Regulator genes of angiogenesis, pharmaceutical preparations containing them and their applications

Abstract

Nucleotide sequences and the corresponding polypeptide sequences implicated in the regulation of angiogenesis are identified. The nucleotide and polypeptide sequences, or pharmaceutical composition made from such sequences can be used in the clinical study of the angiogenesis process, the prognosis, diagnosis and treatment of pathologies linked to angiogenesis, and in the implementation of pharmaceutical, pharmacogenomic and drug identification trials.

1 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an active agent comprising at least one substance selected from the group consisting of: (i) a nucleic acid molecule, the expression of which is induced in an endothelial cell by an angiostatic agent, or a complementary sequence or a fragment or derivative thereof; (ii) a polypeptide sequence coded by said nucleic acid molecule or the complement fragment or derivative thereof, (iii) an antisense nucleic acid molecule that inhibits the expression of a nucleic acid molecule according to (i); and (iv) an antibody capable of binding to a polypeptide sequence according to (ii). 
     
     
         2 . The pharmaceutical composition according to  claim 1 , wherein the nucleic acid molecule is selected from among the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30. 
     
     
         3 . The pharmaceutical composition according to  claim 1 , wherein the antisense nucleic acid molecule is an angiogenesis inhibitory sequence and comprises a nucleic acid sequence selected from among the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30. 
     
     
         4 . The pharmaceutical composition according to  claim 1 , wherein the antisense sequence is selected from the group consisting of SEQ ID No. 11, SEQ ID NO. 12, SEQ ID No. 13 and SEQ ID No. 
     
     
         5 . The pharmaceutical composition according to  claim 1 , wherein the polypeptide sequence is selected from the group consisting of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 34. 
     
     
         6 . The pharmaceutical composition according to  claim 1 , comprising at least one antibody having an affinity for one or more polypeptide sequences identified by the numbers SEQ ID. No. 6 to SEQ ID. No. 10 or by the numbers SEQ ID. No. 31 to SEQ ID No. 34, or fragments of said one or more polypeptide sequences. 
     
     
         7 . An antisense nucleotide sequence comprising at least 10 nucleotides of a nucleotide sequence selected from among the sequences identified by the numbers SEQ ID. No. 1 to SEQ ID No. 5 and SEQ ID No. 27 to SEQ ID No. 30. 
     
     
         8 . The antisense sequence according to  claim 7  having an identity of at least about 85%, wherein the antisense sequence is selected from the group consisting of SEQ ID. No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14. 
     
     
         9 . A mammalian expression vector comprising at least one antisense sequence according to  claim 7 . 
     
     
         10 . The mammalian expression vector according to  claim 9 , wherein the vector is selected from the group consisting of vectors GS-V1, GS-V2, GS-V3 and GS-V4. 
     
     
         11 . A diagnostic and/or prognostic method for an angiogenic pathology in a mammal, comprising detecting in vitro in cells from said mammal the overexpression or the underexpression of 
       one or more nucleotide sequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 5 and SEQ ID No. 27 to SEQ ID No. 30 or 
       one or more polypeptide sequences identified by the numbers SEQ ID No. 6 to SEQ ID No. 10 or SEQ ID No. 31 to SEQ ID No. 34. 
     
     
         12 . The method according to  claim 11 , further comprising: detecting the expression of 
       one or more of said nucleotide sequences SEQ ID No. 1 to SEQ ID No. 5 and SEQ ID No. 27 to SEQ ID No. 30 or 
       one or more of said polypeptide sequences SEQ ID No. 6 to SEQ ID No. 10 or SEQ ID No. 31 to SEQ ID No. 34 
       in a mammalian cell population; detecting the expression of one or more of said nucleotide sequences in a reference cell population, the angiogenic state of which is known; and identifying the differences in the level of expression of said nucleotide sequences in the mammalian and reference cell populations. 
     
     
         13 . The diagnostic or prognostic method according to  claim 11 , wherein the angiogenic disorder is selected from the group consisting of: tumor vascularization, retinopathies, rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometriosis associated with neovascularization, restenosis due to balloon angioplasty, tissue overproduction due to cicatrization, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and phenomena, aneurism, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, tissue cicatrization and repair, ischemia, angina, myocardial infarction, chronic heart disease, cardiac insufficiencies such as congestive heart failure, age-related macular degeneration and osteoporosis. 
     
     
         14 . The diagnostic and/or prognostic method according to  claim 11 , wherein the detection of the expression of the nucleotide sequences is performed after contacting the endothelial cells with a biological fluid from a patient. 
     
     
         15 . A method for the verification of the therapeutic efficacy of an angiogenic treatment in a mammal, comprising identifying in vitro in a cell population from said mammal the overexpression or the underexpression of at least one gene implicated in an angiogenic disorder, wherein said gene is identified by one of the nucleotide sequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 5 and SEQ ID No. 27 to SEQ ID No. 30. 
     
     
         16 . The method for the verification of therapeutic efficacy according to  claim 15 , further comprising the following steps: detecting the expression of one or more of said nucleotide sequences in a cell population isolated from a mammal, to which mammal was administered a composition comprising a test compound intended to treat an angiogenic disorder; detecting the expression of said nucleotide sequences in a reference cell population, the angiogenic state of which is known; and identifying the differences in the level of expression of said sequences in the mammalian and reference cell populations. 
     
     
         17 . The method for the verification of therapeutic efficacy according to  claim 15 , wherein the angiogenic disorder is selected from the group consisting of: tumor vascularization, retinopathies, rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of the ovary, psoriasis, endometriosis associated with neovascularization, restenosis due to balloon angioplasty, tissue overproduction due to cicatrization, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and phenomena, aneurism, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, tissue cicatrization and repair, ischemia, angina, myocardial infarction, chronic heart disease, cardiac insufficiencies such as congestive heart failure, age-related macular degeneration and osteoporosis. 
     
     
         18 . The method for the verification therapeutic efficacy according to  claim 15 , wherein the detection of the expression of the sequences is performed after contacting the endothelial cells with a biological fluid from a patient. 
     
     
         19 . A method for screening for compounds useful for the treatment of an angiogenic disorder of a mammal, comprising: a) detecting the expression of one or more nucleotide sequences SEQ ID No. 1 to SEQ ID No. 5 and SEQ ID No. 27 to SEQ ID No. 30 in a mammalian cell population contacted with a test compound; b) detecting the expression of said nucleotide sequences in a reference cell population, the angiogenic state of which is known; c) identifying the differences in the level of expression of said nucleotide sequences in the mammalian and reference cell populations, wherein a difference in expression of said nucleotide sequences in the mammalian and reference cell population indicates that the test compound has a therapeutic effect on an angiogenic disorder. 
     
     
         20 . The screening method according to  claim 19 , wherein the detection of the expression of the sequences is performed after contacting the endothelial cells with a biological fluid from a patient. 
     
     
         21 . A device comprising a support comprising one or more specific probes of one or more nucleotide sequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 5 and SEQ ID. No. 27 to SEQ ID. No. 30. 
     
     
         22 . A kit for measuring the differential display of genes implicated in angiogenic disorders, comprising a device according to  claim 21 ; and specific primers and accessories required for the amplification of the sequences extracted from a sample, hybridization with the probes of the device and the performance of differential display measurements.