Patent Application
20080317741
This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to aspects of gene expression induced by administration of anti-Nogo-A antibody.
Nogo-A plays an important role in inhibition of neurite outgrowth. Antibodies against Nogo-A have been shown to result in axonal regeneration and functional recovery after spinal cord injury.
A number of microarray gene expression profiling studies have addressed molecular changes after spinal cord injury. For a review, see Bareyre F M & Schwab M E, Trends Neurosci. 26:555-563 (2003). However, there continues to be a need in the art for early peripheral biomarkers for efficacy of the anti-Nogo-A antibody treatment. Such biomarkers would be useful in differentiating the responders from non-responders as well as guiding the dosing in a clinical setting.
The invention provides a description of the molecular changes resulting from inhibition of Nogo-A function using anti-Nogo-A antibodies. Genes and functional pathways affected by inhibition or reduction of Nogo-A have been identified in an in vivo system using a genomics approach.
The invention also relates to novel molecular targets to enhance central nervous system recovery, to enhance regeneration of neuronal connections and to enhance neuronal and synaptic plasticity in clinical conditions such as but not exclusively injury such as trauma or stroke, neurodegenerative disorders such as but not exclusively Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, depression and any other disorder where axonal or dendritic pathology is part of the disease process or result of the disease, such as but not exclusively any demyelinating disorders, such as multiple sclerosis. It also relates to novel indications for targeting Nogo-A and/or genes and pathways affected as a result of inhibition of Nogo-A such as but not exclusively neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS) depression and any other disorder where axonal or dendritic pathology is part of the disease process or result of the disease, such as but not exclusively any demyelinating disorders, such as multiple sclerosis.
In particular, the present invention relates to a method for predicting the response of a subject to a medicament comprising an anti-Nogo-A antibody, wherein the expression of at least one gene of Table 25 is assessed before and after administration of said medicament comprising an anti-Nogo-A antibody and wherein said expression of said at least one gene of Table 25 after administration of said medicament comprising an anti-Nogo-A antibody is compared to the expression of said gene prior to said administration of the medicament comprising an anti-Nogo-A antibody. In a particular embodiment, a dysregulation of said expression of at least one gene of Table 25 after administration of the medicament comprising an anti-Nogo-A antibody as compared to the expression of said gene prior said administration of the medicament comprising an anti-Nogo-A antibody is indicative of a positive response (responder) to said administration of the medicament comprising an anti-Nogo-A antibody. In another embodiment, the lack of a dysregulation of said expression of at least one gene of Table 25 after administration of the medicament comprising an anti-Nogo-A antibody as compared to the expression of said gene prior said administration of the medicament comprising an anti-Nogo-A antibody is indicative of a lack of response (non-responder) to said administration of the medicament comprising an anti-Nogo-A antibody. In a preferred embodiment, said dysregulation of said expression of at least one gene of Table 25 after administration of the medicament comprising an anti-Nogo-A antibody is a change in expression that is larger or equal to 1.2 fold and statistically significant (p<0.05, Student's t-test) as compared to the expression of said gene prior said administration of the medicament comprising an anti-Nogo-A antibody. In a most preferred embodiment, the expression of at least one gene of each of the groups of adhesion genes, cytoskeleton genes and signalling genes is assessed, wherein said group of adhesion genes consists of cadherin 11, cadherin 2, cadherin 8, cadherin 22, Eph receptor A3, Eph receptor A4, Ephrin A3, Ephrin B2, Eph receptor B2, semaphorin 4A, semaphorin 4D, semaphorin 4F, semaphorin 6A, semaphorin 6B, semaF cytoplasmic domain associated protein 3 and Plexin B2, wherein said group of cytoskeleton genes consist of capping protein (actin filament) gelsolin-like, casein kinase 1 delta, centractin, gelsolin, microtubule-associated protein tau and neurofilament 68, and wherein said group of signalling genes consists of Rho-GDP-dissociation inhibitor 1, dihydropyrimidinase related protein 2, dihydropyrimidinase related protein 1, dihydropyrimidinase related protein 5. In another embodiment, the expression of all the genes of Table 25 is assessed.
In one embodiment of the present invention, a dysregulation of the expression of at least one gene of Table 25 after administration of the medicament comprising an anti-Nogo-A antibody as compared to the expression of said gene prior said administration of the medicament comprising an anti-Nogo-A antibody is indicative of indicates central nervous system regeneration.
The methods of the invention can be performed in vitro.
Also encompassed with the present invention is the use of an anti-Nogo-A antibody in the manufacture of a medicament for the treatment of central nervous system injury in a patient population, wherein the patient population is selected as described herein.
Preferably, the anti-Nogo-A antibody is a fully human monoclonal antibody (IgG4/□) that binds to the epitope of human Nogo-A fragment from amino acid 342-357.
The present invention also relates to methods for treating a central nervous system injury in a subject with an anti-Nogo-A antibody, as well as methods for diagnosing central nervous system regeneration in a subject after administering of an anti-Nogo-A.
Moreover, the present invention also encompasses a kit for performing the methods described herein, said kit comprising at least two probes, each probe being capable of specifically detecting the expression of one gene of Table 25, wherein said at least two probes do not detect the expression of the same gene.
Genes and molecular pathways affected by inhibition of Nogo-A can by themselves be therapeutically targeted for similar disorders as those treatable by Nogo-A antibody therapy. Alternatively, novel therapeutics designed for the genes and pathways affected by inhibition of Nogo-A can be used as add-on therapies to enhance the therapeutic effect of Nogo-A inhibition. In addition, the genes and pathways affected by inhibition of Nogo-A provide therapeutic indications for inhibition of Nogo-A such as but not exclusively conditions where neuronal or synaptic plasticity has been challenged such as cognitive impairments related neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, Huntington's disease) and psychiatric disorders.
The drawing figures depict preferred embodiments by way of example, not by way of limitations. In the figures, like reference numerals refer to the same or similar elements.
It is to be appreciated that certain aspects, modes, embodiments, variation and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. In general, such disclosure provides useful biomarkers for the diagnosis and treatment of subjects in need thereof. Accordingly, the various aspects of the present invention relate to diagnostic/theranostic methods and kits to identify individuals predisposed to disease or to classify individuals with regard to drug responsiveness, side effects, or optimal drug dose. The methods and kits are useful for studying the aetiology of diseases, studying the efficacy of drug targeting, predicting individual susceptibility to diseases, and predicting individual responsiveness to drugs targeting the gene product. Accordingly, various particular embodiments that illustrate these aspects follow.
Polynucleotides and Polypeptides of the Invention. Gene expression profiling in a rat spinal cord injury model was undertaken after mouse monoclonal anti-Nogo-A antibody 11C7-treatment and compared to control mouse anti-plant lectin IgG after seven and 14 days of treatment in different tissues, resulting in 12 different comparisons. The datasets were subjected to the following analyses: (1) statistical restriction (Welch t-test p<0.05) and ranking by fold change; and (2) gene set enrichment analysis (GSEA), which is a pathway centric view of the data first introduced by Mootha V K et al., Nat. Genet. 34:267-273 (2003) and recently by Subramanian et al. Proc. Natl. Acad. Sci. USA 102(43): 15545-50 (2005). The analysis resulted in identification of 24 pathways significantly affected by the treatment in three or more of the tissues at either timepoint.
Ranked by the treatment effect size based on the number of significantly differentially expressed genes and the fold change of the top 100 significantly changed transcripts in each treatment group, spinal cord distal to the site of lesion (L1-5), the site of the lesion (T8) and blood were the most affected tissues after one week of treatment. L1-5, motor-somatosensory cortex and spinal cord proximal to the site of lesion (T1-7) were the most affected regions after two weeks of treatment. At either timepoint, only minimal effect in the frontal cortex was observed.
GSEA identified immunity and defence, protein metabolism and phosphorylation, nucleoside, nucleotide and nucleic acid metabolism, neuronal activities and Jak-stat cascade as the most widely affected pathways overall. All of these pathways were affected in three to four tissues concomitantly.
Anti-Nogo-A treatment applied intrathecally after spinal cord injury in rat has the largest effect in spinal cord. Genes promoting axon guidance and neurite outgrowth were upregulated, inhibitory cues downregulated in spinal cord after anti-Nogo-A treatment. Of the neurite outgrowth/axon guidance related pathways, GSEA pointed the slit-robo mediated axon guidance pathway as most frequently affected by 11C7 treatment. Cxcl12 and Cxc4r, two members of this pathway were upregulated by 11C7 in a concerted fashion after one week of treatment in all segments of the spinal cord studied. Cxcl12 and Cxc4r were recently identified as key players in defining the initial trajectory of mammalian motor axons during development by Lieberam I et al., Neuron 47:667-679 (2005). This finding suggests that this pathway is affected by 11C7 treatment and may thus contribute to the mechanism of action of anti-Nogo A during regeneration.
At the site of the lesion, the EGF-receptor mediated signalling pathway was upregulated by 11C7 after one week of treatment but downregulated after two weeks of treatment. In the motor cortex, the EGF-receptor mediated signalling pathway was downregulated by 11C7 after one week and after two weeks of treatment. Altogether 24 pathways with significant enrichment (q<0.001) were identified to be affected by anti-Nogo-A treatment in three or more tissues at either timepoint. The most widely affected pathways overall were related to immunity and defence, protein metabolism and phosphorylation and neuronal activities. Upregulation of synaptic transmission related probesets in lumbar spinal cord after two weeks of anti-Nogo-A treatment.
The results confirm at the level of gene expression the injured spinal cord and motor cortex as the primary sites of action of the anti-Nogo-A antibody treatment applied intrathecally. The analysis identified novel molecular and pathways candidates as possible targets of anti-Nogo-A treatment, such as myocilin and the split-robo pathway. The results also pointed to strong involvement of immune defence related pathways in the treatment effect.
TAQMAN analysis confirmed selected findings concerning the secreted proteins Sfrp4, Mmp9 and myocilin.
Anti-Nogo antibodies. Published PCT patent application WO 00/31235 discloses several antibodies raised against Nogo proteins and derivatives thereof. For examples of anti-Nogo antibodies, including monoclonal antibodies and fragments thereof, and of methods of their use, see Bregman B S et al., Nature 378:498-501 (1995); Brosamle C et al., J. Neurosci. 20:8061-8068 (2000); Bareyre F M et al, J. Neurosci. 22:7097-7110 (2002); Chen et al., Nature 403:434-439 (2000); Fiedler M et al., Protein Eng. 15:931-941 (2002); Merkler D et al., J. Neurosci. 21:3665-3673 (2001); Oertle T et al., J. Neurosci. 23:5393-5406 (2003); Papadopoulos C M et al., Ann. Neurol. (2002); and Von Meyenburg J et al, Exp. Neurol. 154:583-594 (1998). See also, Wiessner C et al., In Pharmacology of Cerebral Ischemia, Krieglstein J & Klumpp S, eds. (2003) pp. 343-353; and Wiessner C et al., J. Cereb. Blood Flow & Metab. 23: 154-165 (2003) for the use of anti-Nogo antibodies in a stroke model. Doses of anti-Nogo A antibody used in the EXAMPLES have been shown to result in functional recovery in the same model. Liebscher et al., Ann. Neurol. 58:706-719 (2005). Published PCT patent application WO 00/31235 also discloses two antisera raised against Nogo A sequences, AS Bruna and AS 472. See also published PCT patent application WO 2000/05364A1, which discloses antibodies to Nogo protein fragments. In the EXAMPLES, anti Nogo-A antibody 11C7: Mouse monoclonal antibody (mAb) 11C7, raised against a 18aa peptide Nogo-A corresponding to rat sequence amino acids 623-640; used at a concentration of 3 mg/ml in PBS. The control antibody was a mouse monoclonal IgG directed against plant lectin used at a concentration of 3 mg/ml in PBS. The biochemical and neutralizing properties of both antibodies are described in Oertle T et al., J. Neurosci. 23:5393-5406 (2003). In one embodiment of the invention, the anti-Nogo antibody is a fully human monoclonal antibody (IgG4/κ) generated from mice which are genetically reconstituted with human immunoglobulin genes and which binds to the epitope of human Nogo-A fragment from aa342-357. See Published PCT patent applications WO 90/05191 and WO 00/31235.
Accordingly, the invention is relevant to ischemic brain injury (stroke), traumatic brain injury (head injury), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease. The invention is also relevant to axonal regeneration and improved sprouting after nerve fibre damage; various diseases of the peripheral and central nervous system, neurodegenerative diseases such as Alzheimer disease, Parkinson disease, ALS, Lewy like pathologies or other dementia in general, diseases following cranial, cerebral or spinal trauma, stroke or a demyeliating disease including multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, pontine myelmolysis, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease; degenerative ocular disorders involving the degeneration of retinal or corneal cells including ischemic retinopathies, anterior ischemic optic neuropathy, optic neuritis, age-related macular degeneration, diabetic retinopathy, cystoid macular oedema, retinitis pigmentosa, Stargardt's disease, Best's vitelliform retinal degeneration, Leber's congenital amaurosis and other hereditary retinal degenerations, pathologic myopia, retinopathy of prematurity, Leber's hereditary optic neuropathy, the after effects of corneal transplantation or of refractive corneal surgery, herpes keratitis.
Definitions. The definitions of certain terms as used in this specification are provided below. Definitions of other terms may be found in the glossary provided by the U.S. Department of Energy, Office of Science, Human Genome Project (http://www.ornl.gov/sci/techresources/Human_Genome/glossary/). In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover D, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, eds. (1985); Transcription and Translation, Hames & Higgins, eds. (1984); Animal Cell Culture, Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Methods in Enzymol. (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, eds. (Cold Spring Harbor Laboratory, New York, 1987); and Methods in Enzymology, Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.
As used herein, the term “antibody” includes, but is not limited to, e.g., polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments sufficient for binding of the antibody fragment to the protein. In an embodiment of the invention, the antibody is an anti-Nogo antibody.
The term “biological sample” is intended to include, but is not limited to, e.g., tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. In the EXAMPLES, the biological samples are central nervous system samples. However, the use of other biological samples is envisioned. Suitable “biological samples” are for instance blood, serum, lymph, endothelial cells, sputum, urine, faeces or semen. Particularly suited for the methods of the invention are central nervous system (CNS) interstitial fluid and/or cerebrospinal fluid (CSF).
As used herein, the term “clinical response” means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
As used herein, the term “clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes, but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
As used herein, the term “effective amount” of a compound is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of, or a decrease in the symptoms associated with, a disease that is being treated. The amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount of the compounds of the present invention, sufficient for achieving a therapeutic or prophylactic effect range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. A preferred dosage ranges from about 0.0001 mg per kilogram body weight per day to about 1,000 mg per kilogram body weight per day. Another preferred dosage ranges from about 0.01 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. The compounds of the present invention can also be administered in combination with each other, or with one or more additional therapeutic compounds. In the EXAMPLES, doses of anti-Nogo A antibody used in the EXAMPLES have been shown to result in functional recovery in the same model. Liebscher et al., Ann. Neurol. 58:706-719 (2005). See also published PCT patent application WO 2000/05364A1, which discloses antibodies to Nogo protein fragments.
As used herein, “expression” includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and mRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
As used herein, the term “genotype” means an unphased 5′ to 3′ sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual. As used herein, genotype includes a full-genotype and/or a sub-genotype.
As used herein, the term “locus” means a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
As used herein, the term “isogene” means the different forms of a given gene that exist in the population.
As used herein, the term “mutant” means any heritable variation from the wild-type that is the result of a mutation, e.g., single nucleotide polymorphism. The term “mutant” is used interchangeably with the terms “marker”, “biomarker”, and “target” throughout the specification.
As used herein, the term “medical condition” includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
As used herein, the term “nucleotide pair” means the nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.
As used herein, the term “polymorphic site” means a position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
As used herein, the term “population” may be any group of at least two individuals. A population may include, e.g., but is not limited to, a reference population, a population group, a family population, a clinical population, and a same sex population.
As used herein, the term “phased” means, when applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, the combination of nucleotides present at those polymorphic sites on a single copy of the locus is known.
As used herein, the term “polymorphism” means any sequence variant present at a frequency of >1% in a population. The sequence variant may be present at a frequency significantly greater than 1% such as 5% or 10% or more. Also, the term may be used to refer to the sequence variation observed in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
As used herein, the term “polynucleotide” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
As used herein, the term “polypeptide” means any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well-known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
As used herein, the term “reference standard population” means a population characterized by one or more biological characteristics, e.g., drug responsiveness, genotype, haplotype, phenotype, etc.
As used herein, the term “reference standard gene expression profile” is the pattern of expression of one or more gene observed in either a reference standard population or a single subject prior to administration of a compound.
As used herein, the term “subject” means that preferably the subject is a mammal, such as a human, but can also be an animal, including but not limited to, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkeys such as cynmologous monkeys, rats, mice, guinea pigs and the like).
As used herein, a “test sample” means a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue, or isolated nucleic acid or polypeptide derived therefrom.
As used herein, the term “dysregulation” means a change that is larger or equal to 1.2 fold and statistically significant (p<0.05, Student's t-test) from the control. For example, a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 fold change.
As used herein, the administration of an agent or drug to a subject or patient includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Amplifying a Target Gene Region. The target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR). (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA, 88:189-193 (1991); published PCT patent application WO 90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al, Science, 241:1077-1080 (1988)). Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymorphic site. Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems. (U.S. Pat. No. 5,130,238; EP 0 329 822; U.S. Pat. No. 5,169,766, published PCT patent application WO 89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci., USA, 89:392-396 (1992).
Hybridizing Allele-Specific Oligonucleotide to a Target Gene. Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lysine, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking, baking, etc. Allele-specific oligonucleotide may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibres, chips, dishes, and beads. The solid support may be treated, coated or derivatised to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
The genotype or haplotype for the gene of an individual may also be determined by hybridization of a nucleic sample containing one or both copies of the gene to nucleic acid arrays and subarrays such as described in WO 95/11995. The arrays would contain a battery of allele-specific oligonucleotides representing each of the polymorphic sites to be included in the genotype or haplotype.
See, also, Molecular Cloning A Laboratory Manual, Second Ed., Sambrook, Fritsch & Maniatis, ed. (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II, Glover D N ed. (1985); Oligonucleotide Synthesis, Gait M J ed. (1984); Nucleic Acid Hybridization, Hames B D & Higgins S J, eds., 1984).
Computer System for Storing or Displaying Gene Expression or Polymorphism Data. The invention also provides a computer system for storing and displaying data determined for the gene. Polymorphism data is information that includes, but is not limited to, e.g., the location of polymorphic sites; sequence variation at those sites; frequency of polymorphisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene. The computer system comprises a computer processing unit, a display, and a database containing the polymorphism data. The polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for a given gene in a reference population. In a preferred embodiment, the computer system is capable of producing a display showing gene expression pattern organized according to their evolutionary relationships.
In addition, the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information, relating to the gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymorphism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, and gene expression pattern for one or more populations). The polymorphism data described herein maybe stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymorphism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.
In the EXAMPLE below, the data normalization was performed as follows: Values below 0 were set to 0.1. Each measurement was divided by the 50.0th percentile of all measurements in that sample. Finally, per gene normalization was performed by normalizing to the expression value of the median of naïve samples.
In EXAMPLE 1, differentially expressed genes between the vehicle and the treatments were identified within each experiment based on the following restrictions: (1) Prefiltering restrictions: Probe sets included in further analysis had to flagged present in 4/6 of replicates in any condition. Raw data signal intensity had to be minimum 50 in at least one of the treatment groups. (2) Statistical restriction: p<0.05 (Welch t-test (parametric)). Similar statistical restriction was always applied to different groups to be compared and is mentioned in each comparison.
In EXAMPLE 1, the Gene Set Enrichment Analysis (GSEA) method was used to analyze microarray data. Genes with expression levels below 100 on more than 75% of the chips are discarded as low- or non-expressed. Microarray results are then analyzed in a series of pairwise comparisons between sets of condition (e.g. treated vs. control). Each gene's relative expression level under condition1 and condition2 is computed as an expression ratio ri
where μi,j is the average expression value for gene i under conditionj. The genes are then sorted according to their expression ratios such that those genes with higher expression under condition1 than condition2 are at the top of the list. Next, the collection of available gene sets is projected onto the sorted list. This step in essence applies a priori biological knowledge to the experimental data to identify functionally related genes that are expressed in a coordinated fashion. Gene sets are processed one at a time. For gene set G each expression ratio ri is labelled ‘in’ the gene set if genei∈G and ‘out’ of the gene set if genej∉G. A two-tailed Wilcoxon rank-sum test is calculated to determine if the genes labelled ‘in’ gene set G are enriched at either the top or bottom of the sorted list. The false discovery rate method of Storey J D & Tibshirani R, Proc Natl Acad Sci USA 100:9440-9445 (2003) is applied to transform p-values to multiple testing corrected q-values. The output from GSEA is a list of q-values (q1, q2, . . . , qN) and labels (l1, l2, . . . , lN), li∈(top, bottom) that correspond to the N available gene sets. A small q-value qi indicates that the genes in gene set Gi are significantly enriched at either the top or bottom of the list of expression ratios.
EXAMPLE 2 also provides a description of a GSEA analysis method.
Kits of the Invention. It is to be understood that the methods of the invention described herein generally may further comprise the use of a kit according to the invention. The invention provides nucleic acid and polypeptide detection kits useful for haplotyping and/or genotyping the gene in an individual. Such kits are useful to classify subjects. Generally, the methods of the invention may be performed ex-vivo, and such ex-vivo methods are specifically contemplated by the present invention. Also, where a method of the invention may include steps that may be practised on the human or animal body, methods that only comprise those steps which are not practised on the human or animal body are specifically contemplated by the present invention.
The kits of the invention are useful for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample, e.g., any body fluid including, but not limited to, e.g., serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue. For example, the kit can comprise a labelled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide.
For antibody-based kits, the kit can comprise, e.g., (1) a first antibody, e.g., attached to a solid support, which binds to a polypeptide corresponding to a marker or the invention; and, optionally; (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
For oligonucleotide-based kits, the kit can comprise, e.g., (1) an oligonucleotide, e.g., a detectably-labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention.
The kit can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. In a preferred embodiment, such kit may further comprise a DNA sample collecting means. The kits of the invention may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, e.g., to use the biomarkers of the present invention in determining a strategy for preventing or treating a medical condition in a subject. In several embodiments, the use of the reagents can be according to the methods of the invention. In one embodiment, the reagent is a gene chip for determining the gene expression of relevant genes.
Correlating a Subject to a Standard Reference Population. To deduce a correlation between clinical response to a treatment and a gene expression pattern, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, i.e., a clinical population. This clinical data maybe obtained by retrospective analysis of the results of a clinical trial(s). Alternatively, the clinical data may be obtained by designing and carrying out one or more new clinical trials. The analysis of clinical population data is useful to define a standard reference populations which, in turn, is useful to classify subjects for clinical trial enrolment or for selection of therapeutic treatment. In a preferred embodiment, the subjects included in the clinical population have been graded for the existence of the medical condition of interest. Grading of potential subjects can include, e.g., a standard physical exam or one or more lab tests. Alternatively, grading of subjects can include use of a gene expression pattern. For example, gene expression pattern is useful as grading criteria where there is a strong correlation between gene expression pattern and disease susceptibility or severity. Such standard reference population comprising subjects sharing gene expression pattern profile characteristic(s). For example, biomarker gene expression characteristic(s), are useful in the methods of the present invention to compare with the measured level of one or more gene expression product in a given subject. This gene expression product(s) useful in the methods of the present invention include, but are not limited to, e.g., characteristic mRNA associated with that particular genotype group or the polypeptide gene expression product of that genotype group. In one embodiment, a subject is classified or assigned to a particular genotype group or class based on similarity between the measured levels of a one or more biomarkers in the subject and the level of the one or more biomarkers observed in a standard reference population.
In one embodiment of the invention, a therapeutic treatment of interest is administered to each subject in a trial population, and each subject's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses, and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
Statistical analysis methods, which may be used, are described in Fisher L D & vanBelle G, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, New York, 1993). This analysis may also include a regression calculation of which polymorphic sites in the gene contribute most significantly to the differences in phenotype.
An alternative method for finding correlations between haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms, one of which is a genetic algorithm (Judson R, “Genetic Algorithms and Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp 1-73, Lipkowitz K B and Boyd D B, eds, (VCH Publishers, New York, 1997). Simulated annealing (Press et al., Numerical Recipes in C: The Art of Scientific Computing, Ch. 10 (Cambridge University Press, Cambridge, 1992), neural networks (Rich E & Knight K, Artificial Intelligence, 2nd Edition, Ch. 10 (McGraw-Hill, New York, 1991), standard gradient descent methods (Press et al., supra Ch. 10), or other global or local optimization approaches can also be used.
Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymorphic sites in the gene. ANOVA is used to test hypotheses about whether a response variable is caused by, or correlates with, one or more traits or variables that can be measured. See, Fisher L D & vanBelle G, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, New York, 1993), Ch. 10.
After both the clinical and polymorphism data have been obtained, correlations between individual response and genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their genotype or haplotype (or haplotype pair) (also referred to as a polymorphism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism group are calculated.
The skilled artisan can construct a mathematical model that predicts clinical response as a function of genotype or haplotype from the analyses described above. The identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug. The diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymorphic sites in the gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying genotype or haplotype. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above.
Predictive Medicine. The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to treat prophylactically a subject. Accordingly, one aspect of the invention relates to diagnostic assays for determining biomarker molecule expression as well as biomarker molecule activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant biomarker molecule expression or activity.
The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with biomarker molecule expression or activity. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with a biomarker polypeptide.
The levels of certain polypeptides in a particular tissue (or in the blood) of a subject may be indicative of the toxicity, efficacy, rate of clearance or rate of metabolism of a given drug when administered to the subject. The methods described herein can also be used to determine the levels of such polypeptides in subjects to aid in predicting the response of such subjects to these drugs. Another aspect of the invention provides methods for determining mutant polypeptide activity in an individual to thereby select appropriate therapeutic or prophylactic compounds for that individual. Methods of the present invention allow for the selection of compounds (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular compound.)
Prognostic Assays. The binding of a prognostic compound to a biomarker molecule, e.g., biomarker polypeptide or nucleic acid encoding a biomarker polypeptide, can be utilized to identify a subject having or at risk of developing a disorder associated with biomarker polypeptide expression or activity (which are described above). A prognostic compound is any compound which binds to or associates with a biomarker molecule, including, but not limited to, e.g., anti-biomarker polypeptide antibody, small molecule, nucleic acid, polypeptide, oligosaccharide, lipid, or combination thereof. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing the disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with biomarker expression or activity in which a test sample is obtained from a subject and prognostic compound binding or activity is detected, wherein the presence of an alteration of prognostic compound binding or activity is diagnostic for a subject having, or at risk of developing, a disease or disorder associated with biomarker expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue, or isolated nucleic acid or polypeptide derived therefrom.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered a compound (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a biomarker-associated disease or disorder. As used herein, the administration of a compound to a subject or patient includes self-administration and the administration by another. In one embodiment, the prognostic assays described herein are used to determine if a subject will be responsive to a compound. For example, such methods can be used to determine whether a subject can be effectively treated with a therapeutic compound for a biomarker-associated disorder (i.e., biomarker-associated medical condition). Thus, the invention provides methods for determining whether a subject can be effectively treated with a compound for a disorder associated with biomarker expression or activity in which a test sample is obtained and biomarker molecule is detected using prognostic compound (e.g., wherein the presence, or altered level of expression of, the biomarker molecule compared with the level of expression of the biomarker in a reference is diagnostic for a subject that can be administered the compound to treat a biomarker-associated disorder.
There are a number of diseases in which the degree of overexpression (or underexpression) of certain biomarker molecules, i.e., biomarker-associated disease or medical condition, is known to be indicative of whether a subject will develop a disease. Thus, the method of detecting a biomarker in a sample can be used as a method of predicting whether a subject will develop a disease. The level of a one or more biomarkers in a suitable tissue or blood sample from a subject at risk of developing the disease is determined and compared with a suitable control, e.g., the level in subjects who are not at risk of developing the disease. The degree to which the one or more biomarkers is overexpressed (or underexpressed) in the sample compared with the control may be predictive of likelihood that the subject will develop the disease. The greater the overexpression (or underexpression) relative to the control, the more likely the subject will development the disease.
The methods described herein can be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe reagent, e.g., anti-biomarker polypeptide antibody described herein, which can be conveniently used, e.g., in clinical setting to diagnose patients exhibiting symptoms or family history of a disease or illness involving a biomarker of the invention. Furthermore, any cell type or tissue in which a biomarker of the invention is expressed can be utilized in the prognostic assays described herein.
Monitoring Clinical Efficacy. Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a biomarker (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied in basic drug screening and in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase biomarker gene expression, protein levels, or upregulate biomarker activity, can be monitored in clinical trials of subjects exhibiting decreased biomarker gene expression, protein levels, or downregulated biomarker activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease biomarker gene expression, protein levels, or downregulate biomarker activity, can be monitored in clinical trials of subjects exhibiting increased biomarker gene expression, protein levels, or upregulated biomarker activity. In such clinical trials, the expression or activity of a biomarker and, preferably, other genes that have been implicated in, for example, a proliferative disorder and cancers, can be used as a “read out” or marker of the responsiveness of a particular cell.
For example, genes, including genes encoding a biomarker of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates a biomarker activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of a biomarker and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
Gene Expression and Subject Classification. Standard control levels of a gene expression product are determined by measuring gene expression in different control groups. The control group gene expression levels are then compared with the measured level of a gene expression product in a given subject. This gene expression product could be the characteristic mRNA associated with that particular genotype group or the polypeptide gene expression product of that genotype group. The subject can be classified or assigned to a particular genotype group based on how similar the measured levels were compared to the control levels for a given group.
As one of skill in the art will understand, there will be a certain degree of uncertainty involved in making this determination. Therefore, the standard deviations of the control group levels can be used to make a probabilistic determination and the method of this invention are applicable over a wide range of probability-based genotype group determinations. Thus, for example, and not by way of limitation, in one embodiment, if the measured level of the gene expression product falls within 2.5 standard deviations of the mean of any of the control groups, then that individual may be assigned to that genotype group. In another embodiment if the measured level of the gene expression product falls within 2.0 standard deviations of the mean of any of the control groups then that individual may be assigned to that genotype group. In still another embodiment, if the measured level of the gene expression product falls within 1.5 standard deviations of the mean of any of the control groups then that individual may be assigned to that genotype group. In yet another embodiment, if the measured level of the gene expression product is 1.0 or less standard deviations of the mean of any of the control groups levels then that individual may be assigned to that genotype group.
Thus, this process allows determination, with various degrees of probability, which group a specific subject should be placed in, and such assignment to a genotype group would then determine the risk category into which the individual should be placed.
Detection of Biomarker Gene Expression. An exemplary method for detecting the presence or absence of mutant polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound, or a compound capable of detecting mutant polypeptide or nucleic acid (e.g., mRNA, genomic DNA) that encodes mutant polypeptide of the invention, such that the presence of mutant gene is detected in the biological sample. A compound for detecting mutant mRNA or mutant genomic DNA is a labelled nucleic acid probe capable of hybridizing to mutant mRNA or mutant genomic DNA. The nucleic acid probe can be, for example, a full-length mutant nucleic acid or a portion thereof, such as an oligonucleotide of at least 5,15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mutant mRNA or mutant genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein. An example of a compound for detecting a mutant polypeptide of the invention is an antibody raised against mutant polypeptide of the invention, capable of binding to the mutant polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labelled”, with regard to the probe or antibody, is intended to encompass direct labelling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labelling of the probe or antibody by reactivity with another compound that is directly labelled. Examples of indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin. That is, the detection method of the invention can be used to detect mutant mRNA, polypeptide, or genomic DNA of the invention in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mutant mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of mutant polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of mutant genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of mutant polypeptide include introducing into a subject a labelled anti-mutant polypeptide antibody. For example, the antibody can be labelled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In practicing the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, New York, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, eds., respectively. Methods to detect and measure mRNA levels (i.e., gene transcription level) and levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of nucleotide microarrays and polypeptide detection methods involving mass spectrometers and/or antibody detection and quantification techniques. See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., New York, 1999).
Techniques for the detection of gene expression of the genes described by this invention include, but are not limited to Northern blots, RT-PCT, real time PCR, primer extension, RNase protection, RNA expression profiling and related techniques. Techniques for the detection of gene expression by detection of the protein products encoded by the genes described by this invention include, but are not limited to, e.g., antibodies recognizing the protein products, western blots, immunofluorescence, immunoprecipitation, ELISAs and related techniques. These techniques are well known to those of skill in the art. Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2000). In one embodiment, the technique for detecting gene expression includes the use of a gene chip. The construction and use of gene chips are well known in the art. See, U.S. Pat. Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795,716 and 5,800,992. See also, Johnston M, Curr. Biol., 8:R171-174 (1998); Iyer V R et al., Science, 283:83-87 (1999) and Elias P, “New human genome ‘chip’ is a revolution in the offing” Los Angeles Daily News (Oct. 3, 2003).
In EXAMPLE 1 below, microarray hybridizations were conducted as recommended by the manufacturer of the microarray system (Affymetrix, Santa Clara, Calif.; Expression analysis technical manual). Six samples from each treatment group were individually hybridized (no pooling) on the rat genome RAE230 2.0 gene expression probe array set containing >31 000 probe sets (Affymetrix, Inc., Santa Clara, Calif., USA).
Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript Choice System (Invitrogen Life Technologies) in the presence of a T7-(dT)24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labelling Kit (ENZO) in the presence of biotinylated ribonucleotides form biotin labelled cRNA. The labelled cRNA was then purified on an affinity resin (RNeasy, Qiagen), quantified and fragmented. An amount of approximately 10 μg labelled cRNA was hybridized for approximately 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes) using the GeneChip Fluidics Workstation 400 (Affymetrix). The array was then scanned twice using a confocal laser scanner (GeneArray Scanner, Agilent) resulting in one scanned image. This resulting “.dat-file” was processed using the MAS5 program (Affymetrix) into a “.cel-file”. Raw data was converted to expression levels using a “target intensity” of 150.
Determination of Marker Gene Transcription. The determination of the level of the expression product of a marker gene in a biological sample, e.g., the tissue or body fluids of an individual, may be performed in a variety of ways. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells. See, e.g., Ausubel et al., ed., Curr. Prot. Mol. Biol. (John Wiley & Sons, NY, 1987-1999).
In one embodiment, the level of the mRNA expression product of a marker gene is determined. Methods to measure the level of a specific mRNA are well-known in the art and include Northern blot analysis, reverse transcription PCR and real time quantitative PCR or by hybridization to a oligonucleotide array or microarray. In other more preferred embodiments, the determination of the level of expression may be performed by determination of the level of the protein or polypeptide expression product of the gene in body fluids or tissue samples including but not limited to blood or serum.
In a particular embodiment, the level of mRNA corresponding to a marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. Additionally, large numbers of tissue samples can readily be processed using techniques well-known to those of skill in the art, such as, e.g., the single-step RNA isolation process of U.S. Pat. No. 4,843,155.
The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, PCR analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, e.g., a full-length cDNA, or a portion hereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth by Mullis, U.S. Pat. No. 4,683,232); ligase chain reaction, Barany (1991), supra; self-sustained sequence replication, Guatelli et al., Proc, Natl. Acad. Sci, USA, 87:1874-1878 (1990); transcriptional amplification system, Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173-1177 (1989); Q-Beta Replicase, Lizardi et al., Biol. Technolog, 6: 1197 (1988); rolling circle replication, U.S. Pat. No. 5,854,033; or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of the nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10-30 nucleotides in length and flank a region from about 50-200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
As noted above, RT-PCR (real-time quantitative PCR) is one way to assess gene expression levels, e.g., of genes of the invention (e.g., those containing SNPs and polymorphisms of interest). The RT-PCR assay utilizes an RNA reverse transcriptase to catalyze the synthesis of a DNA strand from an RNA strand, including an mRNA strand. The resultant DNA may be specifically detected and quantified and this process may be used to determine the levels of specific species of mRNA. One method for doing this is known under the Trademark TAQMAN (PE Applied Biosystems, Foster City, Calif.) and exploits the 5′ nuclease activity of AMPLITAQ GOLD™ DNA polymerase to cleave a specific form of probe during a PCR reaction. This is referred to as a TAQMAN™ probe. See Luthra et al., Am. J. Pathol., 153: 63-68 (1998)). The probe consists of an oligonucleotide (usually ≈20 mer) with a 5′-reporter dye and a 3′-quencher dye. The fluorescent reporter dye, such as FAM (6-carboxyfluorescein), is covalently linked to the 5′ end of the oligonucleotide. The reporter is quenched by TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) attached via a linker arm that is located at the 3′ end. See Kuimelis et al, Nucl. Acids Symp. Ser., 37: 255-256 (1997) and Mullah et al, Nucl. Acids Res., 26(4):1026-1031 (1998)). During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter.
The accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. See Heid et al., Genome Res., 6(6): 986-994 (1996). Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of nucleic acid target, the sooner a significant increase in fluorescence is observed, (Gibson et al., Genome Res., 6: 995-1001 (1996)).
When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer. See Lakowicz et al, J. Biol. Chem., 258:4794-4801 (1983)). During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5′-3′ nucleolytic activity of the AMPLITAQ GOLD™ DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The 3′ end of the probe is blocked to prevent extension of the probe during PCR.
The passive reference is a dye included in the TAQMAN™ buffer and does not participate in the 5′ nuclease assay. The passive reference provides an internal reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescent fluctuations due to changes in concentration or volume.
Normalization is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the passive reference to obtain a ratio defined as the Rn, (normalized reporter) for a given reaction tube.
The threshold cycle or Ct value is the cycle at which a statistically significant increase in ΔRn, is first detected. On a graph of Rn vs. cycle number, the threshold cycle occurs when the sequence detection application begins to detect the increase in signal associated with an exponential growth of PCR product.
To perform quantitative measurements, serial dilutions of a cRNA (standard) are included in each experiment in order to construct a standard curve necessary for the accurate and fast mRNA quantification. In order to estimate the reproducibility of the technique, the amplification of the same cRNA sample may be performed multiple times.
Other technologies for measuring the transcriptional state of a cell produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers (see, e.g., EP 0 534858 A1), or methods selecting restriction fragments with sites closest to a defined mRNA end. See, e.g., Prashar et al., Proc. Natl. Acad. Sci., USA, 93(2):659-663 (1996)).
Other methods statistically sample cDNA pools, such as by sequencing sufficient bases, e.g., 20-50 bases, in each of multiple cDNAs to identify each cDNA, or by sequencing short tags, e.g., 9-10 bases, which are generated at known positions relative to a defined mRNA end pathway pattern. See, e.g., Velculescu, Science, 270:484-487 (1995). The cDNA level(s) in the samples are quantified and the mean, average and standard deviation of each cDNA is determined using by standard statistical means well-known to those of skill in the art. Bailey NTJ, Statistical Methods In Biology, Third Edition (Cambridge University Press, 1995).
Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker gene, the level of expression of the marker is determined for 10 or more samples of normal versus disease biological samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level. Preferably, the samples used in the baseline determination will be from subjects who do not have the polymorphism. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is specific (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data.
Determination of Biomarker Gene Translation. In another embodiment of the present invention, a polypeptide corresponding to a marker is detected. The detection of the biomarker polypeptide (a.k.a., biomarker, marker, marker protein or marker polypeptide) expression product of the biomarker gene in body fluids or tissues can be used to determine the presence or absence of the polymorphism, and the relative level of the biomarker polypeptide expression product can be used to determine if the polymorphism is present in a homozygous or heterozygous state (and hence the risk category of the individual). That is, in another embodiment of the present invention, a polypeptide corresponding to a marker (i.e., biomarker polypeptide) is detected. The level of this biomarker polypeptide gene expression product in body fluids or tissue sample may be determined by any means known in the art.
Immunological Detection Methods. Expression of the protein encoded by the gene(s) of the invention can be detected by a probe which is detectably labelled, or which can be subsequently labelled. Generally, the probe is an antibody that recognizes the expressed protein. A variety of formats can be employed to determine whether a sample contains a biomarker protein that binds to a given antibody. Immunoassay methods useful in the detection of biomarker polypeptides of the present invention include, but are not limited to, e.g., dot blotting, western blotting, protein chips, competitive and non-competitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), immunohistochemistry, fluorescence activated cell sorting (FACS), and others commonly used and widely-described in scientific and patent literature, and many employed commercially. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express a marker of the present invention and the relative concentration of that specific polypeptide expression product in blood or other body tissues. Proteins from individuals can be isolated using techniques that are well-known to those of skill in the art. The protein isolation methods employed can, e.g., be such as those described in Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, N.Y., 1988)).
An intact antibody, or a fragment thereof, e.g., Fab or F(ab′)2 can be used. Antibody fragments, which recognize specific epitopes, may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed (see Huse et al., Science, 246:1275-1281 (1989)), to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
The term “labelled”, with regard to the probe or antibody, is intended to encompass direct-labelling of the probe or antibody by coupling, i.e., physically linking, a detectable substance to the probe or antibody, as well as indirect-labelling of the probe or antibody by reactivity with another reagent that is directly-labelled. Examples of indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin.
Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler & Milstein, Nature, 256:495-497 (1975); and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique of Kosbor et al., Immunol. Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci., USA, 80:2026-2030 (1983); and the EBV-hybridoma technique, Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96 (Alan R. Liss, Inc., 1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgG and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titres of mAbs in vivo makes this the presently preferred method of production.
In addition, techniques developed for the production of “chimaeric antibodies” (see Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Neuberger et al, Nature, 312: 604-608 (1984); and Takeda et al., Nature, 314:452-454 (1985)), by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimaeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived form a murine mAb and a human immunoglobulin constant region.
Alternatively, techniques described for the production of single chain antibodies, U.S. Pat. No. 4,946,778; Bird, Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Ward et al., Nature, 334:544-546 (1989), can be adapted to produce differentially expressed gene single-chain antibodies. Single-chain antibodies are formed by linking the heavy- and light-chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide.
More preferably, techniques useful for the production of “humanized antibodies” can be adapted to produce antibodies to the proteins, fragments or derivatives thereof. Such techniques are disclosed in U.S. Pat. Nos. 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661,016; and 5,770,429. Antibody fragments, which recognize specific epitopes, may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed (see Huse et al., Science, 246:1275-1281 (1989)), to allow rapid aid easy identification of monoclonal Fab fragments with the desired specificity.
In one format, antibodies or antibody fragments can be used in methods, such as Western blots or immunofluorescence techniques, to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite.
The extent to which the known proteins are expressed in a biological sample is determined by immunoassay methods that utilize the antibodies described above. Particularly preferred, for ease of detection, is the sandwich ELISA, of which a number of variations exist, all of which are intended to be used in the methods and assays of the present invention. For example, in a typical forward assay, unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule after a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex. At this point, a second antibody, labelled with a reporter molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and added to the unlabelled surface bound antibody. These techniques are well-known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, “sandwich assay” is intended to encompass all variations on the basic two-site technique. For the immunoassays of the present invention, the only limiting factor is that the labelled antibody must be an antibody that is specific for the protein expressed by the gene of interest.
Two-Dimensional Gel Electrophoresis. Proteins can be separated by two-dimensional gel electrophoresis systems and then identified and/or quantified. Two-dimensional gel electrophoresis is well-known in the art and typically involves isoelectric focusing along a first dimension followed by SDS PAGE electrophoresis along a second dimension. (See, e.g., Hames et al., Gel Electrophoresis of proteins: A Practical Approach (IRL Press, NY, 1990); Shevchenko et al., Proc Natl. Acad. Sci. USA, 93:14440-14445 (1996); Sagliocco et al., Yeast, 12:1519-1533 (1996); and Lander, Science 274: 536-539 (1996)). The resulting electropherograms can be analyzed by numerous techniques, including mass spectrometric techniques, western blotting and immunoblot analysis using polyclonal and monoclonal antibodies, and internal and N-terminal micro-sequencing. Using these techniques, it is possible to identify a substantial fraction of all the proteins produced under given physiological conditions, including in cells, e.g., in yeast, exposed to a drug, or in cells modified by, e.g., deletion or over-expression of a specific gene.
Mass Spectroscopy. The identity and the expression level of biomarker polypeptide can both be determined using mass spectroscopy technique (MS). MS-based analysis methodology is use for analysis of isolated biomarker polypeptide as well as analysis of biomarker polypeptide in a biological sample. MS formats for use in analyzing a biomarker polypeptide include ionization (I) techniques, such as, but not limited to, MALDI, continuous or pulsed ESI and related methods, such as ionspray or thermospray, and massive cluster impact (MCI). Such ion sources can be matched with detection formats, including linear or non-linear reflectron TOF, single or multiple quadrupole, single or multiple magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion trap and combinations thereof such as ion-trap/TOF. For ionization, numerous matrix/wavelength combinations (MALDI) or solvent combinations (ESI) can be employed. Sub-attomole levels of protein have been detected, e.g., using ESI MS (Valaskovic et al., Science, 273:1199-1202 (1996)) and MALDI MS (Li et al, J. Am. Chem. Soc., 118:1662-1663 (1996)).
For MS analysis, the biomarker polypeptide can be solubilised in an appropriate solution or reagent system. The selection of a solution or reagent system, e.g., an organic or inorganic solvent, will depend on the properties of the biomarker polypeptide and the type of MS performed, and is based on methods well-known in the art. See, e.g., Vorm et al., Anal. Chem., 61:3281 (1994) for MALDI; and Valaskovic et al., Anal. Chem., 67:3802 (1995), for ESI. MS of peptides also is described, e.g., in International PCT Application No. WO 93/24834 and U.S. Pat. No. 5,792,664. A solvent is selected that minimizes the risk that the biomarker polypeptide will be decomposed by the energy introduced for the vaporization process. A reduced risk of biomarker polypeptide decomposition can be achieved, e.g., by embedding the sample in a matrix. A suitable matrix can be an organic compound such as a sugar, e.g., a pentose or hexose, or a polysaccharide such as cellulose. Such compounds are decomposed thermolytically into CO2 and H2O such that no residues are formed that can lead to chemical reactions. The matrix can also be an inorganic compound, such as nitrate of ammonium, which is decomposed essentially without leaving any residue. Use of these and other solvents is known to those of skill in the art. See, e.g., U.S. Pat. No. 5,062,935.
Electrospray MS has been described by Fenn et al, J. Phys. Chem., 88:4451-4459 (1984); and in PCT Application No. WO 90/14148; and current applications are summarized in review articles. See Smith et al, Anal. Chem., 62:882-89 (1990); and Ardrey, Spectroscopy, 4:10-18 (1992). With ESI, the determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks, all of which can be used for mass calculation.
Matrix Assisted Laser Desorption (MALDI) is one preferred method among the MS methods herein. Methods for performing MALDI are well-known to those of skill in the art. Numerous methods for improving resolution are also known. For example, resolution in MALDI-TOF-MS can be improved by reducing the number of high energy collisions during ion extraction. See, e.g., Juhasz et al., Analysis, Anal. Chem., 68:941-946 (1996); see also, e.g., U.S. Pat. Nos. 5,777,325; 5,742,049; 5,654,545; 5,641,959; 5,654,545, and 5,760,393 for descriptions of MALDI and delayed extraction protocols. MALDI-TOF: MS has been described by Hillenkamp et al., Burlingame & McCloskey, eds., pp. 49-60 (Elsevier Science Publ., 1990).
In a preferred embodiment, the level of the biomarker protein in a biological sample, e.g., body fluid or tissue sample, maybe measured by means of mass spectrometric (MS) methods including, but not limited to, those techniques known in the art as matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF-MS) and surfaces enhanced for laser desorption/ionization, time-of-flight mass spectrometry (SELDI-TOF-MS) as further detailed below.
MASLDI-TOF-MS Protein Detection Technique. In some preferred embodiments, the detection of specific proteins or polypeptide gene expression products in a biological sample, e.g., body fluid or tissue sample, is performed by means of MS, especially matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MASLDI-TOF-MS). These techniques have been used to analyze macromolecules, such as proteins or biomolecules and utilize sample probe surface chemistries that enable the selective capture and desorption of analytes, including intact macromolecules, directly from the probe surface into the gas (vapour phase), and in the most preferred embodiments without added chemical matrix.
In other embodiments a variety of other techniques for marker detection using mass spectroscopy can be used. See Bordeaux Mass Spectrometry Conference Report, Hillenkamp, ed., pp. 354-362 (1988); Bordeaux Mass Spectrometry Conference Report, Karas & Hillenkamp, Eds., pp. 416-417 (1988); Karas & Hillenkamp, Anal Chem., 60:2299-2301 (1988); and Karas et al., Biomed Environ Mass Spectrum, 18:841-843 (1989). The use of laser beams in TOF-MS is shown, e.g., in U.S. Pat. Nos. 4,694,167; 4,686,366; 4,295,046; and 5,045,694, which are incorporated herein by reference in their entireties. Other MS techniques allow the successful volatilization of high molecular weight biopolymers, without fragmentation, and have enabled a wide variety of biological macromolecules to be analyzed by mass spectrometry.
Surfaces Enhanced for Laser Desorption/Ionization (SELDI). In a preferred embodiment of the present invention, other techniques are used which employ new MS probe element compositions with surfaces that allow the probe element to actively participate in the capture and docking of specific analytes, described as Affinity Mass Spectrometry (AMS). Several types of new MS probe elements have been designed with Surfaces Enhanced for Affinity Capture (SEAC). See Hutchens & Yip, Rapid Commun. Mass Spectrom., 7:576-580 (1993). SEAC probe elements have been used successfully to retrieve and tether different classes of biopolymers, particularly proteins, by exploiting what is known about protein surface structures and biospecific molecular recognition.
In another preferred embodiment of the present invention, the method of detection to be used with the methods of this invention uses a general category of probe elements, i.e., sample presenting means with surfaces enhanced for laser desorption/ionization (SELDI). See SELDI U.S. Pat. Nos. 5,719,060; 5,894,063; 6,020,208; 6,027,942; 6,124,137; and US. Patent Application No. U.S. 2003/0003465.
A polypeptide of interest can be attached directly to a support via a linker. Any linkers known to those of skill in the art to be suitable for linking peptides or amino acids to supports, either directly or via a spacer, may be used. For example, the polypeptide can be conjugated to a support, such as a bead, through means of a variable spacer. Linkers, include, Rink amide linkers (see, e.g., Rink, Tetrahedron Lett., 28:3787 (1976)); trityl chloride linkers (see, e.g., Leznoff, Ace Chem. Res. 11:327 (1978)); and Merrifield linkers. (See, e.g., Bodansky et al., Peptide Synthesis, Second Edition (Academic Press, New York, 1976)). For example, trityl linkers are known. (See, e.g., U.S. Pat. Nos. 5,410,068 and 5,612,474). Amino trityl linkers are also known, (See, e.g., U.S. Pat. No. 5,198,531). Other linkers include those that can be incorporated into fusion proteins and expressed in a host cell. Such linkers may be selected amino acids, enzyme substrates or any suitable peptide. The linker may be made, e.g., by appropriate selection of primers when isolating the nucleic acid. Alternatively, they may be added by post-translational modification of the protein of interest.
Use of a Pin Tool to Immobilize a Polypeptide. The immobilization of a polypeptide of interest to a solid support using a pin tool can be particularly advantageous. Pin tools include those disclosed herein or otherwise known in the art. See, e.g., U.S. application Ser. Nos. 08/786,988 and 08/787,639; and International PCT Application No. WO 98/20166. A pin tool in an array, e.g., a 4×4 array, can be applied to wells containing polypeptides of interest. Where the pin tool has a functional group attached to each pin tip, or a solid support, e.g., functionalized beads or paramagnetic beads, are attached to each pin, the polypeptides in a well can be captured (1 pmol capacity). Polypeptides of interest, particularly biomarker polypeptides, can be immobilized due to contact with the pin tool. Further immobilization can result by applying an electrical field to the pin tool. See, e.g., Juhasz et al., Analysis, Anal. Chem., 68:941-946 (1996), and see also, e.g., U.S. Pat. Nos. 5,777,325; 5,742,049; 5,654,545; 5,641,959; and 5,760,393 for descriptions of MALDI and delayed extraction protocols. Pin tools can be useful for immobilizing polypeptides of interest in spatially addressable manner on an array. Such spatially addressable or pre-addressable arrays are useful in a variety of processes, including, for example, quality control and amino acid sequence diagnostics. The pin tools described in the U.S. application Ser. Nos. 08/786,988 and 08/787,639 and International PCT Application No. WO 98/20166 are serial and parallel dispensing tools that can be employed to generate multi-element arrays of polypeptides on a surface of tie solid support.
Other Aspects of the Biological State. In various embodiments of the present invention, aspects of the biological activity state, or mixed aspects can be measured in order to obtain drug and pathway responses. The activities of proteins relevant to the characterization of cell function can be measured, and embodiments of this invention can be based on such measurements. Activity measurements can be performed by any functional, biochemical or physical means appropriate to the particular activity being characterized. Where the activity involves a chemical transformation, the cellular protein can be contacted with natural substrates, and the rate of transformation measured. Where the activity involves association in multimeric units, e.g., association of an activated DNA binding complex with DNA, the amount of associated protein or secondary consequences of the association, such as amounts of mRNA transcribed, can be measured. Also, where only a functional activity is known, e.g., as in cell cycle control, performance of the function can be observed. However known and measured, the changes in protein activities form the response data analyzed by the methods of this invention. In alternative and non-limiting embodiments, response data may be formed of mixed aspects of the biological state of a cell. Response data can be constructed from, e.g., changes in certain mRNA abundances, changes in certain protein abundances and changes in certain protein activities.
The following EXAMPLES are presented in order to more fully illustrate the preferred embodiments of the invention. These EXAMPLES should in no way be construed as limiting the scope of the invention, as defined by the appended claims.
Purpose. The purpose of this EXAMPLE is to show gene expression changes resulting from anti-Nogo-A antibody-treatment after spinal cord injury in rats in order to identify biomarker candidates of treatment efficacy, mechanism of action or of any potential adverse effects.
Study design. The in life part of the EXAMPLE was performed as follows: A total of 40 adult female Lewis rats (Rattus norwegicus, 160-190 g) were obtained from a Specific Pathogen Free (SPF) breeding colony (R. Janvier, Le Genest-St-Isle, France) and kept as groups of 4-6 animals in standardized cages (type 4, Macrolon, Indulab, Hanstedt, Germany) on a 12 hour light/dark cycle on a standard regime with food and water ad libitum.
The rats were randomized to five groups: Two of 16 underwent spinal hemisection and received either IgG or anti-Nogo A antibody (11C7). The third group, a naïve group of eight, did not undergo surgery and did not receive any treatment, as follows:
Treatment Groups:
Animals were coded with random numbers and the experimenters were blind with regard to the treatments throughout all the steps and phases of the experiment. All the treatments, surgical procedure, and sacrifice and the initial data-analysis was carried out in blinded manner. The antibodies were coded “orange” and “yellow”.
Antibodies. Anti Nogo-A antibody 11C7: Mouse monoclonal antibody (mAb) 11C7, raised against a 18aa peptide Nogo-A corresponding to rat sequence aa 623-640; used at a concentration of 3 mg/ml in PBS. The control antibody was a mouse monoclonal IgG directed against plant lectin used at a concentration of 3 mg/ml in PBS. The biochemical and neutralizing properties of both antibodies are described in Oertle T et al., J. Neurosci. 23:5393-5406 (2003).
Surgical procedures. Animals were anesthetized with a subcutaneous injection of Hypnorm (120 μl/200 g body weight Janssen Pharmaceutics, Beerse, Belgium), and Dormicum (0.75 mg in 150 μl per 200 g body weight Roche Pharmaceuticals, Basle, Switzerland). Vitamin A containing eye ointment (Blache, Chauvin Novopharm AG, Switzerland) was applied to protect the eyes from dehydration during the relatively long operation procedure.
A T-shaped lesion to include the dorsal half of the spinal cord with the main as well as the dorso-lateral and ventro-medial projections of the CST with iridectomy scissors and a sharp, pointed blade was made at thoracic level T8.
A fine intrathecal catheter (32 gauge from RECATHCO, Allison Park, Pa., USA) was inserted from the lumbar level L2/L3 and pushed up to T9, delivering antibodies by osmotic minipumps (5 μl/h, 3.1 μg/μl, Alzet©2ML2, Charles River Laboratories, Les Oncins, France) to the lesion site for 2 weeks. After surgery, the animals were kept on a thermostatically regulated heating pad until completely awake. No pain killers or antibiotics were given in order not to influence the results. Ringer solution (Fresenius Kabi AG, Stans, Switzerland) was given subcutaneously when animals showed signs of dehydration.
Sacrifice. After 1 and 2 weeks respectively, the rats were slightly anesthetized with Isoflurane and decapitated. The naïve animals were sacrificed together with the one week group.
1 ml of whole blood was collected into an EDTA tube, mixed, diluted with 1 ml NaCl 0.9% transferred to a tube containing Fas. The mixture was frozen on dry ice. Approx. 1 ml of whole blood was collected in a Lith/Hep tube, mixed and kept on ice before centrifuged at 2000×g for 10 min (cooled). The supernatant (plasma) was frozen on dry ice.
Brain and spinal cord were exposed, the specific tissue domains were sampled and immediately frozen on dry ice.
Experimental animals. Number of animals per group and sex: 8 females/group, total 40. Age: 8-9 weeks. Weight: 160-190 g.
Tissue sampling. The following tissues were sampled:
Samples were stored on dry ice and subsequently in a deep-freezer at −80° C. until further use. The following tissue samples were processed for gene expression profiling and analyzed:
The brain was divided into two hemispheres and left was kept intact for further confirmation of the microarray findings using in situ hybridization/immunohistochemistry while the right one to be used for dissection.
RNA extraction and purification. Briefly, total RNA was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol, Invitrogen Life Technologies) from each frozen tissue section and the total RNA was then purified on an affinity resin (Rneasy, Qiagen) according to the manufacturer's instructions. and quantified. Total RNA was quantified by the absorbance at λ=260 nm (A260nm), and the purity was estimated by the ratio A260nm/A280nm. Integrity of the RNA molecules was confirmed by non-denaturing agarose gel electrophoresis using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., USA). An aliquot of each individual RNA sample was kept for confirmation of microarray finding by real-time, fluorescence-based PCR (TAQMAN; Applera). RNA was stored at −80° C. until analysis.
Microarray experiment. All microarray hybridizations were conducted as recommended by the manufacturer of the microarray system (Affymetrix, Santa Clara, Calif.; Expression analysis technical manual). Six samples from each treatment group were individually hybridized (no pooling) on the rat genome RAE230 2.0 gene expression probe array set containing >31 000 probe sets (Affymetrix, Inc., Santa Clara, Calif., USA).
Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript Choice System (Invitrogen Life Technologies) in the presence of a T7-(dT)24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalkohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labelling Kit (ENZO) in the presence of biotinylated ribonucleotides form biotin labelled cRNA. The labelled cRNA was then purified on an affinity resin (RNeasy, Qiagen), quantified and fragmented. An amount of approximately 10 μg labelled cRNA was hybridized for approximately 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes) using the GeneChip Fluidics Workstation 400 (Affymetrix). The array was then scanned twice using a confocal laser scanner (GeneArray Scanner, Agilent) resulting in one scanned image. This resulting “.dat-file” was processed using the MAS5 program (Affymetrix) into a “.cel-file”. Raw data was converted to expression levels using a “target intensity” of 150.
Data analysis. Initial data-analysis of the dataset for spinal cord tissues T8 (at the level of injury) and proximal to the injury, T1-7 was performed blindly. Analysis resulted in identifying samples coded “orange” as the 11C7-treated group after which the code was broken and the sample identity confirmed. Remaining of the analysis was not blinded.
Quality control. The following quality measures were studied for each sample: Scaling factor, background, percent present calls, AFFX-GAPDH 3′:AFFX-GAPDH 5′-ratio, AFFX-GAPDH 3′ variance, AFFX-Beta-actin 3′:AFFX-Beta-actin 5′-ratio. Attention was paid to the homogeneity of the data. Average and standard deviation of the background noise level determined the raw data restriction value used in the consequent analysis. GAPDH 3′ variance is a measure of variation among individual samples and can be used as a guideline for a reliable fold difference.
Principal component analysis. Principal component analysis (PCA) including all probe sets on Rat Genome 2.0 (n=15 866) as variables was performed to identify outlier microarrays after log-transformation and centralization of the data using Simca-P 10.0 software (Umetrics, Umea, Sweden). After removal of technical outliers, PCA was repeated using GeneSpring (Silicon Genetics, Redwood City, Calif., USA) version 7.0.
Data normalization. After QC, MAS5 normalized microarray data was imported to GeneSpring version 7.0. (Silicon Genetics). Individual experiments were generated for each tissue separately. Each experiment was normalized as follows: Values below 0 were set to 0.1. Each measurement was divided by the 50.0th percentile of all measurements in that sample. Finally, per gene normalization was performed by normalizing to the expression value of the median of naïve samples.
Identification of differentially expressed genes. Differentially expressed genes between the vehicle and the treatments were identified within each experiment based on the following restrictions: (1) Prefiltering restrictions: Probe sets included in further analysis had to flagged present in 4/6 of replicates in any condition. Raw data signal intensity had to be minimum 50 in at least one of the treatment groups. (2) Statistical restriction: p<0.05 (Welch t-test (parametric)). Similar statistical restriction was always applied to different groups to be compared and is mentioned in each comparison.
Gene Set Enrichment Analysis (GSEA). An in-house implementation of the Gene Set Enrichment Analysis method was used to analyze microarray data. Genes with expression levels below 100 on more than 75% of the chips are discarded as low- or non-expressed. Microarray results are then analyzed in a series of pairwise comparisons between sets of condition (e.g. treated vs. control). Each gene's relative expression level under condition1 and condition2 is computed as an expression ratio ri
where μi,j is the average expression value for gene i under conditionj. The genes are then sorted according to their expression ratios such that those genes with higher expression under condition1 than condition2 are at the top of the list. Next, the collection of available gene sets are projected onto the sorted list. This step in essence applies a priori biological knowledge to the experimental data to identify functionally related genes that are expressed in a coordinated fashion. Gene sets are processed one at a time. For gene set G each expression ratio ri is labelled ‘in’ the gene set if genei∈G and ‘out’ of the gene set if genej∉G. A two-tailed Wilcoxon rank-sum test is calculated to determine if the genes labelled ‘in’ gene set G are enriched at either the top or bottom of the sorted list. The false discovery rate method of Storey J D & Tibshirani R, Proc Natl Acad Sci USA 100:9440-9445 (2003) is applied to transform p-values to multiple testing corrected q-values. The output from GSEA is a list of q-values (q1, q2, . . . , qN) and labels (l1, l2, . . . , lN), li∈(top, bottom) that correspond to the N available gene sets. A small q-value qi indicates that the genes in gene set Gi are significantly enriched at either the top or bottom of the list of expression ratios.
Results. Initial data-analysis of the dataset for spinal cord tissues T8 (at the level of injury) and proximal to the injury, T1-7 was performed blindly. Analysis resulted in identifying samples coded “orange” as the 11C7-treated group after which the code was broken and the sample identity confirmed. Remaining of the analysis was not blinded.
Spinal cord T8 (At the level of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 643 and 449 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.93±1.06 after one week of treatment and 1.31±0.07 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in TABLE 4 and after two weeks of treatment, in TABLE 5 in EXAMPLE 2.90% of the top 20 transcripts were downregulated at one week after 11C7 treatment (whereas of the total differentially expressed ones, 41% were downregulated). Interestingly, among those there were 7 transcripts encoding for proteins related to extracellular matrix (ECM) and wound healing and/or scarring (asporin precursor, dermatopontin, collagen), 2 secreted frizzled-like proteins (Sfrl2 and 4), two IGF-binding proteins (Igfbp 5 and 6, negative regulators of IGF) and myocilin/TIGR, which has been recently shown to inhibit neurite outgrowth and to be upregulated in chronic glial scar after CNS injury. Jurynec M J et al, Mol. Cell. Neurosci. 23:69-80 (2003).
Gene Set Enrichment Analysis (GSEA) identified altogether 30 pathways with significant enrichment of differentially enriched transcripts after one week of treatment (TABLE 16 in EXAMPLE 3). Most significant enrichment was observed in immunity and defence-related transcripts (
After two weeks of treatment, fold changes were significantly smaller than after 1 week of treatment. Only one transcript was >1.5 fold significantly differentially regulated (p53-responsive gene 3, 1.6 fold upregulated after 11C7). GSEA identified 19 pathways in which significant enrichment of differentially expressed transcripts were observed. Oxidative phosphorylation (
Spinal cord T1-7 (Proximal to the site of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 566 and 579 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.43±0.17 after one week of treatment and 1.56±0.98 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in TABLE 18 and after two weeks of treatment, in TABLE 19 in EXAMPLE 3.
The largest changes at one week after 11C7 treatment replicated the theme observed at the site of injury: eight of the top 20 changes were related to ECM (lumican, collagens 1a1-2 and 5a1, fibulin 2, tetranectin, Matrix glycoprotein SC1/ECM2) and downregulated after treatment with 11C7. After two weeks of treatment, fold changes were slightly larger than after 1 week of treatment. Some of the largest changes were related to downregulation of transcripts encoding for proteins expressed in lymphocytes
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in five pathways after one week of treatment (TABLE 18, EXAMPLE 3). No pathways were significantly affected (q<0.001) after two weeks of treatment. The most significantly affected pathways after one week were ECM-mediated signalling, lipid, fatty acid and sterol metabolism and growth factor homeostasis (
Spinal cord L1-5 (Distal to the site of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 1303 and 1301 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.72±0.5 after one week of treatment and 1.91±2.0 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in Table 1-5 and after two weeks of treatment, in TABLE 21 in EXAMPLE 3.
The largest changes at one week after 11C7 treatment were related to transcripts expressed by lymphocytes (Similar to Ig gamma-2C chain C region (LOC362795), mRNA, secretory leukocyte protease inhibitor, lymphocyte selectin, lipocalin 2, thrombomodulin, chemokine (C—X—C motif) ligand 12) and as upregulated, could imply an increased lymphocyte trafficking into the tissue after 11C7 treatment. Also, Sfrp4 and ephrin B1 were upregulated after 11C7. After two weeks of treatment, top significantly changed transcripts included nuclear receptor MrgA10 RF-amide G protein-coupled receptor (Mrga10) and nuclear receptor coactivator 3 as well as immunity related transcripts which were downregulated after 11C7. A large number of significant changes were related to synaptic transmission or synaptic vesicle cycling (Synaptogenesis-related mRNA sequence 6, synaptic vesicle glycoprotein 2 b, synaptoporin) and were upregulated after 11C7.
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in 58 pathways after one week of treatment (TABLE 19, EXAMPLE 3), and 48 pathways (TABLE 20, EXAMPLE 3) after two weeks of treatment. The most significantly affected pathways were immunity and defence, signal transduction and cell communication after one week of treatment (all upregulated in 11C7;
Motor-Somatosensory Cortex. Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 574 and 910 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.42±0.19 after one week of treatment and 1.46±0.09 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in TABLE 20 and after two weeks of treatment, in TABLE 21 in EXAMPLE 3.
70% of the top 100 changes in the motor/somatosensory cortex after one week treatment were ESTs thus complicating interpretation of the data. Among the top changed known transcripts were however S100 calcium-binding protein A9 (calgranulin B, expressed by macrophages, 3 fold upregulated after 11C7) and Crmp5 (Collapsin response mediator protein 5, upregulated after 11C7). Collapsin-response mediator proteins (CRMPs) are highly expressed in the developing brain where they take part in several aspects of neuronal differentiation. In adult, they are expressed in areas of persistent neurogenesis. Veyrac A et al., Eur. J. Neurosci. 21:2635-2648 (2005). After two weeks of treatment, 80% of the top 100 changes were ESTs.
Based on multiple testing corrected analysis, GSEA identified no pathways with significant enrichment of differentially expressed transcripts after one week of treatment. After two weeks of treatment, the oxidative phosphorylation pathway showed a significant enrichment of differentially expressed genes (q<0.001; TABLE 21, EXAMPLE 3). Interestingly, the Huntington's disease, EGF-, FGF-, and NGF-signalling pathways were all affected but escaped the recommended level of significance (q<0.04 vs q<0.001). All were downregulated after 11C7 treatment (
Frontal Cortex. Welch t-test comparing the IgG-treated group to the 11C7-treated group resulted in 657 and 275 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.3±0.3 after one week of treatment and 1.2±0.05 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in Table 1-9 and after two weeks of treatment, in Table 1-10 in Annex-1. Only 13 transcripts after one week and 10 after two weeks of treatment were >1.3 fold differentially expressed, thus indicating a very weak gene expression response to the treatment.
Among >1.3 fold changes were S100 calcium-binding protein A9 (calgranulin B) expressed by macrophages, c-fos oncogene, Dusp6 and Egr-1 related to cell differentiation after one week and stathmin 1, Nr2f2, G protein-coupled receptor 27 and myelin-associated oligodendrocytic basic protein (Mobp; 1.28 fold upregulated after 11C7) after two weeks of treatment.
GSEA was not performed for the frontal cortex dataset due to small number of significant changes.
Blood. Welch t-test comparing the IgG-treated group to the 11C7-treated group resulted in 389 and 427 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 2.1±0.56 after one week of treatment and 1.80±0.40 after two weeks of treatment. The top 100 gene expression changes after one week of treatment are listed in Table 1-11 and after two weeks of treatment, in Table 1-12 in Annex-1.
Among the largest changes at one week after 11C7 treatment were upregulation of matrix metalloproteinases Mmp8 and Mmp9, Hipk3, secretory leukocyte protease inhibitor (also upregulated after one week in L1-5) and calgranulin A. After two weeks of treatment, Similar to beta-amyloid binding protein (LOC362545), mRNA and Creb-binding protein were downregulated after 11C7 and neuroprotective mGluR8 and apoptosis-related Sfrp4 upregulated after 11C7.
Based on multiple testing corrected analysis, GSEA identified six pathways with significant enrichment of differentially expressed transcripts after one week of treatment (q<0.001; Annex-2, Table 1-7). Endocytosis, intracellular protein traffic, receptor mediated endocytosis (
Discussion. The purpose of this EXAMPLE was to identify treatment-related changes in rat after spinal cord hemisection after one week and two weeks of treatment with monoclonal mouse anti-Nogo-A antibody 11C7 in comparison to control treatment, mouse IgG antibody against plant lectin.
After one week of treatment, the most significant gene expression changes in terms of number and magnitude were observed distal to the site of injury (L1-5) followed by the site of injury (T8) and blood, whereas frontal cortex, motor-somatosensory cortex and spinal cord proximal to the site of injury (T1-7) were clearly less affected (TABLE 2). After two weeks of treatment, the largest effect size in terms of gene expression was observed at L1-5 followed by a relatively similar effect on motor-somatosensory cortex, spinal cord proximal to the site of injury (T1-7) and blood. Clearly less effect by the treatment was observed in T8 and in the frontal cortex after two weeks of treatment (TABLE 2).
A very strong effect by 11C7 was observed at the site of lesion down-regulating transcripts related to extracellular matrix and wound healing after one week of treatment. Asporin precursor, dermatopontin, microfibril-associated glycoprotein-2 and several collagens were among the top downregulated changes as well as two secreted frizzled related proteins Sfrp2 and Sfrp4 whose expression has been found to correlate with apoptosis. Myocilin/TIGR, a secreted glycoprotein with upregulated expression in chronic glial scar after CNS injury and neurite outgrowth inhibiting effect on dorsal root ganglia neurons in vivo (Jurynec M J et al, Mol. Cell. Neurosci. 23:69-80 (2003)) was found to be 2.67 fold downregulated after one week of 11C7-treatment. Myocilin is suggested to be a novel neurite outgrowth inhibiting molecule inhibited by anti-Nogo-A-treatment.
Other neurite outgrowth/axon guidance related changes included the slit-robo mediated axon guidance pathway related transcripts encoding for chemokine (C—X—C motif) ligand 12 and chemokine (C—X—C motif) receptor 4 identified by GSEA (q<0.02). Cxcl12 and CXCR4 showed a concerted upregulation in all spinal cord segments studied after one week of treatment with 11C7 (
At the level of individual genes but not identified by GSEA, were changes related to semaphorin-collapsin mediated pathway: sema A/semaphorin 3A and collapsing response proteins 4 and 5 Crmp4/5 mediating repulsive cues to the migrating growth cones were seen downregulated after 1 week of treatment in T8 and in motor-somatosensory cortex.
GSEA was first described by Mootha V K et al., Nat. Genet. 34:267-273 (2003) as a method to identify coordinated transcriptional changes among functionally related groups of genes in microarray data. The gene set enrichment analysis method has been implemented in-house with several refinements to the original methodology [RD-2005-50762]. Often in the microarray data, changes at the level of single transcripts remain insignificant due to small fold changes while a large number of such changes affecting a whole pathway would be of significance. Due to small fold changes observed in nervous system in general (most likely due to a large gene dilution effect of heterogeneous cell populations), GSEA approach would be particularly interesting when interpreting data originating from nervous tissues. Pathway information introduced in the GSEA in this study has been collected from a variety of sources, including publicly available databases (KEGG) and proprietary (Celera, Pathart). Summary of the 24 pathways with significant (q<0.001) gene set enrichment in three or more tissues is presented in TABLE 3.
The most widely affected pathways overall were immunity and defence (4 tissues), protein metabolism and phosphorylation (4), nucleoside, nucleotide and nucleic acid metabolism (4) neuronal activities (4) and Jak-stat cascade (4).
GSEA revealed in this study a very clear effect in the immune defence pathways, including B- and T-cell mediated signalling, B-cell activation, macrophage-, NK-cell mediated as well as neutrophil mediated immunity, toll-like receptor pathway and cytokine and chemokine mediated signalling pathways. Interestingly, the immunity and defence mediated pathway was enriched in the direction of 11C7 after one week of treatment but in the direction of IgG after two weeks of treatment. Same pattern was observed also in all other immune mechanism-related pathways, such as B-cell, T-cell, macrophage and NK-cell mediated immunity pathways. Significant effect on the immunity-related pathways was observed most commonly in the spinal cord at the site of lesion (T8) and distal to it (L1-5) and in the blood, where the enrichment direction paralleled that of the spinal cord tissues. Although not studied in detail microscopically, this suggests an increase in the lymphocytes, macrophages and NK-cells after one week of treatment with 11C7 both in blood and in the injured spinal cord in comparison to the IgG-treated animals and possibly an increased trafficking of lymphocytes into the injured spinal cord. As antibodies targeting the extracellular portion of Nogo-A (Nogo-66) has been suggested to be of therapeutic potential in an animal model of multiple sclerosis (Karnezis T et al., Nat. Neurosci. 7:736-744 (2004); Fontoura P et al., J. Immunol. 173:6981-6992 (2004)), the possible involvement of immune related mechanisms in the compound action are of special interest.
Other significantly enriched pathways affected in more than three tissues studied include apoptosis and apoptosis signalling pathway, blood clotting/coagulation, cell adhesion-mediated signalling, extracellular matrix protein-mediated signalling, growth factor homeostasis, oncogene, oxidative phosphorylation and synaptic transmission. The enrichment direction in most of the pathways was similar to that observed in the immune related pathways, towards 11C7 after one week of treatment but in the direction of IgG after two weeks of treatment. An interesting exception is the synaptic transmission pathway, where after one week of treatment the pathway is downregulated after 11C7 treatment but upregulated after two weeks of treatment. Neuronal activities- and nerve-nerve-synaptic transmission pathways followed the same pattern and were significantly affected in spinal cord at the level of T8 and L1-5.
Identification of the several growth factor pathways, including EGF, FGF, NGF, PDGF and TGF beta-signalling pathways in the action of anti-Nogo-A antibody is of interest from several points: The EGF-receptor activation was recently reported to be the mediator of the inhibitory signals from myelin and chondroitin sulphate in axon regeneration and inhibition of the EGF receptor signalling resulted in regeneration resulted in regeneration of optic nerve after injury. He Z & Koprivica V, Annu. Rev. Neurosci. 27:341-368 (2004); Koprivica V et al., Science 310:106-110 (2005). In current dataset, EGF-receptor mediated signalling pathway was upregulated in blood and L1-5 after 1 week of treatment with 11C7 but interestingly downregulated in motor-somatosensory cortex after 2 weeks of 11C7 treatment. PDGF signalling pathway was concomitantly upregulated after one week of treatment by 11C7 in spinal cord at all three levels studied (T8, T1-7, L1-5).
Conclusion. The results confirm at the level of gene expression the injured spinal cord and motor cortex as the primary sites of action of the anti-Nogo-A antibody treatment applied intrathecally. The analysis identified novel molecular and pathways candidates as possible targets of anti-Nogo-A treatment, such as myocilin and the slit-robo pathway. The results also pointed to strong involvement of immune defence related pathways in the treatment effect.
The secreted proteins Sfrp4, Mmp9 and myocilin were selected to be further studied as candidate markers of treatment effect.
TAQMAN confirmation of selected findings was performed. All selected transcripts were confirmed (Sfrp2, Sfrp4, myocilin, asporin precursor, dermatopontin, Mmp9).
Gene Set Enrichment Analysis (GSEA). Gene set enrichment analysis (GSEA) was performed as described by Mootha V K et al., Nat. Genet. 34:267-273 (2003). Shortly, GSEA determines if the members of a given gene set are enriched among the most differentially expressed genes between two classes. First, the genes are ordered on the basis of a difference metric. It can be the difference in means of the two classes divided by the sum of the standard deviations of the two diagnostic classes but other difference metrics can also be used.
For each gene set, an enrichment measure called the ES is made. This is a normalized Kolmogorov-Smirnov statistic. Consider the genes R1, . . . , RN that are ordered on the basis of the difference metric between the two classes and a gene set S containing G members. We define
if Ri is not a member of S, or
if Ri is a member of S. A running sum across all N genes is then computed. The ES is defined as
or the maximum observed positive deviation of the running sum. ES is measured for every gene set considered. Gene sets are based on pathway information from Celera, Pathart and KEGG. To determine whether any of the given gene sets shows association with the class phenotype distinction, the class labels are permuted 1,000 times, each time recording the maximum ES over all gene sets. In this regard, a single hypothesis is being tested. The null hypothesis is that no gene set is associated with the class distinction.
Results. Initial data-analysis of the dataset for spinal cord tissues T8 (at the level of injury) and proximal to the injury, T1-7 was performed blindly. Analysis resulted in identifying samples coded “orange” as the 11C7-treated group after which the code was broken and the sample identity confirmed. Remaining of the analysis was not blinded.
Spinal cord T8 (At the level of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 643 and 449 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.93±1.06 after one week of treatment and 1.31±0.07 after two weeks of treatment. The top 20 gene expression changes after one week of treatment are listed in TABLE 4 and after two weeks of treatment, in TABLE 5.90% of the top 20 transcripts were downregulated at one week after 11C7 treatment (whereas of the total differentially expressed ones, 41% were downregulated). Interestingly, among them there were 7 transcripts encoding for proteins related to extracellular matrix (ECM) and wound healing and/or scarring (asporin precursor, dermatopontin, collagen), 2 secreted frizzled-like proteins (Sfrl2 and 4), two Igf-binding proteins (Igfbp 5 and 6, negative regulators of Igf) and myocilin/TIGR, which has been recently shown to inhibit neurite outgrowth and to be upregulated in chronic glial scar after CNS injury. Jurynec M J et al, Mol. Cell. Neurosci. 23:69-80 (2003).
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in immunity and defence-related transcripts, cytokine and chemokine mediated signalling pathway, Jak-stat cascade, inhibition of apoptosis and in 90 other pathways after one week of treatment with 11C7 (TABLE 4). Of nervous system related pathways, neuronal activities, neurogenesis and nerve-nerve synaptic transmission were downregulated and slit-robo-mediated axon guidance upregulated in the 11C7-treated animals.
norvegicus:disease:atherosclerosis:aif
norvegicus:disease:atherosclerosis:ifngamma
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:physiology:growth and
norvegicus:physiology:apoptosis:tnf
norvegicus:disease:rheumatoid
norvegicus:disease:atherosclerosis:nfkb
norvegicus:disease:atherosclerosis:ldl
norvegicus:physiology:others:fcer1 signalling
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After two weeks of treatment, fold changes were significantly smaller than after 1 week of treatment. Only one transcript was >1.5 fold significantly differentially regulated (p53-responsive gene 3, 1.6 fold upregulated after 11C7). GSEA identified 45 pathways in which significant enrichment of differentially expressed transcripts were observed. Oxidative phosphorylation, electron/ion/cation transport, mRNA processing and synaptic transmission were among the most significantly affected pathways (TABLE 6).
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Spinal cord T1-7 (Proximal to the site of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 566 and 579 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.43±0.17 after one week of treatment and 1.56±0.98 after two weeks of treatment. The top 20 gene expression changes after one week of treatment are listed in TABLE 8 and after two weeks of treatment, in TABLE 9.
The largest changes at one week after 11C7 treatment replicated the theme observed at the site of injury: eight of the top 20 changes were related to ECM (lumican, collagens 1a1-2 and 5a1, fibulin 2, tetranectin, Matrix glycoprotein SC1/ECM2) and downregulated after treatment with 11C7. After two weeks of treatment, fold changes were slightly larger than after 1 week of treatment. Some of the largest changes were related to downregulation of transcripts encoding for proteins expressed in lymphocytes
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in 35 pathways after one week of treatment (TABLE 10), and 3 pathways (TABLE 11; q<0.05; 32 p<0.05) after two weeks of treatment. The most significantly affected pathways were ECM-mediated signalling, lipid metabolism and growth factor homeostasis after one week, and ion transport, growth factor homeostasis and mRNA transcription termination after two weeks of treatment.
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Spinal cord L1-5 (Distal to the site of injury). Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 1303 and 1301 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.72±0.5 after one week of treatment and 1.91±2.0 after two weeks of treatment. The top 20 gene expression changes after one week of treatment are listed in TABLE 12 and after two weeks of treatment, in TABLE 13.
The largest changes at one week after 11C7 treatment replicated the theme observed at the site of injury: eight of the top 20 changes were related to ECM (lumican, collagens 1a1-2 and 5a1, fibulin 2, tetranectin, Matrix glycoprotein SC1/ECM2) and downregulated after treatment with 11C7. After two weeks of treatment, fold changes were slightly larger than after 1 week of treatment. Some of the largest changes were related to downregulation of transcripts encoding for proteins expressed in lymphocytes
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in 151 pathways after one week of treatment (TABLE 14), and 116 pathways (TABLE 15) after two weeks of treatment. Very interestingly, immunity and defence-related pathway was highly significantly enriched in the direction of IgG-treated (downregulated after 11C7-treatment) after two weeks of treatment, whereas transcripts in synaptic transmission, neuronal activities and neurotransmitter release-related pathways were significantly enriched (upregulated) after 11C7-treatment.
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:disease:obesity:responsive genes
norvegicus:disease:atherosclerosis:ldl signalling
norvegicus:physiology:apoptosis:TGF beta induced
norvegicus:physiology:others:fcer1 signalling
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norvegicus:disease:atherosclerosis:tnf signalling
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norvegicus:physiology:apoptosis:FGF signalling
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norvegicus:disease:atherosclerosis:linoleic acid
norvegicus:disease:atherosclerosis:aif mediated
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norvegicus:physiology:apoptosis:wnt signalling
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norvegicus:disease:alzheimers:NGF signalling
norvegicus:disease:alzheimers:icam1 signalling
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:physiology:others:fcer1 signalling
norvegicus:disease:atherosclerosis:tnf signalling
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norvegicus:disease:obesity:responsive genes
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Motor-Somatosensory Cortex. Welch T-test comparing the IgG-treated group to the 11C7-treated group resulted in 1303 and 1301 differentially expressed genes after one week and two weeks of treatment, respectively. The average fold change of the top 100 largest fold changes was 1.72±0.5 after one week of treatment and 1.91±2.0 after two weeks of treatment. The top 20 gene expression changes after one week of treatment are listed in TABLE 12 and after two weeks of treatment, in TABLE 13.
The largest changes at one week after 11C7 treatment replicated the theme observed at the site of injury: eight of the top 20 changes were related to ECM (lumican, collagens 1a1-2 and 5a1, fibulin 2, tetranectin, Matrix glycoprotein SC1/ECM2) and downregulated after treatment with 11C7. After two weeks of treatment, fold changes were slightly larger than after 1 week of treatment. Some of the largest changes were related to downregulation of transcripts encoding for proteins expressed in lymphocytes
Gene Set Enrichment Analysis (GSEA) identified a significant enrichment in 151 pathways after one week of treatment (TABLE 14), and 116 pathways (TABLE 15) after two weeks of treatment. The most significantly affected pathways were ECM-mediated signalling, lipid metabolism and growth factor homeostasis after one week, and ion transport, growth factor homeostasis and mRNA transcription termination after two weeks of treatment.
norvegicus:disease:atherosclerosis:aif
norvegicus:disease:atherosclerosis:ifngamma
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:physiology:growth and
norvegicus:physiology:cell
norvegicus:physiology:apoptosis:tnf
norvegicus:disease:rheumatoid
norvegicus:disease:atherosclerosis:nfkb
norvegicus:disease:atherosclerosis:ldl
norvegicus:physiology:others:fcer1
norvegicus:disease:atherosclerosis:thrombomodulin
norvegicus:physiology:apoptosis:TGF
norvegicus:disease:multiple
norvegicus:physiology:apoptosis:trail
norvegicus:disease:alzheimers:igf1
norvegicus:disease:atherosclerosis:il1beta
norvegicus:disease:atherosclerosis:tnf
norvegicus:disease:alzheimers:amyloidbeta-peptide
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:disease:obesity:responsive genes
norvegicus:disease:atherosclerosis:ldl signalling
norvegicus:physiology:apoptosis:TGF beta induced
norvegicus:physiology:others:fcer1 signalling
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norvegicus:disease:atherosclerosis:tnf signalling
norvegicus:disease:atherosclerosis:insulin
norvegicus:disease:atherosclerosis:il1 signalling
norvegicus:physiology:apoptosis:NGF signalling
norvegicus:physiology:apoptosis:FGF signalling
norvegicus:disease:alzheimers:igf1 signalling
norvegicus:disease:atherosclerosis:linoleic acid
norvegicus:disease:atherosclerosis:aif mediated
norvegicus:disease:atherosclerosis:PDGF
norvegicus:disease:alzheimers:hydrogen peroxide
norvegicus:physiology:inflammation:il1 signalling
norvegicus:physiology:apoptosis:wnt signalling
norvegicus:disease:atherosclerosis:ifngamma
norvegicus:disease:alzheimers:NGF signalling
norvegicus:disease:alzheimers:icam1 signalling
norvegicus:disease:atherosclerosis:angiotensin
norvegicus:physiology:others:fcer1 signalling
norvegicus:disease:atherosclerosis:tnf signalling
norvegicus:disease:atherosclerosis:ldl signalling
norvegicus:disease:obesity:responsive genes
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norvegicus:physiology:apoptosis:TGF beta induced
norvegicus:disease:atherosclerosis:insulin
norvegicus:disease:atherosclerosis:linoleic acid
norvegicus:physiology:inflammation:il1 signalling
Nogo-A (200 kDa, 1163 aa) differs from Nogo-B (55 kDa, 357 aa) by the insertion of a large 787 aa exon (exon 3). A Nogo-A knock-out mouse was generated by homologous recombination as described by Simonen et al. (2003). The chimeric Nogo-A knock-out mice were backcrossed to either Sv129 mice or BL/6 mice for at least 10 generations. The speed congenics strain marker analysis (Markel et al., 1997) was used during backcrossing. Speed congenic breeding, or marker-assisted congenic production, uses microsatellite markers to follow the inheritance of the chromosomal segments of each strain. Optimal breeder mice are selected by the highest level of markers for each strain. The mice used in the present study had a 100% pure C57BL/6 background according to their marker profile, and a >99% pure background for the 129X1/SvJ strain.
Lumbar spinal cords from three naive, non-injured, wild-type, and knock-out male mice (3 months of age) per strain and genotype were dissected and immediately frozen in liquid nitrogen. For lesion microarray experiment, five female mice (6-7 weeks old) of each genotype and strain underwent a lesion of the spinal cord with the help of fine iridectomy scissors to produce a bilateral lesion of the dorsal and the dorsolateral funiculi and the dorsal horn. Six days after the lesion, a Basso Mouse Scale behavioral analysis for open-field locomotion was performed and four of the five mice per category with the most similar score were selected for microarray analysis. One week after the lesion, 1 cm of the spinal cord was dissected with the lesion site in the middle and immediately froze it in liquid nitrogen. For probe preparation, procedures described in the Affymetrix (Santa Clara, Calif.) GeneChip Analysis manual were followed. Biotinylated cRNA was hybridized onto Affymetrix Mouse Genome 430 2.0 arrays, which represent >45,000 probe sets, in the Affymetrix fluidics station 450, and the chips were then scanned with the Affymetrix Scanner 3000. Each chip was used for hybridization with cRNA isolated from one spinal cord sample from a single animal in a total number of 28 samples. Results were subsequently analyzed using the Affymetrix Microarray Suite 5, followed by the Genespring 7.2 (Silicon Genetics, Redwood City, Calif.).
To identify genes that are differentially expressed in the spinal cords of Sv129 and BL/6 mice of naive and knock-out spinal cords of injured and non-injured animals 1 week after a spinal cord lesion, a statistical filter (ANOVA p<0.05) and fold change thresholds (>1.2/<0.66 or >2/<0.5) were applied following a prefiltering for present calls
Pathways and gene groups commonly affected one week after spinal cord injury in knock-out Sv129 mice and BL/6 mice and in the rat SCI model were identified by comparing the differentially expressed genes identified in two way comparisons between the knock-out and naïve animals and in the rat SCI model, between the control (IgG)-treated and 11C7 anti-Nogo A antibody-treated animals.
113 commonly affected gene groups were identified. They are listed in Table 24.
The above data were further confirmed by 2D-gels and/or isotope-coded affinity tag (ICAT).
A list of the genes differentially regulated after inhibition or downregulation of Nogo-A considered to be the most relevant ones is provided in Table 25.
The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/068457 | 11/14/2006 | WO | 00 | 5/14/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60737410 | Nov 2005 | US |
