EP1836304A2

EP1836304A2 - Genes involved in the regulation of angiogenesis, pharmaceutical preparations containing them and uses thereof

Info

Publication number: EP1836304A2
Application number: EP06709059A
Authority: EP
Inventors: Sylvie Colin; Salman Al-Mahmood
Original assignee: GENE SIGNAL INT SA; Gene Signal International SA
Current assignee: Gene Signal International SA
Priority date: 2005-01-10
Filing date: 2006-01-10
Publication date: 2007-09-26
Also published as: CA2593464A1; WO2006075084A3; FR2880631A1; WO2006075084A2; US20100135985A1

Abstract

The invention pertains to the area of pharmaceutical compositions that are useful for treating pathologies resulting from a deregulation of the angiogenesis mechanism. The invention particularly concerns new genes whose function had not been identified before now as well as genes that are known but whose involvement in the angiogenesis mechanism has been shown for the first time.

Description

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 deregulation of the mechanism of angiogenesis.

In particular, it relates to new genes whose function was not identified to date, as well as to known genes whose involvement in the mechanism of angiogenesis has been demonstrated for the first time by the Applicant.

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, i.e. it is triggered for a short period, a few days, and then completely inhibited. However, several pathologies are related to invasive and uncontrolled angiogenesis. Arthritis, for example, is a condition caused by cartilage damage by invasive neovessels. In diabetic retinopathy, invasion of the retina by neovessels leads to the blindness of 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 therefore oxygen and other nutritive and biological factors necessary for survival. affected tissues.

The formation of neovessels by endothelial cells involves the migration, growth, and differentiation of endothelial cells. The regulation of these biological phenomena is directly related to gene expression. In terms of angiogenesis, an ever increasing number of studies show that the regulation of angiogenesis is done through an equilibrium between factors acting directly on the endothelial cell. These factors can be stimulating, on the one hand, such as, among others, VEGF, FGFs, IL-8, HGF / SF, PDGF. They can also be inhibiting, as, among others, I ¹ IL-10, IL-12, gro-α and β, platelet factor 4, angiostatin, the human chondrocyte - derived inhibitor, thrombospondin, the 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; al., Cancer Res., 1997, 57, 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 therefore 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 development of the new therapies intended to treat the angiogenesis. 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 angiogenesis stimulating or inhibiting agents, or 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. FR 2798674 and of an international patent application PCT published under No. 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 agents. angiogenesis regulators, i.e. angiogenic factors, angiostatic factors, as well as the various 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 for inhibiting angiogenesis wherein the endothelial cells are stimulated by one or more angiogenic factors and brought into the presence of one or more angiostatic factors; and 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 encoding the cellular constituents involved in the regulation of angiogenesis. angiogenesis, including positive regulators and 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 highlight and identify genes encoding cellular constituents involved in the regulation of angiogenesis. Moreover, 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 endothelial cells. angiogenesis.

The identification of the 34 genes reported below was carried out according to the methodology described above, using the angiogenic and angiostatic factors, as well as the type I collagen as a component of the extracellular matrix to reproduce the four experimental conditions. The Applicant has also proved the involvement of these 34 new genes identified by the sequences SEQ ID No. : l to SEQ ID No. : 34 in the attached sequence listing, in the mechanism of regulation of angiogenesis.

Thus, the invention relates to a nucleic acid molecule, characterized in that it comprises or consists of i) one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 2 , 4, 5, 9 to 11, 13, 17 to 19, 27 to 29 and 34 or its complement or a fragment of said sequences or an equivalent sequence; ii) an antisense sequence of one of the sequences of i), identified in the sequence listing provided in the annex under the numbers SEQ ID 62, 63, 65, 67, 69, 73, 74, 81, 82 and 85 or its complementary or a fragment of said sequences or an equivalent sequence; (iii) the antisense sequence identified in the sequence listing provided in the Annex under the number SEQ ID No. 86 or its complement or a fragment of said sequence or an equivalent sequence.

For the purposes of the present invention, nucleotide sequences having one or more structural modifications, minor (s), not modifying their function, should be considered as sequences equivalent to the sequences described above (see (s) the deletion, mutation or addition of bases, the identity of which is at least 90% with the nucleotide sequences identified as SEQ ID Nos. 1 to 34 in the attached sequence listing.

For the purposes of the present invention, the term "fragment" is intended to mean a sequence of the 1Orner type, preferably of the 15-mer type and particularly preferably of the 2Orner type.

The interest of all the sequences described in the present text lies in the fact that an antisense of these sequences, when introduced into a cell, influences the phenomena of angiogenesis, that is to say that its introduction into the cell leads to an activation or inhibition of angiogenesis.

According to another aspect, the invention relates to a polypeptide or fragment of said polypeptide, characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5. , 13, 17, 18, 19 and 29.

In a particular embodiment of the invention, said polypeptide comprises or consists of one of the polypeptide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID No. 35,

37, 38, 43, 47, 48, 49 or 57.

According to yet another aspect, the subject of the invention is an expression vector, characterized in that it comprises: i) a nucleic acid molecule as described previously; ii) a nucleic acid molecule characterized in that it comprises or consists of a) one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 3, 6, I ₁ 8 , 12, 14 to 16, 20 to 26 and 30 to 33 or its complement or a fragment of said sequences or an equivalent sequence; b) an antisense sequence of one of the sequences of a), identified in the sequence listing provided in the annex under the numbers SEQ ID NO: 64, 66, 67, 68, 70 to 72, 75 to 81 and 84 or its complementary or a fragment of said sequences or an equivalent sequence; c) the antisense sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 86 or its complement or a fragment of said sequence or an equivalent sequence. More particularly, said vector is chosen from the group of vectors GS-VI to GS-V23, identified by their sequence, bearing the numbers SEQ ID No. 87 to SEQ ID No. 109 in the attached sequence listing.

Said vectors can be constructed by any method known to those skilled in the art. In particular, the method described in the patent application WO

03/074073 with the primer sequences, described in this patent, GS-PGS-F and GS-PGM-R for fragments cloned in sense orientation in the bacterial plasmid, namely the primers GS-PGS-F and GS-PGM-R for cloned fragments in sense orientation in the bacterial plasmid.

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

According to another aspect, the invention relates to an antibody characterized in that it has an affinity for one of the polypeptide sequences encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1 to 34, or a fragment of said sequences, particularly coded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5, 13, 17, 18, 19 and 29 or a fragment thereof. sequences, or having an affinity for a polypeptide comprising or consisting of one of the polypeptide sequences identified in the sequence listing provided in the appendix as SEQ ID NO ^: 35 to 61, or a fragment thereof, particularly for a polypeptide comprising or consisting of in one of the polypeptide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID No. 35, 37, 38 , 43, 47, 48, 49 or 57 or a fragment of said sequences.

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 according to the invention, or the one of their fragments preserving the 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 introduction of said nucleotide sequences identified in the attached sequence listing under the numbers SEQ ID No. 1 to 34 and SEQ ID No. 62 to 86 (sense and antisense) or said vectors identified in the attached sequence listing under the numbers SEQ ID No. 87 to SEQ ID No. 109 or the one of their homologues, and the subsequent insertion of said vectors into mammalian cells makes it possible to obtain cell lines over-expressing or under-expressing the genes involved in the mechanism of angiogenesis.

Thus, according to yet another aspect, the invention relates to a genetically modified cell, characterized in that it over-expresses or under-expresses at least one gene involved in the angiogenesis chosen from the genes identified in the sequence listing. appended under the numbers SEQ ID No. 1 to SEQ ID No. 34, particularly the genes identified in the attached sequence listing under the numbers SEQ ID No. 1, 2, 4, 5, 9 to 11, 13, 17 to 19, 27 to 29 and 34.

The genetically modified cells may be constructed by any method known to those skilled in the art. In particular, they can be constructed by the method described in the patent application WO 03/074073 and which comprises:

(a) introducing a gene for resistance 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 selection of viable cells.

In yet another aspect, the invention relates to the drug use of a nucleic acid molecule, a polypeptide, an expression vector, an antibody, or a genetically modified cell as previously described.

According to yet another aspect, the invention relates to a pharmaceutical composition comprising as active agent at least one substance selected from: i) a nucleic acid molecule characterized in that it comprises or that it corresponds to a) one of the nucleotide sequences identified in the sequence listing provided appended under the numbers SEQ ID No. 1 to 34 or its complement or a fragment of said sequences or an equivalent sequence; b) an antisense sequence of one of the sequences of a), identified in the sequence listing provided as SEQ ID Nos. 64, 66, 67, 68, 70 to 72, 75 to 81 and 84, or its complementary or a fragment of said sequences or an equivalent sequence; (c) the antisense sequence identified in the sequence list provided in the Annex under the number

SEQ ID No. 86 or its complementary or a fragment of said sequence or an equivalent sequence. ii) a polypeptide characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1 to 34 or that it comprises or consists of a polypeptide identified in the list sequence provided in the annex under the numbers SEQ

ID No. 35 to 61 or a fragment of said polypeptides;

Hi) an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv) an antibody characterized in that it has an affinity for one of the polypeptide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID Nos. 35 to 61 or for one of the sequences encoded by the nucleic acid sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID NO ^: 2, 9-11, 27, 28 and 34; v) a genetically modified cell characterized in that it over-expresses or under-expresses at least one gene chosen from those identified in the attached sequence listing under the numbers SEQ ID No. l to 34. Particularly, the pharmaceutical composition according to the invention may be intended for the diagnosis, prognosis and / or treatment of pathologies linked to I ¹ angiogenesis.

According to one particular embodiment of the invention, the pharmaceutical composition may be intended for the treatment of pathologies related to angiogenesis chosen from: cancers, particularly through vascularization and / or proliferation of tumors, retinopathies, rheumatoid arthritis, Crohn 's disease, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometrium associated with neovascularization, restenosis due to balloon angioplasty, tissue superproduction due to cicatrization , 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 insufficiency congestive heart failure, age - related macular degeneration and osteoporosis.

According to another aspect, the invention relates to the use in the preparation of a pharmaceutical composition for inhibiting angiogenesis, characterized in that it comprises an active agent angiogenesis inhibitor selected from: i. a nucleic acid molecule characterized in that it comprises or that it corresponds to a. a nucleotide sequence identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 4 or 5 or a fragment of said sequence or an equivalent sequence; b. an antisense sequence of the nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ

ID Nos. 1 to 3, 6 to 34, nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ ID No. 62 to 64 or 66 to 85 or a fragment of said sequence or an equivalent sequence; vs. or the antisense sequence identified in the sequence listing provided in the appendix under No. SEQ ID No. 86 or a fragment of said sequence or an equivalent sequence; ii. a polypeptide characterized in that it is encoded by a nucleotide sequence identified in the sequence listing provided in the annex under the number SEQ ID No. 4 or 5 or that it comprises or consists of a polypeptide identified in the sequence listing provided in the appendix under the number SEQ ID No. 37 or 38 or a fragment of said polypeptides; iii. an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv. an antibody characterized in that it has an affinity for one of the polypeptide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID No. 37 or 38 or a fragment of said polypeptides; v. a genetically modified cell characterized in that it overexpresses at least the gene identified in the attached sequence listing under the number SEQ ID No. 4 or 5 or that it sub-expresses at least one gene selected from those identified in the attached sequence list under the numbers SEQ ID No. 1 to 3 and 6 to 34.

According to another aspect, the subject of the invention is the use in the preparation of a pharmaceutical composition intended to activate angiogenesis, characterized in that it comprises an activating active agent for angiogenesis chosen from: i) a nucleic acid molecule characterized in that it comprises or that it corresponds to a) one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1 to 3 or 6 to 34 or a fragment of said sequence or an equivalent sequence; b) the antisense sequence of one of the nucleotide sequences identified in the sequence listing provided in the annex under the number SEQ ID No. 4 or 5, sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 65 , or a fragment of said sequence or an equivalent sequence; ii) at least one polypeptide characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ ID No. 1 to 3 or 6 to 34 or that it comprises or consists of one of the polypeptides identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 35, 36, 39 to 61 or a fragment of said polypeptides; iii) an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv) an antibody characterized in that it has an affinity for the polypeptide sequence identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 35, 36, 39 to 61 or a fragment of said polypeptides; v) a genetically modified cell characterized in that it overexpresses at least one gene chosen from those identified in the attached sequence listing under the numbers SEQ ID No. 1 to 3 and 6 to 34 or that it sub- expresses a gene or sub-expresses at least the gene identified in the attached sequence listing under the number SEQ ID No. 4 or 5.

According to a particular aspect, another subject of the invention concerns any nucleic acid sequence (in the form of DNA or RNA) comprising or consisting of at least 10 months of a nucleotide sequence chosen from the sequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 34 in the sequence listing in the appendix, or complementary to such a sequence, preferably at least 15 seas.

Most preferably, the subject of the invention is the sequences having an identity of at least 85%, preferably 95% and particularly preferably 100% with a sequence chosen from the sequences identified under the numbers SEQ ID No. 62 to SEQ ID No. 86 in the attached sequence listing. In particular, the invention relates to RNAi (RNA interference) and more specifically to a siRNA (small interfering RNA) comprising or consisting of a double - stranded nucleotide sequence in the form of RNA, of at least 10 mers, complementary to a corresponding mRNA. at one of nucleotide sequences identified as SEQ ID NO: 1 to SEQ ID NO: 34.

Thus, the subject of the invention is the use of such an siRNA of at least 10 mers, preferably at least 15 mers comprising or consisting of an RNA complementary to one of the nucleotide sequences identified under the numbers SEQ ID No. : 1 to SEQ ID No: 34 in the attached sequence listing for the preparation of a medicament for the treatment of pathological conditions related to angiogenesis. 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. 34 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. 34, 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 the possible differences in the level of expression of that (s). ) same sequence (s) by both 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 numbers SEQ ID No. : 35 to SEQ ID NO. : 61 in the sequence list in Annex .

According to a preferred embodiment, said method comprises the following steps: a) the detection of the expression of one or more of said polypeptide sequences SEQ ID No. : 35 to SEQ ID

No. By a mammalian cell population, b) the detection of the expression of the same polypeptide sequence (s) by a reference cell population whose angiogenic state is known, c) 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, characterized in that it comprises the identification

"In vitro" in a cell population of said mammal, overexpression or under-expression of at least one gene involved in an angiogenic disorder identified by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID NO: 1 to SEQ ID NO: 34.

Such a method for verifying the therapeutic efficacy comprises the following steps: the detection of the expression by an isolated cell population of a mammal to which a therapeutic composition for treating an angiogenic disorder is administered, of at least one of the nucleotide sequences identified in the list sequences provided in the annex under the number SEQ ID N ⁰ 1 to SEQ ID N ⁰ 34; detecting the expression of said nucleotide sequence by a reference cell population whose angiogenic state is known, the identification of a possible difference in the level of expression of said sequence 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 cells, particularly the endothelial cells, in the presence of a biological fluid resulting 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: a) the detection of expression by an isolated cell population of a mammal placed in the presence of a compound likely to have a therapeutic effect on an angiogenic disorder, at least one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID N ⁰ 1 to SEQ ID No. 34, b) the detection of the expression of the same nucleotide sequence by a reference cell population whose angiogenic state is known, c) the identification of any 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. : 35 to SEQ ID No. 61 in the attached sequence listing 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 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 the 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 putting the cells, particularly the endothelial cells, in the presence of a biological fluid originating 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: cancers, particularly through the vascularization and / or proliferation 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 congestive heart failure, age - related macular degeneration and osteoporosis.

The subject of the invention is also 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: 34 in the attached sequence listing for the setting implementation of the screening method of the invention, particularly one or more nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5, 13, 17, 18, 19 and 29, or its complement or a fragment of said sequences.

In the context of the present invention, the term "probe" is understood to mean any fragment of single-stranded DNA whose sequence is complementary to a desired sequence: this fragment may, for example, be detected by hybridization with a labeled probe (for example by incorporation of radioactive atoms or fluorescent groups), which plays the role of 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: 34 in the attached sequence listing.

The invention also relates to 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 performing 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: 34 in the attached sequence listing, or one of their fragments as a reference cell population and the means necessary for measuring said differential display.

The invention also relates to a kit for measuring the differential display of genes 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: 34 in the attached sequence listing, or a fragment thereof, as the reference cell population and the means necessary for measuring said differential display.

Verification of the involvement of the 34 identified genes and their homologs in the mechanism of angiogenesis was performed according to the methodology described in the Material and Methods section.

This involvement is illustrated with the aid of Figures 1 to 11 in the appendix, in which:

Figure 1 shows that the expression of GS-V1, GS-V2, GS-V3 and GS-V5 in human endothelial cells inhibit capillary tube formation and expression of GS-V4 in human endothelial cells stimulates formation of capillary tubes: IA-transfected endothelial cells) GS-V1 encoding the antisense transcript specific for GS-N1; IB) GS-V2 encoding the specific antisense transcript of GS-N2; IC) GS-V3 encoding the GS-N3 specific antisense transcript; ID) GS-V4 encoding the specific antisense transcript of GS-N4 and its counterpart GS-N5; IE) GS-V5 encoding the GS-N6 specific antisense transcript and its GS-N7 counterpart, and the GS-N8 specific antisense transcript and its GS-N9, GS-N10 and GS-NII counterparts; IF) the empty vector (Control). Figure 2 shows that the expression of GS-V6 _/ GS-V7, GS-V8, GS-V9 and GS-V10 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 2A) GS-V6 coding for the specific antisense transcript of GS-N12; 2B) GS-V7 encoding the specific antisense transcript of GS-N13; 2C) GS-V8 encoding the specific antisense transcript of GS-N14; 2D) GS-V9 encoding the specific antisense transcript of GS-N15; 2E) GS-VlO encoding the antisense transcript specific for GS-16; 2F) the empty vector (Control). Figure 3 shows that the expression of GS-VI1, GS-V12, GS-V13, GS-V14, GS-V15, in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 3A) GS-VII encoding the specific antisense transcript of GS-N17; 3B) GS-V12 encoding the antisense transcript specific for GS-N18 and its counterpart GS-N19; 3C) GS-V13 encoding the antisense transcript specific for GS-N20; 3D) GS-V14 coding for the antisense transcript specific for GS-N21; 3E) GS-V15 encoding the specific antisense transcript of GS-N22; 3F) the empty vector (Control).

Figure 4 shows that the expression of GS-V16, GS-V17, GS-V18, GS-V19, GS-V20 in human endothelial cells inhibits the formation of capillary tubes: endothelial cells transfected with 4A) GS-V16 coding for the antisense transcript specific for GS-N23; 4B) GS-V17 encoding the specific antisense transcript of GS-N24; 4C) GS-V18 encoding the GS-N25 specific antisense transcript; 4D) GS-V19 encoding the specific antisense transcript of GS-N26 and its counterparts GS-N27 and GS-N28; 4E) GS-V20 encoding the specific antisense transcript of GS-N29 and 4F) the empty vector (Control).

Figure 5 shows that the expression of GS-V21, GS-V22,

GS-V23 in human endothelial cells inhibits capillary tube formation: endothelial cells transfected with 5A) GS-V21 encoding GS-N30 specific antisense transcript; 5B) GS-V22 coding for the antisense transcript specific for GS-N31, and its counterparts

GS-N32 and GS-N33; 5C) GS-V23 encoding the antisense transcript specific for GS-N34; 5D) the empty vector

(Control).

MATERIAL AND METHODS

1.Culture of Cells and Angiogenesis Test Umbilical vein endothelial cells (HUVEC) under said four culture conditions are then used to identify the genes encoding the cellular constituents 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 USA, 83 (19), 727-301). The cells are first and foremost 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 between 0, 1 and 5 μg / ml, preferably between 0.5 μg / ml and 1 μg / 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; Ang-2: at concentrations of between 20 ng / ml and 800 ng / ml, preferably between 200 ng / ml and 400 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, having an effect on the expression of the identified genes in correlation with the formation of neovessels or the inhibition of neovessels respectively, used, by way of example, in the context of the present invention, are illustrated in FIG. - below. VEGF = vascular endothelial growth factor. FGF2 ≈ Basic growth factor of the fibroblast. PF4 ≈ Platelet factor 4. Ang-2 = Angiopoietin 2 IFN-γ = Interferon gamma. TNF-α = tumor necrosis factor alpha TNF-α 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). In our model of 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 the cells

HUVEC grown on a collagen gel in the presence of the various factors used, according to the RNeasy Mini kit method (Qiagen) by integrating a DNase I digestion step 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 the αP ³³ -ATP in isotopic dilution in the course of time. 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 out, reamplified, cloned in a PGEM plasmid. easy vector, Promega), sequences and identified by questioning the BLAST bank.

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

Gene functionality test

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

For the construction of these vectors, the amplification of the cloned fragment in the bacterial plasmid is carried out by means of particular primers, selected from GS-PGS-F, GS-PGM-R or GS-PGM-F and GS-PG sequences. PGS-R hybridizing to the regions of the plasmid flanking the cloned gene and also comprising in their ends the restriction sites (SalI and MIuI sites), not contained in the cloned fragment, and present in the multisite region 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.

These primers (see international patent application published under No. WO 01/218312) are indicated in Table I below: TABLE I

Controls carried out with these primers, which can be considered as universal primers, in the absence of the cloned gene (empty plasmid) have shown that the fragment Amplified plasmid (40 base pairs) 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.

These primers contain at each of their ends a site of a different restriction enzyme (SalI: GTCGAC or MIuI: 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 MIuI 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 general, the vectors that can be used to demonstrate 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 may also be used are indicated in the non-exhaustive list below:

Vectors marketed by the Promega Company; vectors with a "strong" promoter for a high level mammalian cells (pCI Mammalian expression vector expression expression expression vector cloning vector pALTER (R) * -MAX), vectors marketed by Invitrogen: (pcDNA3.1, / hygro, - / Zeo , pcDNA4 / HisMAx, -E, base pairsCE4, pRcRSV, pRcCMV2, pSecTag2, - / hygro secretion vectors, vectors pEBVHis A, B and C), mammalian expression vectors marketed by Clontech (pIRES, pIRES-EYFP pIRES2-EGFP, pCMV-Myc and pCMV-HA), Epitope-tagged pTRE, VP16 Minimal Domain vectors (ptTA 2, ptTA 3 and ptTA 4), bidirectional Tet expression vectors (base pairs, pairs) base-IEGFP, base pairs G, base pairsI-L), pRevTRE, pTRE2, pLEGFP-N1 Retroviral vector pLEGFP-C1, Adeno-X adenoviral expression systems, pCMS-EGFP, pdlEGFP-N1, pd2ECFP -N1, pd2EYFP-N1, pEGFP (-Cl, -C2, -C3, -N1, -N2, -N3), pEYFP-C1, -N1.

Each vector comprising said antisense fragment is then produced in E. CoIi, extracted, purified and quantified. A jug 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 a selection marker for an antibiotic (a gene of antibiotic resistance) to select transfected cells stably expressing the vector comprising the nucleic acid cloned in said vector and, either in the same vector or in a vector cotransfected ^2nd.

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.

To do this, 24h after transfection performed under the conditions described above, the BAEC endothelial cells are trypsinized and seeded at a rate of 80,000 cells / well in six-well plate in the presence of 700 μg / ml of antibiotic G418. (Promega) A control well is inoculated with untransfected cells.

The medium is changed every three days with a refill of the antibiotic. 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 identified in the sequence listing provided in the appendix by the numbers SEQ ID No. 1, 2, 4, 5, 9 to 11, 13, 17 to 19, 27 to 29 and 34 and the proteins identified in the sequence listing provided by SEQ ID Nos. 35, 37, 38, 43, 47 to 49 and 57 have not previously been identified as having a biological role and still less as having a role in the process of angiogenesis or the 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:

GS-N1: mRNA of 1041 base pairs, 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 BC002759.

The sequence of this mRNA has a coding sequence from nucleotide 213 to nucleotide 482. A protein, GS-P1, resulting from the translation of this mRNA (SEQ ID No. 35 in the attached sequence listing) has been identified. This protein is composed of 89 amino acids.

GS-N2: 4275 base pair mRNA 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 BC040192. GS-N3: 6104 bp mRNA 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 accession number XM_497078.

The sequence of this mRNA has a coding sequence of nucleotide 438 to nucleotide 5687. A protein, GS-P2, (SEQ ID No. 36 in the attached sequence listing), resulting from the translation of this mRNA, has been identified. This protein is composed of 1749 amino acids, is called Nucleoporin 188. Nucleoporine 188kDa is part of the family of about thirty proteins called nucleoporins which constitute on the nuclear double membrane large protein structures forming nuclear pores and serving as sites of translocation of macromolecules between the nucleus and cytoplasm. Studies have shown that a nucleoporin has a unique role in the regulation of nuclear pore function and the transport of proteins and RNAs. Their role has been clearer with the demonstration of their association with specific diseases (review: Cronshaw and Matunis, Trends Endocrinol Metab 2004 Jan-Feb; 15 (1): 34-9.). For example, overexpression of NUP88 nucleoporin has been associated with high aggression in breast cancer (Agudo et al, Int J Cancer, 2004 May 1, 109 (5): 717-20). Specific roles have also been demonstrated by other types of studies, for example, a specific role has been suggested for nucleoporin 98kDa (NUP98): the repression of the expression of this protein by the RNai technique. Some authors have been able to demonstrate a specific alteration of the structure of the nuclear pore and of certain functions such as the entry of the cDNA of the HIV virus into the nucleus, suggesting that this protein participates in the entry of the cDNA of the virus into the nucleus ( Ebina et al, Microbes Infect, 2004 Jul, 6 (8): 715-24). As another example, Nucleoporin P62 has been implicated in the transport of transcriptional activating factors (STAT3) to the nucleus of neurons when these cells are stimulated by angiotensin II (Lu et al, Neurosci, 1998 Feb 15; 18 (4)). : 1329-36). As regards the NUP188 protein, no specific role has yet been demonstrated, its role particularly in the regulation of angiogenesis has not yet been described.

GS-N4: mRNA of 1768 bp, identified by the sequence SEQ ID No: 4 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under accession number BC002509.

The sequence of this mRNA (GS-N4) has a coding sequence from nucleotide 176 to nucleotide 1387. A protein, GS-P3, (SEQ ID No. 37 in the attached sequence listing) resulting from the translation of this mRNA has been identified. This protein is composed of 403 amino acids.

This sequence is homologous to the GS-N5 sequence.

GS-N5: 1552 base pair mRNA identified by the sequence SEQ ID No: 5 in the attached sequence listing.

A BLAST search based on GENBANK sequences makes it possible to identify it under accession number BC008630.

The sequence of this mRNA (GS-N5) has a partial coding sequence from nucleotide 1 to nucleotide 949. A protein, GS-P4, (SEQ ID No. 38 in the attached list of sequences), resulting from the translation of this mRNA. , homologous to the GS-P3 protein, composed of 315 amino acids has been identified.

This new 44kDa protein contains a PHD Zinc finger domain, a motif mainly found in proteins involved in transcriptional regulation in eukaryotes (reviewed Trends Biochem Sci., 1995 Feb; 20 (2): 56-9).

GS-N6: 3181 base pair 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 accession number NM_170707.

The sequence of this mRNA (GS-N6) has a coding sequence of nucleotide 213 to nucleotide 2207. A protein, GS-P5, (SEQ ID No. 39 in the list of sequences in the appendix), called lamin A / C isoform 1, resulting from the translation of this mRNA, composed of 664, has been identified.

This sequence is homologous to the GS-N7 sequence.

GS-N7: 2404 base pair mRNA 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 accession number X03444.

The sequence of this mRNA (GS-N7), has a coding sequence of nucleotide 211 to nucleotide 2319. A protein, GS-P6, (SEQ ID No. 40 in the attached sequence listing), resulting from the translation of this mRNA, composed of 702 amino acids, called laminamine precursor A, has been identified. The LMNA gene codes for a protein called lamin A / C isoform 1. The lamins are the main components of the nuclear lamina. These proteins are important in a variety of cellular functions such as nuclear assembly, replication, transcription, and nuclear integrity (reviewed: Curr Opin Cell Biol 2002 Jun, 14 (3): 357-64). Mutations in lamin A / C have been linked to several diseases such as muscular dystrophies or cardiovascular diseases (reviewed: Trends Cardiovasc Med 2001 Oct; ll (7): 280-5); loss of expression of this gene has been reported in a form of dilated cardiopathy (Virchows Arch 2003 Nov, 443 (5): 664-71, Epub 2003 JuI 26). But to date, no implication of this gene has been described in the regulation of angiogenesis.

GS-N8: mRNA of 2570 base pairs 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 the accession number AB005047.

The sequence of this mRNA (GS-N8) has a coding sequence from nucleotide 64 to nucleotide 1341. A protein, GS-P7, (SEQ ID No. 41 in the attached sequence listing), resulting from the translation of this mRNA was identified. This protein is composed of 425 amino acids and is called SAB protein.

The SAB protein is an SH3 domain binding protein, its role in the signaling voice of Bruton tyrosine kinase (Btk) has been suggested by the demonstration of its binding with the SH3 domain of this kinase (Biochem Biophys Res Commun 1998 Apr 17; 245 (2): 337-43). Its role in the signaling voice of the c-Jun protein N-terminal kinase has also been suggested (Biochem J. 2002 Nov 1; 367 (Pt 3): 577-85). Moreover, c-Jun N-terminal kinase has been shown to be involved in angiogenesis (Jimenez et al, Oncogene, 2001 Jun 7; 20 (26): 3443-8) and more recently, augmented expression of Btk was observed during in vivo angiogenesis (2004, Zippo et al, Blood).

However, to date no involvement of the SAB protein has been demonstrated in angiogenesis.

This sequence has sequence homology with GS-N9, GS-N10, GS-NII sequences less than 90%. However these four sequences exhibit a conserved sequence which I ¹ antisense, identified in the sequence listing provided in the annex under the number SEQ ID NO: 67, allows the inhibition of the expression. GS-N9: 2190 base pair 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 AK090524.

GS-N10: mRNA of 5593 base pairs 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 AL133111

GS-NI1: 2774 base pair 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 BX641159.

GS-N12: 8974 base pair mRNA identified by the sequence SEQ ID No: 12 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number NM_015382

The sequence of this mRNA (GS-N12) has a coding sequence of nucleotide 324 to nucleotide 8162. A protein, GS-P8, (SEQ ID No. 42 in the attached sequence listing), resulting from the translation of this mRNA was identified. This protein is composed of 2612 amino acids, and is called HECT domain containing protein 1 (HECTDl).

The HECTD1 protein is stored by its conserved domain in the HECT family of proteins that function as E3 ubiquitin ligase proteins, targeting specific proteins for ubiquitin-mediated proteolysis (Callaghan et al., Oncogene, 1998 Dec 31; 17 (26)). ): 3479-91). By way of example, the Smurfl protein, another member of this family, plays a specific role in osteoblast cell differentiation and bone formation in vivo by inhibiting this differentiation (Zhao et al., J. Biol Chem. 26; 279 (13): 12854-9). The Nedd4 protein, also a member of the HECT family, has been described as regulating the stability and therefore the activity of the growth factor receptor IGF-I (Molecular Insulin-like Growth Factor I) (Mol CeIl Biol 2003 May; 9): 3363-72 The specific role of HECTD1 is not yet described in the literature, and in particular no role has been described in the regulation of angiogenesis for this protein - GS-N13: 5346 base pairs identified under sequence number SEQ ID No. 13 in the attached sequence listing A BLAST search in the GENBANK sequence database identifies it as accession number XM_291344. This mRNA has a coding sequence from nucleotide 1 to nucleotide 3522. A protein, GS-P9, (SEQ ID No. 43 in the attached sequence listing), resulting from the translation of this mRNA has therefore been identified.This protein is composed of 1173 amino acids. protein still e unknown is characterized by a catalytic tyrosine kinase domain.

GS-N14: mRNA of 3769 base pairs identified under sequence number SEQ ID No. 14 in the attached sequence listing. A BLAST search in the database sequences GENBANK allows to identify it under accession number NM__181847.

This mRNA has a coding sequence of the nucleotide

469 at nucleotide 2037. A protein, GS-PlO, (SEQ ID No. 44 in the attached sequence listing), resulting from the translation of this mRNA was therefore identified. This protein is composed of 522 amino acids and is called AMIG02.

The Amigo 2 protein was recently discovered and has been classified into a new gene family encoding type I transmembrane proteins that contain a secretion signal sequence and a transmembrane domain

(Kuja-Panula et al., J. Biol. 2003; 160 (6): 963-73). These authors have suggested that they are new adhesion molecules, they are expressed on the fibers of neurons and participate in their formation.

The Amigo 2 protein is still poorly known, it has never been described as implicated in angiogenesis.

GS-N15: mRNA of 7407 base pairs identified under the number SEQ ID No. 15 in the attached sequence listing. A BLAST search in the GENBANK sequence database makes it possible to identify it under accession number NM_005650 s

This mRNA has a coding region of nucleotide 135 to nucleotide 6017. It has therefore been identified a protein, GS-PlI, (SEQ ID No. 45 in the attached sequence listing), resulting from the translation of this mRNA. This protein is composed of 1960 amino acids, and is called transcription factor 20, isoform 1 (TCF20).

This protein, also known as SPBP, was originally described as a transcription factor that controls the expression of stromolysin, a metalloproteinase involved in tumor invasion and metastasis (Sanz et al., Cell Biol. 15 (6), 3164-3170 (1995). More recently, it has been reported that this protein Although nuclear contains several functional domains and stimulates the transcriptional activity of various transcription factors such as Ets1 or C-Jun, it is suggested to be a transcriptional coactivator (Rekdal et al., J Biol Chenu 2000 Dec 22; 275 (51)). : 40288-300).

To date, no involvement in the regulation of angiogenesis has been described for this protein.

GS-N16: 2104 base pair mRNA identified under the number SEQ ID No. 16 in the attached sequence listing. A BLAST search in the GENBANK sequence database makes it possible to identify it under the accession number NM_016408.

This mRNA has a coding sequence from nucleotide 125 to nucleotide 1888. Thus, a GS-P12 protein (SEQ ID No. 46 in the attached list of sequences) has been identified, resulting from the translation of this mRNA. This protein is composed of 587 amino acids, and is called protein 1 associated with the CDK5 regulatory subunit (CDK5RAP1).

This protein also called C42, or HSPC167, has been isolated and shown to associate with an activating subunit (p25nck5a derived from p35 ^nck5a ) of a cell cycle-associated protein kinase called cyclin-dependent protein kinase, CDK5 (Ching et al. Wang, Gene, 2000 Jan 25, 242 (1-2): 285-94). In dividing cells, cdks regulate proliferation, differentiation, senescence, and apoptosis. Neuronal CDK5 has been implicated in the regulation of differentiation and neuronal migration. The activity of Cdk proteins is regulated by complex mechanisms including phosphorylation of proteins, association with specific inhibitors. For the Cdk5, two activators were identified p35 " ^σA5a , and its isoform, p39 ^πck5ai.The protein CDK5RAP1 was shown to inhibit the kinase activity of neuronal CDK5 by associating with p35 ^nσ * ^5a fChing et al. J. Biol Chem, Vol. 277, Issue 18, 15237-15240, May 3, 2002). On the other hand, recently, increased expression of CDK5 and its role in proliferation and apoptosis in bFGF-stimulated endothelial cells (BAE) have been reported (Sharma et al., J. Biochem. 2004 Feb. 91 (2): 398-409).

However, to date, no involvement of CDK5RAP1 has been demonstrated in the regulation of angiogenesis - GS-N17: 5859 base pair mRNA identified as SEQ ID No 17 in the attached sequence listing . A BLAST search in the GENBANK sequence database makes it possible to identify it under the accession number AK074056.

This mRNA (GS-N17) has a partial coding sequence from nucleotide 45 to nucleotide 935.

A new protein, GS-P13, (SEQ ID No. 47 in the attached sequence listing) has thus been identified, resulting from the translation of this mRNA. This protein is composed of 296 amino acids. This unknown protein, of 33kDa, contains BTB / POZ and Kelch domains, the BTB / POZ domain is a conserved domain of protein-protein interaction (Xu et al, Int JMed Med 2004 Jan; 13 (1): 193 However, the proteins containing these domains constitute a large family whose physiological functions are still poorly understood (Ohmachi et al, Genes Cells, 1999). Jun; 4 (6): 325-37 A member of this family has already been described involved in a process such as directed cell migration in Drosophila (Development, 2001 Aug; 128 (15): 3001-15) .

GS-N18: 1694 base pair mRNA identified under the number SEQ ID No. 33 in the attached sequence listing. A BLAST search in the GENBANK sequence database allows identify it under accession number BC001792.

This mRNA, (GS-N18), has a coding sequence of nucleotide 164 to nucleotide 448. It has thus been identified a protein, GS-P14, (SEQ ID No. 48 in the attached sequence listing), resulting from the translation. of this

MRNA. This protein is composed of 94 amino acids.

This 10kDa protein has no particular domain but contains a signal sequence of secretion and a transmembrane helix. This sequence is homologous to the GS-N19 sequence.

GS-N19: 2437 base pair mRNA identified by the sequence SEQ ID No: 19 in the attached sequence listing. A BLAST search based on GENBANK sequences makes it possible to identify it under the accession number AF370373. This mRNA (GS-N19) has a coding sequence of nucleotide 628 to nucleotide 1533. A protein, GS-P15 (SEQ ID No. 49 in the attached sequence listing), resulting from the translation of this mRNA has thus been identified. . This protein, isoform of the GS-P14 protein, is composed of 301 amino acids.

This isoform is characterized by a domain called ubiquitin, a family of ubiquitins, molecules involved in the proteolysis of proteins that regulates the "turnover of proteins", in order to control the progression of the cell cycle.

These 2 isoforms are also characterized by a signal sequence of excretion.

GS-N20: a 1714 base pair mRNA identified under the number SEQ ID No. 20 in the attached sequence listing. A BLAST search can identify it under accession number BC022870 in the GENBANK sequence database.

The sequence of this mRNA has a coding sequence from nucleotide 90 to nucleotide 1424. It was thus identified a GS-P16 protein resulting from the translation of this mRNA. This protein is composed of 444 amino acids. It is identified as SEQ ID No. 50 in the attached sequence listing, called interferon-induced protein 44 (IFI44).

The gene encoding a novel antigen protein, p44, was originally isolated from chimpanzees infected with hepatitis virus (Takahashi et al., 1990, J Gen Virol., 71 (Pt 9): 2005-ll). . Subsequently, it has been identified in humans as inducible by interferon (Kitamura et al, Eur J Biochem 1994 Sep 15; 224 (3): 877-83). To date, this protein has not been described as being involved in angiogenesis.

GS-N21: 1715 base pair mRNA identified under the number SEQ ID No. 21 in the attached sequence listing. A BLAST search can identify it under the accession number NM_004905 in the GENBANK sequence database.

This mRNA, GS-N21, has a coding sequence of nucleotide 52 to nucleotide 726. It has thus been identified a protein, GS-Pl7, (SEQ ID No. 51 in the attached sequence listing), resulting from the translation of this MRNA, composed of 224 amino acids, called peroxiredoxin 6 (PRDX6).

Peroxiredoxin 6 protein (also known as antioxidant protein 2, non-selenium glutathione peroxidase, acidic calcium-independent phospholipase A2, 1-Cys peroxiredoxin) belongs to the growing and ubiquitous family of peroxiredoxins which are multifunctional enzymes with peroxidase in vitro, and In vivo are involved in many cellular processes known to be responsive to reactive oxygen such as pathophysiological processes including oxygen adaptation, atherosclerosis, cancer, cell differentiation (WAGNER et al, Biochem J. (2002)). 366): 777-785. A recent study describes that PRDX6 is a unique non-redundant antioxidant that works independently of other peroxiredoxins and antioxidant proteins. Very abundant in epithelial cells, PRDX6 was found in the cytosol (Wang et al., J. Biol Chem., Vol 278, Issue 27, 25179-25190, JuIy 4, 2003). It has also been found in endothelial cells only in the cytosol (Stuhlmeier et al, Eur J Biochem, 2003 Jan; 270 (2): 334-41).

To date, this protein has not been described as having a role in the regulation of angiogenesis.

GS-N22: 5978 base pair mRNA identified under the number SEQ ID No. 22 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AF220037 in the GENBANK sequence database. This mRNA has a coding sequence from nucleotide 1801 to nucleotide 3933. Thus, a protein, GS-P18 (SEQ ID No. 52 in the attached list of sequences), resulting from the translation of this mRNA composed of 710 acids has been identified. amines and called tripartite isoform beta motif protein, TRIM9.

The TRIM9 protein belongs to a family characterized by a conserved domain, the tripartite motif (TRIM). The TRIM is composed of three zing binding domain, a RING (R), a B-box type 1 (B1) and a B-box type 2 (B2) followed by a coiled-coil region. These proteins share a common function by virtue of homo-multimerization, each of which is associated with a specific compartment of the cell; the TRIM9 protein has been reported to be cytoplasmic (EMBO J. 2001 May 1; 20 (9) : 2140-51). The questioning of the NCBI database on conserved domains shows that the TRIM9 protein also contains a fibronectin type III domain, a modulus present in both extracellular and intracellular proteins. Genes of the TRIM family have been involved in processes various, such as cell growth and growth and oncogenesis and other pathologies (EMBO J. 2001 May 1; 20 (9): 2140-51; Berti et al., Mech Dev. 2002 May; 113 (2): 159 -62.) • TRIMII, for example, would play a role in regulating the level of intracellular expression of a neuroprotective peptide that specifically suppresses neurotoxicity related to Alzheimer's disease through the protein-controlled pathway of protein degradation. ubiquitins (Niikura et al, Eur J Neurosci, 2003 Mar; 17 (6): 1150-8 TRIM9 is still poorly known, at present it has been reported that TRIM9 is mainly confined to the central nervous system.

Its role is not yet defined and so far no role in the regulation of angiogenesis has been described for TRIM9.

GS-N23: 5858 bp mRNA identified as SEQ ID No. 23 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AF213987 in the GENBANK sequence database. This mRNA has a coding sequence from nucleotide 66 to nucleotide 2213. Thus, a protein, GS-P19 (SEQ ID No. 53 in the attached list of sequences), resulting from the translation of this mRNA, has been identified. This protein is composed of 715 amino acids and is called MCEF protein. The MCEF protein has been described as a new member of the family of AF4 transcription factors involved in lymphoblastic leukaemias Estable et al., J Biomed Sci. 2002 May-Jun; 9 (3) -.234-45).

The MCEF protein is still very little studied and to date, no role in the regulation of angiogenesis has been described for this protein.

GS-N24: 3907 base pair mRNA identified under the number SEQ ID No. 24 in the attached sequence listing. A search BLAST makes it possible to identify it under the number accession number NM_012318 in the GENBANK sequence database.

This mRNA has a coding sequence of nucleotide 298 to nucleotide 2517. A protein, GS-P20, (SEQ ID No. 54 in the attached sequence listing), of 739 amino acids, called transmembrane protein 1 containing a "leucine zipper" domain. EF-hand "(LETMl) has been identified.

The LETM1 protein contains two "EF-Hand" domains, a transmembrane domain, a leucine Zipper domain and several coiled-coil domains. Based on its possible calcium binding property and its involvement in the calcium signaling pathway, this protein has been suggested to be implicated in a mental illness, Wolf-Hirschhorn syndrome, this protein being deleted in many patients with this syndrome (Endele et al., Genomics, 1999 Sep 1; 60 (2): 218-25. A recent study has shown a mitochondrial localization of this protein (Schlickum et al, Genomics, 2004 Feb; 83 (2)). : 254-61).

To date, no role in angiogenesis is described for this protein. GS-N25: a mRNA of 7190 base pairs identified under the number SEQ ID No. 41 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number NM_020119 in the GENBANK sequence database. This mRNA has a coding sequence of nucleotide 389 to nucleotide 3097. Thus, a protein, GS-P21 (SEQ ID No. 55 in the attached list of sequences), resulting from the translation of this mRNA, has been identified. This protein is composed of 902 amino acids and is called zinc finger antiviral protein (ZAP).

ZAP protein is a CCCH type Zinc finger protein. His current role is in the prevention of retrovirus infection. The expression of this ZAP protein has been shown to cause a deep and specific loss of viral mRNAs in the cytoplasm of the cell without affecting nuclear mRNAs suggesting a role in the inhibition of viral replication of infected cells (Gao et al., Science 2002 Sep 6; 297 (5587): 1703-6 ). No other role is known at present and no role in the regulation of angiogenesis has been described so far for this protein.

GS-N26: 2359 base pair mRNA identified as SEQ ID No. 26 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number NM_022777 in the GENBANK sequence database.

This mRNA, GS-N26, has a coding sequence of nucleotide 41 to nucleotide 598. It has thus been identified a protein, GS-P22, (SEQ ID No. 56 in the attached sequence listing), resulting from the translation of this mRNA. This protein is composed of 185 amino acids and is called RABL5 protein.

The gene encoding the RABL5 protein has recently been described (Ota et al., Nat Genet 36 (1), 40-45 (2004) and the protein has been classified by its conserved domains as a member of the Rab GTPAse family. belonging to the superfamily of GTP-binding Ras proteins, which have emerged as cell-membrane regulators, particularly formation, vesicular transport, and membrane fusion (reviews: Prekeris R, Scientific World Journal, 2003 Sep 15; 3: 870-80, Stenmark H and Olkkonen VM., Genome Biol 2001; 2 (5)). This superfamily comprises over 80 highly conserved proteins that are involved in multiple voices of intracellular signaling and function as switches. of signal transduction from membrane receptors, from an inactive state that binds GDP to an active state that binds GTP and can thus act on different effector molecules (review: Coxon FP and Rogers MJ., Calcif Tissue Int. 2003 Jan; 72 (1): 80-4. ). Members of the Ras family have been extensively involved in oncogenesis either by mutation or overexpression of the protein (reviewed: Oxford and Theodorescu, Cancer Lett, 2003 Jan 28, 189 (2): 117-28). Rab has already been described in angiogenesis, referred to as VRP (Yonekura et al, Nucleic Acids Res, 1999 JuI 1; 27 (13) .2591-600.

On the other hand, the RABL5 protein is still poorly known and its exact role has not yet been discovered, no role in angiogenesis has been described to date.

This sequence, GS-N26, has a sequence homology with the GS-N27 and GS-N28 sequences of less than 90%. However, these three sequences have a conserved sequence which I ¹ antisense, identified in the sequence listing provided in the annex under the number SEQ ID NO: 81, allows the inhibition of the expression.

- GS-N27: 1782 base pair mRNA, identified under the number SEQ ID No. 27 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number BC050531 in the GENBANK sequence database.

GS-N28: mRNA of 2587 base pairs, identified under the number SEQ ID No. 28 in the attached sequence listing. A BLAST search can identify it under the accession number AL157469 in the GENBANK sequence database.

GS-N29: 2520 bp mRNA identified under the number SEQ ID No. 29 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number XM_211534 in the GENBANK sequence database. This mRNA (GS-N29) has a coding sequence of nucleotide 484 to nucleotide 792. It has thus been identified a protein, GS-P23, (SEQ ID No. 57 in the attached sequence listing), resulting from the translation of this mRNA. This protein is composed of 102 amino acids. This new 11 kDa protein (102 amino acids) has no particular domain, is extracellular and has a secretory sequence of secretion. GS-N30: mRNA of 7300 base pairs identified under the number SEQ ID No. 30 in the attached sequence listing. A BLAST search makes it possible to identify it under accession number NM__003023 in the GENBANK sequence database.

This mRNA (GS-N30), has a coding sequence of nucleotide 262 to nucleotide 1947. It has thus been identified a protein, GS-P24 (SEQ ID No. 58 in the attached sequence listing), resulting from the translation of this mRNA. This protein is composed of 561 amino acids and is called SH3 domain binding protein 2 (SH3BP2). This protein has been identified in bladder cancer and by its structure it has been suggested to play a role in signaling and could be a negative regulator of the abl oncogene (Bell et al, Genomics, 1997 Sep 1; (2): 163-70 Recently, this protein has been described as an adapter protein in signaling pathways by promoting the transcriptional activity of two NFAT / AP-1 transcription factors (known to be involved in gene transcription). interleukin 2) in T cells through activation of Ras - and calcineurin - dependent pathways (Foucault et al., J Biol Chem 2003 Feb 28, 278 (9): 7146-53). described as a positive regulator of tyrosine phosphorylation of PLC-gamma in basophilic cells leading to their degranulation (Sada et al, Blood, 2002 Sep 15, 100 (6): 2138-44). this gen It has recently been described that may have a role in a hereditary multi-cell cystic pathology, Cherubism (Ueki et al., Nat. Genet. 2001 Jun, 28 (2): 125-6; Lo et al, Am J Med Genet. , 2003 Aug 15; 121A (I): 37- 40). To date, no role in the regulation of angiogenesis has been described for this protein.

GS-N31: 1701 base pair mRNA identified under the number SEQ ID No. 31 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AF380162 in the GENBANK sequence database.

This mRNA (GS-N31) has a coding sequence from nucleotide 19 to nucleotide 1542. Thus, a protein, GS-P25 (SEQ ID No. 59 in the attached sequence listing), resulting from the translation of this

MRNA. This protein is composed of 507 amino acids and is called FAPP2 protein.

This mRNA sequence (GS-N31) is homologous to GS-N32 and GS-N33 sequences. GS-N32: 2836 base pair mRNA identified under the number SEQ ID No. 32 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AK023180 in the GENBANK sequence database.

This mRNA has a coding sequence of nucleotide 323 to nucleotide 1441. Thus, a protein, GS-P26 (SEQ ID No. 60 in the attached sequence listing), resulting from the translation of this mRNA, composed of 372, has been identified. amino acids and called Protein similar to FAPP2 protein. The gene coding for the protein FAPP2 was identified in 2002 by Strausberg (Proc Natl Acad Sci U.S.A 99 (26), 16899-16903), it is highly similar, moreover, to a protein NY-BR-86 described as a breast cancer antigen (Scanlan et al., 2001, Cancer Immun.1,4). This protein has a conserved glycolipid transfer domain and a pleckstrin homology domain. This domain is shared by a group of novel proteins that have binding specificities with the phosphorylated derivatives of inositol. These Proteins are described as potentially adapter proteins because they do not have catalytic domains. These molecules may be key mediators of cellular responses that are specifically regulated by the second messenger (the phosphorylated derivative of inositol) (Dowler et al., Biochem J. (2000) 351, 19-31).

The exact role of FAPP2 is not yet known, its role in angiogenesis has not yet been described.

This mRNA sequence (GS-N32) is homologous to GS-N31 and GS-N33 sequences.

GS-N33: mRNA of 2004 base pairs identified under the number SEQ ID No. 33 in the attached sequence listing. A BLAST search can identify it under accession number BC002838 in the GENBANK sequence database. This mRNA has a coding sequence from nucleotide 92 to nucleotide 1414. Thus, a protein, GS-P27 (SEQ ID No. 61 in the attached sequence listing), resulting from the translation of this mRNA, composed of 440 acids, has been identified. amino is called Protein linked to the proliferation potential.

This mRNA sequence (GS-N33) is homologous to GS-N31 and GS-N32 sequences.

GS-N34: 1789 base pair mRNA identified under the number SEQ ID No. 34 in the attached sequence listing. A BLAST search makes it possible to identify it under the accession number AK057491 in the GENBANK sequence database.

Expression of the above identified mRNAs is observed in endothelial cells that 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 (Table II). Table II

It thus appears that there is a direct correlation between the expression of each of the GS-N1 GS-N34 genes and the angiogenic state of the 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 antisense oligonucleotide specific for each of the identified genes, chosen from the oligonucleotides identified by the sequences SEQ ID No. 62 to SEQ ID. : 86 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-V23 identified by their sequence SEQ ID No: 87 to SEQ ID No: 109 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:

repression of the expression of the genes SEQ ID N. 1 to SEQ ID No. 3, SEQ ID No. 6 to SEQ ID NO. 34 inhibits the formation of neovessels by endothelial cells;

the repression of the genes SEQ ID No. 4 and SEQ ID NO. Stimulates the formation of neovessels by endothelial cells; and this despite the presence of different angiogenic factors.

These results are illustrated in the corresponding figures 1 to 5 in the appendix. TABLE III

K)

Claims

A nucleic acid molecule characterized in that it comprises or consists of i) one of the nucleotide sequences identified in the sequence listing provided in the appendix under numbers SEQ ID No. 1, 2, 4, 5, 9 to 11, 13, 17 to 19, 27 to 29 and 34 or its complement or a fragment of said sequences or an equivalent sequence; ii) an antisense sequence of one of the sequences of i), identified in the sequence listing provided in the annex under the numbers SEQ ID 62, 63, 65, 67, 69, 73, 74, 81, 82 and 85 or its complementary or a fragment of said sequences or an equivalent sequence; iii) the antisense sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 86 or its complement or a fragment of said sequence or an equivalent sequence.

2. Polypeptide or fragment of said polypeptide characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5, 13, 17, 18, 19 and 29.

3. Polypeptide according to claim 2, characterized in that it comprises or that it consists of one of the polypeptide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 35, 37, 38, 43, 47 , 48, 49 or 57.

4. Expression vector, characterized in that it comprises i) a nucleic acid molecule according to claim 1; H) a nucleic acid molecule characterized by what it comprises or consists of a) one of the nucleotide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID Nos. 3, 6, 7, 8, 12, 14 to 16, 20 to 26 and 30 to 33 or its complement or a fragment of said sequences or an equivalent sequence; (b) an antisense sequence of one of the sequences of (a), identified in the sequence list provided in the Annex under the numbers SEQ ID No

64, 66, 67, 68, 70 to 72, 75 to 81 and 84 or its complement or a fragment of said sequences or an equivalent sequence; (c) the antisense sequence identified in the sequence list provided in the Annex under the number

SEQ ID No. 86 or its complementary or a fragment of said sequence or an equivalent sequence.

An antibody, characterized in that it has an affinity for one of the polypeptide sequences encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID Nos. 1 to 34, or a fragment of said sequences, particularly coded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5, 13, 17, 18, 19 and 29 or a fragment of said sequences, or having an affinity for a polypeptide comprising or consisting of one of the polypeptide sequences identified in the sequence listing appended as SEQ ID Nos. 35 to 61, or a fragment thereof, particularly for a polypeptide comprising or consisting of one of the polypeptide sequences identified in the sequence list provided in the annex under the numbers SEQ ID No. 35, 37, 38, 43, 47, 48, 49 or 57 or a fragment of said sequences.

6. Genetically modified cell, characterized in that it over-expresses or under-expresses at least one gene involved in the angiogenesis chosen from the genes identified in the attached sequence listing under the numbers SEQ ID No. 1 to SEQ ID No. 34, particularly the genes identified in the attached sequence listing under the numbers SEQ ID Nos. 1, 2, 4, 5, 9 to 11, 13, 17 to 19, 27 to 29 and 34.

Pharmaceutical composition comprising as active agent at least one substance chosen from: i) a nucleic acid molecule characterized in that it comprises or that it corresponds to a) one of the nucleotide sequences identified in the list of sequences provided in the appendix under the numbers SEQ ID No. 1 to 34 or its complement or a fragment of said sequences or an equivalent sequence; b) an antisense sequence of one of the sequences of a), identified in the sequence listing provided as SEQ ID Nos. 64, 66, 67, 68, 70 to 72, 75 to 81 and 84, or its complementary or a fragment of said sequences or an equivalent sequence; (d) the antisense sequence identified in the sequence list provided in the Annex under the number

SEQ ID No. 86 or its complementary or a fragment of said sequence or an equivalent sequence; il) a polypeptide characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID Nos. 1 to 34 or that it comprises or consists of a polypeptide identified in the sequence listing provided in the appendix under the numbers SEQ

ID No. 35 to 61 or a fragment of said polypeptides;

Hi) an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv) an antibody characterized in that it has an affinity for one of the polypeptide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 35 to 61 or for one of the sequences encoded by the acid sequences nucleic acids identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 2, 9 to 11, 27, 28 and 34; v) a genetically modified cell characterized in that it over-expresses or under-expresses at least one gene chosen from those identified in the list of sequences in the appendix under the numbers SEQ ID No. 1 to 34.

8. Use, for the preparation of a pharmaceutical composition for inhibiting angiogenesis, of an angiogenesis inhibiting active agent selected from: i. a nucleic acid molecule characterized in that it comprises or that it corresponds to a. a nucleotide sequence identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 4 or 5 or a fragment of said sequence or an equivalent sequence; b. an antisense sequence of the nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ ID No. 1 to 3, 6 to 34, nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ ID No. 62 to 64 or 66 to 85 or a fragment of said sequence or an equivalent sequence; vs. or the antisense sequence identified in the sequence listing provided in the appendix under No. SEQ ID No. 86 or a fragment of said sequence or an equivalent sequence; ii. a polypeptide characterized in that it is encoded by a nucleotide sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 4 or 5 or that it comprises or consists of a polypeptide identified in the sequence listing provided in the appendix under the number SEQ ID No. 37 or 38 or a fragment of said polypeptides; iii. an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv. an antibody characterized in that it has an affinity for one of the polypeptide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID No. 37 or 38 or a fragment of said polypeptides; v. a genetically modified cell characterized in that it over-expresses at least the gene identified in the list of sequences in the appendix under the number SEQ ID No. 4 or 5 or that it sub-expresses at least one gene chosen from those identified in the attached sequence listing under numbers SEQ ID Nos. 1 to 3 and 6 to 34.

9. Use, for the preparation of a pharmaceutical composition for activating angiogenesis, of an angiogenesis activating active agent selected from: i. a nucleic acid molecule characterized in that it comprises or that it corresponds to a. one of the nucleotide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID No. 1 to 3 or 5 to 34 or a fragment of said sequence or an equivalent sequence; b. the antisense sequence of the nucleotide sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 4 or 5, sequence identified in the sequence listing provided in the appendix under the number SEQ ID No. 65, or a fragment of said sequence or an equivalent sequence; ii. at least one polypeptide characterized in that it is encoded by one of the nucleotide sequences identified in the sequence listing provided in the appendix under the number SEQ ID Nos. 1 to 3 or 4 to 34 or that it comprises or consists of one of the polypeptides identified in the sequence listing provided in the annex under number SEQ

ID No. 35, 36, 38-61 or a fragment of said polypeptides; iii. an expression vector, characterized in that it comprises a nucleic acid molecule according to i) of the present claim; iv. an antibody characterized in that it has an affinity for one of the polypeptide sequences, identified in the sequence listing provided in the annex under the numbers SEQ ID No. 35, 36, 38 to 61 or a fragment of said polypeptides; v. a genetically modified cell characterized in that it overexpresses at least one gene chosen from those identified in the attached sequence listing under the numbers SEQ ID No. 1 to 3 and 5 to 34 or that it under-expresses a gene or sub- expresses at least the gene identified in the attached sequence listing under the number SEQ ID No. 4.

10. Sequence of nucleic acids (in the form of DNA or RNA) comprising or consisting of at least 10 months of a nucleotide sequence selected from the sequences identified by the numbers SEQ ID No. l to SEQ ID No. 34 in the list of sequences in the appendix, or complementary or antisense of such a sequence.

11. siRNA comprising or consisting of a double-stranded nucleotide sequence in the form of RNA, at least 10 mers, preferably at least 15 mers, complementary to an mRNA corresponding to one of the nucleotide sequences identified under the numbers SEQ ID No: 1 to SEQ ID No: 34.

12. Use of an siRNA according to claim 11 for the preparation of a medicament for the treatment of pathologies related to angiogenesis.

13. 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 cell population of said mammal, overexpression or of the sub-expression of at least one gene involved in angiogenic disorder identified by one of the nucleotide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID N ⁰ 1 to SEQ ID N ⁰ 34.

14. Method for verifying the therapeutic efficacy according to claim 13, characterized in that it comprises the following steps: the detection of the expression by a cell population isolated from a mammal to which a therapeutic composition for administering a therapeutic composition is administered. treating an angiogenic disorder, at least one of the nucleotide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID N ⁰ 1 to SEQ ID N ⁰ 34; the detection of the expression of said nucleotide sequence by a reference cell population whose angiogenic state is known, the identification of a possible difference in the level of expression of said sequence by the two cell populations.

15. The method of verification according to any one of claims 13 or 14, characterized in that the detection of the expression of said sequence is performed after putting the cell population in the presence of a biological fluid from a patient.

16. A method of screening compounds useful for the treatment of an angiogenic disorder of a mammal, including a human being, characterized in that it comprises the following steps: a) the detection of expression by a population cell isolated from a mammal placed in the presence of a compound capable of having a therapeutic effect on an angiogenic disorder, of at least one of the nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1 to SEQ ID NO: 34, b) the detection of the expression of the same nucleotide sequence by a reference cell population whose angiogenic state is known, c) the identification of possible differences in the level of expression of (s) ) the same nucleotide sequence (s) by the two cell populations.

17. Screening method according to claim 16, characterized in that the detection of the expression of the Sequences are performed after placing the cells in the presence of a biological fluid from a patient.

18. Device comprising a support comprising one or more probes specific for one or more nucleotide sequences identified in the sequence listing provided in the annex under the numbers SEQ ID N ^0: 1 to SEQ ID NO: 34, for the implementation of a screening method according to claims 16 or 17.

19. Device according to claim 18, characterized in that the probe is specific for one or more nucleotide sequences identified in the sequence listing provided in the appendix under the numbers SEQ ID No. 1, 4, 5, 13, 17, 18. , 19 and 29, or its complement or a fragment of said sequences.

20. A kit for measuring the differential display of genes involved in angiogenic disorders comprising a device according to any one of claims 18 or 19, specific primers and accessories necessary for amplifying sequences extracted from a sample, their hybridization with the probes of the device and the realization of the differential display measurements.

21. Kit according to claim 20, characterized in that it further comprises a stable line of genetically modified cells, according to claim 6, as the reference cell population.