WO2003064663A1 - Polymorphismes de genes d'elastine, de fibrilline et de genes lies predisposant a une restenose et a une atherosclerose - Google Patents

Polymorphismes de genes d'elastine, de fibrilline et de genes lies predisposant a une restenose et a une atherosclerose Download PDF

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WO2003064663A1
WO2003064663A1 PCT/US2003/003001 US0303001W WO03064663A1 WO 2003064663 A1 WO2003064663 A1 WO 2003064663A1 US 0303001 W US0303001 W US 0303001W WO 03064663 A1 WO03064663 A1 WO 03064663A1
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fibrillin
elastin
restenosis
genes
atherosclerosis
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PCT/US2003/003001
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Stephen E. Epstein
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Medstar Research Institute
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Priority to US10/502,984 priority Critical patent/US20050282163A1/en
Priority to EP03706019A priority patent/EP1478758A4/fr
Priority to JP2003564254A priority patent/JP2005531287A/ja
Publication of WO2003064663A1 publication Critical patent/WO2003064663A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Coronary artery disease is a disease that is endemic in Western society. In this disease the arteries that supply blood to the heart muscle become narrowed by deposits of fatty, fibrotic, or calcified material on the inside of the artery. The build up of these deposits is called atherosclerosis. Atherosclerosis reduces the blood flow to the heart, which starves the heart muscle of oxygen, leading to either/or angina pectoris (chest pain), myocardial infarction (heart attack), and congestive heart failure.
  • PTC A percutaneous transluminal coronary angioplasty
  • restenosis begins soon after angioplasty, wherein the increased size of the vascular lumen (the open channel inside the artery) becomes gradually occluded by the proliferation of smooth muscle cells. Approximately 20 to 30% of all angioplasty patients experience restenosis to the extent that they must undergo repeated angioplasty or even coronary bypass surgery.
  • Restenosis has a complex pathology, triggered by the stretch-induced injury of the vessel walls during balloon inflation.
  • the injury involves several components of the vessel wall, including the elastin fibers.
  • the injury stimulates smooth muscle cell migration and proliferation, and thereby leads to neointimal accumulation (which constitutes the restenotic lesion). Additional processes contributing to restenosis include inflammation and accumulation of extracellular matrix. Remodeling of the vessel wall, leading to narrowing of the vessel, is a critically important component of restenosis. However, this is totally eliminated by the emplacement of a stent at the site of angioplasty, which prevents the vessel from remodeling. Stenting has become almost routine, being performed in many centers in over 70% of all angioplasty procedures. Restenosis also occurs in the arteries supplying the legs when these vessels are narrowed by atherosclerosis and are treated by angioplasty.
  • restenosis is diagnosed by visualizing the narrowed vessel through the injection of radioopaque dye into the vessel being examined and performing a cineangiogram (angiography).
  • angiography angiography
  • restenosis is diagnosed if the post- angioplasty diameter of the vessel narrows to less than 50%, a process, when it occurs, that usually within 2-6 months following the procedure.
  • brachytherapy intravascular radiation
  • angioplasty a procedure normally reserved for patients who are now identified as being at high risk of restenosis using a rather blunt assessment — they already have had multiple episodes of restenosis.
  • new and improved methods for detecting and treating restenosis are greatly to be desired.
  • restenosis shares, with atherosclerosis, many common and overlapping processes and mechanisms.
  • One of the key differences in these two conditions is the speed with which functionally important narrowing of the involved artery develops.
  • restenosis can be used as an efficient model to understand many of the mechanisms responsible for atherosclerosis, and the genetic factors contributing to restenosis also probably contribute to atherosclerosis.
  • the inventor has identified a set of genes that encode proteins present in the extracellular matrix of the artery, and which are injured during the acute injury imposed on the vessel during angioplasty, and during the chronic injury imposed on the vessel during the process of atherogenesis.
  • the genes identified are those involved in the formation of the elastin fiber network that is present in vessel walls.
  • genes consist of, but are not limited to, the genes encoding elastin/tropoelastin, fibrillin-1 and fibrillin-2, lysyl oxidase, and microfibril-associated glycoprotein (MAGP), and the genes encoding the dermatan sulphate proteogl yeans, biglycan, osteopontin (Eta-1), and decorin.
  • the protein products of these genes contribute to the formation of elastin fibers, briefly, as follows; tropoelastin is secreted by cells, and is laid down along a cytoskeletal matrix support structure composed of microfibrils, which are in turn formed mainly by fibrillin-1 and 2.
  • the soluble tropoelastin transforms into the insoluble elastin. This is in part accomplished by the enzyme lysyl oxidase, which has been shown to incorporate soluble tropoelastin into insoluble elastin fibers with the formation of elastin crosslinks.
  • the dermatan sulphate proteoglycans, biglycan, and decorin have been found to bind to tropoelastin, but their exact role in the function of elastic fibers is not known.
  • Osteopontin is a constitutive component of normal elastic fibers in human skin and aorta (L.J. van't Veer et al., Nature 415:530-536, January 31 , 2002). It is present in human coronary artherosclerotic plaques, but not in non-diseased vessels.
  • elastin fibers not only forms a structural supporting network for the vessel, but, when intact, elastin induces the quiescent phenotype of SMCs residing within the vessel media.
  • SMCs When elastin is disrupted or absent, SMCs no longer retain the quiescent phenotype — instead they proliferate and migrate to form neointimal hyperplasia, the key process involved in both restenosis and atherosclerosis.
  • Genetic mutations of elastin have been found to lead to supravalvular aortic stenosis, characterized by narrowing (stenosis) of the aorta as it emerges from the heart just distal to the aortic valve.
  • the stenosis is caused by migration and proliferation of SMCs located in the media of the aorta — this causes neointimal hyperplasia and ultimately narrowing of the aortic lumen such that obstruction to blood flow develops (supravalvular aortic stenosis).
  • Genetic mutations of fibrillin proteins also can lead to vascular diseases. It is the concept of this invention that polymo ⁇ hisms of the genes encoding proteins involved in the formation of the elastin fiber network, which alter the functional integrity of elastin, will predispose to SMC migration and proliferation, and thereby to obstruction of the coronary, cerebral, or peripheral arteries. These processes could occur in the context of angioplasty and predispose to restenosis, or chronic vascular injury, predisposing to atherosclerosis.
  • the inventor recognizes that since the differential expression of these genes is involved in the healing response to vascular injury, whereby intact elastin fibers are necessary for SMCs to resume their quiescent phenotype, changes in the degree of expression, or in the length of time during which elastin and genes contributing to the functional integrity of elastin (as noted above) are differentially expressed, could lead to abnormal patterns of healing. Analogous to a keloid scar, in which a genetic precondition leads to excessive fibrous tissue developing on the skin in response to cutaneous injury, in the context of injury to the vessel wall (either acute as in restenosis or chronic as in atherosclerosis) the excessive healing response would contribute to the development of restenosis or atherosclerosis.
  • the inventor further recognizes that changes in the degree of gene expression, or in the length of time during which the genes are expressed, can be caused by polymo ⁇ hisms in the gene or in the regulatory components of the gene. Such polymo ⁇ hisms, conveying an increased risk of disease development, have already been identified for several genes associated with several diseases.
  • This invention therefore, identifies the genes encoding elastin/tropoelastin, fibrillin-1 and fibrillin-2, lysyl oxidase, and microfibril-associated glycoprotein (MAGP), and the genes encoding the dermatan sulphate proteoglycans, biglycan, osteopontin, and decorin as genes in which polymo ⁇ hisms can convey susceptibility to the development of restenosis or atherosclerosis.
  • the invention is not limited to these specific genes, as it involves all genes whose protein products influence the integrity of elastin fibers. Subsequent reference, therefore, to prediction of restenosis (or atherosclerosis-see below), relate to polymo ⁇ hisms of the genes identified by this invention, or of their regulatory units.
  • Methods to treat may include gene therapy to increase the expression of genes down-regulated during the disease. Treatment may also include methods to decrease the expression of genes up-regulated during restenosis/atherosclerosis. Treatment to decrease gene expression may include, but is not limited to, the expression of anti-sense mRNA, triplex formation or inhibition by co-expression.
  • the inventor further recognizes that identification of the genes encoding elastin/tropoelastin, fibrillin-1 and fibrillin-2, lysyl oxidase, and microfibril- associated glycoprotein (MAGP), and the genes encoding the dermatan sulphate proteoglycans, biglycan and decorin (and any other genes whose protein products influence the functional integrity of elastin), which influence the development of restenosis/atherosclerosis, makes possible an identification of proteins that may effect the development of restenosis/atherosclerosis. Identification of such proteins makes possible the use of methods to affect their expression or alter their metabolism.
  • MAMP microfibril- associated glycoprotein
  • Methods to alter the effect of expressed proteins include, but are not limited to, the use of specific antibodies or antibody fragments that bind the identified proteins, specific receptors that bind the identified protein, or other ligands or small molecules that inhibit the identified protein from affecting its physiological target and exerting its metabolic and biologic effects.
  • those proteins that are down-regulated during the course of restenosis may be supplemented exogenously to ameliorate their decreased synthesis.
  • the inventor realizes that identification of the genes encoding elastin/tropoelastin, fibrillin-1 and fibrillin-2, lysyl oxidase, and microfibril- associated glycoprotein (MAGP), and the genes encoding the dermatan sulphate proteoglycans, biglycan, osteopontin, and decorin (and any other genes whose protein products influence the functional integrity of elastin), which influence the development of restenosis/atherosclerosis, makes possible the prophylactic use of methods to affect gene expression or protein function, and such methods may be used to treat individuals at risk for the development of restenosis/atherosclerosis.
  • MAGP microfibril- associated glycoprotein
  • the inventor further recognizes that different polymo ⁇ hisms of the genes encoding elastin/tropoelastin, fibrillin-1 and fibrillin-2, lysyl oxidase, and microfibril-associated glycoprotein (MAGP), and the genes encoding the dermatan sulphate proteoglycans, biglycan, osteopontin, and decorin (and any other genes whose protein products influence the functional integrity of elastin, which influence the development of restenosis/atherosclerosis, may play a role in the development of restenosis/atherosclerosis in different patients.
  • This invention makes possible an identification of specific abnormalities that are characteristic of a specific patient.
  • the inventor has discovered, using transcriptional profiling (DNA arrays) in their rat carotid injury model that the genes encoding elastin, fibrillin-1, and fibrillin-2 are differentially upregulated following injury, indicating that these genes the elastin or fibrillin genes, or any of the other genes contributing to the functional integrity of elastin as noted above, are important in the response to vascular injury.
  • the invention therefore predicts that abnormal transcriptional regulation of these genes, which could occur as a result of polymo ⁇ hisms in the coding or regulatory regions of the genes, will predispose to the development of restenosis/atherosclerosis.
  • identification of polymo ⁇ hisms in the elastin gene or fibrillin genes, or any of the other genes contributing to the functional integrity of elastin as noted above, may be predictive of restenosis/atherosclerosis.
  • identification of these polymo ⁇ hisms can help predict the patient at risk, and thereby identify patients who should be treated more aggressively to prevent the development of restenosis/atherosclerosis.
  • the inventor has discovered that the expression of the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) are altered during the healing response to acute vascular injury, and therefore during the course of atherosclerosis.
  • Methods to treat atherosclerosis may include gene therapy to increase the expression of genes down-regulated during the healing response to acute vascular injury and therefore during atherosclerosis. Treatment may also include methods to decrease the expression of genes up-regulated during the healing response to acute vascular injury and therefore during atherosclerosis. Treatment to decrease gene expression may include, but is not limited to, the expression of anti-sense mRNA, triplex formation or inhibition by co-expression.
  • the inventor further recognizes that identification that the elastin and fibrillin genes (and any of the other genes contributing to the functional integrity of elastin as noted above) are involved in the healing response to acute vascular injury and therefore in the development of restenosis/atherosclerosis makes possible an identification of proteins that may effect the development of atherosclerosis. Identification of such proteins makes possible the use of methods to affect their expression or alter their metabolism. Methods to alter the effect of expressed proteins include, but are not limited to, the use of specific antibodies or antibody fragments that bind the identified proteins, specific receptors that bind the identified protein, or other ligands or small molecules that inhibit the identified protein from affecting its physiological target and exerting its metabolic and biologic effects.
  • those proteins that are down-regulated during the healing response to acute vascular injury and therefore during the course of restenosis/atherosclerosis may be supplemented exogenously to ameliorate their decreased synthesis.
  • the inventor realizes that the identification of the elastin and fibrillin genes
  • the inventor further recognizes that different polymo ⁇ hisms may play a role in the development of restenosis/atherosclerosis in different patients.
  • the inventor therefore recognizes that this invention makes possible an identification of specific abnormalities that are characteristic of a specific patient.
  • the inventor recognizes that this would allow for greater specificity of treatment.
  • a regime that may be efficacious in one patient with a specific polymo ⁇ hism profile may not be effective in a second patient with a different polymo ⁇ hism profile.
  • Such a profiling would also allow treatment to be individualized so that unnecessary side effects of a treatment strategy that would not be effective for a specific patient can be avoided.
  • Figure 1 shows the differential expression of the tropoelastin gene and the fibrillin-1 gene following acute vascular injury of the rat carotid artery.
  • the invention provides new and improved methods for prediction, prevention, and treatment of restenosis and of atherosclerosis.
  • the elastin and fibrillin genes (and any of the other genes contributing to the functional integrity of elastin as noted above) have been identified as having altered expression levels during the healing response to acute vascular injury, and therefore during restenosis and during atherosclerosis.
  • differential expression of these genes are involved in the healing response to vascular injury, changes in the degree of their expression, or in the length of time during which they are expressed, would lead to abnormal patterns of healing.
  • the excessive healing response would contribute to the development of either restenosis or atherosclerosis.
  • Changes in the degree of gene expression, or in the length of time during which the genes are differentially expressed are caused by polymo ⁇ hisms in the gene or in the regulatory components of the gene.
  • This invention therefore, identifies the elastin gene, or any of the other genes contributing to the functional integrity of elastin as noted above, in which polymo ⁇ hisms can convey susceptibility to the development of either restenosis or atherosclerosis.
  • the identification of the elastin and fibrillin genes (and any of the other genes contributing to the functional integrity of elastin as noted above) as being involved in the healing response to acute vascular injury allows for identification of these genes whose changed degree or duration of expression caused by polymo ⁇ hisms of the gene, as targets to identify genetic abnormalities that convey altered risk of restenosis or atherosclerosis.
  • identification of the elastin and fibrillin genes provides new methods for preventing, ameliorating, or treating the disease by targeted inhibition of the expression of a suitable set or subset of those genes.
  • the invention therefore also allows risk profiling of individuals for the development of atherosclerosis prior to the actual development of clinically significant atherosclerosis; i.e. prior to the development of detectable or significant narrowing of the relevant cardiac artery or peripheral arteries. This information therefore allows prophylactic intervention to prevent atherosclerosis, and prompt detection to allow delay or amelioration of the disease process.
  • the invention also allows the identification of the elastin gene, or any of the other genes contributing to the functional integrity of elastin as noted above, as genes to be analyzed for polymo ⁇ hisms that predispose to atherosclerosis risk. Because different polymo ⁇ hisms play a role in the development of atherosclerosis in different patients, the invention allows identification of specific abnormalities that are characteristic of a specific patient. The invention therefore allows for greater specificity of treatment. A regime that may be efficacious in one patient with a specific polymo ⁇ hism profile may not be effective in a second patient with a different polymo ⁇ hism profile. Such a profiling also allows treatment to be individualized so that unnecessary side effects of a treatment strategy that would not be effective for a specific patient can be avoided. Elucidation of Changes in Gene Expression in Restenosis/atherosclerosis
  • the rat is a widely accepted model for the human for vascular studies, and results obtained in the rat are considered highly predictive of results in humans. Accordingly, it is expected that, as found in the rat, there will be differences in gene expression of the elastin and fibrillin genes, (and some of the other genes contributing to the functional integrity of elastin as noted above), in humans during the healing response to acute or chronic vascular injury. Exaggerated changes in the degree of expression in these genes, or in the length of time during which the genes are differentially expressed, will predispose to restenosis/atherosclerosis.
  • the rat genes identified as being differentially regulated during the healing response to acute vascular injury will be the homologous human genes in which such polymo ⁇ hisms will be found to convey susceptibility to restenosis. Because restenosis shares many of the processes and mechanisms as atherosclerosis, and since both result from vascular injury, then the genes identified in the rat model of the healing response to acute vascular injury will also be the genes whose abnormal expression will predisponse to atherosclerosis.
  • the specific abnormalities will be determined by identifying polymo ⁇ hisms of the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) that are associated with restenosis/atherosclerosis.
  • Such polymo ⁇ hisms will also serve indicate which gene should serve as the target for therapeutic interventions — those genes whose polymo ⁇ hisms cause an upregulation of gene transcription can be targeted by therapy designed to decrease gene expression or function of the proteins encoded by these genes; those genes in which identified polymo ⁇ hisms cause down-regulation of transcription can be targeted by therapy designed to increase gene expression or function of the proteins encoded by these genes.
  • the invention provides new compositions that can be used to inhibit, slow, or prevent restenosis and atherosclerosis. Dysregulation of the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above)
  • Antibodies for use in such ELISA methods either are commercially available or may be prepared using well known methods.
  • Other methods of quantitative analysis of multiple proteins include, for example, proteomics technologies such as isotope coded affinity tag reagents, MALDI TOF/TOF tandem mass spectrometry, and 2D-gel/mass spectrometry technologies. These technologies are commercially available from, for example, Large Scale Proteomics Inc. (Germantown MD) and Oxford Glycosystems (Oxford UK).
  • quantitative mRNA amplification methods such as quantitative RT-PCR
  • quantitative RT-PCR can be used to measure changes in gene expression at the message level.
  • Systems for carrying out these methods also are commercially available, for example the TaqMan system (Roche Molecular System, Alameda, CA) and the Light Cycler system (Roche Diagnostics, Indianapolis, IN).
  • Methods for devising appropriate primers for use in RT-PCR and related methods are well known in the art.
  • a number of software packages are commercially available for devising PCR primer sequences.
  • Nucleic acid arrays offer are a particularly attractive method for studying the expression of multiple genes.
  • arrays provide a method of simultaneously assaying expression of a large number of genes.
  • Such methods are now well known in the art and commercial systems are available from, for example, Affymetrix (Santa Clara, CA), Incyte (Palo Alto, CA), Research Genetics (Huntsville, AL) and Agilent (Palo Alto, CA). See also US Patent Nos. 5,445,934, 5,700,637, 6,080,585, 6,261,776 which are hereby inco ⁇ orated by reference in their entirety.
  • the inventor further recognizes that changes in the degree of gene expression, or in the length of time during which the genes are differentially expressed, can be caused by polymo ⁇ hisms in the gene or in the regulatory components of the gene.
  • polymo ⁇ hisms conveying an increased risk of disease development, have already been identified for several genes associated with several diseases.
  • This invention therefore, identifies the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) in which polymo ⁇ hisms can convey susceptibility to the development of restenosis/atherosclerosis. Subsequent reference, therefore, to prediction of restenosis (or atherosclerosis-see below), relate to polymo ⁇ hisms of these genes, or of their regulatory units.
  • Polymo ⁇ hisms can be identified by several methods including sequencing, short tandem repeat association studies, single nucleotide polymo ⁇ hism association studies, etc. These methods are well-known to anyone well-versed in the art. Gene expression can also be studied at the protein level. While each cell nucleus carries a complete set of genes only those genes expressed in each cell are transcribed into mRNA which is then translated into proteins. Consequently, gene expression is tissue or even cell specific. Generally, it is thought that the greater the number of RNA molecules transcribed the greater the number of protein molecules translated from them and, accordingly, the results obtained using protein analysis should be the same, at least in terms of relative changes in levels of gene expression.
  • RNA Expression may therefore be directed at the quantity of a particular mRNA transcript or the amount of protein translated from it.
  • gene polymo ⁇ hisms are detected reliably with tissue derived from any source, including peripheral blood, assay of the mRNA or protein encoded by the elastin and fibrillin genes (and any of the other genes contributing to the functional integrity of elastin as noted above) to determine relevant changes in the level of gene expression is critically dependent on tissue sampled. While some idea of altered gene expression occurring at the site of developing restenosis or of atherosclerosis can be obtained from sampling and testing peripheral blood, much more reliable estimates of altered gene expression would be obtained from sampling the actually artery developing restenosis or of atherosclerosis.
  • cells or tissue are lysed and the lysed cells centrifuged to remove the nuclear pellet. The supernatant is then recovered and the nucleic acid extracted using phenol/chloroform extraction followed by ethanol precipitation.
  • RNA can be quantified by measurement of optical density at 260-280 nM.
  • mRNA can be isolated from total RNA by exploiting the "PolyA" tail of mRNA by use of several commercially available kits.
  • QIAGEN mRNA Midi kit (Cat. No. 70042); Promega PolyATtract ® mRNA Isolation Systems (Cat. No. Z5200).
  • the QIAGEN kit provides a spin column using Oligotex Resin designed for the isolation of poly A mRNA and yields essentially pure mRNA from total
  • RNA within 30 minutes.
  • the Promega system uses a biotinylated oligo dT probe to hybridize to the mRNA poly A tail and requires about 45 minutes to isolate pure mRNA.
  • Microarray technology is an extremely powerful method for assaying the expression of multiple genes in a single sample of mRNA.
  • Gene Chip® technology commercially available from Affymetrix Inc. (Santa Clara, Ca) uses a chip that is that is plated with probes for over thousands of known genes and expressed sequence tags (ESTs).
  • Biotinylated cRNA linearly amplified RNA
  • Complementary sequences are then visualized and the intensity of the signal is commensurate with the number of copies of mRNA expressed by the gene.
  • Quantitative PCR quantitative PCR
  • Quantitative PCR employs the co-amplification of a target sequence with serial dilutions of a reference template. By inte ⁇ olating the product of the target amplification with that a curve derived from the reference dilutions an estimate of the concentration of the target sequence may be made.
  • Quantitative reverse transcription PCR may be carried out on mRNA using kits and methods that are commercially available from, for example, Applied BioSystems (Foster City, CA) and Stratagene (La Jolla, CA) See also Kochanowsi, Quantitative PCR Protocols" Humana Press, 1999.
  • total RNA may be reverse transcribed using random hexamers and the TaqMan Reverse Transcription Reagents Kit (Perkin Elmer) following the manufacturer's protocols.
  • the cDNA is amplified using TaqMan PCR master mix containing AmpErase UNG dNTP, AmpliTaq Gold, primers and TaqMan probe according to the manufacture's protocols.
  • the TaqMan probe is target-gene sequence specific and is labeled with a fluorescent reporter (FAM) at the 5' end and a quencher (e.g. TAMRA) at the 3' end.
  • Standard curves for both endogenous control and the target gene may be constructed and the comparison of the ration of CT (threshold cycle number) of target gene to control in treated and untreated cells is determined. This technique has been widely used to characterize gene expression.
  • FAM fluorescent reporter
  • Standard curves for both endogenous control and the target gene may be constructed and the comparison of the ration of CT (threshold cycle number) of
  • Gene expression may also be studied at the protein level.
  • Target tissue is first isolated and then total protein is extracted by well known methods. Quantitative analysis is achieved, for example, using ELISA methods employing a pair of antibodies specific to the target protein.
  • a subset of the proteins encoded by genes related to the functional integrity of elastin are soluble or secreted. In such instances the proteins may be found in the blood, plasma or lymph and an analysis of those proteins may be afforded by any of those methods described for the analysis of proteins in such tissues. This provides a minimally invasive means of obtaining patient samples for estimate of risk of developing restenosis or of atherosclerosis. Treatment of Restenosis
  • the identification of the set of genes encompassing the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) having altered expression during the healing response to vascular injury, provides new opportunities to treat restenosis or atherosclerosis.
  • elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) that exhibit decreased expression during the healing response to vascular injury, it is possible to ameliorate or prevent restenosis or atherosclerosis by enhancing expression of one or more of these genes.
  • Gene transcription may be deliberately modified in a number of ways. For example, exogenous copies of a gene may be inserted into the genome of cells in vascular tissue by genomic transduction via homologous recombination. While expression by genomic transduction is relatively stable it also is of low efficiency.
  • An alternative method is transient transduction where the gene is inserted within a vector allowing for its transcription independent of the genomic allele making use of a vector specific promoter. Yet another method is transfection with naked DNA.
  • the present invention also affords an ability to negatively affect the expression of the elastin or fibrillin genes (or any of the other genes contributing to the functional integrity of elastin as noted above) that are up-regulated during the healing response to vascular injury.
  • Methods for down regulating genes are well known. It has been shown that antisense RNA introduced into a cell will bind to a complementary mRNA and thus inhibit the translation of that molecule. In a similar manner, antisense single stranded cDNA may be introduced into a cell with the same result.
  • RNAi short interfering RNA
  • stable triple-helical structures can be formed by bonding of oligodeoxyribonucleotides (ODNs) to polypurine tracts of double stranded DNA.
  • ODNs oligodeoxyribonucleotides
  • Triplex formation can inhibit DNA replication by inhibition of transcription of elongation and is a very stable molecule.
  • Such antibodies may be obtained through the use of conventional hybridoma technology or may be isolated from libraries commercially available from Dyax (Cambridge, MA), Mo ⁇ hoSys (Martinsried, Germany), Biosite (San Diego, CA) and Cambridge Antibody Technology (Cambridge, UK).
  • proteins usually exert their cellular effects by ligating to cellular receptors. Identification of the receptors to which proteins, which are implicated by the current invention as contributing to restenosis or atherosclerosis, bind will allow the design of specific ligand antagonists that block pathways mediating the effects leading to the development of restenosis or atherosclerosis. The identification of genes that are down regulated during the healing response to acute vascular injury leads to the ability to identify their protein products. Down-regulated proteins may then be supplemented, thereby ameliorating the effect of their decreased synthesis.
  • the methods of the present invention may be used prophylactically to prevent the development of restenosis or atherosclerosis in at risk individuals.
  • the present invention also provides kits having chips containing the DNA of the biologically important polymo ⁇ hisms for the elastin and fibrillin genes (and any of the other genes contributing to the functional integrity of elastin as noted above).
  • chips permit the rapid detection of the polymo ⁇ hisms, providing a convenient means for the rapid detection of those individuals at high or at low risk of developing restenosis or of atherosclerosis.
  • the detection of specific polymo ⁇ hisms in specific patients will allow highly specific and individualized treatment strategies to be devised for each patient to prevent or attenuate restenosis and or atherosclerosis.
  • the present invention thus generally described, will be understood more readily by reference to the following example, which is provided by way of illustration and is not intended to be limiting of the present invention.

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  • Biophysics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Vascular Medicine (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés pour évaluer le risque de développer une resténose chez un individu, en détectant la présence de polymorphismes, importants d'un point de vue biologique, de gènes impliqués dans la formation du réseau de fibres d'élastine. La détection de polymorphismes de gènes d'élastine, de fibrilline, de fibrilline-1, de fibrilline-2, de lysyl oxydase, de glycoprotéine associée aux microfibrilles, de biglycane, d'ostéopontine et/ou de décorine permet de prédire rapidement et objectivement le risque de développer une resténose. La présente invention concerne également des procédés pour traiter une resténose et pour réduire sa récurrence.
PCT/US2003/003001 2002-02-01 2003-02-03 Polymorphismes de genes d'elastine, de fibrilline et de genes lies predisposant a une restenose et a une atherosclerose WO2003064663A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/502,984 US20050282163A1 (en) 2003-02-03 2003-02-03 Polymorphisms in elastin, fibrillin and related genes as predisposing to restenosis and to a therosclerosis
EP03706019A EP1478758A4 (fr) 2002-02-01 2003-02-03 Polymorphismes de genes d'elastine, de fibrilline et de genes lies predisposant a une restenose et a une atherosclerose
JP2003564254A JP2005531287A (ja) 2002-02-01 2003-02-03 再狭窄およびアテローム性動脈硬化症の発症素因としてのエラスチン、フィブリリンおよび関連遺伝子の多型

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35288502P 2002-02-01 2002-02-01
US60/352,885 2002-02-01

Publications (1)

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WO2003064663A1 true WO2003064663A1 (fr) 2003-08-07

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PCT/US2003/003001 WO2003064663A1 (fr) 2002-02-01 2003-02-03 Polymorphismes de genes d'elastine, de fibrilline et de genes lies predisposant a une restenose et a une atherosclerose

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EP (1) EP1478758A4 (fr)
JP (1) JP2005531287A (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4019035A1 (fr) * 2020-12-23 2022-06-29 Institut National De La Sante Et De La Recherche Medicale - Inserm Nouveaux fragments recombinants de fibrilline-1 et leurs procédés d'utilisation

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
HU0700024D0 (en) * 2007-01-11 2007-03-28 Mta Szegedi Biolog Koezpont Use of enhancers in biglycan activuty in the preparation of pharmaceutical compositions having utility in cardiac diseases
CN104207991B (zh) * 2010-06-30 2017-04-12 雅芳产品公司 用于刺激magp‑1以改善皮肤外观的组合物和方法

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Publication number Priority date Publication date Assignee Title
US6903244B1 (en) * 1999-02-26 2005-06-07 University Of Utah Research Foundation Mice which are +/− or −/− for the elastin gene as models for vascular disease

Non-Patent Citations (3)

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Title
KINGWELL ET AL.: "Large atery stiffness: structural and genetic aspects", CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, vol. 28, 2001, pages 1040 - 1043, XP002965034 *
SAJID ET AL.: "PIA polymorphism of integrin beta3 differentially modulates cellular migration on extracellular matrix proteins", ARTERIOSCLER. THROMB. VASC. BIOL., vol. 22, 2002, pages 1984 - 1989, XP002965035 *
See also references of EP1478758A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4019035A1 (fr) * 2020-12-23 2022-06-29 Institut National De La Sante Et De La Recherche Medicale - Inserm Nouveaux fragments recombinants de fibrilline-1 et leurs procédés d'utilisation

Also Published As

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EP1478758A4 (fr) 2006-08-30
JP2005531287A (ja) 2005-10-20
EP1478758A1 (fr) 2004-11-24

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