ SEQ ID No. 76 SEQ ID No. SEQ 25 SEQ ID NO 25 (GS-P24) 1 SEQ ID NO 172 6B 6F (GS-N24) (GS-P24) (381 bp) (GS-V24)

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 77 <SEP> SEQ <SEP> ID <SEP> No
<tb> 26 <SEP> SEQ <SEP> ID <SEP> No <SEP> 26 <SEP> (GS-P25) <SEP> 127 <SEP> SEQ <SEP> ID <SEP> No <SEP> 173 <SEP > 6C <SEP> 6F
<tb> (GS-N25) <SEP> transp.ac <SEP> (395 <SEP> pb) <SEP> (GS-V25)
<tb> sial.-CMP1 <SEP>'<SEP>' ~~~~~~~~~~~~~~~~~~~~~~~~~
<Tb>

SEQ SEQ ID No 78 SEQ ID No SEQ 27 SEQIDNO 27 SE%1DJ* 18 128 SEQIDNO 174 6D 27 (GS-N26) (GS-P26) 128 (GS-V26) 6D 6F

SEQ SEQ ID No 78 SEQ ID No SEQ 27 SEQ ID NO 27 SE% 1DJ * 18 128 SEQ ID NO 174 6D 27 (GS-N26) (GS-P26) 128 (GS-V26) 6D 6F

<Tb>
<tb> (GS-N26) <SEP> CBL-b <SEP> (381 <SEP> pb) <SEP> (GS-V26)
<Tb>

SEQ ID No SEQ ID No 79 SEQ ID No SEQ ID No175 28 (GS-N27) (GS-P27) 129 (GS-V27) 6E 6F

SEQ ID No SEQ ID No 79 SEQ ID No SEQ ID No175 28 (GS-N27) (GS-P27) 129 (GS-V27) 6E 6F

<Tb>
<tb> (GS-N27) <SEP> H2AV <SEP> (298 <SEP> pb) <SEP> (GS-V27)
<Tb>

SEQ SEQ ID No 80 SEQ ID No SEQ 2g SEQIDNO 29 SEQ#TB)S S"0 SEQIDNO 176 7A (GS-N28) (GS-P28) 130 (GS-V28) (GS-N28) CSNK2B (413 pb) (GS-V28)

SEQ SEQ ID No 80 SEQ ID No SEQ 2g SEQ ID NO 29 SEQ # TB) SS "0 SEQ ID NO 176 7A (GS-N28) (GS-P28) 130 (GS-V28) (GS-N28) CSNK2B (413 bp) (GS -V28)

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> 30 <SEP> SEQ <SEP> ID <SEP> No <SEP> 81 <SEQ> SEQ <SEP> ID <SEP> No <SEQ> SEQ <SEP > ID <SEP> No <SEP> 177 <SEP> 7F
<tb> (GS-N29) <SEP> (GS-P29) <SEP> 131 <SEP> (GS-V29)
<Tb>

<Desc / Clms Page number 89>

<Tb>
<tb> Genes <SEP> Protein <SEP> Sequences <SEP> Vector <SEP> with <SEP> Fig
<Tb>

NOM Gènes Protéine antisens l'antisens Fig contrôle NOM SÉQ. ID SEQ. ID antisens insere Fig contrôle ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~insère~~~~~~~~~~~~~~~~

NAME Genes Antisense Protein Antisense Fig Control NAME SEQ. ID SEQ. Antisense ID inserts Fig control ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ inserts ~ ~~~~~~~~~~~~~~~

<Tb>
<tb> Hemicentin <SEP> (564 <SEP> bp)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP > 178
<tb> SEQ <SEP> ID <SEP> No <SEP> (GS-P30) <SEP> 132 <SEP> (GS-V30)
<tb> 31 <SEP> (GS-30) <SEP> (GS <SEP> SYNE-2 <SEP> (414 <SEP> pb) <SEP> (414)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP > 179 <SEP>
<Tb>

(GS-N31 (GS-P31) 133 (GS-V31) (GS-N31) Séladine (298 pb) (GS#V31) ~~~~'~~~~~' Séladine-1 (298 pb) ~~~~~ ~~~~~~~

(GS-N31 (GS-P31) 133 (GS-V31) (GS-N31) Seladine (298bp) (GS # V31) ~~~~~~~~~ Seladin-1 (298bp) ~~ ~~~~~~~~~~

<Tb>
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP > 180 <SEP>
<tb> (GS-N32) <SEP> (GS-P32) <SEP> 134 <SEP> (GS-V32)
<tb> 33 <SEP> (GS-N32) <SEP> (GS <SEP> CHD2 <SEP> CHD2 <SEP> (365 <SEP> pb) <SEP> (GS-V32)
<tb> 34 <SEP> SEQ <SEP> ID <SEP> No <SEP> 34 <SEP> SEQ <SEP> ID <SEP> No <SEP> 85 <SEP> SEQ <SEP> ID <SEP> No <SEP > SEQ <SEP> ID <SEP> No <SEP> 181 <SEP> 8A <SEP> 8F
<tb> (GS-N33) <SEP> BRD2 <SEP> (270 <SEP> pb) <SEP> (GS-V33)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP > 182 <SEP>
<tb> (GS-N34) <SEP> (GS-P34) <SEP> 136 <SEP> (GS-V34)
<tb> (GS-N34) <SEP> Syntaxin <SEP> 3A <SEP> (298 <SEP> pb) <SEP> (SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 183 <SEP>
<tb> 36 <SEP> (GS-P35) <SEP> 137 <SEP> 8C <SEP> 8F
<tb> GS-N35) <SEP> (GS-V35)
<tb> SHARP <SEP> (117 <SEP> pb)
<tb> SEQ <SEP> ID <SEP> No <SEP> 88 <SEP> SEQ <SEP> ID <SEP> nO
<tb> SEQ <SEP> ID <SEP> No <SEP> 37 <SEP> SEQ <SEP> ID <SEP> nO <SEP> 184
<tb> 37 <SEP> (GS-P36) <SEP> 138 <SEP> 10C <SEP> 10F
<tb> (GS-N36) <SEP> (GS-P36) <SEP> 138 <SEP> (GS-V36) <SEP> 10C
<tb> 37 <SEP> (GS-N36) <SEP> (GS-P36) <SEP> PLPP <SEP> (96 <SEP> pb) <SEP> (GS-V36)
<Tb>

SEQ SEQ SEQ SEQ SEQ ID N038 SEQ ID No 89 SEQ ID No SEQ ID No 185 8D 8F (GS-N37) (GS-P37) 139 (GS-V37)

SEQ SEQ SEQ SEQ ID SEQ ID NO: SEQ ID NO. 89 SEQ ID No SEQ ID No. 185 8D 8F (GS-N37) (GS-P37) 139 (GS-V37)

<Tb>
<tb> (GS-N37) <SEP> (GS-P37) <SEP> (393 <SEP> pb) <SEP> (GS-V37)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP > 186 <SEP>
<tb> (GS-N38) <SEP> (GS-P38) <SEP> 140 <SEP> (GS-V38)
<tb> (GS-N38) <SEP> NUP88 <SEP> (100 <SEP> pb) <SEP> (GS-V38)
<Tb>

SEQ N040 SEQ N091 SEQ SEQ SEQ N 40 SEQ ID N 91 SEQ ID SEQ 187 10D SEQ ID N 40 (GS-P39) 141 (GS-V39)

SEQ N040 SEQ N091 SEQ SEQ SEQ N 40 SEQ ID N 91 SEQ ID SEQ 187 10D SEQ ID No. 40 (GS-P39) 141 (GS-V39)

<Tb>
<tb> (GS-N39) <SEP> FKPB1A <SEP> (90 <SEP> pb) <SEP> (GS-V39)
<tb> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> N 92 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 188 <SEP> 9A
<Tb>

(GS-N40) (GS-P40) 142 (GS-V40) 41 (GS-N40) SALF (144 pb) (GS-V40) SALF (144 pb) '~~~~~'~~~~~~~~~~~~ SEQ SEQ N093 SEQ SEQ SEQ ID No 42 SEQ ID N 93 SEQ ID No SEQ ID No 189 10E lOF (GS-N41) (GS-p4l) 143 (GS-V41) 10E

(GS-N40) (GS-P40) 142 (GS-V40) 41 (GS-N40) SALF (144bp) (GS-V40) SALF (144bp) ~~~~~~~~~~ SEQ SEQ SEQ SEQ SEQ SEQ ID SEQ SEQ ID SEQ ID SEQ ID No SEQ ID No. SEQ ID No 189 SEQ ID No 189 10E (GS-N41) (GS-p4l) 143 (GS-V41) 10E

<Tb>
<tb> (GS-N41) <SEP> Homol.P29 <SEP> (113 <SEP> pb) <SEP> (GS-V41)
<tb> SEQ <SEP> ID <SEP> No <SEP> 43 <SEP> SEQ <SEP> ID <SEP> N 94 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 190
<Tb>

(GS-N42) (GS-P42) 144 (GS-V42) fG-N4? (GS s"tz; TMEM2 (180 r"Vd7 ~~~~~(GS-N42) TMEM2 (180 pb) (GS-V42)~~~~~~~~~~~~~~ SEQ SEQ Nc>95 SEQ SEQ SEQ ID N044 SEQ ID N 95 SEQ ID No SEQ ID No 191 9C (GS-N43) (GS-P43) 145 (GS-V43)

(GS-N42) (GS-P42) 144 (GS-V42) fG-N4? (GS s "tz; TMEM2 (180 rd) Vd7 ~~~~~ (GS-N42) TMEM2 (180 bp) (GS-V42) ~~~~~~~~~~~~~~ SEQ SEQ Nc> SEQ SEQ ID SEQ ID NO: SEQ ID NO: 95 SEQ ID No. SEQ ID No. 191 9C (GS-N43) (GS-P43) 145 (GS-V43)

<Tb>
<tb> (GS-N43) <SEP> Dorfine <SEP> (507 <SEP> pb) <SEP> (GS-V43)
<Tb>

SEQ SEQ N-96 SEQ SEQ SEQ ID N045 SEQ ID N 96 SEQ ID No SEQ ID No 192 9D (GS-N44) (GS-P44) 146 (GS-V44)

SEQ SEQ N-96 SEQ SEQ SEQ ID N045 SEQ ID N 96 SEQ ID No. SEQ ID No. 192 9D (GS-N44) (GS-P44) 146 (GS-V44)

<Tb>
<tb> (GS-N44) <SEP> (GS <SEP> TM4SF2 <SEP> (632 <SEP> pb) <SEP> (632 <SEP> (GS-V44)
<Tb>

SEQ SEQ N097 SEQ SEQ SEQ SEQ ID N 97 SEQ ID SEQ 193 (GS-N45 (GS-P45) 147 (GS-V45)

SEQ SEQ N097 SEQ SEQ SEQ ID SEQ ID 97 SEQ ID SEQ 193 (GS-N45 (GS-P45) 147 (GS-V45)

<Tb>
<tb> (GS-N45) <SEP> Ecto-ATPase <SEP> I <SEP> (704 <SEP> pb) <SEP> (GS-V45)
<Tb>

SEQ ID N-98 SEQ SEQ (GS-P46) 148 SEQ ID No 194 11A SEQIDNo 47 (GS-P46) 148 (GS-V46)

SEQ ID N-98 SEQ SEQ (GS-P46) 148 SEQ ID No. 194 11A SEQ ID NO 47 (GS-P46) 148 (GS-V46)

<Tb>
<tb> (GS-N46) <SEP> Selenoprotein <SEP> 14 <SEP> pb) <SEP> (GS-V46) <SEP> 11A
<tb> N
<tb> SEQ <SEP> ID <SEP> No <SEP> 48 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 171 <SEP>
<tb> 48 <SEP> (GS-N47) <SEP> - <SEP> 125 <SEP> (GS-V23) <SEP> 10B <SEP> 10F
<Tb>

(228 SEQ SEQ ID N 99 SEQ ID No SEQ ID No 171 lOB

(228 SEQ SEQ ID N 99 SEQ ID No SEQ ID No. 171 lOB

<Tb>
<tb> 49 <SEP> SEQ <SEP> ID <SEP> No <SEP> 49 <SEP> (GS-P48) <SEP> 125 <SEQ> SEQ <SEP> ID <SEP> No <SEP> 171 <SEP > 10B <SEP> 10F
<Tb>

(GS N48) MLL (228 SEQ 50 SEQ ID N 100 SEQ ID No SEQ ID No 172

(GS N48) MLL (228 SEQ 50 SEQ ID N 100 SEQ ID No SEQ ID No 172

<Tb>
<tb> 50 <SEP> SEQ <SEP> ID <SEP> No <SEP> 50 <SEP> (GS-P49) <SEP> 126 <SEP> SEQ <SEP> ID <SEP> No <SEP> 172 <SEP > 6B <SEP> 6F
<Tb>

<Desc / Clms Page number 90>

<Tb>
<tb> NAME <SEP> Genes <SEP> Protein <SEP> Sequences <SEP> Vector <SEP> with <SEP> Fig
<tb> NAME <SEP> SEQ.ID <SEP> SEQ. <SEP> ID <SEP> antisense <SEP> antisense <SEP> Fig <SEP> control
<tb> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ insert ~~~~ ~~
<tb> SEQ <SEP> ID <SEP> No <SEP> 51 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> No175 <SEP>#######
<tb> 51 <SEP> (GS-N51) - <SEP> 129 <SEP> (GS-V27) <SEP> 6E <SEP> 6F
<tb> (298 <SEP> pb) <SEP> (GS-V27)
<Tb>

SEQ SEQ ID N 101 SEQ ID No SEQ SEQIDNo 52 (GS-P52) 131 SEQ ID No 177 52 (GS-N52) (GS-P52) 131 ( 7B 7F

SEQ SEQ ID NO: 101 SEQ ID No. SEQ SEQ ID NO 52 (GS-P52) 131 SEQ ID No. 177 52 (GS-N52) (GS-P52) 131 (7B-7F)

<Tb>
<tb> @ <SEP> Fibulin <SEP> 6 <SEP> (564 <SEP> pb) <SEP> (GS-V29)
<tb> SEQ <SEP> ID <SEP> No <SEP> 53 <SEP> SEQ <SEP> ID <SEP> N 102 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 179
<Tb>

(GS-N53) (GS-P53) 133 fGS-V3H (GS-N53) (GS-P53) 133 (GS-V31)

(GS-N53) (GS-P53) 133 fGS-V3H (GS-N53) (GS-P53) 133 (GS-V31)

<Tb>
<tb> @ <SEP> Seladine <SEP> 1 <SEP> (298 <SEP> pb) <SEP> (GS-V31)
<tb> SEQ <SEP> ID <SEP> No <SEP> 225 <SEP> SEQ <SEP> ID <SEP> No <SEP> SEQ <SEP> ID <SEP> No <SEP> 173
<Tb>

(GS-N50) 127 (GS-V25) (GS-N50) (395 pb) (GS-V25)

(GS-N50) 127 (GS-V25) (GS-N50) (395bp) (GS-V25)

Claims (37)

A pharmaceutical composition inhibiting angiogenesis characterized in that it comprises as active agent at least one substance chosen from: (i) a nucleic acid molecule of a gene of an endothelial cell, of which expression is induced by an angiogenic factor and is inhibited by an angiostatic agent, selected from the sequences SEQ ID N 1 to SEQ ID N 5, SEQ ID N 7, SEQ ID N 9, SEQ ID N 11 to SEQ ID N 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, a complementary sequence or a fragment thereof, (ii) a coded polypeptide sequence by said nucleic acid molecule, chosen from the polypeptide sequences identified under the numbers SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID N 62, SEQ ID N: 64 to SEQ ID N 67, SEQ ID N 68 to SEQ ID N 102 and SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing, or a fragment thereof (iii) a mol antisense nucleic acid molecule of one of the sequences identified under the numbers SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 16 in the attached sequence listing, comprising at least 10 seas; a nucleic acid sequence chosen from the nucleic acid sequences identified under the numbers SEQ ID No. 108, SEQ ID No. 110 and SEQ ID No. 112 in the attached sequence listing, or (iv) an antibody capable of binding on a polypeptide sequence encoded by one of the nucleic acid sequences identified under the numbers SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 16 in the attached sequence listing.  2. Pharmaceutical composition according to claim 1 for the diagnosis and / or treatment of pathologies related to angiogenesis. <Desc / Clms Page number 92>  3. Antibody characterized in that it has an affinity for one of the polypeptide sequences identified under the numbers SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID N 62, SEQ ID N: 64 to SEQ ID N 67, SEQ ID N 68 to SEQ ID N 102 and SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing.  4. Method for preparing an antibody according to claim 3, characterized in that it comprises the immunization in vivo or in vitro, of an immunocompetent cell of an animal, in particular a vertebrate and preferably of a mammal, with at least one of the polypeptide sequences identified as SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID N 62, SEQ ID N: 64 to SEQ ID N 67 and SEQ ID N 68 to SEQ ID N 102 or among the polypeptide sequences identified under the numbers SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing.  5. Antibody according to claim 3, characterized in that it is a monoclonal antibody.  6. Antibody according to claim 3, characterized in that it is a polyclonal antibody.  7. Pharmaceutical composition for the diagnosis and / or treatment of pathologies related to angiogenesis, characterized in that it comprises as active compound one or more antibodies according to any one of claims 3,5 or 6 or prepared according to the method of claim 4, or a fragment thereof.  An antisense nucleic acid sequence of one of the nucleic acid sequences identified as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ. ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, comprising at least 10 mer of a selected nucleic acid sequence. from <Desc / Clms Page number 93>  sequences identified as SEQ ID N 103 to SEQ ID SEQ ID N 107, SEQ ID N 109, SEQ ID N 111 and SEQ ID No.113 to SEQ ID N 148 in the attached sequence listing.  An antisense nucleic acid sequence according to claim 8 having an identity of at least 85%, preferably 95%, with a sequence selected from the sequences identified as SEQ ID N 103 to SEQ ID SEQ ID N 107. , SEQ ID N 109, SEQ ID N 111 and SEQ ID No.113 to SEQ ID N 148 in the attached sequence listing.  A mammalian expression vector comprising at least one antisense sequence defined in any one of claims 8 or 9.  Vector according to claim 10, characterized in that it is chosen from the group of vectors GS-V1 to GS-V5, GS-V7, GS-V9, GS-VII to GS-V15, GSV16 to GS-V46, identified by their sequence bearing numbers SEQ ID N 149 to SEQ ID N 153, SEQ ID N 155, SEQ ID N 157, SEQ ID N 159 to SEQ ID N 163 and SEQ ID N 164 to SEQ ID N 194 in the list of sequences in annex.  A mammalian expression vector comprising at least one nucleic acid sequence selected from the set of sequences identified as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID. N 11 to SEQ ID N 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, or a fragment thereof.  13. A method for preparing a genetically modified cell, overexpressing or under-expressing a gene involved in an angiogenic disorder, characterized in that it comprises inserting, in a mammalian cell, a vector defined at any of claims 10 to 12. <Desc / Clms Page number 94>  14. Genetically modified cell, characterized in that it overexpresses at least one gene involved in the angiogenesis identified by a nucleic acid sequence chosen from the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the sequence listing. annex or one of their fragments.  15. Genetically modified cell, characterized in that it under-expresses at least one gene involved in the angiogenesis identified by a nucleic acid sequence chosen from the set of sequences identified under the numbers SEQ ID N 1 to SEQ ID N 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225, and SEQ ID NO: 284 to SEQ ID NO: 290 in the list. sequences attached.  16. A method for preparing a cell line stably expressing an expression vector defined in any one of claims 11 or 13 characterized in that: (a) a resistance gene is introduced into at least one antibiotic in a genetically modified cell according to claims 14 or 15, b) culturing the cells obtained in step (a) in the presence of said antibiotic, c) selecting the viable cells.  17. Therapeutic composition for the treatment and / or diagnosis of an angiogenic disorder in a mammal, in particular a human, characterized in that it comprises a genetically modified cell according to any one of claims 14 or 15 or prepared according to the method of claim 16. <Desc / Clms Page number 95>  18. A method for preparing a recombinant protein comprising the steps of: a) constructing an expression vector comprising at least one nucleic acid sequence chosen from the sequences identified under the numbers SEQ ID N 1 to SEQ ID N 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225, and SEQ ID NO: 284 to SEQ ID NO: 290 in the list. sequences attached, or one of their fragments. b) introducing said vector into a cellular host, c) culturing said cells in a suitable medium, d) purifying the expressed proteins or a fragment thereof.  19. A recombinant protein or a peptide fragment obtained by the process according to claim 18.  20. Therapeutic composition comprising a recombinant protein according to claim 19.  21. The therapeutic composition as claimed in claim 1, wherein the angiogenic disorder is selected from: vascularization of tumors, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometrium associated with neovascularization, restenosis due to angioplasty of the balloon, tissue superproduction due to scarring, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and phenomena, aneurysm, arterial restenosis, thrombophlebitis, lymphagyte, lymphodema, scarring and tissue repair, ischemia, angina, myocardial infarction , chronic heart disease, heart failure such as <Desc / Clms Page number 96>  congestive heart failure, age-related macular degeneration, and osteoporosis.  22. A method for diagnosis and / or prognosis of an angiogenic pathology in a mammal, in particular in a human being, characterized in that the cells of said mammal are detected in vitro in which overexpression or under-expression is detected. one or more nucleic acid sequences identified as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID N 53, SEQ ID N 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing.  23. The method of claim 22 characterized in that it comprises the following steps: - the detection of the expression of one or more of said nucleic acid sequences, identified under the numbers SEQ ID N 1 to SEQ ID N 5 , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the Sequence Listing. in the appendix, by a cell population of a mammal, - the detection of the expression of one or more of said nucleic acid sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID N 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, by a cell population reference whose angiogenic state is known, - the identification of any differences in the level of expression of one or more of said nucleic acid sequences among the SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID N 290 in the list of sequences attached by the two cell populations.  24. Method of diagnosis and / or prognosis of an angiogenic pathology in a mammal, in particular <Desc / Clms Page number 97>  in a human being, consisting in detecting in vitro in the cells of said mammal the overexpression or the under-expression of one or more polypeptide sequences identified under the numbers SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID N 62 , SEQ ID N: 64 to SEQ ID N 67, SEQ ID N 68 to SEQ ID N 102 and SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing.  25. Method according to claim 24, characterized in that it comprises: a) the detection of the expression of one or more of said polypeptide sequences identified under the numbers SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID NO: 62, SEQ ID NO: 64 to SEQ ID NO: 67, SEQ ID NO: 68 to SEQ ID NO: 102 and SEQ ID NO: 291 to SEQ ID NO: 297 in the attached sequence listing, by a first cell population of a mammal, b) detecting the expression of one or more of said polypeptide sequences identified under the numbers SEQ ID N 54 to SEQ ID N 58, SEQ ID N 60, SEQ ID N 62, SEQ ID N: 64 to SEQ ID N 67, SEQ ID N 68 to SEQ ID N 102 and SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing, by a second reference cell population whose angiogenic state is known, c) the identifying any differences in the level of expression of said polypeptide sequences identified as SEQ ID N 54 to SEQ ID N 58, S EQ ID N 60, SEQ ID N 62, SEQ ID N: 64 to SEQ ID N 67, SEQ ID N 68 to SEQ ID N 102 and SEQ ID N 291 to SEQ ID N 297 in the attached sequence listing, by said first and second cell populations.  26. Method of diagnosis and / or prognosis according to any one of claims 22 to 25, characterized in that the angiogenic disorder is selected from: vascularization of tumors, retinopathies, rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometrium associated with neovascularization, <Desc / Clms Page number 98>  restenosis due to balloon angioplasty, tissue superproduction due to scarring, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and phenomena, aneurysm, arterial restenosis, thrombophlebitis , lymphagyte, lymphodema, scarring and tissue repair, ischemia, angina, myocardial infarction, chronic heart disease, heart failure such as congestive heart failure, macular degeneration related to age and osteoporosis.  27. Method of diagnosis and / or prognosis according to any one of claims 22 to 26, characterized in that the detection of the expression of said sequences is performed after putting the endothelial cells in the presence of a biological fluid from a patient.  28. A method for verifying the therapeutic efficacy of an angiogenic treatment in a mammal, in particular in a human being, characterized in that it comprises the identification in vitro in a cellular population of said mammal, overexpression or of the sub-expression of at least one gene involved in an angiogenic disorder identified by one of the nucleic acid sequences identified under the numbers SEQ ID N 1 to SEQ ID N 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID N 11 to SEQ ID N 15, SEQ ID N 17 to SEQ ID N 53, SEQ ID N 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing.  29. Method for verifying the therapeutic efficacy according to claim 28, characterized in that it comprises the following steps: the detection of the expression of one or more of said nucleic acid sequences chosen from the sequences SEQ ID N 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 to SEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. <Desc / Clms Page number 99>  $ N 53, SEQ ID N 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing, by a first cell population of a mammal to which a therapeutic composition for treating an angiogenic disorder is administered; detecting one or more of said nucleic acid sequences selected from the sequences identified as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, by a second reference cell population whose angiogenic state is known; identification of the possible differences in the level of expression of one or more of said nucleic acid sequences chosen from the sequences identified under the numbers SEQ ID N 1 to SEQ ID N 5, SEQ ID N 7, SEQ ID N 9, SEQ ID No. 11 to SEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 SEQ ID NO: 290 in the Sequence Listing appended by said first and second cell populations.  30. A method of verifying the therapeutic efficacy according to any one of claims 28 to 29, characterized in that the angiogenic disorder is selected from: vascularization of tumors, retinopathies, rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometrium associated with neovascularization, restenosis due to angioplasty of the balloon, tissue superproduction due to scarring, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and phenomena, aneurysm, arterial restenosis, thrombophlebitis, lymphagyte, lymphodema, scarring and tissue repair, ischemia, angina, myocardial infarction , chronic heart disease, heart failure such as <Desc / Clms Page number 100>  congestive heart failure, age-related macular degeneration, and osteoporosis.  31. The method of verification according to any one of claims 28 to 30, characterized in that the detection of the expression of said sequences is performed after putting the endothelial cells in the presence of a biological fluid from a patient.  32. A method for screening compounds that are useful for the treatment of an angiogenic disorder of a mammal, in particular a human being, characterized in that it comprises the following steps: a) the detection of the expression of a or more of said nucleic acid sequences selected from the sequences identified as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 at SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to SEQ ID NO: 290 in the attached sequence listing, by a first cell population of a mammal placed in the presence of a compound likely to have an effect. therapeutic on an angiogenic disorder, b) detecting the expression of one or more of said nucleic acid sequences selected from the sequences identified as SEQ ID N 1 to SEQ ID N 5, SEQ ID N 7, SEQ ID N 9, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 53, SEQ ID NO: 225 and SEQ ID NO: 284 to S. EQ ID N 290 in the attached sequence listing, by a second reference cell population whose angiogenic state is known, c) identifying possible differences in the level of expression of one or more of said acid sequences nucleic acids chosen from the sequences identified under the numbers SEQ ID N 1 to SEQ ID N 5, SEQ ID N 7, SEQ ID N 9, SEQ ID N 11 to SEQ ID N 15, SEQ ID N 17 to SEQ ID N 53, SEQ ID N 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing, by said first and second cell populations. <Desc / Clms Page number 101>  33. Screening method according to claim 32, characterized in that the detection of the expression of said sequences is performed after putting the endothelial cells in the presence of a biological fluid from a patient.  34. Device comprising a support comprising one or more probes specific for one or more nucleic acid sequences chosen from the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 to SEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing, for the implementation of a screening method according to claims 32 or 33.  35. Device according to claim 34 characterized in that the support is selected from a glass membrane, a metal membrane, a polymer membrane, a silica membrane.  36. A kit for measuring the differential display of genes involved in angiogenic disorders comprising a device according to any one of claims 34 or 35, specific primers and accessories necessary for the amplification of the sequences extracted from a sample, their hybridization with the probes of the device and the realization of the differential display measurements.  37. A kit for measuring the differential display of genes involved in angiogenic disorders comprising a stable line of genetically modified cells, according to claims 14 or 15, as a reference cell population, one or more sequence-specific probes. chosen from the sequences identified under the numbers SEQ ID No. 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 to SEQ ID No. 15, <Desc / Clms Page number 102>  SEQ ID N 17 to SEQ ID N 53, SEQ ID N 225 and SEQ ID N 284 to SEQ ID N 290 in the attached sequence listing, and the means necessary for the detection and measurement of said differential display.