Patent Application
20080311185
The invention relates to the field of molecular diagnostics and medicine. The invention in particular relates to the deregulation of genes and/or processes in arthritis. The invention utilizes this deregulation in a number of ways.
The invention is exemplified by means of Rheumatoid arthritis (RA), however, the invention is not limited to this disease but is applicable to all inflammatory arthritis diseases. RA is a chronic destructive arthropathy that affects 1-3% of the general population. RA imposes substantial personal, social and economic costs. It is characterized by prolonged inflammation of the joints, eventually leading to destruction of the cartilage and bone. Inflammation is initially localized in the synovial lining, a monolayer of synovial cells that lines diarthoidal joints. In RA, the synovial lining becomes markedly thickened due to synovial cell proliferation and infiltration by inflammatory cells. This proliferative mass, the pannus, invades and destroys articular cartilage and bone, leading to irreversible destruction of joint structure and function [1]. Current therapies of RA rely mainly on symptomatic treatment with non-steroidal anti-inflammatory drugs and/or with disease-modifying anti-rheumatic drugs. However, even the best available treatments (like targeting TNF and TNF signaling) do not cure the disease and do not even sufficiently retard progression in the majority of the patients, while they often exhibit adverse side effects [2].
Despite intense efforts, the etiology and pathogenesis of RA remain poorly understood. Traditional paradigms for RA have implicated a variety of mechanisms that contribute to the initiation and perpetuation of synovial inflammation, including autoantibodies and immune complexes, T-cell mediated antigen-specific responses, persistence of cytokine networks and other pro-inflammatory molecules, genetic bias and sex predisposition, as well as proliferative behaviour of the arthritic synovium [3]. Animal models of RA share many clinical features with the human disease and hence constitute valuable tools in deciphering the pathogenetic mechanisms that govern disease activation and perpetuation [4]. Among them, the TNF-Tg mouse [5] has been instrumental in demonstrating the role of TNF in the development of the disease and foreshadowed the introduction and success of anti-TNF therapies that transformed the effective management of the disease [6]. In this model, chronic over expression of human TNF results in a chronic, erosive, symmetric polyarthritis, with 100% phenotypic penetrance, timed disease onset and progressive histological symptoms that resemble closely human RA [5-7].
The completion of the Human Genome Project and the emergence of functional genomics and high-throughput technologies offer new possibilities for research into the etiology of RA. Amongst them, expression profiling creates a molecular fingerprint of the disease status, by the simultaneous quantification of the expression levels of thousand of genes. Similarly, reverse genetics (the genetic modification of a particular gene in a search of its function) allow for the creation of animal models of disease, valuable tools for the dissection of pathogenetic mechanisms in vivo.
Expression profiling, the relative quantification of the expression levels of thousand of genes simultaneously, is an approach for understanding mechanisms of differentiation, development and disease. However, the small number of samples employed limits significantly statistical analysis and precludes the use of False Discovery Rates methodologies, as well as of multivariate techniques, which would be appropriate for polygenic diseases such as RA. As it is, results of expression profiling need to be confirmed by an independent biological method, which largely diminishes the high throughput nature and discovery rate of any given approach.
To overcome these problems the invention in one aspect provides a novel, twin high throughput approach, composed of subtractive cDNA libraries and oligonucleotide DNA microarray hybridizations. Both methodologies, due to experimental setup, are governed by different constraints: random chance for the libraries, robotic clone selection and chip design for the microarrays. Consequently, both methodologies employ different, independent statistical analyses, are completely uncorrelated towards the propagation of error and conceptually have entirely different False Discovery Rates. As a result of this, the intersection of their statistically significant deregulated gene lists has a very low false discovery rate. To illustrate this and to exhibit the validity of the twin high throughput approach, a number of representative genes from the commonly selected list was confirmed with a number of different methods, such as automated literature search, semi-quantitative and quantitative RT-PCR in both mouse and human samples. Thus is illustrated that coupling of two different high throughput approaches renders independent confirmation unnecessary, although it can of course still be done in addition. Consequently the number of likely deregulated genes is increased. In one embodiment the invention thus provides a method for identifying genes involved in a disorder, comprising performing separately a subtractive cDNA library methodology and a oligonucleotide DNA microarray hybridization methodology and optionally another high throughput expression profiling methodology on a sample of an individual and then taking an intersection of results of said methodologies.
To validate the generated deregulated gene list even further, as well as to bridge two different animal models of RA, expression profiling in the invention was combined with genetic linkage analysis. All differentially expressed genes were mapped to the chromosomes, together with the known QTLs for an induced animal model of Arthritis, Collagen Induced Arthritis (CIA)[14]. Eight deregulated genes map to CIA QTLs (
Deregulated genes express their one or more gene products (or their enzymatic activities) either at a lower or at a higher level when compared to “normal cells” obtained from a healthy individual. This altered expression can be used to scrutinize samples and determine whether a sample is from an individual suffering from inflammatory arthritis (IA) or is at risk of suffering from IA. The invention thus provides a method for determining whether a sample is derived from an individual suffering from inflammation arthritis or from an individual at risk of suffering from IA, said method comprising measuring in said sample the level of expression or enzymatic activity of at least one gene product of a gene of table A, table B, or table 4 and comparing said expression level with a reference value. The mentioned tables indicate whether in IA, expression or enzymatic activity of said gene product is higher than “normal” or lower than “normal”. Thus by measuring the expression level or enzymatic activity of at least one gene product, and comparing it with a reference value, one can identify the sample as being derived from an individual suffering from IA, or at risk of suffering from IA, or derived from an individual that is not suffering from IA or is not at risk of suffering from IA. It is possible to identify a sample in this way by analyzing expression of gene products or their enzymatic activity of one of said genes. In this case, a sample identifies as being derived from an individual suffering from IA, or at risk of suffering from IA when the level of expression or enzymatic activity is altered in the indicative direction given in table A, B, 3, 4 and/or C by at least a factor of 1,5. Thus a gene is said to be deregulated in the context of the invention when the expression level or enzymatic activity of a gene product of said gene is altered in the indicative direction as given in tables A, B, 3, 4 and/or C by a factor of at least 1,5. When expression levels or enzymatic activity of gene products of more than one gene of table A, table B, table 3 or table 4 are determined, the accuracy of the diagnostic method increases allowing comparison of patterns of expression levels in the sample with patterns of a reference. In these cases, samples are identified as being derived from an individual suffering from IA, or at risk of suffering from IA when the pattern of expression levels or enzymatic activity matches the pattern of a reference derived from an individual suffering from IA, or at risk of suffering from IA. Or alternatively, identifies as normal, when the pattern matches the pattern of a reference. A reference as used in the invention is preferably from an arthritic sample, however, a normal sample can also be used. Preferably the reference is derived from at least an arthritic sample and one from a normal sample. Statistical methods to analyze patterns of expression levels are available in the art. A non-limiting statistical method for determining similarity of expression profiles, that can be used advantageously in the present invention is referred to as the Rosetta Error Model (Hughes T R, Marton M J, Jones A R et al. Functional discovery via a compendium of expression profiles. Cell. 2000; 102:109-126). A practical case is described on page 6 line 3-page 7 line 19 of WO 02/103320.
It is of course possible to enlarge the set of genes from which the expression level or enzymatic activity of gene products is determined. In a preferred embodiment, expression levels of gene products of at least 2 and more preferably at least 5 genes of table A, table B, or table 4 are analyzed. Analyzing said 5 genes provides a good correlation with the absence of presence of IA. Increasing the number of analyzed genes of table A or table B, further increases the correlation. Thus in a particularly preferred embodiment gene products or enzymatic activity of at least 10 genes of table A, B, or 4 are analyzed. In a preferred embodiment at least one gene product or enzymatic activity is of a gene of table A and at least one other gene product or enzymatic activity is of a gene of table B or C. In a particularly preferred embodiment said one or more, said at least 2, said at least 5 or said at least 10 genes are genes of table A, optionally supplemented by gene products or enzymatic activity of one or more genes of table B or table C. As mentioned, the set of genes analyzed may be expanded to other genes, preferably genes known to be deregulated in IA. Thus in a preferred embodiment said analysis includes the analysis of gene products of at least one gene of table C. The direction in which expression is affected in the arthritic samples is indicated in tables A, B and C(SF UP, SF down, WJ up, WJ down, where SF means that expression as indicated for synovial fibroblast cells and WJ means that expression is indicated for whole joint cells.
Genes can have several different gene products. Typically, though not necessarily, a gene is transcribed in mRNA, which in turn is translated into protein. A gene may be transcribed in several mRNAs which in turn can be translated into several proteins. As used herein, the term “gene product” encompasses both RNA and protein products. In a preferred embodiment a gene product in a method of the invention comprises RNA. Expression levels of RNA are typically determined with a probe that specifically hybridises with said RNA (Northern blot) and/or with a set of primers that specifically anneal to said RNA (Real Time RT-PCR). Expression levels of proteins are typically determined either directly with antibodies that specifically bind to the protein encoded by the gene or indirectly by the enzymatic/catalytically activity of the protein of interest. Many different kinds of probes are presently known in the art. Probes are often nucleic acids, however, alternatives having the same binding specificity in kind, not necessarily in amount are available to the person skilled in the art, such alternatives include but are not limited to peptide nucleic acids (PNA). Similarly, many different specific binding bodies are available. Of old, antibodies are used. However, currently many different parts, derivatives and/or analogues of antibodies are in use. Non-limiting examples of such parts, derivatives and/or analogues are, single chain Fv-fragments, monobodies, VHH, Fab-fragments and the like. A common denominator of such specific binding bodies is the presence of an affinity region (a binding peptide) that is present on a structural body that provides the correct structure for presenting the binding peptide. Binding peptides are typically derived from or similar to CDR sequences (typically CDR3 sequences) of antibodies, whereas the structure providing body is typically derived from or similar to framework regions of antibodies. For review of suitable probes and specific binding bodies see any of the following references:
Diagnosing early-onset rheumatoid arthritis: the role of anti-CCP antibodies, Vasishta A, Am Clin Lab. 2002 August-September; 21(7):34-6; Keys to unlocking the mysteries of rheumatic autoimmune disease, Moser K L, Gaffney P, Peterson E, Behrens T, Minn Med. 2004 May; 87(5):46-51; A new tool for rheumatology: large-scale analysis of gene expression Lequerre T, Coulouarn C, Derambure C, Lefebvre G, Vittecoq O, Daveau M, Salier J P, Le Loet X, Joint Bone Spine, 2003 August; 70(4):248-56.
Expression levels of gene products can be altered as result of a variety of mechanisms. These include but are not limited to, altered level of transcription/translation, altered stability, altered degradation of the protein and/or RNA. The altered expression level can also be the result of a combination of processes in a cell. The sample to be analyzed is typically derived from an individual suspected of suffering from IA or suspected of being at risk of suffering from IA. The sample can be either synovial membrane or synovial fluid isolated during an arthroscopy, and/or serum isolated from the blood of said individual.
An individual is typically a human although the expression level or enzymatic activity of the genes of interest could be determined in animal models to determine drug efficacy. Therefore an individual is preferably a mammal, preferably a rodent or a primate. More preferably a human.
A particular class of deregulated genes in the present invention is involved with cytoskeleton arrangement. The invention therefore in one aspect provides a method for determining whether a sample is derived from an individual suffering from inflammatory arthritis, preferably rheumatoid arthritis, or from an individual at risk of suffering thereof, said method comprising measuring in said sample the level of expression of at least one gene product of a gene involved in cytoskeleton arrangement and comparing said expression level with a reference value.
Cytoskeleton arrangement in a cell comprises both the structural processes involved with assembly and organisation of cytoskeleton and the signaling machinery in a cell that directs the structural processes. Assembly includes both gain and loss of cytoskeleton. Organisation typically includes the apparent three dimensional appearance of the cytoskeleton in a cell. The signaling machinery involved in cytoskeleton organisation has an extracellular component and an intracellular component, thus in a preferred embodiment said gene is involved in upstream/downstream extracellular or intracellular signaling to the cytoskeleton. A preferred embodiment of the invention is particularly related to deregulation of genes involved in assembly of actin cytoskeleton. Thus in a preferred embodiment, said gene is involved in binding and/or rearrangement of actin cytoskeleton. In a preferred embodiment said gene is involved in cytoskeleton arrangement in a synovial fibroblast. In a particularly preferred embodiment said gene is any one of the following: tubulin alpha 1, RAB14, Gelsolin, phosphatidylinositol membrane-associated, matrix metalloproteinase 3, matrix metalloproteinase 13, tissue inhibitor of matrix metalloproteinase 13, cathepsin S, matrix gamma-carboxylglutamate, integrin alpha X, myosin light chain, troponin T1 skeletal slow, troponin I skeletal fast 2, VASP, Gesolin and/or table 4. The individual is suffering from inflammatory arthritis when there is at least a differences of at least 1.5 in the level of expression of said gene product of a gene involved in cytoskeleton arrangement in said sample and said reference value. Preferably, said method comprises measuring in said sample the level of expression of a gene product of at least two of said genes involved in cytoskeleton arrangement and comparing said expression levels with reference values. In a particularly preferred embodiment the level of expression of a gene product of at least five of said genes involved in cytoskeleton arrangement is measured and compared with a reference value.
While measuring at least one gene products expression level or enzymatic activity and comparing it with a reference value, according to a method of the invention, one can determine a magnitude of a difference between said expression level and said reference level. In one embodiment a method according to the invention is provided, wherein a magnitude of a difference between said expression level and said reference value is normative for a degree in which said inflammation arthritis is present. A magnitude of a difference as used herein comprises at least a significant difference between the measured value and the reference value. A magnitude of difference can for example be determined by measuring fluorescence intensities. A difference between said expression level and said reference value is normative in that it ascertains a degree in which inflammatory arthritis is present. A degree in which inflammatory arthritis is present can either be a stage of IA that said disease has reached in an individual and/or it can be a measurement of severity of IA in an individual.
Deregulation of genes is a process that occurs in many different diseases. Each of those diseases has a characteristic gene or multiple genes that are deregulated in said diseases. IA is such a disease. IA is a generic term, which includes specific types of arthritis. Specific types of arthritis are typically associated with different characteristic genes that are deregulated. Deregulation of a gene will result in alteration of an expression level of a gene product of a gene and thus in an altered amount of a gene product or in alteration of a composition of a gene product. In one embodiment the invention provides the use of an expression level of a gene product of a gene of table A, table B, or table 4 for typing inflammatory arthritis in an individual. In a preferred embodiment expression levels of gene products of at least 2 and more preferably at least 5 genes of table A, table B, or table 4 are analyzed. Analyzing said 5 genes gives a good picture of the involvement of characteristic genes of a type of IA. Increasing the number of analyzed genes of table A, table B, or table 4, further increases the certainty about the type of IA concerned. Thus in a particularly preferred embodiment gene products of at least 10 genes of table A, table B, or table 4 are analyzed. In another preferred embodiment a gene product of a gene of table A is analyzed. In a further preferred embodiment expression levels of gene products of at least 2 and more preferably at least 5 genes of table A are analyzed. In a particularly preferred embodiment gene products of at least 10 genes of table A are analyzed. A sample is typed as derived from an individual or non-human animal suffering from or at risk of suffering from IA when the expression level of the tested genes is changed in the direction as indicated in tables A, B or C(SF UP, for genes that are over-expressed in synovial fibroblast cells in IA and SF-down for genes that are under-expressed in said cells in IA, WJ UP, for genes that are over-expressed in whole joint cells in IA and WJ down for genes that are under-expressed in whole joint cells in IA).
Inflammatory arthritis includes a number of specific arthritis conditions such as but not limited to Rheumatoid Arthritis, Systemic Lupus Erythematosus, Juvenile Rheumatoid Arthritis, Relapsing Polychondritis, Spondyloarthropathie, Ankylosing Spondylitis, Reiter's Syndrome, Psoriatic Arthritis, Enteropathic Arthritis, Crystal Deposition Disease, Gout Chondrocalcinosis, Calcium Hydroxyaptite Crystal Deposition Disease. In a preferred embodiment the invention is concerned with Rheumatoid Arthritis (RA). Thus when the invention refers to inflammatory arthritis in general, the preferred application is in rheumatoid arthritis.
Typing IA according to the invention preferably comprises determining a pattern of expression levels of gene products (i.e. higher than normal SF UP, WJ up or lower than normal SF down, WJ down in tables A, B and C), as a pattern of measured values is more specific than individual measured values. The invention thus provides a use according to the invention, wherein said typing comprises determining a pattern of expression levels of gene products of at least two genes in a sample and wherein at least one of said genes is a gene of table A, table B, or table 4. In a preferred embodiment at least one gene product is of a gene of table A and at least one other gene product is of a gene of table B or C. In a particularly preferred embodiment said one or more, said at least 2, said at least 5 or said at least 10 genes are genes of table A, optionally supplemented by gene products of one or more genes of table B or table C. As mentioned, the set of genes analyzed may be expanded to other genes, preferably genes known to be deregulated in IA. Thus in a preferred embodiment said analysis includes the analysis of gene products of at least one gene of table C.
In one embodiment the invention provides a use according to the invention, wherein said typing comprises indicating one of the following types: Rheumatoid Arthritis, Systemic Lupus Erythematosus, Juvenile Rheumatoid Arthritis, Relapsing Polychondritis, Spondyloarthropathie, Ankylosing Spondylitis, Reiter's Syndrome, Psoriatic Arthritis, Enteropathic Arthritis, Crystal Deposition Disease, Gout Chondrocalcinosis, Calcium Hydroxyaptite Crystal Deposition Disease. In a particularly preferred embodiment of the invention said typing comprises indicating an inflammatory arthritide. Said types of arthritis are all included in the generic term Inflammatory Arthritis as used in the invention.
In one embodiment of the invention genes and/or the expression level of gene products encoded thereby are detected. The expression level of their products or their enzymatic activity are determined with a probe or a set of probes or appropriate enzymatic assays. The invention thus provides a kit of parts comprising a probe or a set of probes or enzymatic assays, wherein at least one of said probes is a probe specific for one of the genes listed in table A, table B, or table 4. Preferably a set of probes comprises two or more probes that are specific for one of the genes listed in table A, table B, or table 4. In another embodiment the invention provides a kit of parts according to the invention, comprising a probe or a set of probes, wherein at least one of said probes is a probe specific for one of the genes listed in table A. The invention further provides a kit of parts according to the invention, comprising at least two probes, wherein at least one of said probes is a probe specific for one of the genes listed in table A, table B, or table 4 and at least one of said probes is specific for one of the genes listed in table C. Also provided by the invention is a kit of parts according to the invention, comprising at least two probes, wherein at least one of said probes is a probe specific for one of the genes listed in table A and at least one of said probes is specific for one of the genes listed in table B or in table C. Any of the probe sets according to the invention can comprise probes specific for a gene that is not listed in table A, B or C. Such a probe is preferably specific for a gene that is known to be involved in inflammatory arthritis, more preferably known to be involved in Rheumatoid Arthritis. In another preferred embodiment such a probe is specific for a gene known to be involved in cytoskeleton arrangement.
The present invention has surprisingly found that in IA particular genes involved with cytoskeleton arrangement are deregulated. The invention thereby presents a novel means of therapy. In one embodiment the invention hereto provides the use of a compound for altering cytoskeleton or its organization in a cell for the manufacture of a medicament for alleviating at least one symptom of inflammatory arthritis in an individual. An individual with IA can present either one symptom or multiple symptoms. Alleviating at least one symptom will make the condition of an individual with IA better, thereby improving a patient's quality of life. When reference is made to alleviating a symptom of a disease, it is meant that the severity of a symptom is at least reduced.
Rheumatoid arthritis is a polygenic disease. Studying multiple genes therefore provides more valuable information about RA than concentrating on one gene. Thus, in the invention concerted gene functions and cellular processes were studied in order to gather valuable clues and to prioritize targets for therapeutic intervention of RA. In this context, deregulated processes were determined as these are formalized through the controlled vocabulary of the Gene Ontology Consortium [17]. GO term frequencies were calculated in the selected gene list and their statistical significance was estimated. The resulting table (Supplementary Table 3) includes a number of functions that encompass accumulated knowledge about the pathogenesis of the disease, such as collagen catabolism, complement activation, immune and stress response. More importantly, 5 out of 26 predicted deregulated GO functions concerned (directly or indirectly) the actin cytoskeleton. In order to increase the experimental evidence F-actin stress fibers were visualized in vitro on arthritic as well as wt SFs, both primary and immortalized. As evident in
The invention thus surprisingly found that deregulation of genes involved in cytoskeleton arrangement in a cell is a prominent feature in arthritic SF's. A new therapeutic and diagnostic entrance is hereby established. In one embodiment the invention provides the use of a compound for altering cytoskeleton or its organization in a cell for reducing stress fibers in a synovial cell.
Recent experiments have shown that the cytoskeleton plays a critical role in tumorigenesis [31]. Since the role of the cytoskeleton in cancer has been established, compounds that are directed to the cytoskeleton have been developed for the treatment of cancer. In one embodiment the invention provides the use of a drug for the treatment of cancer for altering cytoskeleton or its organization in a cell for the manufacture of a medicament for alleviating at least one symptom of inflammatory arthritis in an individual. In another embodiment the invention provides the use of a drug for the treatment of cancer for altering cytoskeleton or its organization in a cell for reducing stress fibers in a synvovial cell or for the treatment of inflammatory arthritis. Non-limiting examples of drugs that are directed to the cytoskeleton are: Cytochalasin D, Phalloidin, Jasplakinolide, Chondramides, Dolostatin 11, Latrunculin A, Swinholide A, Misakinolide A, Mycalolide B, Shinxolide C, Scytophycins, Goniodomin A, Pseudopterolide, Lysophosphatidylcholine, Lysophosphatidyl acid. In one embodiment the invention provides the use of a compound according to the invention, wherein said compound is one of the following: Cytochalasin D, Phalloidin, Jasplakinolide, Chondramides, Dolostatin 11, Latrunculin A, Swinholide A, Misakinolide A, Mycalolide B, Shinxolide C, Scytophycins, Goniodomin A, Pseudopterolide, Lysophosphatidylcholine, Lysophosphatidyl acid.
Many different animal models are used in studies of RA. An animal model typically represents a patient with RA. In order to make an animal represent an individual suffering from RA different strategic routes can be applied. Disease can for example be induced by injection of an immunogenic substance. Alternatively an animal can be bred that has an altered gene expression. A gene in such an animal can either be knocked-out, over-expressed or under-expressed. The invention provides an animal with an altered gene expression. In one embodiment the invention provides a non-human animal wherein at least one of the genes listed in table A, table B, table 3 or table 4 is knocked-out, over-expressed or under-expressed. In another embodiment the invention provides a non-human animal wherein at least one of the genes listed in table A is knocked-out, over-expressed or under-expressed. The invention further provides a non-human animal wherein at least one of the genes listed in table A, table B, table 3 or table 4 and wherein at least one of the genes listed in table C or table 6 is knocked-out, over-expressed or under-expressed. Furthermore the invention provides a non-human animal wherein at least one of the genes listed in table A and wherein at least one of the genes listed in table B, table C, table 3, table 4 or table 6 is knocked-out, over-expressed or under-expressed. Naturally the invention also provides an animal wherein, besides a gene listed in table A, B, 3, 4, 6 or C, another gene is knocked-out, over-expressed or under-expressed. Said another gene preferably is a gene known to be involved in RA. Another preferred gene in this context is a gene known to be involved in cytoskeleton rearrangement. The invention in one embodiment provides a non-human animal wherein at least one gene known to be involved in cytoskeleton rearrangement is knocked-out, over-expressed or under-expressed.
In RA one of the cell types that plays a very important role in the disease process is the synovial fibroblast. Therefore, in a preferred embodiment, the invention provides a non-human animal according to the invention, wherein said cytoskeleton arrangement is in a synovial fibroblast. Cytoskeleton organization is typically influenced by diverse signals. Therefore the invention in one embodiment provides a non-human animal according to the invention, wherein said gene is involved in extracellular or intracellular signaling to the cytoskeleton. In a preferred embodiment the invention provides a non-human animal according to the invention, wherein said gene is involved in binding and/or rearrangement of the actin cytoskeleton. The invention provides genes that are involved in cytoskeleton arrangement. The invention thus in a preferred embodiment provides a non-human animal according to the invention, wherein said gene is any one of the following: tubulin alpha 1, RAB14, Gelsolin, phosphatidylinositol membrane-associated, matrix metalloproteinase 3, matrix metalloproteinase 13, tissue inhibitor of matrix metalloproteinase 13, cathepsin S, matrix gamma-carboxylglutamate, integrin alpha X, myosin light chain, troponin T1 skeletal slow, troponin I skeletal fast 2, VASP, ADF/Cofilin and/or is gene of table 4. Preferably said gene is Gelsolin, VASP and or ADF/Cofilin. These genes are further discussed in the examples, tables and figures.
An alternative means to create an animal wherein a gene is over-expressed or under-expressed is the introduction of a foreign gene into the genome of said animal. Such an animal is a transgenic animal. The invention in one embodiment provides a non-human animal according to the invention, wherein said animal is transgenic for said gene or homologue thereof that is over-expressed or under-expressed. A foreign gene can be constructed using recombinant DNA methodology. A preferred non-human animal as provided by the invention is rodent or a non-human primate. Preferably, said non-human animal is a mouse. Characteristics of these animals are well known and these animals are practical for laboratory circumstances. Mice and rats are often used in disease models for RA. In a preferred embodiment the invention provides the use of a non-human animal according to the invention as a disease model. Preferably said disease represented by said model is rheumatoid arthritis. Alternatively said disease is a disease in which deregulated genes as determined by the invention are involved. Inflammatory and/or rheumatoid arthritis in an animal can either occur spontaneously, by known or unknown internal causes, or can be induced. In a preferred embodiment the invention provides the use of a non-human animal as a disease model for rheumatoid arthritis, wherein said rheumatoid arthritis is induced. Induction can proceed directly or indirectly. Direct induction for example occurs when an immunogenic composition that causes arthritis is injected. Examples of such immunogenic compositions include but are not limited to collagen. Indirect induction is for example due to a genetic defect, because of which an animal at a certain stage of its life develops arthritis. Preferred examples of such induction are indicated in table 5.
Further preferred is a non-human animal of the invention wherein said at least one of the genes listed in table A, table B, table C, table 3, table 4 or table 6 is knocked-out, over-expressed or under-expressed, in an arthritis model. Preferably a genetic arthritis model, preferably a genetic model as indicated in table 5, and-or in a Tg197 mouse. Provided is preferably a Tg197 mouse wherein Gelsolin, VASP and/or ADF/Cofilin is knocked-out, over-expressed or under-expressed. Preferably specifically in the myeloid lineage. Preferably, Gelsolin and/or ADF/Cofilin are knocked out or under-expressed. VASP is preferably over-expressed.
The invention further provides the use of a non-human animal of the invention as an inflammatory arthritis model. Preferably, said non-human animal is suffering from inflammatory arthritis. This is typically achieved by providing a non-human animal of the invention with a composition that induces said inflammatory arthritis. Such compositions are well known in the art and include but are not limited to collagen (are there other well known examples).
Inflammatory inflammation being a severe disease for which no definite cure has been found, there is an ongoing need for effective medication for diverse types and stages of RA. In the process of searching for effective medication a reliable test for compounds comprised in this medication is essential. The invention provides a method for determining whether a compound is effective in alleviating a symptom of IA, comprising testing said compound in a model system for said symptom, said method characterized in that said compound is a compound for altering the cytoskeleton or its organization in a cell. As in the development of cancer therapy compounds have been found that are involved in cytoskeleton arrangement, said compounds are used in a method of the invention. Thus, in one embodiment the invention provides a method for determining whether a compound is effective in alleviating a symptom of IA, comprising testing said compound in a model system for said symptom, said method characterized in that said compound is a compound for altering the cytoskeleton or its organization in a cell, wherein said compound is effective in cancer therapy. Said compound preferably is one of the following: Cytochalasin D, Phalloidin, Jasplakinolide, Chondramides, Dolostatin 11, Latrunculin A, Swinholide A, Misakinolide A, Mycalolide B, Shinxolide C, Scytophycins, Goniodomin A, Pseudopterolide, Lysophosphatidylcholine, Lysophosphatidyl acid.
In the invention specific genes have been found to be deregulated in IA. In the search process for compounds with therapeutic value for treatment of IA, the expression of these genes is a useful parameter. For other diseases in which the deregulated genes that are found in the invention are deregulated, said parameter can be used as well. The similar deregulation for instance occurs because of a similar causal mechanism that acts alike in these diseases. The invention provides a method for determining whether a compound has an effect on an expression of one or more of the genes listed in table A, table B, or table 4, comprising testing said compound in an environment wherein said gene is expressed and comparing a resulting expression level with a reference value. Preferably reference value indicating a higher than normal expression level for the genes as listed in table A, B or C by SF UP and/or WJ up or a lower than normal expression level as indicated in table A, B or C by SF down, and/or WJ down. The invention further provides a method for determining whether a compound has an effect on an expression of one or more of the genes listed in table A, comprising testing said compound in an environment wherein said gene is expressed and comparing a resulting expression level with a reference value. In one embodiment the invention provides a method for determining whether a compound has an effect on an expression of one or more of the genes listed in table A, table B, or table 4 and on one or more of the genes listed in table C, comprising testing said compound in an environment wherein said genes are expressed and comparing a resulting expression level with a reference value. In another embodiment the invention provides a method for determining whether a compound has an effect on an expression of one or more of the genes listed in table A and on one or more of the genes listed in table B or in table C, comprising testing said compound in an environment wherein said genes are expressed and comparing a resulting expression level with a reference value. Preferably an effect on multiple genes that are listed in table A, B or C is determined.
Genes that have been found to be deregulated in the invention are in one embodiment of the invention used in a disease model of IA. In such an embodiment said genes are deregulated on purpose. In contrast, in an individual with IA, said genes already are deregulated. In this context, the purpose is to regulate said genes in order to improve the condition of said individual. In one embodiment the invention therefore provides a method for alleviating at least one symptom of rheumatoid arthritis in an individual, comprising modifying expression in said individual of a gene that is involved in a cytoskeleton arrangement. Modifying expression as used herein alters the expression of said deregulated genes in said individual in such a way that the condition of said individual is improved. Alleviating a symptom of IA in a patient will usually imply up-regulating a down-regulated gene or down-regulating an up-regulated gene (see table A, B or C SF or WJ down and SF or WJ UP respectively), such that the expression of said gene approaches or equals a reference value of a normal, i.e. healthy, state. In a preferred embodiment the invention provides a method for alleviating at least one symptom of rheumatoid arthritis in an individual, comprising modifying expression in said individual of a gene that is involved in a cytoskeleton arrangement, wherein expression of said gene is modified in a synovial fibroblast. In one embodiment of the invention said gene is involved in extracellular or intracellular signaling to the cytoskeleton. In another embodiment said gene is involved in binding and/or rearrangement of the actin cytoskeleton. The invention discloses genes that are involved in cytoskeleton arrangement and deregulated in IA. In one embodiment the invention thus provides a method according to the invention, wherein said gene is any one of the following: tubulin alpha 1, RAB14, Gelsolin, phosphatidylinositol membrane-associated, matrix metalloproteinase 3, matrix metalloproteinase 13, tissue inhibitor of matrix metalloproteinase 13, cathepsin S, matrix gamma-carboxylglutamate, integrin alpha X, myosin light chain, troponin T1 skeletal slow, troponin I skeletal fast 2, VASP, ADF/Cofilin and/or a gene of table 4. A method of the invention is also applicable in another disorder than IA wherein genes involved in cytoskeleton arrangement, such as aforementioned genes, are deregulated. In a preferred embodiment said gene in a method according to the invention codes for Gelsolin, VASP or ADF/Cofilin. Preferably Gelsolin.
Gelsolin is an actin binding protein [33] that has been implicated, amongst others, in the transduction of signals into dynamic rearrangements of the cytoskeletal architecture. In the presence of calcium, gelsolin severs preexisting actin filaments and caps them, thereby preventing monomer addition to their fast-growing ends. The barbed end cap is highly stable, even in the absence of calcium, unless displaced by interactions with regulatory phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP2). In the presence of a large pool of profilin (another actin binding protein), or under depolymerizing conditions, these gelsolin-capped ends allow the disassembly of populations of actin filaments by subunit loss from the pointed ends [34]. Gsn−/− fibroblasts were found to have excessive stress fibers in vitro [18], very similar to the ones observed in arthritic SFs, where gelsolin was found down-regulated with a variety of methods. Knocking out gsn expression from the arthritic mice resulted in exacerbation of the disease (
The genes that are involved in cytoskeleton arrangement are only one group of genes that, in the invention, are found to be deregulated in IA. Other genes are, amongst others, listed in table A, table B, table 3, table 4 or C. The invention in one embodiment provides a method for alleviating at least one symptom of inflammatory and/or rheumatoid arthritis in an individual, comprising modifying the level of expression of at least one gene product of a gene of table A, table B, table 3, table 4 or C in cells of said individual. In another embodiment said method comprises modifying the level of expression of at least one gene product of a gene of table A in cells of said individual. In a further embodiment said method comprises modifying the level of expression of at least one gene product of a gene of table A, table B, or table 4 and at least one gene product of a gene of table C in cells of said individual. Further the invention provides a method according to the invention, wherein said method comprises modifying the level of expression of at least one gene product of a gene of table A and at least one gene product of a gene of table B or table C in cells of said individual. In preferred circumstances a method according to the invention alleviates two or more symptoms. Preferably the level of expression of two, three or more gene products of genes of table A, B, C, or 4 are modified. The direction in which an expression level is modified is dependent on the direction in which an expression level of a gene product of a gene is deregulated (see table A, B or C). The extent to which modification is applied depends on the degree in which said level of expression of said gene product of said gene is deregulated. The expression level of a gene product of a gene is preferably modified to match or approach a reference value that represents a healthy status, thus a normal level. Alternatively, the expression level of a gene product of a gene is modified to match a physiologically acceptable level of an individual. Physiologically acceptable levels will often be levels that are approximately normal levels. Normal levels are levels that can be found in a healthy individual of the same age and constitution. Physiologically acceptable levels can fall outside a normal range but do still provide a sufficient function for an individual.
A gene product in a method of the invention encompasses both RNA and protein products. In one embodiment the invention provides a method for alleviating at least one symptom of inflammatory and/or rheumatoid arthritis in an individual according to the invention, wherein said gene product comprises protein. A cell in which the level of expression of a gene product of a gene is modified in a method of the invention, is a cell that is affected by the disease process of inflammatory and/or rheumatoid arthritis in an individual. Preferably said cell is a cell that makes contact with a joint of said individual. In a preferred embodiment of the invention, a method according to the invention is provided, wherein said cells are cells of a joint or precursors thereof. These cells can be part of any component of the joint. Precursor cells can already be present in or in the surroundings of said joint or they can for example be derived from the blood. In a preferred embodiment said cells comprise synovial membrane cells or cells of synovial fluid. Preferably in a method according to the invention the level of expression of at least two gene products is modified. In a preferred embodiment a method according to the invention comprises modifying the level of expression of at least one gene product of a gene of table A, table B, or table 4 in cells of said individual and modifying the level of expression of at least one gene product of a gene known to be deregulated in IA other than listed in table A, B or C. Further provided is a method according to the invention, wherein said method comprises modifying the level of expression of at least one gene product of a gene of table A in cells of said individual and modifying the level of expression of at least one gene product of a gene known to be deregulated in IA other than listed in table A, B or C. In a further embodiment said method comprises modifying the level of expression of at least one gene product of a gene of table A or table B and at least one gene product of a gene of table C in cells of said individual and modifying the level of expression of at least one gene product of a gene known to be deregulated in IA other than listed in table A, B or C. Further the invention provides a method according to the invention, wherein said method comprises modifying the level of expression of at least one gene product of a gene of table A and at least one gene product of a gene of table B or table C in cells of said individual and at least one gene product of a gene known to be deregulated in IA other than listed in table A, B or C.
In a preferred embodiment, the invention provides a method for alleviating at least one symptom of inflammatory and/or rheumatoid arthritis in an individual according to the invention, wherein modifying said expression level comprises lowering expression of a gene that is over-expressed in said individual compared to a comparable healthy individual, or elevating expression of a gene that is under-expressed in said individual compared to a comparable healthy individual (table A, B or C).
Raising expression of an under-expressed gene is for instance effected by the addition of a transcription factor and/or by activating an enhancer-sequence. Lowering expression of an over-expressed gene can for example be realized by applying antisense therapy or by administering a repressor protein that binds to the promoter of said aberrantly expressed gene. In a preferred embodiment, the invention provides a method according to the invention, wherein said lowering said expression comprises utilizing an antisense-sequence. There are many different anti-sense approaches to down-regulate production of a gene product available to a person skilled in the art. Antisense technology exploits oligonucleotide analogs to bind to target RNAs via Watson-Crick hybridization. After binding, the antisense agent either disables or induces the degradation of the target RNA. Antisense agents can also alter splicing. The basic mechanisms of antisense technology have been extensively explored in the last ten years. Much has been learned about the medicinal chemistry and the pharmacologic, pharmacokinetic, and toxicologic properties of antisense molecules. Antisense technology has proven valuable in gene functionalization and target validation.
Non-limiting examples of antisense approaches are interference RNA (RNAi), microRNA and splice interference techniques such as exon-skipping. An antisense sequence can be administered as a single stranded molecule or be part of a hairpin construct-molecule. The anti-sense sequence can be administered directly or be produced in a cell by means of an expression cassette. Today RNA interference (RNAi), is a technique in which exogenous, double-stranded RNAs (dsRNAs) that are complimentary to known mRNA's, are introduced into a cell to specifically destroy that particular mRNA, thereby diminishing or abolishing gene expression. The technique has proven effective in Drosophila, Caenorhabditis elegans, plants, and recently, in mammalian cell cultures. For research purposes in cultured mammalian cells, small interfering RNAs (siRNAs) are delivered into the cell. SiRNAs are dsRNAs of approximately 21-25 nucleotides. This is done with transfection reagents, solutions optimized for allowing DNA and RNA to be absorbed by cultured cells. In a preferred embodiment the invention provides a method according to the invention, wherein lowering said expression comprises utilizing RNAi. Preferably siRNA is used for the induction of RNAi. In another embodiment shRNA is used. A classical antisense sequence can be complementary to at least 10% and preferably at least 25 of the mRNA it is specific for. SiRNA, miRNA, RNAi and exon-skipping antisense sequence have the normal overlap that is typically used in the field. They typically have between 15-40 bases overlap, preferably between 15-30. SiRNAs have typically 21-25 nucleotides. The antisense can be provided as single-stranded or double stranded molecules, depending on their effectivity as is commonly known in the art. The antisense sequence can be comprised in a nucleic acid or a functional equivalent thereof.
An anti-sense sequence can for example be used in a compound that is applied to modify an expression level of a gene product of a gene. In a preferred embodiment the invention provides the use of a compound for the manufacture of a medicament for alleviating at least one symptom of inflammatory and/or rheumatoid arthritis in an individual, said compound comprising an antisense-sequence of one of the genes listed in table A or table B. In another embodiment the invention provides a use according to the invention, wherein said compound comprises an antisense-sequence of one of the genes listed in table A. Further provided is a use according to the invention, wherein said compound comprises an antisense-sequence of one of the genes listed in table A or table B and an antisense-sequence of one of the genes listed in table C. In another use according to the invention, said compound comprises an antisense-sequence of one of the genes listed in table A and an antisense-sequence of one of the genes listed in table B or in table C. Preferably antisense-sequences of two or more genes listed in table A, B or C are comprised in a use of the invention. In addition to antisense-sequences of aforementioned genes, antisense-sequences of other genes known to be involved in IA can be comprised in said compound. In preferred embodiment an antisense is provided specific for mRNA from a gene that is identified in table A, B or C by SF UP or WJ up.
Before a suitable drug can be developed, a drug target has to be identified and validated. Drug targets are mainly proteins, DNA or RNA. The invention provides such drug targets as it has identified genes and gene products that are deregulated in IA. Thus in one embodiment the invention provides the use of a protein encoded by a gene of table A, table B, or table 4 as a drug target for selecting candidate drugs for a treatment of inflammatory arthritis. Further provided is a use according to the invention, wherein said protein is encoded by a gene of table A. In one embodiment a first protein encoded by a gene of table A or table B is used as a first drug target and a second protein encoded by a gene of table C is used as a second drug target. In another embodiment said first protein encoded by a gene of table A is used as a first drug target and a second protein encoded by a gene of table B or table C is used as a second drug target. By a use of the invention said drug targets are thus validated. After validation the drugs are tested further, first in a pre-clinical stage and then in a clinical setting.
As already mentioned, the invention provides probes specific for deregulated genes in IA. Probes thus provided, are also applicable for disorders other than IA in which said genes are deregulated. In order to make the addressing of a gene more specific, more then one probe is preferentially used. More then one probe, as used in the invention, is preferably comprised in an array. An array is used to analyze one or more samples at the same time for one or more genes or gene products. An array can be specific for a whole genome, but in the invention preferentially is specific for a part of a genome, i.e. a subgenome. Another preference of the invention is an array specific for a transcriptome or a part of a transcriptome, i.e. a subtranscriptome. A transcriptome of an organism as used herein, are all DNA transcripts, mostly mRNA's, being produced at a certain point. A certain point is specified in its location and/or in time. Thus in a preferred embodiment of the invention, an array is a subgenome array or a subtranscriptome array. In a preferred embodiment the invention provides an array comprising at least two probes, wherein at least one probe is specific for at least one gene listed in table A, table B, or table 4. The invention further provides an array according to the invention, wherein at least one probe is specific for at least one gene listed in table A. Also provided by the invention is an array according to the invention, wherein at least one probe is specific for at least one gene listed in table A or table B and wherein at least one probe is of at least one gene listed in table C. In another embodiment the invention provides an array according to the invention, wherein at least one probe is specific for at least one gene listed in table A, and wherein at least one probe is specific for at least one gene listed in table B or in table C. In a preferred embodiment an array of the invention is a microarray. Said probes specific for said genes are thus preferably comprised in a microarray. Said microarray preferably comprises oligonucleotides or a functional equivalent thereof. Probes or oligonucleotides are preferably between 15-80 nucleotides or functional equivalents thereof long. More preferably between 15-40.
An array as provided by the invention is in one embodiment used in a method of the invention. Provided is the use of an array according to the invention in a method according to the invention for determining whether a sample is derived from an individual suffering from inflammatory and/or rheumatoid arthritis or from an individual at risk of suffering there from, said method comprising measuring in said sample the level of expression of at least one gene product of a gene of table A or table B and comparing said expression level with a reference value. Further provided is the use of an array according to the invention for determining whether an individual is suffering from IA or is at risk of suffering from IA.
Protein expression levels can be measured in various ways such as ELISA, Western blotting or another sort of antibody based calorimetric or fluorescent quantitation of protein. In a preferred embodiment the invention provides an array comprising at least two binding bodies, wherein at least one binding body is specific for at least one gene product of a gene of table A, table B, or table 4. In on embodiment said binding body is an antibody. Said gene product is RNA or a protein. In one embodiment the invention provides an array according to the invention, wherein said gene product is a protein. In a preferred embodiment an array according to the invention comprises at least one binding body that is specific for at least one gene product of a gene of table A. Further provided is an array according to the invention, wherein at least one binding body is specific for at least one gene product of a gene of table A. In one embodiment the invention provides an array according to the invention, wherein at least one binding body is specific for at least one gene product of a gene of table A, table B, or table 4, and wherein at least one gene product is of a gene of table C. In another embodiment the invention provides an array according to the invention, wherein at least one binding body is specific for at least one gene product of a gene of table A, and wherein at least one gene product is of a gene of table B or table C. Said array according to the invention preferably is a microarray. Said array is in one embodiment used in a method according to the invention. Provided is the use of an array according to the invention in a method according to the invention for determining whether a sample is derived from an individual suffering from inflammatory and/or rheumatoid arthritis or from an individual at risk of suffering there from, said method comprising measuring in said sample the level of expression of at least one gene product of a gene of table A or table B and comparing said expression level with a reference value. Further provided is the use of an array according to the invention for determining whether an individual is suffering from IA or is at risk of suffering from IA.
Where in the description reference is made to table A or table B this reference also includes the reference to tables 3 and 4. In these cases a preference is indicated for tables A, B or table 3. Preferred is Table A or table B, Preferred is at least table A.
The invention further provides a method for determining whether a compound, a (poly)peptide or a nucleic acid molecule or functional equivalent thereof affects a phenotype of synovial fibroblast cells of an individual or non-human animal suffering from inflammatory arthritis or at risk of suffering thereof, comprising culturing synovial fibroblast cells in vitro from a sample obtained from an affected joint or a joint at risk of being affected from said individual or non-human animal, in the presence of said compound and/or providing said synovial fibroblast cells with said nucleic acid or said functional equivalent thereof and determining whether a phenotype of said cultured synovial fibroblast cells is altered. Preferably, wherein said synovial fibroblast cells are obtained from a joint of a mouse comprising the genetic alteration of the Tg197 mouse, a non-human animal according to the invention and/or a genetic model as depicted in table 5. In a preferred embodiment said compound, nucleic acid or functional equivalent thereof is a compound for altering cytoskeleton or its organization in a cell. Preferably said compound is effective in cancer therapy, as described herein above. Preferably said compound is one of the following:
Cytochalasin D, Phalloidin, Jasplakinolide, Chondramides, Dolostatin 11, Latrunculin A, Swinholide A, Misakinolide A, Mycalolide B, Shinxolide C, Scytophycins, Goniodomin A, Pseudopterolide, Lysophosphatidylcholine, Lysophosphatidyl acid. In a preferred embodiment said nucleic acid comprises an antisense-sequence, preferably an RNAi, an siRNA, or an shRNA. Said antisense sequence can be provided in a nucleic acid or a functional equivalent thereof. Said nucleic acid or functional equivalent thereof preferably comprises a morpholino, locked nucleic acid (LNA) or a peptide nucleic acid (PNA). In a particularly preferred embodiment said antisense-sequence is an antisense sequence of a gene listed in table A, B, C, or table 4, wherein said gene is identified in either of said tables by expression SF Up or WJ Up or said gene is VASP. In a particularly preferred embodiment said antisense sequence is specific for a gene of table A indicated in table A by expression SF UP or WJ up. It is preferably determined whether stress fiber formation, existence is affected. In another preferred embodiment said phenotype comprises adhesion, migration, proliferation, wound-healing and/or apoptosis of said synovial fibroblast cells. The present in vitro assay is particularly suited for high throughput screening, particularly for screening candidate drugs and/or nucleic acids of the invention for potential therapeutic effect in IA. In a preferred embodiment said nucleic acid codes for a protein produced by a gene of table A, B, C, or 4, wherein said gene is identified in table A, B or C by SF down and/or WJ down or said gene is gelsolin or ADF/Cofilin. An antisense sequence is preferably specific for a gene that is identified in table A, B or C by SF or WJ UP, or VASP. Said (poly)peptide is preferably an antibody or functional equivalent thereof such as but not limited to a single chain antibody, a monobody, a FAB fragment or the like. A peptide preferably comprises at least 6, and preferably at least 8 amino acids in peptide linkage. A peptide is preferably not longer than 40 amino acids. A polypeptide comprises 41 or more amino acids in peptidic linkage. A (poly)peptide may of the invention may comprise modifications such as chemical modifications and/or post translational modifications such as in a protein. A (poly)peptide may be linear or circular.
The present invention further provides the use of a gene delivery vehicle comprising the coding region of a gene of table A, B or C identified by SF and/or WJ down, Gelsolin or ADF/Cofilin, for delivering said gene to cells of a joint. Preferably said gene is delivered to a synovial fibroblast cell. In another aspect the invention provides the use of a gene delivery vehicle comprising the coding region of a gene of tables A, B or C identified by SF or WJ down, Gelsolin or Cofilin, for the preparation of a composition for delivering said gene to cells of a joint (WJ down) and/or a synovial fibroblast cell (SF down). Preferably said gene delivery vehicle is used for the treatment of inflammatory arthritis. In a particularly preferred embodiment said gene is Gelsolin, ADF/Cofilin. The invention thus further provides a gene delivery vehicle comprising a coding region for Gelsolin and/or ADF/Cofilin. Further provided is the use of a gene delivery vehicle comprising a nucleic acid of a functional equivalent thereof comprising an antisense-sequence for a gene of table A, B or C identified by SF and/or WJ UP or VASP, or comprising an expression cassette therefore, for delivering said nucleic acid or functional equivalent thereof to cells of a joint, or to a synovial fibroblast cell. Preferably wherein said antisense-sequence comprises an shRNA, RNAI and/or siRNA. Preferably wherein said gene delivery vehicle comprises a liposome, or a viral vector. Preferably an adenovirus, an adeno-associated virus vector or a lentivirus vector. The gene delivery vector may be delivered/administered systemically. In a particularly preferred embodiment said gene delivery vehicle is administered locally, preferably intrarticular. Said expression cassette preferably comprises a promoter to drive expression of the coding region and/or antisense sequence. Preferably said promoter is a pol II promoter or a pol III promoter.
Animals. All transgenic mice were bred and maintained on a mixed CBAxC57BL/6 genetic background and kept at the animal facilities of the B.S.R.C. “Al. Fleming” under specific pathogen-free conditions, in compliance with the Declaration of Helsinki principles. (Temperature 20-22° C., Humidity 55±5%, 12 hours light/dark cycle, Water ad libitum). “Arthritic” transgenic mice (Tg197, hTNF+/−), carried the human TNF gene where the 3′ UTR was replaced by the corresponding one from b-globin [5]. “Arthritic” knock in mice (TNFΔARE/+ on 129/C57BL6) expressed the endogenous mTNF gene, where 69 bp encompassing the TNF ARE (AU-rich elements) at the 3′ UTR have been deleted, resulting in increased message stability and translational efficiency [8]. Overall, 86 mice were used for the experiments shown in this study (since some experiments were only representative ones), as listed explicitly where appropriate.
Cell isolation and culture. Synovial Fibroblasts (VCAM+) were isolated from 6-8 weeks old mice essentially as previously described [7,40]. Fibroblasts were selected by continuous culturing for at least 21 days and a minimum of four passages. No macrophage markers could be detected by FACS analysis. Cells were grown at 37° C., 5% CO2 in complete DMEM medium (Gibco/BRL) supplemented with 10% FBS and 100 Units/ml of penicillin/streptomycin. The creation and culture conditions (33° C., 10 Units/ml of murine rIFN-γ) of conditionally immortalized synovial fibroblast cell lines and has been described previously [7].
RNA extraction. Total RNA was extracted from sub-confluent (70-80%) cultured Synovial Fibroblasts (primary or conditionally immortalized) with the RNAwiz reagent (Ambion Inc.), followed by single passage through an RNeasy column (QIAGEN, Hilden, Germany) according to manufacturer's instructions. Total RNA was extracted from whole joints using the Guanidinium isothiocynate/acid phenol protocol [41], followed by single passage through an RNeasy column. RNA integrity was assessed by electrophoresis on denaturing 1.2% agarose/formaldehyde gels. RNA quantity/quality was calculated based on OD readings at 260/280 nm.
Generation of full-length cDNA libraries. 1st strand cDNA synthesis was performed using SUPERSCRIPT II (Life technologies/Invitrogen) at 56° C. in the presence of trehalose and sorbitol. The cap structure at the 5′ end of mRNAs was biotinylated and full length cDNAs were selected, after RNAse I treatment, using streptavidin-coated beads [42]. 2nd strand synthesis was primed by the single-strand linker ligation method (SSLLM), where a double stranded DNA linker (with random 6-bp, dN6 or dGN5, 3′ overhangs) is ligated to the single-stranded cDNA [43]. The 2nd strand is made by primer extension using mixtures of long-range thermostable polymerases, followed by restriction digestion (BamHI nd XhoI) and ligation to the λ-FLCI phagemid vector [43,44]. After packaging, cDNA libraries were amplified on solid phase, as previously described [45].
cDNA libraries normalization and subtraction (outlined in Supplementary
High-throughput sequencing and sequence analysis. Colony picking, cDNA sequencing and sequence analysis were performed essentially as previously described [46-48]. Sequences were filtered for primer and vector sequences [48] and masked for rodent specific and mammalian wide repeats with RepeatMasker (http://www.phrap.org). EST clustering was performed with stackPACK and d2_cluster (word size 6, similarity cutoff 0.98, minimum sequence size 50, window size 200)[49,50]. Homology searches with known genes were performed with BLAST [51,52] at Unigene (http://www.ncbi.nlm.nih.gov/UniGene), FANTOM2 (http://fantom2.gsc.riken.go.jp) [53] and SWISSPROT (http://www.ebi.ac.uk/swissprot/) databases. BLAST results were associated with Gene Ontology (GO) terms (http://www.geneontology.org) at http://source.stanford.edu/cgi-bin/sourceSearch (for Unigene), ftp://ftp.ebi.ac.uk/pub/databases/GO/goa/SPTR/gene_association.goa_sptr.gz (for SWISSPROT) and http://fantom2.gsc.riken.go.jp (for FANTOM2). The detailed results (including clone numbers, clusters/genes, blast results and E values, accession numbers and GO assignments) can be found in the corresponding author's web site, at http://www.fleming.gr/en/investigators/Aidinis/data.html. Most of the sequencing data, have been already submitted to public databases [47], in the context of the ongoing FANTOM (Functional annotation of the mouse) project [53,54].
Differential gene expression statistical analysis of subtractive libraries. The differential gene expression/abundance among the different subtracted cDNA libraries was calculated with a single statistical test, designed especially for this purpose [55]. Essentially, the used formula is the entropy of a partitioning of genes among cDNA libraries and is described by an R-value, which is the log likelihood ratio statistic, that follows (2Rj) an asymptotic χ2 distribution[55]. The formula for the statistic Rj for the jth gene is given by the expression:
where m is the number of cDNA libraries, Ni is the total number of clones sequenced in the ith library, χi,j the counts (transcript copies) of the jth gene in the ith library, and fi is the frequency of gene transcripts copies of the jth gene in all the libraries.
The significance of the R statistic was established utilizing a re-sampling method, which tries to establish an “optimal” cut-off value using simulated library datasets based on the observed counts. The method proceeds as follows: 1) For each and every gene, the common gene transcript frequency, fj is calculated with the help of the formula above. This number corresponds to the expected frequency of gene transcripts under the null hypothesis of no difference in the abundance of gene j across all libraries. 2) The parameters of the Poisson distributions giving the sampling distribution of clone abundance for each gene and library are calculated under the null hypothesis. This parameter is equal to Ni fj for gene j in library i, where Ni is the total number of clones (taking into account ALL genes) sequenced in library i and fj was calculated in Step 1. This value is equal to the expected absolute number of clones of the jth gene in the ith library under the null hypothesis. 3) Then, for each library compared and each gene, a random number is generated from the Poisson distribution of step 2. This number is a simulated count compatible with the null hypothesis i.e. gene frequency for a particular gene is the same across all libraries. This step uses the random generating functions of the computer to create an artificial dataset corresponding to performing the actual experiment of library creation and analysis for each of the libraries compared under the null hypothesis. 4) For gene j in the artificial dataset of Step 3 we calculated the test statistic R by substituting for χi,j the random number generated in Step 3, for fj the frequency calculated in Step 1 and for Ni the total number of clones sequenced. 5) The R values calculated in Step 4 are sorted in descending order, and for a range of values for specificity (i.e. the true negative rate) we find the corresponding R statistic value. These are by definition true negatives since they were obtained from libraries under the null hypothesis. 6) Steps 3-5 are repeated 1000 times allowing us to construct the histogram shown in Supplementary
High density oligonucleotide array hybridization. cRNA probes were generated from 5 μg of total RNA and hybridized to the Mu11K (subunits A and B) chip set according to the manufacturer's instructions (Affymetrix, Santa Clara, Calif., USA) and as previously described [7]. The chip set is designed to collectively recognize 13179 distinct murine transcripts, where the expression level of any given gene is interrogated by 40 oligonucleotides, 20 with a perfect match sequence (PM) and 20 carrying a single mismatch (MM). Hybridized chips were washed and stained in a Fluidics Station (Affymetrix) following standard protocols; subsequently fluorescence intensities were read by an Affymetrix scanner. All MIAME-compliant microarray data can be downloaded from the ArrayExpress database (accession number: E-MEXP-255), as well as from the corresponding author's web site (http://www.fleming.gr/en/investigators/Aidinis/data.html).
Microarray data pre-processing and normalization (outlined in Supplementary
Microarray data analysis. Comparisons of Affymetrix chips hybridizations and real time PCR have indicated that chip analyses are accurate, reliable and underestimate differences in gene expression [58]. Nevertheless, in order to assess the system's performance, the reproducibility of the (technical) duplicate samples was examined, in terms of Pearson correlation coefficient of normalized Signals, as well as Detection concordance (expressed as the percentage of probe sets that were consistently detected with a “Present” or an “Absent” call in both chips). As it can be seen in Supplementary
As previously reported [59,60], the significance of fold changes (that are commonly used as a measure of differential expression) is highly dependent on the expression level of the corresponding genes. Therefore, in order to identify genes that are significantly differentially expressed, we decided not to use a fixed fold change threshold, but instead model intensity-dependent fold changes between replicated chips using a variant of a previously described approach [61]. Briefly, we first calculated fold changes in pair-wise comparisons of replicated chips representing the transcriptome of the same sample type (SF or WJ) measured under the same experimental condition using the following definition:
FC=|x
1
−x
2|/min(x1, x2)
where xi was the normalized expression level of a given gene in the ith chip. Genes were then ranked according to the minor of their two expression values (i.e. min(x1, x2)) and the overall expression range was partitioned into 10 intervals, each containing an equal number of probe sets. In each partition the median of all (min(x1, x2)) values as well as the 90th and the 95th percentile fold change were determined, thus obtaining 10 distinct modeling points for each sample type (SF or WJ) and for each of the two significance levels (90% or 95%). Based on the observation that measurement variability of high-density oligonucleotide microarrays depends on signal intensity following a power law [11], a continuous Fold Change Model (FCM) was derived from the 10 modeling points using a least squares linear fit in log-log plots. The following modeling parameters have been obtained for the two FCM (Supplementary
FC
90%=min(x1, x2)a90%*eb90%
FC
95%=min(x1, x2)a95%*eb95%
For each of the 4 data matrices (i.e. Tg197/SF, TNFΔARE/SF, Tg197/WJ, TNFΔARE/WJ) an observed average fold change between experimental conditions (diseased or normal) was calculated for each gene in the following way:
FC
obs=|μ1−μ2|/min(μ1, μ2)
where μi was the average expression level of a given gene in the ith experimental condition. If no replicates were available, the single expression value of the given gene was used instead of the average. For each gene, this observed fold change was then compared to the fold change that was expected at 90% (or 95%) significance level using the corresponding “sample type”-specific FCM, given the observed values of μi of that gene:
FC
90%=min(μ1, μ2)a90%*eb90%
FC95%=min(μ1, μ2)a95%*eb95%
Finally, genes with FVobs>FC90% (or >FC95%) were selected as differentially expressed at a significance level of 90% (or 95%, respectively).
Visualization of quantitative trait loci (QTL) and gene-expression data was performed with Expression view [62] at http://ensembl.pzr.uni-rostock.de/Mus_musculus/expressionview. QTL data were derived from Serrano-Fernadez et al [63].
Functional clustering and determination of statistical significance. Biological annotation, in the form of Gene Ontology (GO) “Biological process” term [17], for each of the genes (probe sets) in table 2, was obtained from the NetAffx portal (http://www.affymetrix.com). We then calculated the observed GO term frequencies, as means to discover deregulated functions (Supplementary Table 3). The statistical significance for GO term frequencies was determined essentially as has been previously described [64]. Briefly, the hypergeometric distribution was used to obtain the chance probability of observing a given number of genes annotated in NetAffx with a particular GO term and thus calculating appropriate p-values. More specifically, the probability of observing at least (k) probe sets annotated with a particular GO term within a list of selected probe sets of size (n) was calculated [64] as:
where (f) was the total number of genes within a functional category and (g) was the total number of probe sets on the chip set (13,179).
Adhesion assays were performed on Cytometrix adhesion strips (Chemicon) coated with human fibronectin, vitronectin, laminin and collagen I, according to the manufacturer instructions.
Immunofluorescence. Cells were fixed using 4% paraformaldehyde/PBS and stained by standard methods. For visualizing F-actin cells were stained with Alexa594-phalloidin (Molecular Probes). P-tyrosine was detected using the 4G10 antibody (Upstate Biotechnology), FAK was detected using a monoclonal antibody (clone 77, Transduction Laboratory). Antibodies against gelsolin were raised by immunizing rabbits with recombinant mouse gelsolin, immune serum was used at 1:500 dilution. For western blot analysis, 10 μg of total cell protein was separated by SDS/PAGE, transferred to Immobilion-P and probed for gelsolin and re-probed for actin as an internal loading control.
RT-PCR. Total RNA were extracted from synovial fibroblast and whole joints tissue using TRIzol reagent (Life Technologies, Rockville, Md.) in accordance with the manufacturer's instructions. RNA yield and purity were determined spectro-photometrically at 260/280 nm. 1st strand cDNA synthesis was performed using the MMLV reverse transcriptase and oligo-dT15 (Promega).
Semi-quantitative PCR was performed by 20-25 cycles of denaturation at 95° C. for 30 s, annealing at 57-62° C. (depending on the Tm of each individual set of primers) for 30 s and extension at 72° C. for 1 min, in a final volume of 20 μl. The products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide. Product intensity was quantified with GelWorcs 1D Advanced (v.4.01) and normalized to the intensity of B2m and/or L32. The primers were selected to span two exons, while the two control primers were chosen from the Primer Bank database (http://pga.mgh.harvard.edu/primerbank/). Primer sequences (5′->3′, s: sense; as: anti-sense) and product sizes (in bp) were as follows:
Quantitative real-time PCR was performed using the iCycler iQ Real-Time detection system and the IQ™ SYBR Green Supermix (Bio-Rad Laboratories), according to the manufacturer's instructions, for 4 min at 94° C. and 40 cycles of denaturation\ annealing (50 s at 95° C.; 50 s at 57-62° C.). Primers were chosen from exons separated by large introns, and the PCR quality and specificity was verified by melting curve analysis and gel electrophoresis. Values were normalized to B2m and/or L32 (using the same primers as for the semi-quantitative PCR). Mouse (m) and human (h) primer sequences and expected lengths were as follows:
Cycle threshold (Ct; the first cycle that amplification can be detected) values were obtained from the iCycler iQ software for each gene of interest (GOI) and control housekeeping genes (HKG; L32 and/or b2m), together with amplification efficiencies (η; 80-120%). For the mouse samples, we calculated the relative expression of the samples (S) to wt controls as reference samples (R) using the gene expression—relative quantification Microsoft® Excel add-on macro (Bio-Rad Laboratories) that utilizes the following formulas: Relative expression=2·(SΔCt·RΔCt), where ΔCt=GOI Ct-HKG Ct. For the human samples, Ct values were converted to concentration values (ng/ml) utilizing the standard curve made by serial dilutions (in duplicates) of a reference sample. Values were normalized to the corresponding values of the reference (housekeeping) gene(s) and presented (in logarithmic scale for visualization purposes) as expression index.
Arthritic score and Histopathology Paraffin-embedded joint tissue samples were sectioned and stained with haematoxylin and eosin. Arthritic histopathology was assessed (in a blinded fashion) separately for synovial hyperplasia, existence of inflammatory sites, cartilage destruction and bone erosion using a semi-quantitative (0-5) scoring as described previously [65].
Transmission electron microscopy. Ankle joints (dissected from the right hind leg of each mouse—3 Tg197 and 3 wt) were split open longitudinally through the midline between the tibia and the talus, were prefixed with 2.5% glutaraldehyde in PBS (pH 7.4) O/N. After postfixing with 1% osmium tetroxide in PBS for 2 h, they were dehydrated in a graded series of ethanol and processed into Araldite. Semithin sections (1.0μ) were cut and stained with methylene blue to observe the orientation under the light microscope. Ultrathin sections (80-90 nm) were then cut with an ultramicrotome (Riechert-Jung Ultracut E), mounted on copper grids, counterstained with uranyl acetate and lead citrate, and evaluated through electron microscope (CM120 Biotwin, FEI Company).
To discover genes and cellular pathways that participate in the pathogenesis of RA on a large scale, we performed a twin high throughput approach, comprising of two entirely different methodologies, governed by different constrains and analyzed by different statistics. Total RNA samples were extracted from whole joints (WJ) and synovial fibroblast (SF) ex vivo cultures, isolated from 6 weeks old arthritic (RA; disease severity index 3, see
Subtractive cDNA libraries and large scale sequencing. Full-length, cDNA libraries were constructed with the cap-trapper method, by cloning in the λ-FLCI vector, as described in Materials and Methods. Following solid phase amplification and plasmid excision, the four different cDNA libraries (RA SF, wt SF, RA WJ, wt WJ) were normalized and subtracted to each other, as outlined in Supplementary
Oligonucleotide microarray hybridizations. Fluorescent-labeled cRNA probes made from similar (biological replicates, see above) samples of total RNA (RA SF, wt SF, RA WJ, wt WJ) were utilized to hybridize (in duplicates) the Affymetrix Mu11K oligonucleotide DNA chipset (8 chipsets, 16 chips total). Furthermore, similar samples (equimolar amounts of RNA pooled from 4-2 male and 2 female—6 weeks old arthritic mice; severity index 3) of total RNA (from SF and WJ) from another animal model of Arthritis (spontaneous, knock-in, TNFΔARE+/−)[8] were used for additional chip hybridizations (4 chipsets, 8 chips total). The MIAME-compliant [9] microarray data (which can be found at the ArrayExpress database [10], accession number: E-MEXP-255) were normalized and analyzed as outlined in Supplementary
Cross platform comparison and validation. Both differential expression analysis methods presented above produced lists of deregulated genes of high statistical significance. In order to validate the results independently and in order to avoid performing numerous RT-PCRs, differential expression results by both platforms of analysis were compared to each other, for the same animal model (Tg197, hTNF+/−) and sample type (WJ, SF), as well as for the same direction of deregulated expression (up or down). The comparison, at significance levels 99% for the libraries and 90% for the microarrays (selected based on similar output gene numbers), was performed through the NetAffx database (www.affymetrix.com). Remarkably (since there have been very few examples of expression profiling cross-platform overlaps[12,13]), 46 (15 for SF; 31 for WJ) genes were commonly predicted as up/down-regulated by both methods (Table 2) (with a combined p value of 0.001).
In order to verify the validity of the twin high throughput approach and to prove that the reported gene list is self-validated, a number of the predicted deregulated genes, were confirmed with different methods. 40 genes were also predicted deregulated in the knock-in disease model (TNFΔARE+/−, Table 2). 20 genes were previously reported in the literature to be associated with RA (Supplementary
In an attempt to combine gene expression analysis with genetic linkage analysis, all differentially expressed genes were mapped to the chromosomes, together with the known Quantitative Trait Loci (QTLs, chromosomal regions/genes segregating with a quantitative trait) for an induced animal model of Arthritis, Collagen Induced Arthritis (CIA)[14]. As graphically represented in
Arthritic synovial fibroblasts have a rearranged cytoskeleton. The twin high throughput expression profiling approach described above, yielded a large number of disease implicated, deregulated genes, of high statistical significance. Furthermore and in order to: i) prove the validity and extend the utility of the expression data analysis even further, ii) to infer deregulated biological functions from the gene expression data and iii) to define functional criteria for further gene selection, the selected genes were annotated in the form of the Gene Ontology (GO)[17] term “Biological process”. GO term frequencies in the selected gene lists were then calculated and their statistical significance was estimated, as described in Materials and Methods. As shown in Supplementary Table 3, predicted deregulated functions in SFs include, as expected, collagen catabolism, complement activation, immune and stress response. Interestingly, 5 out of 26 statistical significant (p<0.01) deregulated GO functions concerned (directly or indirectly) the actin cytoskeleton, suggesting that arthritic SFs have a rearranged actin cytoskeleton. In order to confirm the prediction, F-actin was visualized in vitro on arthritic as well as wt SFs (both primary and immortalized). As evident from
Stress fibers within fibroblasts allow them to exert tension on the Extra Cellular Matrix (ECM) surrounding them—an essential process in wound healing. It is well expected that differences in the actin cytoskeleton reflect altered ECM attachment properties and/or vice versa. Indeed, arthritic SFs were shown to adhere to different proteins of the ECM (fibronectin, vitronectin, laminin and collagen) with increased affinity in vitro (
Attachment to the ECM (and associated cytoskeletal changes), mediated mainly through engagement and clustering of transmembrane integrin molecules, largely define cell shape and morphology, as well as their behavior and fate. Increased adhesion to the ECM is expected to lead to a more elongated shape. In order to confirm the increased adhesion of the arthritic fibroblasts in vivo, we examined ankle joints from arthritic or wt littermate mice on an ultrastructural level with transmission electron microscopy (
We thus found a new pathogenic mechanism in RA: The promotion of actin polymerization and rearrangement of the actin cytoskeleton. Gelsolin (Gsn) is a gene that maps in one of the CIA QTLs (
Unless mentioned herein under example 2, the materials and methods of example 1 where used.
Details on cell culture, RNA extraction from both whole joint tissue and ex-vivo synovial fibroblast cell cultures and RT-PCR conditions can be found at Aidinis et al, 2005. The primers specific for mCofilin 1 were s:TGGGTGAAACTCTGGGAGAT as: CCATAGTGGAAGCCTGATGC, 399 bp and for mVASP s: CTTGCCAAGGATGAAGTCG as: GCTTCACCCTCTCCAAGT, 98 bp.
Cells cultured at confluency (3×105 per well) were seeded in 6-well plates (in DMEM supplemented with 10% FCS and 100 Units/ml of penicillin/streptomycin at 37° C., 5% CO2) and allowed to adhere for 24 h. The following day cells were treated with 10 μg/ml Mitomycin C (Sigma) in culture medium at 37° C. for 2 h, to block cell proliferation. Assays of wound healing were performed by scrapping the cell monolayer with the edge of a pipette tip, forming a straight line. Cells were then allowed to continue to grow and pictures were taken at 0, 24 and 48 h, after the scrapping.
Confluent mouse synovial fibroblasts were washed twice with phosphate-buffered saline (PBS) and placed in DMEM without serum for 24 h. The following day 1.5×104 cells were plated into each well of a 96-well flat-bottom plate (Costar, Corning Inc.) in 100 μl of media plus serum. After 24 h, cells were treated with 12.5-100 ng/ml TNFα plus 0.5-3 μg/ml cyclohexamide (CHX), 30-300 μM Etoposide and 0.01-1 μM Staurosporine, for 24 h at 37° C. overnight. Cell survival was determined using the crystal violet assay. The absorbance at 570 nm was determined on a microplate reader (SPECTRAmax PLUS384, Molecular Devices, Sunnyvale, Calif., USA), as previously described (Aidinis et al, 2003). Results are expressed as the means±S.E. of one representative experiment performed in triplicate. Statistical analysis was performed using Student's t test, with a level of significance set at p<0.05.
Disruption of actin cytoskeleton was induced with 4 μg/ml of Latrunculin A (Sigma) for 2 h at 37° C. as previously described (Spector et al, 1989). As control of TNFα involvement in actin-cytoskeleton organisation 1 μg/ml of anti-TNF (Infliximab) was added on the cells for the same period of time.
Total RNA was extracted from 5-8 Tg197 mice (from both whole joint tissue and ex-vivo synovial fibroblast cell cultures) and pooled. For the comparison group, total RNA was extracted from wild-type litter mates (from both whole joint tissue and ex-vivo synovial fibroblast cell cultures) and pooled. The results are depicted in table 3.
Preferred proteins that affect cytoskeleton arrangement include actin binding proteins, controlling the nucleation, assembly, disassembly, crosslinking of actin filaments and/or the size, localization and dynamics of the un-polymerized actin. A protein of table 4 is preferred in the present invention. A particularly preferred protein includes a protein expressed by the gelsolin gene, VASP and/or Cofilin (Galler et al, 2006; Hotulainer et al, 2005; Paavilainen et al, 2004). Others, like Autotaxin and SHIP2 (Song et al, 2005; von Wichert et al, 2003). These proteins are major players in signaling pathways leading to re-organization of the actin cytoskeleton.
Cofilin 1 (non-muscle cofilin) promotes actin dynamics by depolymerising and severing actin filaments, in a similar manner like gelsolin (Hotulainer et al, 2005). As
In order to evaluate the gene expression status of VASP in Tg SF's we have performed Q-RT-PCR in both whole joint samples and ex-vivo synovial fibroblast cells culture (same material as for the SQ-RT-PCR). Surprisingly, VASP is found to be over-expressed in the whole joint of Tg mice compare to wild-type littermates, while it is under-expressed in the SF's (
KO mice for genes known to be involved in the cytoskeleton or crossed with Tg197 and/or with Collagen VI-Cre knockout with the purpose of (i) exacerbate, (ii) ameliorate the disease and (iii) make the disease specific for synovial fibroblasts.
VASP is a key player in the actin cytoskeleton as it is shown in vitro to promote F-actin formation while in vivo regulates and promotes actin polymerization (Hauser et al, 1999). In the present invention a cross of VASP KO with Tg197 is expected to lead to amelioration of the disease.
Cofilin depolymerises and severs actin filaments, in a similar manner like gelsolin, in the present invention a crossing of Cofilin KO with Tg197 is expected to result in exacerbation of the disease (Gurniak et al, 2005).
LST-1 was found to be over-expressed in whole joints from Tg197 (Aidinis et al, 2005), while in vitro studies have correlated this gene with a function in cytoskeleton, by creation of long filopodia and thus accumulation of F-actin (Raghunathan et al, 2001). Creation of a KO of this gene and crossing with Tg197 provides further evidence of the involvement of this gene in cytoskeleton-related rheumatoid arthritis. RhoA, Rac and Cdc42 belong to the Rho family of GTP-binding proteins known to induce actin polymerization (Lamarche et al, 1996). Conditional ablation of these genes (Chrostek et al, 2006) and crossing with ColVI-Cre and Tg-197 would ameliorate the disease specific for synovial fibroblasts. A similar outcome is predicted from crosses with the serum response factor (SRF) KO, which is a transcription factor regulating the expression of several actin-cytoskeleton genes (RhoA, Vinculin, Actin) and it's ablation was shown to result in low F-actin levels and a reduced adhesive and migratory capacity (Wiebel et al, 2002).
A conditional KO model for Autotaxin (ATX) will be also utilized to further support the role of cytoskeleton re-modeling in RA. Activated hyperplastic RA Synovial Fibroblasts (SFs) exhibit reorganization of their actin cytoskeleton and deregulated ECM adhesion that could explain the tumor-like behavior of the arthritic synovium. Lysophosphatidic acid (LPA), the enzymatic product of autotaxin (ATX)—a lysophospholipase D highly expressed in a variety of tumors, is an important lipid mediator that has been found to induce cytoskeletal reorganization, proliferation and migration in many cell types. Preliminary studies have shown that the expression of ATX is upregulated in SFs from Tg197, and ex vivo cultured RA SFs secreted significantly more ATX protein than their wild-type counterparts.
It is provided here that Tg SF's exhibit distinct phenotype compared to those of wild type mice. It was shown that Tg SFs have rearranged actin cytoskeleton and exhibit pronounced stress fibers. We also showed that Tg SF's adhere with increased affinity to the proteins of the ECM in vitro, while they possess greater motility compared with wild-type mice. This increased adhesion leads to a more elongated shape of the SF's. For this reason a number of in vitro assays were developed/standardised, including Adhesion, Migration, Proliferation, Wound Healing and Apoptosis (
These in vitro assays demonstrate the distinct properties of Tg SFs, including increased adhesion, migration and apoptosis resistance compare to Wt SF's, while proliferation is increased. Targeting the expression cytoskeleton related genes (such as preferably Gelsolin, VASP, Cofilin 1 and/or Aquaporin 1) affects cytoskeleton arrangement. Knocking down the expression of such genes has an effect in the actin cytoskeleton machinery of the SF's and this is revealed by the above assays.
In vitro analysis on the effect of Latrunculin A (Lat-A), an inhibitor of F-actin polymerization (Giganti and Friederich, 2003), has indicated the potential of actin-drugs in altering the cytoskeleton properties of Tg197 synovial fibroblasts. In detail, treatment with Lat-A has resulted in decreased adhesion of TgSF's, while migration seems to be increased compare to Wt (
The patent states that “analyzing by quantitative RT-PCR (Q-RT-PCR) DEGs selected from tables A, B and C may provide a good correlation with the absence or presence of inflammatory arthritis (IA). For this purpose a number of genes were selected (i.e. C3, CstB, Dusp1, LSP1, PITPNM, RAB14, SHIP2, TGFB1I4, TIMP1, TIMP3, TUBA1, C1QA, SHIP, LST1) based on the following criteria: a) DEGs that showed the greatest level of differential expression following validation in the Tg197 mouse model, by SQ-RT-PCR analysis; b) DEGs identified in the Tg197 mouse microarray data and are key players in the regulation of actin cytoskeleton and c) candidate genes of interest that were not identified by the microarray experiments (SHIP and SHIP2) but are known to be involved in the mechanism of actin cytoskeleton.
In order to carry out the above task a collaboration was established with Dr Wilson's lab at the University of Sheffield, UK, who agreed to provide us with sufficient amounts of cDNA from biopsies of RA and OA patients (Ethics no: SSREC 01/333; Project title “Regulation of cytokine genes in RA”. In detail, synovial tissue was obtained during joint replacement surgery from patients with RA (n=21), defined according to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association). Synovial tissue was also obtained from patients with osteoarthritis (OA) (n=12), who were undergoing knee joint replacement surgery. Patient's informed consent was obtained according to the national ethical rules, as these were implemented by the corresponding local ethic commission.
Quantitative real-time PCR was performed as previously described (Aidinis et al, 2005). Primers were chosen from either exons separated by large introns or exon boundaries and the PCR quality and specificity was verified by melting curve analysis and gel electrophoresis. Values were normalized to both B2M and L32. Human primer sequences and expected lengths were as follows (listed in the 5′ to 3′ direction and designated as s; sense and as; antisense): hC3 (s, GGCCTTTGTTCTCATCTCGCT; as, ATAGGATGTGGCCTCCACGTT, 274 bp), hCSTB (s, CCTGTGTTTAAGGCCGTGTCAT; as, GGAGAGATTGGAACACTCGCAG, 115 bp), hDUSP1 (s, ACAAGGCAGACATCAGCTCCTG; as, TAAGCAAGGCAGATGGTGGCT, 130 bp), hLSP1 (s, CAACAATTAAGAGCACCCCATC; as, TGGCAACAGGAAGCAACTTCT, 223 bp), hRAB14 (s, GCTTATTGTTCCTCGAAGCGAG; as, ATCCAAGCTTCCATCCTGAATG, 107 bp), hSHIP2 (s, GGCTCGGAGGAACCAAAACTAC; as, TGAGGTCCCCAAACCAGAAGA, 119 bp), hTGFB114 (s, TAGCTCCTCTGGTGCAAGTGTG; as, TCCACTTCTTCTCTGACCGCA, 102 bp), hLST1 (s, CGTGATGAGGAACTTGAGG; as, GCGATAACATTAGGTCAGGG, 114 bp).
Of the 8 cytoskeleton-related DEGs studied, 5 showed the same pattern of deregulated expression in human samples and therefore merit further investigation (
Streptococcus pyogenes
| Number | Date | Country | Kind |
|---|---|---|---|
| 20050100526 | Oct 2005 | GR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/010228 | 10/19/2006 | WO | 00 | 8/5/2008 |
