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WO2001050133A1 - Expression genique et maladies de la cornee - Google Patents

Expression genique et maladies de la cornee Download PDF

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Publication number
WO2001050133A1
WO2001050133A1 PCT/DK2000/000745 DK0000745W WO0150133A1 WO 2001050133 A1 WO2001050133 A1 WO 2001050133A1 DK 0000745 W DK0000745 W DK 0000745W WO 0150133 A1 WO0150133 A1 WO 0150133A1
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WIPO (PCT)
Prior art keywords
gene
expression
genes
cells
corneal
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PCT/DK2000/000745
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English (en)
Inventor
Torben F. ØRNTOFT
Thomas Thykjaer
Karin DEMTRÖDER
Niels Ehlers
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Aros Applied Biotechnology Aps
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Priority to AU23523/01A priority Critical patent/AU2352301A/en
Publication of WO2001050133A1 publication Critical patent/WO2001050133A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • This invenention relates to the field of corneal diseases, more specifically it relates to methods of using gene expression to catagorize and detect corneal diseases, such as keratoconus.
  • Keratoconus is a slowly progressing change of corneal shape leading to the formation of a pyramidal shape of the cornea instead of the normal spherical shape.
  • the disease process takes several years and can untreated lead to blindness. Modern treatment is contact lenses or later on cornea transplantation.
  • Keratoconus is a bilateral noninflammatory corneal ectasia with an incidence of approximately 1 per 2,000 in the general population. It has well- described clinical signs, but early forms of the disease may go undetected unless the anterior comeal topography is studied. Early disease is now best detected with videokeratography.
  • the present invention relates to the numbers of genes that are expressed in the normal cornea to be used as a reference for corneal diseases, to be used for basic understanding of corneal function, and to be used to define markers of genes that identify a tissue as corneal, especially as healthy cornea.
  • the present invention relates to a method of determining the presence or absence of corneal diseases in humans
  • the gene from the first gene group is selected from genes expressed in normal tissue cells in an amount higher than expression in corneal disease cells, and/or
  • a second expression level of at least one gene from a second gene group wherein the second gene group is selected from genes expressed in a normal tissue cells in an amount lower than expression in corneal disease cells
  • the invention relates to a method of determining the stage of a condition in human corneal tissue
  • the methods above may be used for determining single gene expressions, however the invention also relates to a method of determining an expression pattern of a cornea cell sample, comprising:
  • determining the expression level of two or more genes in the sample wherein at least one gene belongs to a first group of genes, said gene from the first gene group being expressed in a higher amount in normal tissue than in corneal disease cells, and wherein at least one other gene belongs to a second group of genes, said gene from the second gene group being expressed in a lower amount in normal tissue than in corneal disease cells, and the difference be- tween the expression level of the first gene group in normal cells and comeal disease cells being at least two-fold, obtaining an expression pattern of the corneal cell sample.
  • Gene expression patterns may rely on one or a few genes, but more preferred gene expression patterns relies on expression from multiple genes, whereby a combined information from several genes is obtained.
  • the invention relates to a method of determining an expression pattern of a corneal cell sample independent of the proportion of stroma, or endothelium cells present, comprising:
  • the expression pattern may be used in a method according to this information, and accordingly, the invention also relates to a method of determining the presence or absence of a corneal disease in human tissue comprising,
  • determining the presence or absence of the corneal disease of said tissue as well as a method for determining the stage of a corneal disease in human tissue, comprising,
  • the invention further relates to a method for reducing biological abnormalities of a cornea cell suffering from keratoconus, said method comprising,
  • the invention relates to a method for reducing biological abnormalities of a cornea cell suffering from keratoconus, said method comprising,
  • the invention relates to a method for reducing biological abnormalities of a cornea cell suffering from keratoconus, said method comprising,
  • At least one nucleotide probe capable of hybridising with at least one gene of a cell from a keratoconus suffering cornea, said at least one gene being selected from genes being expressed in an amount two-fold lower in normal cells than the amount expressed in said keratoconus cell, and introducing said at least one nucleotide probe into the keratoconus cell in a manner allowing the probe to hybridise to the at least one gene, thereby inhibiting expression of said at least one gene.
  • the invention concerns a method for producing antibodies against an expression product of a cell from corneal tissue, said method comprising the steps of
  • the invention furthermore relates to a pharmaceutical composition for the treatment of a corneal disease comprising at least one antibody produced and a vaccine for the prophylaxis or treatment of a corneal disease comprising at least one expression product from at least one gene said gene being expressed.
  • the invention relates to the use of a method producing an assay for diagnosing a corneal disease in human tissue.
  • the invention relates to the use of a peptide as defined above as an expression product and/or the use of a gene as defined above and/or the use of a probe as defined above for the preparation of a pharmaceutical composition for the treatment of a corneal disease in human tissue.
  • the invention relates to an assay for determining the presence or absence of a corneal disease in human tissue, comprising
  • the invention in another aspect relates to an assay for determining an expression pattern of a corneal disease cell, comprising at least a first marker and a second marker, wherein the first marker is capable of detecting a gene from a first gene group, and the second marker is capable of detecting a gene from a second gene group.
  • Figure 1 shows immunostains of the protein p27 / kipl .
  • FIG. 1 shows immunostains of the protein Cytokeratin 13
  • Figure 3 shows immunostains of the protein ILGF-2 - interleukin like growth factor expressed in cell matrix.
  • Figure 4 shows immunostains of the protein junB, nuclear expression; transcription factor.
  • Figure 5 shows immunostains of the protein Metallothionein hMT-le, described in the nucleus and cytoplasm of a subset of astrocytes increased in the spinal cord of patients with amyotrophic lateral sclerosis, resulted from inflammation or gliosis.
  • Figure 6 depicts immunostains of the protein Keratin 6 (KRT6 isoforms A,C,E). It is expressed in the cytosol; KRT6A intermediate filaments of the Cytoskeleton
  • Figure 7 shows immunostains of the protein beta 2-microglobulin. Localized in the cytoplasm of epithelia.
  • Table 1a+1b Comparison of expression of genes between normal and keratoconus pool.
  • Table 2a+2b Comparison of expression of genes between normal and keratoconus pool.
  • Table 3a+3b Comparison of expression of genes between normal and keratoconus pool.
  • Table 4a+4b Comparison of expression of genes between normal and keratoconus pool.
  • Table 5a+5b Comparison of expression of genes between normal and keratoconus pool.
  • Table 6a+6b Comparison of expression of genes between normal and keratoconus pool.
  • Table 7a+7b Comparison of expression of genes between normal and keratoconus pool.
  • Table 8a+8b Comparison of expression of genes between normal and keratoconus pool.
  • Table 9a+9b Comparison of expression of genes between normal and keratoconus pool.
  • Table 10a+10b Comparison of expression of genes between normal and keratoco- nus pool.
  • Table 11a+11b Comparison of expression of genes between normal and keratoconus pool.
  • Table 12a+12b Comparison of expression of genes between normal and keratoconus pool.
  • Table 13a+13b Comparison of expression of genes between normal and keratoconus pool.
  • Table 14 Genes expressed in human cornea.
  • Table 15 Selected genes expressed in human cornea.
  • samples according to the present invention may be any cornea tissue sample, it is however often preferred to conduct the methods according to the invention on epithelial tissue.
  • the sample may be obtained by any suitable manner known to the man skilled in the art, such as a biopsy of the tissue, or a superficial sample scraped from the tissue.
  • the sample may be prepared by forming a cell suspension made from the tissue, or by obtaining an extract from the tissue.
  • the sample comprises substantially only cells from said tissue, such as substantially only cells from stroma of the cornea.
  • the methods according to the invention may be used for determining any condition of the cornea, wherein said condition leads to a change in the expression of at least one gene, and preferably a change in a variety of genes.
  • the expression level may be determined as single gene approaches, i.e. wherein the determination of expression from one or two or a few genes is conducted. It is preferred that expression from at least one gene from a first (normal) group is determined in combination with determination of expression of at least one gene from a second group, said first group representing genes being expressed at a higher level in normal tissue, i.e. suppressors and said second group representing genes being expressed at a higher level in tissue from corneal disease cells than in normal tissue.
  • determination of the expression of a single gene whether belonging to the first group or second group is within the scope of the present invention. In this case it is preferred that the single gene is selected among genes having a very high change in expression level from normal cells to biological condition cells.
  • Another approach is determination of an expression pattern from a variety of genes, wherein the determination of the biological condition in the tissue relies on informa- tion from a variety of gene expression, i.e. rather on the combination of expressed genes than on the information from single genes.
  • the present invention relates to a variety of genes identified either by an EST identi- fication number and/or by a gene identification number. Both type of identification numbers relates to identification numbers of UniGene database, NCBI, build 18.
  • the first gene group relates to genes being expressed in normal tissue cells in an amount higher than expression in corneal disease cells.
  • the term "normal tissue cells" relates to cells from the same type of tissue that is examined with respect to the corneal disease in question.
  • the normal tissue relates to corneal tissue, in particular to corneal epithelium.
  • the tissue may also be selected from corneal stroma or corneal endodermis.
  • the first gene group therefore relates to genes being downregulated in cells suffering from keratoconus, such genes being expected to serve as suppressor genes, and they are of importance as predictive markers for the disease as loss of one or more of these may signal a poor outcome or an aggressive disease course.
  • they may be important targets for therapy as restoring their expression level, e.g. by gene therapy, may suppress the development of keratoconus.
  • a gene from the first gene group is preferably selected independently from the at least one group of genes designated an "I” in the columns called "Diff Call Kp3 vs Np" of the Tables 1b, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, 10b, 11b, 12b or 13b.
  • I is to be understood as genes which are expressed in an amount higher in normal tissue than in tissue of cells suffering from keratoconus.
  • a gene from the first gene group is preferably selected independently from genes comprising a sequence as identified below, wherein the first gene group are selected independently from genes comprising a sequence as identified below
  • genes that are up-regulated (or gained de novo) during the progression of keratoconus from normal tissue may be those genes that create or enhance the development of keratoconus.
  • the expression level of these genes may serve as predictive markers for the disease course, as a high level may signal an aggressive disease course, and they may serve as targets for therapy, as blocking these genes by e.g. anti-sense therapy, or by biochemical means could inhibit, or slow, the keratoconus.
  • Such up-regulated (or gained de novo) genes may be classified according to the present invention as genes belonging to second genes group.
  • genes belonging to the second gene group are preferably selected independently independently from the at least one group of genes designated an "D” in the columns called “Diff Call Kp3 vs Np" of the Tables 1b, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, 10b, 11b, 12b or 13b.
  • D is to be understood as genes which are expressed in an amount lower in normal tissue than in tissue of cells suffering from keratoconus.
  • expression from more genes from both groups are determined, such as determination of expression from at least two genes from either of the gene groups, such as determination of expression from at least three genes from either of the gene groups, such as determination of expression from at least four genes from either of the gene groups, such as determination of expression from at least five genes from either of the gene groups, such as determination of expression from at least six genes from either of the gene groups, such as determination of expression from at least seven genes from either of the gene groups.
  • a pattern of characteristic expression of one gene can be useful in characterizing a cell type source or a stage of disease. However, more genes may be usefully analyzed. Useful patterns include expression of at least one, two, three, five, ten, fifteen, twenty, twenty-five, fifty, seventy-five, one hundred, or several hundred informative genes.
  • a gene is indicated as being expressed if an intensity value of greater than or equal to 20 is shown. Conversely, an intensity value of less than 20 indicates that the gene is not expressed above background levels.
  • Comparison of an expression pattern to another may score a change from expressed to non-expressed, or the reverse. Alternatively, changes in intensity of expression may be scored, either increases or decreases. Any statistically significant change can be used. Typically changes which are greater than 2-fold are suitable. Changes which are greater than 5-fold are highly significant.
  • the present invention in particular relates to methods using genes wherein the ratio of the expression level in normal tissue to biological condition tissue for suppressor genes or vice versa of the expression level in biological condition tissue to normal tissue for condition genes is as high as possible, such as at least two-fold change in expression, such as at least three-fold, such as at least four fold, such as at least five fold, such as at least six fold, such as at least ten fold, such as at least fifteen fold, such as at least twenty fold.
  • the stage of a corneal disease according to the invention indicates the severity of the disease. Cases of varying degrees of progression of for example keratoconus exists. In less severe situations the disease may be diagnosed using a videokerato- graphy or determine irregular astigmatism. This condition may be treated using contact lenses. In more severe situations (stages) the cornea has to be replaced by transplantation.
  • the invention relates to a method as described above further comprising the steps of determining the stage of corneal disease in the human tissue, comprising assaying a third expression level of at least one gene from a third gene group, wherein a gene from said second gene group, in one stage of the disease, is expressed differently from a gene from said third gene group.
  • the difference in expression level of a gene from one stage to the expression level of the gene in another group is preferably at least two-fold, such as at least threefold.
  • the genes selected may be a gene from each gene group being expressed in a significantly higher amount, or a significantly lower amount in that stage than in one of the other stages, such as at least five fold, for example at least ten fold, such as at least fifteen fold, for example twenty fold.
  • a method is provided of determining an expression pattern of a cell sample preferably independent of the proportion of stroma cells or endothelium present. Expression is determined of one or more genes in a sample comprising cells, said genes being selected from the same genes as discussed above and shown in the tables of the examples.
  • characteristic patterns of expression of genes can be used to characterize different types of tissue.
  • gene expression patterns can be used to characterize stages/severity and grades of keratoconus.
  • gene expression patterns can be used to distinguish cells having a corneal origin from other cells.
  • gene expression of cells which routinely contaminate corneal biopsies has been identified, and such gene expression can be removed or subtracted from patterns obtained from corneal biopsies.
  • corneal biopsies are of the comeal epithelium without stroma and endothelium cells.
  • the gene expression patterns of single-cell solutions of corneal epithelium cells have been found to be far from interfering expression of contaminating stroma and endothelial cells that biopsy samples.
  • the one or more genes exclude genes which are expressed in the stroma or endothelial tissue.
  • a pattern of expression is formed for the sample which is independent of the proportion of stroma and endothelial tissue cells in the sample.
  • the invention relates to a method of determining an expression pattern of a corneal cell sample, comprising:
  • determining the expression level of two or more genes in the sample wherein at least one gene belongs to a first group of genes, said gene from the first gene group being expressed in a higher amount in normal tissue than in comeal disease cells, and wherein at least one other gene belongs to a second group of genes, said gene from the second gene group being expressed in a lower amount in normal tissue than in corneal disease cells, and the difference between the expression level of the first gene group in normal cells and corneal disease cells being at least two-fold, obtaining an expression pattern of the corneal cell sample.
  • Another embodiment of the invention provides a method for determining an expression pattern of the two or more genes exclude genes which are expressed in the corneal stroma, or endothelium cells, whereby a pattern of expression is formed for the sample which is independent of the proportion of stroma, or endothelium cells in the sample.
  • the method comprises determining the expression level of one or more genes in the sample comprising predominantly corneal stroma and endothelium cells, obtaining a second pattern, subtracting said second pattern from the expression pattern of the corneal cell sample, forming a third pattern of expression, said third pattern of expression reflecting expression of the corneal cells independent of the proportion of stroma, and endothelium cells present in the sample.
  • the invention concerns a method of determining an expression pattern of a corneal cell sample independent of the proportion of stroma, or endothelium cells present, comprising:
  • a method is provided of determining the presence or absence, or the stage of a corneal disease in human tissue comprising, collecting a sample comprising cells from said tissue and then determining an expression pattern of the cells as defined above, correlating the determined expression pattern to a standard pattern, and determining the presence or absence of the corneal disease of said tissue.
  • a first pattern of expression is determined of one or more genes in a comeal tissue sample suspected of suffering from keratoconus.
  • the first pattern of expression is compared to a second and third reference pattern of expression.
  • the second pattern is of the one or more genes in normal corneal tissue and the third pattern is of the one or more genes in keratoconus.
  • a method for predicting outcome or prescribing treatment of a keratoconus.
  • a first pattern of expression is determined of one or more genes in a keratoconus cell sample.
  • the first pattern is compared to one or more reference patterns of expression determined for keratoconus.
  • the reference pattern which shares maximum similarity with the first pattern is identified.
  • the outcome or treatment appropriate for the grade/severity of the keratoconus may in this way be determined.
  • Another aspect of the invention is a method to aid in diagnosing, predicting outcome, or prescribing treatment of keratoconus. Those genes which are expressed in stroma or endothelial cells are removed from the first set of genes. Those genes which are not expressed in stroma or endothelial tissue are removed from the second set of genes.
  • the invention also relates to methods, wherein the expression pattern of the tissue is independent of the amount of endothelial tissue in the sample.
  • Biopsies do inevitably contain many different cell types, such as cells present in the blood, connective and muscle tissue, endothelium etc.
  • microdissection or laser capture are methods of choice, however the time dependent degradation of RNA makes it difficult to perform manipulation of the tissue for more than a few minutes.
  • studies of expressed sequences may be difficult on the few cells obtained via microdissection or laser capture, as these may have an expression pattern that deviates from the predominant pattern in a tumor due to large intratumoral heterogeneity.
  • high density expression arrays may be used to evaluate the impact of cornea wall components in keratoconus biopsies, and tested preparation of single cell solutions as a means of eliminating the contaminants.
  • the results of these evaluations permit designing methods of evaluating colorectal samples without the interfering background noise caused by ubiquitous contaminating stroma and endothelial cells.
  • the evaluating assays of the invention may be of any type.
  • Gene expression patterns according to the present invention are determined by measuring any gene product of a particular gene, including mRNA and protein. A pattern may be for one or more gene.
  • RNA or protein can be isolated and assayed from a test sample using any techniques known in the art. They can for example be isolated from fresh or frozen biopsy, from formalin-fixed tissue, from body fluids, such as blood, plasma, serum, urine, or sputum.
  • the data provided of expression for stroma and endothelial tissue can be used in at least three ways to improve the quality of data for a tested sample.
  • the genes identified in the data as expressed can be excluded from the testing or from the analysis.
  • the intensity of expression of the genes expressed in the stroma and endothelial tissue can be subtracted from the intensity of expression determined for the tests tissue.
  • Genes identified as changing expression can be used as markers for drug screening. Thus by treating cornea cells with test compounds or extracts, and monitoring the expression of genes identified as changing in the progression of keratoconus, one can identify compounds or extracts which change expression of genes to a pattern which is of an earlier stage or even of normal corneal tissue.
  • Expression of genes may in general be detected by either detecting mRNA from the cells and/or detecting expression products, such as peptides and proteins.
  • the detection of mRNA of the invention may be a tool for determining the developmental stage of a cell type may be definable by its pattern of expression of messenger RNA. For example, in particular stages of cells, high levels of ribosomal RNA are found whereas relatively low levels of other types of messenger RNAs may be found. Where a pattern is shown to be characteristic of a stage, a stage may be defined by that particular pattern of messenger RNA expression.
  • the mRNA population is a good determinant of developmental stage, will be correlated with other structural features of the cell. In this manner, cells at specific developmental stages will be characterized by the intracellular environment, as well as the extracellular environment.
  • the present invention also allows the combination of definitions based, in part, upon antigens and, in part, upon mRNA expression.
  • the two may be combined in a single incubation step.
  • a particular incubation condition may be found which is compatible with both hybridization recognition and non-hybridization recognition molecules.
  • an incubation condition may be selected which allows both specificity of antibody binding and specificity of nucleic acid hybridization. This allows simultaneous performance of both types of interactions on a single matrix.
  • a cell sorter may be used to sort specifically those cells having desired mRNA population patterns.
  • Such methods often involve sample extraction, PCR amplification, nucleic acid fragmentation and labeling, extension reactions, transcription reac- tions and the like.
  • the nucleic acid may be isolated from the sample according to any of a number of methods well known to those of skill in the art.
  • genomic DNA is preferably isolated.
  • expression levels of a gene or genes are to be detected, preferably RNA (mRNA) is isolated.
  • the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and polyA.sup.+ mRNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Mole- cular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-lnterscience, New York (1987)).
  • the sample may be from tissue, blood, saliva, faeces, mucus, sputum, cerebro, or spinal fluid.
  • sample preparation operations will include such manipulations as ex- traction of intracellular material, e.g., nucleic acids from whole cell samples, viruses and the like, amplification of nucleic acids, fragmentation, transcription, labeling and/or extension reactions.
  • sample preparation operations may include such manipulations as ex- traction of intracellular material, e.g., nucleic acids from whole cell samples, viruses and the like, amplification of nucleic acids, fragmentation, transcription, labeling and/or extension reactions.
  • these various operations may be readily incorporated into the device of the present invention.
  • DNA extraction may be relevant if possible mutations in the genes are to be determined additionally. For those embodiments where whole cells, or other tissue samples are being analyzed, it will typically be necessary to extract the nucleic acids from the cells or viruses, prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatments to prepare the sample for subsequent operations, e.g., denaturation of contaminating (DNA binding) proteins, purification, filtration, desalting, and the like.
  • nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatments to prepare the sample for subsequent operations, e.g., denaturation of contaminating (DNA binding) proteins, purification, filtration, desalting, and the like.
  • Liberation of nucleic acids from the sample cells, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods.
  • chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chao- tropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins.
  • nucleic acids and denature DNA binding proteins such as physical protrusions within microchannels or sharp edged particles piercing cell membranes and extract their contents.
  • Combina- tions of such structures with piezoelectric elements for agitation can provide suitable shear forces for lysis.
  • cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current to the sample. More specifically, the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow. Subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture are also possible extraction methods.
  • nucleic acids Following extraction, it will often be desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, salts, and the like. Removal of particulate matter is generally accomplished by filtration, flocculation or the like. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample, passing salts through dialysis membranes, and the like.
  • Suitable solid sup- ports for nucleic acid binding include, e.g., diatomaceous earth, silica (i.e., glass wool), or the like.
  • Suitable gel exclusion media also well known in the art, may also be readily incorporated into the devices of the present invention, and is commercially available from, e.g., Pharmacia and Sigma Chemical.
  • desalting methods may generally take advantage of the high electrop- horetic mobility and negative of DNA compared to other elements.
  • Electrophoretic methods may also be utilized in the purification of nucleic acids from other cell contaminants and debris. Upon application of an appropriate electric field, the nucleic acids present in the sample will migrate toward the positive electrode and become trapped on the capture membrane. Sample impurities remaining free of the mem- brane are then washed away by applying an appropriate fluid flow. Upon reversal of the voltage, the nucleic acids are released from the membrane in a substantially purer form. Further, coarse filters may also be overlaid on the barriers to avoid any fouling of the barriers by particulate matter, proteins or nucleic acids, thereby per- mitting repeated use.
  • the high electrophoretic mobility of nucleic acids with their nega- tive charges may be utilized to separate nucleic acids from contaminants by utilizing a short column of a gel or other appropriate matrix or gel which will slow or retard the flow of other contaminants while allowing the faster nucleic acids to pass.
  • This invention provides nucleic acid affinity matrices that bear a large number of different nucleic acid affinity ligands allowing the simultaneous selection and removal of a large number of preselected nucleic acids from the sample. Methods of producing such affinity matrices are also provided.
  • the methods involve the steps of a) providing a nucleic acid amplification template array comprising a surface to which are attached at least 50 oligonucleotides having different nucleic acid se- quences, and wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said oligonucleotides is greater than about 60 different oligonucleotides per 1 cm.sup.2, and all of said different oligonucleotides have an identical terminal 3' nucleic acid sequence and an identical terminal 5' nucleic acid sequence, b) amplifying said multiplicity of oligonucleotides to provide a pool of amplified nucleic acids; and c) attaching the pool of nucleic acids to a solid support.
  • nucleic acid affinity chromatography is based on the tendency of complementary, single-stranded nucleic acids to form a double-stranded or duplex structure through complementary base pairing.
  • a nucleic acid (either DNA or RNA) can easily be attached to a solid substrate (matrix) where it acts as an immobilized ligand that interacts with and forms duplexes with complementary nucleic acids present in a solution contacted to the immobilized ligand. Unbound components can be washed away from the bound complex to either provide a solution lacking the target molecules bound to the affinity column, or to provide the isolated target molecules themselves.
  • the nucleic acids captured in a hybrid duplex can be separated and released from the affinity matrix by denaturation either through heat, adjustment of salt concentration, or the use of a destabilizing agent such as formamide, TWEEN.TM.-20 denaturing agent, or sodium dodecyl sulfate (SDS).
  • a destabilizing agent such as formamide, TWEEN.TM.-20 denaturing agent, or sodium dodecyl sulfate (SDS).
  • Affinity columns are typically used either to isolate a single nucleic acid typically by providing a single species of affinity ligand.
  • affinity columns bearing a single affinity ligand e.g. oligo dt columns
  • affinity columns bearing a single affinity ligand have been used to isolate a multiplicity of nucleic acids where the nucleic acids all share a common sequence (e.g. a polyA).
  • affinity matrix used depends on the purpose of the analysis. For exam- pie, where it is desired to analyze mRNA expression levels of particular genes in a complex nucleic acid sample (e.g., total mRNA) it is often desirable to eliminate nucleic acids produced by genes that are constitutively overexpressed and thereby tend to mask gene products expressed at characteristically lower levels.
  • the affinity matrix can be used to remove a number of preselected gene products (e.g., actin, GAPDH, etc.). This is accomplished by providing an affinity matrix bearing nucleic acid affinity ligands complementary to the gene products (e.g., mRNAs or nucleic acids derived therefrom) or to subsequences thereof.
  • Hybridization of the nucleic acid sample to the affinity matrix will result in duplex formation between the affinity ligands and their target nucleic acids.
  • the matrix Upon elution of the sample from the affinity matrix, the matrix will retain the duplexes nucleic acids leaving a sample depleted of the overexpressed target nucleic acids.
  • the affinity matrix can also be used to identify unknown mRNAs or cDNAs in a sample.
  • the affinity matrix contains nucleic acids complementary to every known gene (e.g., in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template) in a sample
  • capture of the known nucleic acids by the affinity matrix leaves a sample enriched for those nucleic acid sequences that are unknown.
  • the affinity matrix is used to perform a subtractive hybridization to isolate unknown nucleic acid sequen- ces. The remaining "unknown" sequences can then be purified and sequenced ac- cording to standard methods.
  • the affinity matrix can also be used to capture (isolate) and thereby purify unknown nucleic acid sequences.
  • an affinity matrix can be prepared that con- tains nucleic acid (affinity ligands) that are complementary to sequences not previously identified, or not previously known to be expressed in a particular nucleic acid sample.
  • the sample is then hybridized to the affinity matrix and those sequences that are retained on the affinity matrix are "unknown" nucleic acids.
  • the retained nucleic acids can be eluted from the matrix (e.g. at increased temperature, increa- sed destabilizing agent concentration, or decreased salt) and the nucleic acids can then be sequenced according to standard methods.
  • the affinity matrix can be used to efficiently capture (isolate) a number of known nucleic acid sequences.
  • the matrix is prepared bearing nucleic acids complementary to those nucleic acids it is desired to isolate.
  • the sample is contacted to the matrix under conditions where the complementary nucleic acid sequences hybridize to the affinity ligands in the matrix.
  • the non-hybridized material is washed off the matrix leaving the desired sequences bound.
  • the hybrid duplexes are then denatured providing a pool of the isolated nucleic acids.
  • the different nucleic acids in the pool can be subsequently separated according to standard methods (e.g. gel electrophoresis).
  • the affinity matrices can be used to selectively remove nucleic acids from virtually any sample containing nucleic acids (e.g., in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template, and so forth).
  • the nucleic acids adhering to the column can be removed by washing with a low salt concentration buffer, a buffer containing a destabilizing agent such as formamide, or by elevating the column temperature.
  • the affinity matrix can be used in a method to enrich a sample for unknown RNA sequences (e.g. expressed sequence tags (ESTs)).
  • the method involves first providing an affinity matrix bearing a library of oligonucleotide probes specific to known RNA (e.g., EST) sequences. Then, RNA from undifferentiated and/or unactivated cells and RNA from differentiated or acti- vated or pathological (e.g., transformed) or otherwise having a different metabolic state are separately hybridized against the affinity matrices to provide two pools of RNAs lacking the known RNA sequences.
  • RNA sequences e.g. expressed sequence tags (ESTs)
  • the affinity matrix is packed into a columnar casing.
  • the sample is then applied to the affinity matrix (e.g. injected onto a column or applied to a column by a pump such as a sampling pump driven by an autosampler).
  • the affinity matrix (e.g. affinity column) bearing the sample is subjected to conditions under which the nucleic acid probes comprising the affinity matrix hybridize specifically with complementary target nucleic acids. Such conditions are accomplished by maintaining appropriate pH, salt and temperature conditions to facilitate hybridization as discussed above.
  • the device of the present invention may, in some cases, include an mRNA purification chamber or channel.
  • mRNA purification chamber or channel In general, such purification takes advantage of the poly-A tails on mRNA.
  • poly- T oligonucleotides may be immobilized within a chamber or channel of the device to serve as affinity ligands for mRNA.
  • Poly-T oligonucleotides may be immobilized upon a solid support incorporated within the chamber or channel, or alternatively, may be immobilized upon the surface ⁇ ) of the chamber or channel itself.
  • Immobilization of oligonucleotides on the surface of the chambers or channels may be carried out by methods described herein including, e.g., oxidation and silanation of the surface followed by standard DMT synthesis of the oligonucleotides.
  • the lysed sample is introduced to a high salt solution to increase the ionic strength for hybridization, whereupon the mRNA will hybridize to the immobilized poly-T.
  • the mRNA bound to the immobilized poIy-T oligonucleotides is then washed free in a low ionic strength buffer.
  • the poy-T oligonucleotides may be im- mobiliized upon poroussurfaces, e.g., porous silicon, zeolites silica xerogels, scin- tered particles, or other solid supports.
  • Hybridization Following sample preparation, the sample can be subjected to one or more different analysis operations.
  • analysis operations may generally be performed, including size based analysis using, e.g., microcapillary electrophoresis, and/or sequence based analysis using, e.g., hybridization to an oligonucleotide array.
  • the nucleic acid sample may be probed using an array of oligonucleotide probes.
  • Oligonucleotide arrays generally include a substrate having a large number of positionally distinct oligonucleotide probes attached to the substrate. These arrays may be produced using mechanical or light directed synthesis met- hods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods.
  • the basic strategy for light directed synthesis of oligonucleotide arrays is as follows.
  • the surface of a solid support, modified with photosensitive protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions.
  • a selected nucleotide typically in the form of a 3'-O- phosphoramidite-activated deoxynucleoside (protected at the 5' hydroxyl with a photosensitive protecting group)
  • the substrate is rinsed and the surface is illuminated through a second mask, to expose additional hydroxyl groups for coupling.
  • a second selected nucleotide e.g., 5'- protected, 3'-O-phosphoramidite-activated deoxynucleoside
  • the selective deprotection and coupling cycles are repeated until the desired set of products is obtained. Since photolithography is used, the process can be readily miniaturized to generate high density arrays of oligonucleotide probes. Furthermore, the sequence of the oligonucleotides at each site is known. See, Pease, et al. Mechanical synthesis methods are similar to the light directed methods except involving mechanical direction of fluids for deprotection and addition in the synthesis steps.
  • oligonucleotide arrays may be prepared having all possible probes of a given length.
  • the hybridization pattern of the target sequence on the array may be used to reconstruct the target DNA sequence.
  • Hybridization analysis of large numbers of probes can be used to sequence long stretches of DNA or provide an oligonucleotide array which is specific and complementary to a particular nucleic acid sequence.
  • the oligonucleotide array will contain oligonucleotide probes which are complementary to speci- fie target sequences, and individual or multiple mutations of these. Such arrays are particularly useful in the diagnosis of specific disorders which are characterized by the presence of a particular nucleic acid sequence.
  • nucleic acid portion of the sample is typically subjected to one or more preparative reactions. These preparative reactions include in vitro transcription, labeling, fragmentation, amplification and other reactions. Nucleic acid amplification increases the number of copies of the target nucleic acid sequence of interest.
  • a variety of amplification methods are suitable for use in the methods and device of the present invention, including for exam- pie, the polymerase chain reaction method or (PCR), the ligase chain reaction
  • LCR self sustained sequence replication
  • NASBA nucleic acid based sequence amplification
  • sequence analysis may be carried out using either type of substrate, i.e., complementary to either DNA or RNA.
  • the high density array may then include probes specific to the internal standard for quantification of the amplified nucleic acid.
  • this invention provides for a method of optimizing a probe set for detection of a particular gene.
  • this method involves providing a high density array containing a multiplicity of probes of one or more particular length(s) that are complementary to subsequences of the mRNA transcribed by the target gene.
  • the high density array may contain every probe of a particular length that is complementary to a particular mRNA.
  • the probes of the high density array are then hybridized with their target nucleic acid alone and then hybridized with a high complexity, high concentration nucleic acid sample that does not contain the targets complementary to the probes.
  • the probes are first hybridized with their target nucleic acid alone and then hybridized with RNA made from a cDNA library (e.g., reverse transcribed polyA.sup. ⁇ mRNA) where the sense of the hybridized RNA is opposite that of the target nucleic acid (to insure that the high complexity sample does not contain targets for the probes).
  • a cDNA library e.g., reverse transcribed polyA.sup. ⁇ mRNA
  • Those probes that show a strong hybridization signal with their target and little or no cross-hybridization with the high complexity sample are preferred probes for use in the high density arrays of this invention.
  • PCR amplification generally involves the use of one strand of the target nucleic acid sequence as a template for producing a large number of complements to that sequence.
  • two primer sequences complementary to different ends of a seg- ment of the complementary strands of the target sequence hybridize with their respective strands of the target sequence, and in the presence of polymerase enzymes and nucleoside triphosphates, the primers are extended along the target sequence. The extensions are melted from the target sequence and the process is repeated, this time with the additional copies of the target sequence synthesized in the preceding steps.
  • PCR amplification typically involves repeated cycles of denaturation, hybridization and extension reactions to produce sufficient amounts of the target nucleic acid.
  • the first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complemen- tary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer.
  • the cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
  • strand separation is normally achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase.
  • Typical heat denaturation involves temperatures ranging from about ⁇ O.degree. C. to 105.degree. C. for times ranging from seconds to minutes.
  • Strand separation can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means.
  • Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity.
  • the methods and devices of the present invention are also applicable to a number of other reaction types, e.g., reverse transcription, nick translation, and the like.
  • the nucleic acids in a sample will generally be labeled to facilitate detection in subsequent steps. Labeling may be carried out during the amplification, in vitro trans- cription or nick translation processes. In particular, amplification, in vitro transcription or nick translation may incorporate a label into the amplified or transcribed sequence, either through the use of labeled primers or the incorporation of labeled dNTPs into the amplified sequence.
  • Hybridization between the sample nucleic acid and the oligonucleotide probes upon the array is then detected, using, e.g., epifluorescence confocal microscopy.
  • sample is mixed during hybridization to enhance hybridization of nucleic acids in the sample to nucleoc acid probes on the array.
  • hybridized oligonucleotides may be labeled following hybridization.
  • biotin labeled dNTPs are used in, e.g., amplification or transcription
  • streptavidin linked reporter groups may be used to label hybridized com- plexes.
  • the nucleic acids in the sample may be labeled following amplification.
  • Post amplification labeling typically involves the covalent attachment of a particular detectable group upon the amplified sequences. Suitable labels or detectable groups include a variety of fluorescent or radioactive labeling groups well known in the art. These labels may also be coupled to the sequences using methods that are well known in the art.
  • a fluorescent label is preferred because of its extreme sensitivity and simplicity. Standard labeling procedures are used to determine the positions where interactions between a sequence and a reagent take place. For example, if a target sequence is labeled and exposed to a matrix of different probes, only those locations where probes do interact with the target will exhibit any signal. Alternatively, other methods may be used to scan the matrix to determine where interaction takes place. Of course, the spectrum of interactions may be determined in a temporal manner by repeated scans of interactions which occur at each of a multiplicity of conditions. However, instead of testing each individual interaction separately, a multiplicity of sequence interactions may be simultaneously determined on a matrix.
  • Means of detecting labeled target (sample) nucleic acids hybridized to the probes of the high density array are known to those of skill in the art. Thus, for example, where a colorimetric label is used, simple visualization of the label is sufficient. Where a radioactive labeled probe is used, detection of the radiation (e.g with photographic film or a solid state detector) is sufficient.
  • the target nucleic acids are labeled with a fluorescent label and the localization of the label on the probe array is accomplished with fluorescent microscopy.
  • the hybridized array is excited with a light source at the excitation wavelength of the particular fluorescent label and the resulting fluore- scence at the emission wavelength is detected.
  • the excitation light source is a laser appropriate for the excitation of the fluorescent label.
  • the target polynucleotide may be labeled by any of a number of convenient de- tectable markers.
  • a fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure.
  • Other potential labeling moieties include, radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes. Another method for labeling may bypass any label of the target sequence.
  • the target may be exposed to the probes, and a double strand hybrid is formed at those positions only. Addition of a double strand specific reagent will detect where hybridization takes place.
  • An intercalative dye such as ethidium bromide may be used as long as the probes themselves do not fold back on themselves to a significant extent forming hairpin loops. However, the length of the hairpin loops in short oligonucleotide probes would typically be insufficient to form a stable duplex.
  • Suitable chromogens will include molecules and compounds which absorb light in a distinctive range of wavelengths so that a color may be observed, or emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluo- rescers.
  • Biliproteins e.g., phycoerythrin, may also serve as labels.
  • Suitable dyes are available, being primarily chosen to provide an intense color with minimal absorption by their surroundings.
  • Illustrative dye types include quinoline dyes, triarylmethane dyes, acridine dyes, alizarine dyes, phthale- ins, insect dyes, azo dyes, anthraquinoid dyes, cyanine dyes, phenazathionium dyes, and phenazoxonium dyes.
  • fluorescers may be employed either by themselves or in conjuncti- on with quencher molecules. Fluorescers of interest fall into a variety of categories having certain primary functionalities. These primary functionalities include 1- and 2- aminonaphthalene, p.p'-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone imines, anthracenes, oxacarbocyani- ne, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl ben- zene, 1 ,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, te- tracycline, sterophenol, benzimidzaolylphenylamine, 2-oxo-3-chromen
  • Individual fluorescent compounds which have functionalities for linking or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodami- neisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6- sulfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl, N-methyl 2- aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9'- anthroyl)palmitate; dansyl phosphatidylethanolamine; N,N'-di
  • fluorescers should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound dye may differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent.
  • Fluorescers are generally preferred because by irradiating a fluorescer with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.
  • Detectable signal may also be provided by chemiluminescent and bioluminescent sources.
  • Chemiluminescent sources include a compound which becomes electroni- cally excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor.
  • a diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions.
  • One family of compounds is 2,3-dihydro-1,-4-phthalazinedione.
  • the most popular compound is luminol, which is the 5-amino compound.
  • Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylami- no.calbenz analog.
  • Chemiluminescent analogs include para-dimethylamino and -methoxy substi- tuents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.
  • Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy.
  • exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like.
  • exemplary spin labels include ni- troxide free radicals.
  • amplified sequences may be subjected to other post amplification treatments.
  • analysis operations include, e.g., sequence based analyses using an oligonucleotide array and/or size based analyses using, e.g., microcapillary array electrophoresis.
  • Microcapillary array electrophoresis generally involves the use of a thin capillary or channel which may or may not be filled with a particular separation medium. Electrophoresis of a sample through the capillary provides a size based separation profile for the sample. Microcapillary array electrophoresis generally provides a rapid method for size based sequencing, PCR product analysis and restriction fragment sizing. The high surface to volume ratio of these capillaries allows for the application of higher electric fields across the capillary without substantial thermal variation across the capillary, consequently allowing for more rapid separations. Furthermore, when combined with confocal imaging methods, these methods provide sensitivity in the range of attomoles, which is comparable to the sensitivity of radioactive sequencing methods.
  • the capillaries e.g., fused silica capillaries or channels etched, machined or molded into planar substrates, are filled with an appropriate separation/sieving matrix.
  • sieving matrices include, e.g., hydroxyethyl cellulose, polyacrylamide, agarose and the like.
  • Gel matrices may be introduced and polymerized within the capillary channel. However, in some cases, this may result in entrapment of bubbles within the channels which can interfere with sample separations.
  • Polymerization outside of the channels helps to ensure that no bubbles are formed. Further, the pressure of the welding process helps to ensure a void-free system.
  • the capillary arrays may also be used in sequencing applications.
  • gel based sequencing techniques may be readily adapted for capillary array electrophoresis.
  • expression products from the genes discussed above may be detected as indications of the biological condition of the tissue.
  • Expression products may be detected in either the tissue sample as such, or in a body fluid sample, such as blood, serum, plasma, fae- ces, mucus, sputum, cerebrospinal fluid, and/or urine of the individual.
  • the expression products, peptides and proteins, may be detected by any suitable technique known to the person skilled in the art.
  • the expression products are detected by means of specific antibodies directed to the various expression products, such as immunofluore- scent and/or immunochemical staining of the tissue.
  • the expression products may be detected by means of con- ventional enzyme assay, such as ELISA methods.
  • the expression products may be detected by means of peptide/protein chips capable of specifically binding the peptides and/or proteins assessed. Thereby an expression pattern may be obtained.
  • the invention relates to an assay for determining the presence or absence of a corneal disease in human tissue, comprising
  • the assay comprises at least two markers for each gene group.
  • the marker(s) are preferably specifically detecting a gene as identified herein, in particular in the tables.
  • the marker may be any nucleotide probe, such as a DNA, RNA, PNA, or LNA probe capable of hybridising to mRNA indicative of the expression level.
  • the hybridisation conditions are preferably as described below for probes.
  • the marker is an antibody capable of specifically binding the expression product in question.
  • Patterns can be compared manually by a person or by a computer or other machine.
  • An algorithm can be used to detect similarities and differences.
  • the algorithm may score and compare, for example, the genes which are expressed and the genes which are not expressed.
  • the algorithm may look for changes in intensity of expression of a particular gene and score changes in intensity between two samples. Similarities may be determined on the basis of genes which are expressed in both samples and genes which are not expressed in both samples or on the basis of genes whose intensity of expression are numerically similar.
  • the detection operation will be performed using a reader device external to the diagnostic device. However, it may be desirable in some cases, to incorporate the data gathering operation into the diagnostic device itself.
  • the detection apparatus may be a fluorescence detector, or a spectroscopic detector, or another detector.
  • antibody reagents may also be very useful.
  • Gathering data from the various analysis operations will typically be carried out using methods known in the art.
  • the arrays may be scanned using lasers to excite fluorescently labeled targets that have hybridized to regions of probe arrays mentioned above, which can then be imaged using charged coupled devices ("CCDs") for a wide field scanning of the array.
  • CCDs charged coupled devices
  • another particularly useful method for gathering data from the arrays is through the use of laser confocal microscopy which combines the ease and speed of a readily automated process with high resolution detection.
  • the data will typically be reported to a data analysis operation.
  • the data obtained by the reader from the device will typically be analyzed using a digital computer.
  • the computer will be appropriately programmed for receipt and storage of the data from the device, as well as for analysis and reporting of the data gathered, i.e., interpreting fluorescence data to determine the sequence of hybridizing probes, normalization of background and single base mismatch hybridizations, ordering of sequence data in SBH applications, and the like.
  • the invention also relates to a pharmaceutical composition for the treatment of a corneal disease comprising at least one antibody.
  • the pharmaceutical composition comprises one or more of the peptides as defined above.
  • the peptides may be expression products, suppressor peptides normally lost in disease cell tissue in order to stabilise the development of corneal diseases at a less developed stage.
  • the peptides are peptides capable of eliciting an immune response towards the cells suffering from for example keratoconus.
  • the peptides are bound to carriers.
  • the peptides may suitably be coupled to a polymer carrier, for example a protein carrier, such as BSA.
  • a polymer carrier for example a protein carrier, such as BSA.
  • the pharmaceutical composition comprises genetic material, either genetic material for substitution therapy, or for suppressing therapy as dis- cussed below.
  • the pharmaceutical composition comprises at least one antibody produced as described below.
  • the term pharmaceutical composition is used synonymously with the term medicament.
  • the medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition as defined above in combination with pharmaceutically acceptable additives.
  • Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intrave- nous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration. For most indications a localised or substantially localised application is preferred.
  • Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or which enhance the effectiveness or transportation of the preparation.
  • Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.
  • the preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect.
  • Additional formulations which are suitable for other modes of administration include suppositories, and, in some cases, oral formulations.
  • suppositories traditional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • the preparations are administered in a manner compatible with the dosage formula- tion, and in such amount as will be therapeutically effective.
  • the quantity to be ad- ministered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are of the order of several hundred ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range of from about 1 ⁇ g to 300 ⁇ g, and especially in the range of from about 10 ⁇ g to 50 ⁇ g. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosis would be in the interval 30 mg to 70 mg per 70 kg body weight.
  • the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • additives and carriers will be known in the art.
  • Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc.
  • the present invention relates to a vaccine for the prophylaxis or treatment of a corneal disease comprising at least one expression product from at least one gene said gene being expressed as defined above.
  • vaccines is used with its normal meaning, i.e preparations of immunogenic material for administration to induce in the recipient an immunity to infection or in- toxication by a given infecting agent.
  • Vaccines may be administered by intravenous injection or through oral, nasal and/or mucosal administration.
  • Vaccines may be either simple vaccines prepared from one species of expression products, such as proteins or peptides, or a variety of expression products, or they may be mixed vaccines containing two or more simple vaccines. They are prepared in such a manner as not to destroy the immunogenic material, although the methods of preparation vary, depending on the vaccine.
  • the enhanced immune response achieved according to the invention can be attrib- utable to e.g. an enhanced increase in the level of immunoglobulins or in the level of T-cells including cytotoxic T-cells will result in immunisation of at least 50% of individuals exposed to said immunogenic composition or vaccine, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 92%, such as at least 94%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 98.5%, for example at least 99%, for example at least 99.5% of the individuals exposed to said immunogenic composition or vaccine are immunised.
  • compositions according to the invention may also comprise any carrier and/or adjuvant known in the art including functional equivalents thereof.
  • Functionally equivalent carriers are capable of presenting the same immunogenic determinant in essentially the same steric conformation when used under similar conditions.
  • Functionally equivalent adjuvants are capable of providing similar increases in the effi- cacy of the composition when used under similar conditions.
  • the invention further relates to a method of treating individuals suffering from a corneal disease, in particular keratoconus.
  • the invention relates to a method of substitution therapy, ie. administration of genetic material generally expressed in normal cells, but lost or decreased in cells suffering from keratoconus.
  • substitution therapy ie. administration of genetic material generally expressed in normal cells, but lost or decreased in cells suffering from keratoconus.
  • the invention relates to a method for reducing biological abnormalities of a cornea cell suffering from keratoconus, said method comprising contacting a cell from a keratoconus suffering cornea with at least one peptide expressed by at least one gene selected from genes being expressed in an amount at least two-fold higher in normal cells than the amount expressed in said keratoconus cell.
  • the at least one gene is selected independently from genes being expressed in an amount at least three-fold higher in normal cells than the amount expressed in said keratoconus cell.
  • the keratoconus cell is contacted with at least two different peptides.
  • the invention relates to a therapy whereby genes generally connected to disease are inhibited by one or more of the following methods:
  • a method for reducing biological abnormalities of a cornea cell suffering from keratoconus comprising,
  • the at least one gene is selected independently from genes being expressed in an amount at least three-fold higher in normal cells than the amount expressed in said keratoconus cell.
  • a method for reducing cell tumorigenicity of a cell comprising
  • a method for reducing biological abnormalities of a cornea cell suffering from keratoconus comprising
  • At least one nucleotide probe capable of hybridising with at least one gene of a cell from a keratoconus suffering cornea, said at least one gene being selected from genes being expressed in an amount two-fold lower in normal cells than the amount expressed in said keratoconus cell, and introducing said at least one nucleotide probe into the keratoconus cell in a manner allowing the probe to hybridise to the at least one gene, thereby inhibiting expression of said at least one gene.
  • the probes consists of the sequences identified above.
  • hybridization conditions are of low to moderate stringency. These conditions favour specific interactions between completely complementary sequences, but allow some non-specific interaction between less than perfectly matched se- quences to occur as well.
  • the nucleic acids can be "washed” under moderate or high conditions of stringency to dissociate duplexes that are bound together by some non-specific interaction (the nucleic acids that form these duplexes are thus not completely complementary).
  • the optimal conditions for washing are determined empirically, often by gradually increasing the stringency.
  • the parameters that can be changed to affect stringency include, primarily, temperature and salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency. Washing can be initiated at a low temperature (for example, room temperature) using a solution containing a salt concentration that is equivalent to or lower than that of the hybridization solution. Subsequent washing can be carried out using progressively warmer solutions having the same salt concentration. As alternatives, the salt concentration can be lowered and the temperature maintained in the washing step, or the salt concentration can be lowered and the tem- perature increased. Additional parameters can also be altered. For example, use of a destabilizing agent, such as formamide, alters the stringency conditions.
  • nucleic acids In reactions where nucleic acids are hybridized, the conditions used to achieve a given level of stringency will vary. There is not one set of conditions, for example, that will allow duplexes to form between all nucleic acids that are 85% identical to one another; hybridization also depends on unique features of each nucleic acid. The length of the sequence, the composition of the sequence (for example, the content of purine-like nucleotides versus the content of pyrimidine-like nucleotides) and the type of nucleic acid (for example, DNA or RNA) affect hybridization. An additional consideration is whether one of the nucleic acids is immobilized (for example, on a filter).
  • An example of a progression from lower to higher stringency conditions is the fol- lowing, where the salt content is given as the relative abundance of SSC (a salt solution containing sodium chloride and sodium citrate; 2X SSC is 10-fold more concentrated than 0.2X SSC).
  • Nucleic acids are hybridized at 42°C in 2X SSC/0.1% SDS (sodium dodecylsulfate; a detergent) and then washed in 0.2X SSC/0.1% SDS at room temperature (for conditions of low stringency); 0.2X SSC/0.1% SDS at 42°C (for conditions of moderate stringency); and 0.1X SSC at 68°C (for conditions of high stringency).
  • Washing can be carried out using only one of the conditions given, or each of the conditions can be used (for example, washing for 10-15 minutes each in the order listed above). Any or all of the washes can be repeated. As mentioned above, optimal conditions will vary and can be determined empirically.
  • a method of reducing biological abnormalities of comeal cells suffering from keratoconus relates to the use of antibodies against an expression product of a cell from a corneal tissue.
  • the antibodies may be produced by any suitable method, such as a method comprising the steps of
  • the methods described above may be used for producing an assay for diagnosing a corneal disease in human tissue. Furthermore, the invention relates to the use of a peptide as defined above for preparation of a pharmaceutical composition for the treatment of a corneal disease in human tissue.
  • the invention relates to the use of a gene as defined above for preparation of a pharmaceutical composition for the treatment of a corneal disease in human tissue.
  • the invention relates to the use of a probe as defined above for preparation of a pharmaceutical composition for the treatment of a of a comeal disease in human tissue.
  • the genetic material discussed above for may be any of the described genes or functional parts thereof.
  • the constructs may be introduced as a single DNA molecule encoding all of the genes, or different DNA molecules having one or more genes.
  • the constructs may be introduced simultaneously or consecutively, each with the same or different markers.
  • the gene may be linked to the complex as such or protected by any suitable, system normally used for transfection such as viral vectors or artificial viral envelope, liposomes or micellas, wherein the system is linked to the complex.
  • Vectors containing useful elements such as selectable and/or amplifiable markers, promoter/enhancer elements for expression in mammalian, particularly human, cells, and which may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art. Many are commercially available. Various techniques have been developed for modification of target tissue and cells in vivo. A number of virus vectors, discussed below, are known which allow transfection and random integration of the virus into the host. See, for example, Duben- sky et al. (1984) Proc. Natl. Acad. Sci. USA 81:7529-7533; Kaneda et al., (1989) Science 243:375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86:3594-
  • Routes and modes of administering the vector include injection, e.g intravascularly or intramuscularly, inhalation, or other parenteral administration.
  • adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms.
  • vaccinia virus which can be rendered non-replicating (U.S. Pat. Nos. 5,225,336; 5,204,243; 5,155,020; 4,769,330).
  • AVE artificial viral envelopes
  • the envelope is preferably produced in a two-step dialysis procedure where the "naked" envelope is formed initially, followed by unidirectional insertion of the viral surface glycoprotein of interest. This process and the physical characteristics of the resulting AVE are described in detail by
  • vesicles are constructed which mimic the natural membranes of enveloped viruses in their ability to bind to and deliver materials to cells bearing corresponding surface receptors.
  • AVEs are used to deliver genes both by intravenous injection and by instillation in the lungs.
  • AVEs are manufactured to mimic RSV, exhibiting the RSV F surface glycoprotein which provides selective entry into epithelial cells.
  • F-AVE are loaded with a plasmid coding for the gene of interest, (or a reporter gene such as CAT not present in mammalian tissue).
  • the AVE system described herein in physically and chemically essentially identical to the natural virus yet is entirely "artificial", as it is constructed from phospholipids, cholesterol, and recombinant viral surface glycoproteins. Hence, there is no carryover of viral genetic information and no danger of inadvertant viral infection. Con- struction of the AVEs in two independent steps allows for bulk production of the plain lipid envelopes which, in a separate second step, can then be marked with the desired viral glycoprotein, also allowing for the preparation of protein cocktail formulations if desired.
  • Another delivery vehicle for use in the present invention are based on the recent description of attenuated Shigella as a DNA delivery system (Sizemore, D. R. et al., Science 270:299-302 (1995), which reference is incorporated by reference in its entirety).
  • This approach exploits the ability of Shigellae to enter epithelial cells and escape the phagocytic vacuole as a method for delivering the gene construct into the cytoplasm of the target cell. Invasion with as few as one to five bacteria can result in expression of the foreign plasmid DNA delivered by these bacteria.
  • a preferred type of mediator of nonviral transfection in vitro and in vivo is cationic (ammonium derivatized) lipids. These positively charged lipids form complexes with negatively charged DNA, resulting in DNA charged neutralization and compaction. The complexes endocytosed upon association with the cell membrane, and the DNA somehow escapes the endosome, gaining access to the cytoplasm.
  • Cationic lipid DNA complexes appear highly stable under normal conditions.
  • Studies of the cationic lipid DOTAP suggest the complex dissociates when the inner layer of the cell membrane is destabilized and anionic lipids from the inner layer displace DNA from the cationic lipid.
  • Several cationic lipids are available commercially.
  • the first approach was based on a spreadsheet approach in which the fold change and the pattern of expression being present or absent in the different preparations of RNA was used.
  • the corneal disease keratoconus has been studied by using oligo nucleotide arrays and gene expression changes that define this disease compared to normal cornea and per se have been identified.
  • Protein p27 / kip1 a cyclin-dependent kinase inhibitor, regulates progression from G1 into S phase.
  • Hanne P has abs against p16, p21 , pRB and l-myc, which are in the same pathway as p27.
  • L-myc is NC from Ns to Np and Ks to KP, but is decreasing by a factor of 3 from Np to Kp. See also Figure 1.
  • polypeptide growth factor with growth and differentiation promoting activity Expressed in cell matrix, polypeptide growth factor with growth and differentiation promoting activity; expressed from paternal allele; binds vitronectin (see Figure 3)
  • Protein Metallothionein hMT-le Described in the nucleus and cytoplasm of a subset of astrocytes increased in the spinal cord of patients with amyotrophic lateral sclerosis, resulted from inflammation or gliosis (see Figure 5).
  • Keratin 6 Keratin 6 (KRT6 isoforms A.C.E)
  • Protein beta 2-microglobulin
  • the invention describes the many alterations of gene expressions in keratoconus compared with normal cornea that can be used for: diagnosis of keratoconus disease, establishing a method for diagnosing keratoconus disease, preventing keratoconus disease, establishing a method for preventing keratoconus disease, treat keratoconus disease, establishing a method for treating keratoconus disease, predict the disease course of keratoconus disease, establishing a method for predicting the disease course of keratoconus disease.
  • Keratoconus (S37)-cornea from patient S37 having keratoconus disease Keratoconus (S37)-cornea from patient S37 having keratoconus disease.
  • Enclosure 1 absolute measurement of gene expression in all three samples. In the tables in enclosure one (A, B, C) the absolute level of genes expressed in cornea can be seen. The first column describes the material used, the column marked "Avg Diff" describes the level of the gene transcript.
  • Enclosure 2 1) comparison of gene expression in keratoconus versus normal cornea from individual S28, 2) comparison of gene expression in keratoconus versus normal cornea pool, 3) comparison of gene expression in normal cornea from individual S28 and normal cornea pool.
  • the first column describes the corneas that are compared (see above), the column labelled "fold change” describes the change in expression level. It is clear that several genes changes as a result of keratoconus disease. See for example first line, human GABA receptor increased 3 fold in keratoconus from S37 compared to normal cornea. All the genes that alter more that 2 fold are candidates for exploitation, can be used for the mentioned purposes and are the subjects for patenting.

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Abstract

L'invention concerne un procédé permettant d'une part, de déterminer la présence ou l'absence de maladies de la cornée chez l'homme, et d'autre part, de déterminer le stade de l'affection des tissus cornéens humains par la détermination du schéma d'expression d'un échantillon de cellules cornéennes. En outre, l'invention concerne un procédé permettant de déterminer la présence ou l'absence d'une maladie de la cornée au niveau de tissus humains, de déterminer le stade d'avancement d'une maladie de la cornée au niveau de tissus humains, et de réduire également les anomalies biologiques d'une cellule cornéenne atteinte d'un kératocône. L'invention concerne également un procédé de production d'anticorps dirigés contre le produit d'expression d'une cellule tirée des tissus cornéens. L'invention concerne aussi une composition pharmaceutique, destinée au traitement d'une maladie de la cornée, qui contient au moins un anticorps, ainsi qu'un vaccin prophylactique ou thérapeutique contre une maladie de la cornée. L'invention concerne enfin l'utilisation d'un procédé de production d'un dosage permettant de diagnostiquer une maladie de la cornée dans des tissus humains, l'utilisation d'un peptide, d'un gène ou d'une sonde pour la préparation d'une composition pharmaceutique destinée au traitement d'une maladie de la cornée au niveau de tissus humains, et un dosage servant à déterminer la présence ou l'absence d'une maladie de la cornée au niveau de tissus humains et à déterminer le schéma d'expression d'une cellule touchée par ladite maladie de la cornée.
PCT/DK2000/000745 1999-12-29 2000-12-29 Expression genique et maladies de la cornee WO2001050133A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046567A2 (fr) * 2001-11-23 2003-06-05 Syn. X Pharma, Inc. Marqueurs de biolpolymeres proteiniques permettant de predire la maladie d'alzheimer
EP2507394A1 (fr) * 2009-12-02 2012-10-10 The University of Sydney Méthodes diagnostiques et thérapeutiques pour kératectasie à la suite d'une chirurgie réfractive, d'un kératocône ou d'une dégénérescence pellucide
WO2014106833A1 (fr) * 2013-01-07 2014-07-10 Singapore Health Services Pte Ltd Procédé d'identification de biomarqueurs pour la progression d'un kératocône
ES2564426A1 (es) * 2014-09-19 2016-03-22 Universidad De Oviedo Marcador de patologías oculares

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589451A (en) * 1992-09-21 1996-12-31 Board Of Regents, The University Of Texas System Methods and treaments for corneal healing with hepatocyte and keratinocyte growth factors
WO1998053319A2 (fr) * 1997-05-21 1998-11-26 The Johns Hopkins University Profils d'expression de genes dans des cellules normales et cancereuses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589451A (en) * 1992-09-21 1996-12-31 Board Of Regents, The University Of Texas System Methods and treaments for corneal healing with hepatocyte and keratinocyte growth factors
WO1998053319A2 (fr) * 1997-05-21 1998-11-26 The Johns Hopkins University Profils d'expression de genes dans des cellules normales et cancereuses

Non-Patent Citations (2)

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Title
BROWN D J ET AL: "Differential Gene Expression in Keratoconus Keratocytes", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 38, no. 4, 15 March 1997 (1997-03-15), pages s960, XP002901683 *
SUJATA CHIPLUNKAR ET AL: "Enhanced Expression of a Transmembrane Phosphotyrosine Phosphatase ( LAR ) in Keratoconus Cultures and Corneas", EXP. EYE RES., vol. 68, - 1999, pages 283 - 293, XP002901682 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046567A2 (fr) * 2001-11-23 2003-06-05 Syn. X Pharma, Inc. Marqueurs de biolpolymeres proteiniques permettant de predire la maladie d'alzheimer
WO2003046567A3 (fr) * 2001-11-23 2004-05-27 Syn X Pharma Inc Marqueurs de biolpolymeres proteiniques permettant de predire la maladie d'alzheimer
EP2507394A1 (fr) * 2009-12-02 2012-10-10 The University of Sydney Méthodes diagnostiques et thérapeutiques pour kératectasie à la suite d'une chirurgie réfractive, d'un kératocône ou d'une dégénérescence pellucide
EP2507394A4 (fr) * 2009-12-02 2013-06-12 Univ Sydney Méthodes diagnostiques et thérapeutiques pour kératectasie à la suite d'une chirurgie réfractive, d'un kératocône ou d'une dégénérescence pellucide
WO2014106833A1 (fr) * 2013-01-07 2014-07-10 Singapore Health Services Pte Ltd Procédé d'identification de biomarqueurs pour la progression d'un kératocône
ES2564426A1 (es) * 2014-09-19 2016-03-22 Universidad De Oviedo Marcador de patologías oculares

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