EVALUATING AND TREATING SCLERODERMA

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Serial No. 60/712,998, filed on August 31, 2005, the contents of which are hereby incorporated by reference in their entirety.

SUMMARY

Scleroderma is a condition manifested by the appearance of a hard type skin. The condition includes a group of connective tissue and rheumatic disorders, including localized scleroderma (morphea and linear scleroderma) and systemic scleroderma (limited scleroderma, diffuse scleroderma, and sine scleroderma).

The expression of a number of genes is altered in scleroderma. Therapeutic methods for treating scleroderma can include counteracting the effects of the altered gene expression profile. Further, scleroderma can be diagnosed and monitored by evaluating the expression of one or more of the altered genes.

In one aspect, this disclosure features a method of treating or preventing scleroderma or an other fibrotic disorder. The method includes administering, to a subject, e.g., a human subject, a Wnt signalling antagonist, e.g., in an amount effective to treat or prevent scleroderma. The subject is typically a human, e.g., a human who has or who is at risk for scleroderma or other fibrotic disorder. The subject can be a human who has been identified as having decreased WIFl expression in a skin biopsy.

A Wnt signalling antagonist is an agent that decreases Wnt signalling in a cell of the subject. The antagonist can inhibit a canonical Wnt, e.g., Wntl, Wnt3A, or Wnt8, and/or a non-canonical Wnt, e.g., Wnt4, Wnt5A, or Wntl 1. The antagonist can be a protein or a small molecule. For example, the antagonist can be an agent that inhibits interaction between a Wnt and a cell surface receptor for Wnt, e.g., a canonical Wnt and Frizzled or LRP5/6.

In one embodiment, the Wnt signalling antagonist is a Wnt binding protein, e.g., an antibody that binds to Wnt or a protein at least 90, 95, 97, 98, or 99% identical, or

identical to, to a naturally occurring Wnt binding protein or a functional fragment thereof, e.g., Wnt binding fragment. Examples of a naturally occurring Wnt binding protein, e.g., an sFRP protein (soluble frizzled-related protein), WIF-I, or Cerberus.

In one embodiment, the Wnt signalling antagonist is a protein that binds to a cell surface receptor for Wnt. For example, the protein can be an antibody that binds to a cell surface receptor for Wnt, e.g., a Frizzled protein or an LRP, e.g., LRP5/6. In particular, the antibody can bind to an extracellular region of the cell surface receptor for Wnt. The protein can be at least 90, 95, 97, 98, or 99% identical, or identical to, to a naturally occurring protein that interacts with a cell surface receptor for Wnt. For example, the protein is at least 90, 95, 97, 98, or 99% identical, or identical to, a functional fragment of a Dickkopf protein, e.g., Dkk-1, Dkk-2, Dkk-3, or Dkk-4.

A Wnt signalling antagonist can be a protein (e.g., an artificial transcription factor that represses transcription) or nucleic acid agent (e.g., siRNA, anti-sense, or aptamer) that decreases expression (or activity) of a positively acting component of the Wnt pathway, e.g., a Wnt protein or a Wnt receptor. Alternatively, a Wnt signalling antagonist can be a protein (e.g., an artificial transcription factor that activates transcription) or nucleic acid agent (e.g., gene therapy vector) that increases expression of a negatively acting component of the Wnt pathway, e.g., a Wnt inhibitor such as an artificial or naturally occurring Wnt inhibitor (e.g., an sFRP, WIF, or Cerberus protein). In another aspect, the disclosure features a method of treating or preventing scleroderma. The method includes administering, to a subject, an agent that increases activity or expression of WIF (e.g., WIFl) or an sFRP protein, e.g., in an amount effective to treat or prevent scleroderma. The agent can be, for example, a protein that includes a functional fragment of a WIF or sFRP protein or a nucleic acid that encodes such a protein. In one embodiment, the agent is a Wnt-binding fragment of WIFl . In another embodiment, the agent includes a full-length, mature WIFl .

Li another aspect, the disclosure features a method of treating or preventing scleroderma. The method includes administering, to a subject, an IGF binding agent or an inhibitor of an IGF, in an amount effective to treat or prevent scleroderma. For example, the IGF binding agent binds to IGF I or IGF II. The IGF binding agent can include an IGF binding region of a naturally occurring IGFBP, e.g., IGFBP-3. In one

embodiment, the IGF binding agent includes a full length, mature IGFBP. In one embodiment, the IGF binding agent includes an antibody that binds to IGF I or IGF II.

In another aspect, the disclosure features a method of treating or preventing scleroderma. The method includes: administering, to a subject, an agent that (i) increases expression of a gene encoding gene product in columns 2, 4, or 6 of Table 1, or (ii) increases activity of a gene product encoded by the gene. The agent can be administered in an amount effective to treat or prevent scleroderma. In one embodiment, the agent is a nucleic acid that includes a sequence that encodes a protein that includes a functional fragment of the gene product. For example, the nucleic acid is in a viral vector and delivered using viral particle. In one embodiment, the agent is a protein, e.g., a protein that includes a functional fragment of the gene product. For example, the agent includes the gene product itself.

In still another aspect, the disclosure features a method of treating or preventing scleroderma. The method includes administering, to a subject, an agent that (i) decreases expression of a gene encoding a gene product in columns 1, 3, or 5 of Table 1, or (ii) decreases activity of a gene product encoded by the gene. The agent can be administered in an amount effective to treat or prevent scleroderma. In one embodiment, the agent is a nucleic acid antagonist of gene expression, e.g., an RNAi, e.g., an siRNA. In another embodiment, the agent is an antibody that binds to the gene product.

In another aspect, the disclosure features a method of evaluating a subject. The method includes: obtaining a sample (e.g., a skin biopsy or serum sample) from a subject; and evaluating expression of a gene in Table 1 in cells in the biopsy. An alteration in expression of the gene relative to a reference is indicative of scleroderma or risk for scleroderma. With respect to a gene listed in columns 2, 4, and 6 of Table 1, a decrease in expression of the gene relative to a reference is indicative of sclerodeπna or risk for scleroderma; whereas with respect to a gene listed in columns 1, 3, and 5 of Table 1, an increase in expression of the gene relative to a reference is indicative of scleroderma or risk for scleroderma. Samples can be used without culturing and passaging of cells within the sample.

In one embodiment, the gene encodes WIFl. A decrease in WIF 1 expression relative to a reference is indicative of scleroderma or risk for scleroderma.

The evaluating can include a quantitative or qualitative evaluation of expression levels. For example, the reference is a parameter obtained by evaluating a normal subject who does not have scleroderma.

In one embodiment, a plurality of genes is evaluated. The expression of each of the genes can be compared to corresponding references, e.g. values (quantitative or qualitative values) for the expression of same genes based on a reference sample or for a statistical assessment, e.g., an average of a cohort of matched subjected, e.g., a cohort of subjects who have scleroderma or a cohort of subject who do not have scleroderma, e.g., healthy subjects. Information from evaluating the plurality of genes can be used to obtain a profile of gene expression. The profile can be compared to a corresponding reference profile, e.g., a profile based on a reference sample or for a statistical assessment, e.g., an average of a cohort of matched subjected, e.g., a cohort of subjects who have scleroderma or a cohort of subject who do not have scleroderma, e.g., healthy subjects. Profiles can be compared, e.g., using a distance function.

In another aspect, the disclosure features a method of evaluating a subject. The method includes: obtaining a sample (e.g., a skin biopsy, serum sample, or other sample) from a subject; and evaluating expression of a collagen in cells in the sample, wherein a increase in collagen expression relative to a reference is indicative of scleroderma or risk for scleroderma. Collagen expression can be evaluated by detecting mRNA encoding collagen or by detecting collagen protein (including fragments thereof). The method can include administering a therapy for scleroderma to the subject, if the subject is indicated for scleroderma or risk for scleroderma. For example, the therapy is a therapy described herein. In one embodiment, the collagen is collagen XL The method can include detecting fragments of the collagen, e.g., collagen XI fragments (e.g., N and C propeptides). Fragments can be detected, e.g., using an antibody. The method can further include preparing a report indicating a diagnosis of scleroderma or risk for scleroderma using results of the evaluating. hi another aspect, the disclosure features a computer-readable database that includes a plurality of records. Each of which includes: a) a first field that comprises

information about skin pathology of a subject; and b) a second field that comprises information about expression of a gene in Table 1 in cells from a sample (e.g., a skin biopsy or serum sample) obtained from the subject. For example, each record of the plurality further includes a field that comprises information identifying the subject. Each record of the plurality can further include additional fields that include information about expression of a gene in Table 1 such that each record includes information for a plurality of genes in Table 1, e.g., at least 2, 4, 5, 7, 8, 9, or 10 genes from one or more columns in Table 1, e.g., at least 5, 10, 20, or 25% of the genes in one or more columns of Table 1.

DETAILED DESCRIPTION

Agents that inhibit Wnt signalling or increase IGFBP activity can be used to treat scleroderma or another fibrotic disorder, as can agents that alter the expression or activity of the proteins listed in Table 1. Changes in gene expression that accompany scleroderma also provide a reference for identifying other therapeutic agents and for diagnostic methods of evaluating subj ects .

Wnt signalling

Decreased expression of a Wnt inhibitor protein, WIFl, is characteristic of scleroderma biopsies. Accordingly, therapies that decrease Wnt pathway signalling can be used to treat or prevent scleroderma and other fibrotic disorders. Wnt proteins are secreted glycoproteins that mediate important cell signalling functions. Wnts include canonical Wnts (e.g., Wntl, Wnt3a, and Wnt8). These Wnts can signal by stabilizing β- catenin and activating transcription mediated by Tcf/LEF. Wnts also include noncanonical Wnts, such as Wnt4, Wnt5A, and Wntl 1. The noncanonical Wnts can activate alternative signalling pathways include Ca ²⁺ signals. Many Wnt signals are transduced by cell surface receptors, e.g., receptors of the Frizzled (Fr) family and low- density lipoprotein receptor-related proteins (LRP), particularly LRP5 and LRP6.

A number of naturally occurring proteins function as inhibitors of Wnt signalling. These proteins include proteins that bind directly to Wnt, such as members of the sFRP class of inhibitors, e.g., the sFRP family itself, WIF-I, and Cerberus. Other naturally

occurring proteins that function as inhibitors include the Dickkopf class which includes Dkk-1 through Dkk-4. These proteins interact with LRP5/6 to inhibit Wnt signalling.

IGFBPs and the IGF pathway

Decreased expression of insulin-like growth factor binding protein-3 (IGFBP-3) is characteristic of scleroderma biopsies. Accordingly, therapies that decrease IGF activity can be used to treat or prevent scleroderma and other fibrotic disorders. Insulin-like growth factor binding proteins (IGFBPs) are secreted proteins that bind to and sequester insulin-like growth factor (IGF), e.g., IGF-I or IGF-2. IGFs are secreted growth factors that can act as potent mitogens that activate cell proliferation and differentiation. IGFBPs have high affinity for IGFs, e.g., better than 10 ^'10 M. hi addition to using IGFBP-3 as a therapeutic agent, it is possible to use antibodies or other agents that bind to and inhibit an IGF, e.g., IGF-I or IGF-2.

Functionally Related Proteins

The use of a particular protein described herein can also be implemented using a related, but different protein that provides the same function, e.g., a protein that includes a functional fragment of that particular protein and that is related by sequence homology to the protein, e.g., at least 90, 95, 97, 98, or 99% identical to the particular protein within the region of the functional fragment, or at least 90, 95, 97, 98, or 99% with respect to the full-length of the particular protein (referring usually to the mature version of secreted and/or processed proteins). The protein can differ, e.g., by at least one, 2, 3, 4, 5, or 8 amino acids, and, e.g., by fewer than 25, 20, 15, 12, 10, 8, 7, 6, 4, 3 residues.

Calculations of sequence identity between two sequences are performed by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) and then counting the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The determination of sequence identity is typically calculated using the GAP program in the GCG software package, using a

Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Related proteins may also be encoded by nucleic acids that hybridize to one another, e.g., under medium stringency, high stringency, or very high stringency conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions include: i) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ⁰ C; ii) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and iii) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65 ⁰ C. Further a useful protein may have one or more mutations (e.g., deletions, insertions, or substitutions) relative to a particular protein described herein (e.g., a conservative or non-essential amino acid substitutions), which do not have a substantial effect on function. Whether or not a particular substitution will be tolerated, can be predicted, e.g., by aligning closely related natural proteins to identify conserved and non- conserved positions, by mutagenesis experiments (e.g., alanine scanning), by inspecting structural models, or by consulting tables of related residues, e.g., as described in Bowie, et al. (1990) Science 247:1306-1310. Generally, a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Functionally related proteins can be identified by a variety of methods. For example, a nucleic acid encoding a protein can be subjected to mutagenesis and then evaluated in a functional assay for the protein, e.g., using cultured cells.

Useful methods for mutagenesis include PCR mutagenesis, saturation mutagenesis, cassette mutagenesis, alanine scanning, and oligonucleotide directed mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. PCR mutagenesis can be performed by reducing the fidelity of Taq polymerase so that random mutations are introduced during replication, e.g., by using a dGTP/dATP ratio of five and adding Mn ²⁺ to the PCR reaction. (Leung et al, 1989, Technique 1:11 -15). The pool of amplified DNA fragments can be inserted into appropriate cloning vectors to provide random mutant libraries. Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. A library of variants can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of degenerate sequences can be earned out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues in the amino acid sequence of the protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of these options. Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA. See, e.g., Adelman et al., (DNA 2:183,1983). Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-

1085,1989). Cassette mutagenesis can also be used, e.g., as described in Wells et al. (1985) Gene, 34:315, and can be used to create, e.g., combinatorial libraries of variants.

Screening Methods

Test compounds can be screened to identify compounds useful for the prevention or treatment of scleroderma or other fibrotic disorder. A test compound can be any chemical compound, for example, a macromolecule (e.g., a polypeptide, a protein complex, or a nucleic acid) or a small molecule (e.g., an amino acid, a nucleotide, an organic or inorganic compound). The test compound can have a formula weight of less than about 10,000 grams per mole, less than 5,000 grams per mole, less than 1,000 grams per mole, or less than about 500 grams per mole. The test compound can be naturally occurring (e.g., a herb or a nature product), synthetic, or both. Examples of macromolecules are proteins, protein complexes, and glycoproteins, nucleic acids, e.g., DNA, RNA and PNA (peptide nucleic acid). Examples of small molecules are peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds e.g., heteroorganic or organometallic compounds. A test compound can be the only substance assayed by the method described herein. Alternatively, a collection of test compounds can be assayed either consecutively or concurrently by the methods described herein. Exemplary test compounds can be obtained from a combinatorial chemical library including peptide libraries {see, e.g., US 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)), peptoids (e.g., WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, et al. infra), peptide nucleic acid

libraries {see, e.g., US 5,539,083), antibody libraries (see, e.g., Vaughn et ah, Nature Biotechnology, 14(3):309-314 (1996) and PCTVUS96/10287), carbohydrate libraries (see, e.g., Liang et ah, Science, 274:1520-1522 (1996) and US 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, US5,569,588; thiazolidinones and metathiazanones, US 5,549,974; pyrrolidines, US 5,525,735 and US 5,519,134; morpholino compounds, US 5,506,337; benzodiazepines, US 5,288,514, and the like). Libraries of aptamers can also be evaluated.

The test compound or compounds can be screened individually or in parallel. A compound can be screened by being monitored the level of expression of one or more genes encoding a protein in Table 1. Comparing a compound-associated expression profile to a reference profile can identify the ability of the compound to modulate gene expression in dermal tissue, e.g., to alter fibroblast behavior or otherwise treat or prevent a fibrotic disorder, such as scleroderma. The expression profile can be a profile based on one or more genes mentioned herein, e.g., a gene encoding a protein listed in Table 1. An example of the parallel screening is a high throughput drug screen. A high-throughput method can be used to screen large libraries of chemicals. Such libraries of test compounds can be generated or purchased e.g., from Chembridge Corp., San Diego, CA. Libraries can be designed to cover a diverse range of compounds. For example, a library can include 10,000, 50,000, or 100,000 or more unique compounds. Alternatively, prior experimentation and anecdotal evidence, can suggest a class or category of compounds of enhanced potential. A library can be designed and synthesized to cover such a class of chemicals. A library can be tested on cell lines, such as scleroderma fibroblasts, and gene expression levels can be monitored. Regardless of a method used for screening, compounds that alter the expression level are considered "candidate" compounds or drugs. Candidate compounds are retested on cells from scleroderma samples, or tested on animals. Candidate compounds that are positive in a retest are considered "lead" compounds.

Once a lead compound has been identified, standard principles of medicinal chemistry can be used to produce derivatives of the compound. Derivatives can be screened for improved pharmacological properties, for example, efficacy,

pharmacokinetics, stability, solubility, and clearance. The moieties responsible for a compound's activity in the assays described above can be delineated by examination of structure-activity relationships (SAR) as is commonly practiced in the art. A person of ordinary skill in phaπnaceutical chemistry could modify moieties on a lead compound and measure the effects of the modification on the efficacy of the compound to thereby produce derivatives with increased potency. For an example, see Nagarajan et al. (1988) J. Antibiot. 41 : 1430-8. Furthermore, if the biochemical target of the lead compound is known or determined, the structure of the target and the lead compound can inform the design and optimization of derivatives. Molecular modeling software is commercially available (e.g., Molecular Simulations, Inc.).

Expression Monitoring

Agents for treating sclerodeπna and other disorders described herein can be identified by selecting agents that modulate expression of a gene described herein, e.g., a gene encoding a protein in Table 1, e.g., WIFl, an IGFBP, or collagen XI. Any method can be used to evaluate an agent for its ability to modulate gene expression. For example, a cell is contacted with a candidate compound and mRNA or protein expression is evaluated, e.g., relative to the level of expression in the absence of the candidate compound. When expression is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of a gene expression. Alternatively, when expression is less (e.g., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of gene expression.

Methods for detecting gene expression in a sample include detecting mRNA, detecting cDNA, or detecting protein, e.g., using an antibody or other binding protein, or using an activity assay. Exemplary molecular techniques include RT-PCR and microarray analysis. Many of these techniques can be used to obtain qualitative or quantitative values for gene expression. For example, QT-PCR can be used to provide a quantitative value for expression of a gene of interest. These techniques can be used to evaluate one or more genes encoding a gene product listed in Table 1, e.g., WIF-I, an IGFBP, or collagen XL

Examples of methods of gene expression analysis include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. US A., 2000, 97, 1976- 81), protein arrays and proteomics (Celis, et al., supra; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., supra; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,

203-208), subtractive cloning, differential display (e.g., Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

Reporter genes can be used to evaluate changes in gene expression. Exemplary regulatory sequences include those located within 100, 200, 500, 700, or 1600 basepairs of the mRNA start site of the gene of interest, e.g., WIFl, IGFBP3, or a gene in Table 1.

Reporter genes can be made by operably linking a regulatory sequence to a sequence encoding a reporter gene. A number of methods are available for designing reporter genes. For example, the sequence encoding the reporter protein can be linked in frame to all or part of the sequence that is normally regulated by the regulatory sequence. Such constructs can be referred to as translational fusions. It is also possible to link the sequence encoding the reporter protein to only regulatory sequences, e.g., the 5' untranslated region, TATA box, and/or sequences upstream of the mRNA start site. Such constructs can be referred to as transcriptional fusions. Still other reporter genes can be constructed by inserting one or more copies (e.g., a multimer of three, four, or six copies) of a regulatory sequence into a neutral or characterized promoter.

Reporter genes can be introduced into germline cells of non-human mammals, e.g., to produce transgenic animals. Reporter genes can also be introduced into culture cells, e.g., tissue culture cells, e.g., fibroblasts. Exemplary reporter proteins include chloramphenicol acetyltransferase, green fluorescent protein and other fluorescent proteins (e.g., artificial variants of GFP), beta-

lactamase, beta-galactosidase, luciferase, and so forth. The reporter protein can be any protein other than the protein encoded by the endogenous gene that is subject to analysis. Epitope tags can also be used. The reporter protein is preferably stable and rapidly degraded. Exemplary methods can include evaluating a transgene that includes a reporter gene for the gene of interest of a transgenic mammal for altered expression of a reporter gene (e.g., a GFP or variant protein). The transgenic mammal can be administered a test compound, and, if the compound modulates expression of the reporter gene, the test compound is selected. Similarly, compounds can be screened using a cell-based assay, e.g., using cultures of cells that contain a reporter whose expression is operably linked to a regulatory sequence from the gene of interest (e.g., from a promoter, enhancer, untranslated region, upstream or downstream of the coding sequence).

Antibodies Antibodies can be used to modulate activity of a Wnt signalling pathway.

Particularly useful antibodies bind to a secreted component of a Wnt signalling pathway or an extracellular region of a component of a Wnt signalling pathway. An antibody can be selected based on whether it antagonizes or agonize Wnt signalling.

For example, one class of antibodies includes molecules that bind to Wnt and inhibit Wnt activity, e.g., inhibit Wnt binding to a cell surface receptor, e.g., a frizzled receptor or LRP5/6. Another class of antibodies includes molecules that bind to the extracellular region of a cell surface receptor for Wnt, such as a frizzled receptor or LPR5/6, and reduce or prevent Wnt interaction with the receptor or otherwise reduce receptor signalling. The term "antibody" refers to a protein comprising at least one immunoglobulin variable domains. A typical antibodies includes a heavy chain variable domain and a light chain variable domains, but a camelid antibody may have only a single variable immunoglobulin domain. Immunoglobulin variable domains include into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of

the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. MoI. Biol. 196:901-917). Each variable domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.

The antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system and the first component (CIq) of the classical complement system.

As used herein, the term "immunoglobulin" refers to a protein that includes one or more polypeptides that have a domain that forms an immunoglobulin fold. An immunoglobulin domain is roughly a cylinder (about 4 x 2.5 x 2.5 nm) with two extended protein layers: one layer contains three strands of polypeptide chain and the other contains four. In each layer the adjacent strands are antiparallel and form a β-sheet. The two layers are roughly parallel and are often connected by a single intrachain disulfide bond. An immunoglobulin can include a region encoded by an immunoglobulin gene. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl , IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin genes and gene segments.

The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and

CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et at, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) one or more complementarity determining regions (CDR) that retain antigen-binding ability, in the absence of a complete variable domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et at (1988) Science 242:423-426; and Huston et at (1988) Proc. Natl. Acad. ScL USA 85:5879-5883) that are antigen-binding fragments of an antibody.

An "effectively human" immunoglobulin variable domain is an immunoglobulin variable domain that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable domain does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human. Human and effectively human immunoglobulin variable domains and antibodies can be used as therapeutics for human subjects.

Antibodies can be made by immunizing an animal (e.g., non-human animals and non-human animals include human immunoglobulin genes) with the relevant antigen or a fragment thereof. Such antibodies may be obtained using the entire mature protein as an immunogen, or by using fragments (e.g., soluble fragments and small peptides). The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides include, for example, as in Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); Krstenansky, et al., FEBS Lett. 211, 10 (1987). Antibodies can also be made by selecting antibodies from a protein expression library, e.g., a phage display library.

Human monoclonal antibodies (mAbs) directed against target proteins can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., WO 91/00906, WO 91/10741 ; WO 92/03918; WO 92/03917; Lonberg et al. 1994 Nature 368:856-859; Green, et al. 1994 Nature Genet. 7:13-21; Morrison et al. 1994 Proc. Natl. Acad. Sd. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326). Monoclonal antibodies can also be generated by other methods including methods that use recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al. 1989 PNAS 86:5728; Huse et al. 1989 Science 246: 1275; and Orlandi et al. 1989 PNAS 86:3833). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. The DNA sequence of the variable domains of a diverse population of antibodies can be obtained using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FRl) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable domains from a number of murine antibodies (Larrick et al. , 1991 , Biotechniques 11:152- 156). A similar strategy can also been used to amplify human heavy and light chain variable domains from human antibodies (Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2:106-110).

Chimeric antibodies, including chimeric immunoglobulin chains, can be produced by recombinant DNA techniques. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc. constant region is substituted (see PCT/US86/02269; EP

184,187; EP 171,496; EP 173,494; WO 86/01533; US 4,816,567; EP 125,023; Better et

al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559). An antibody or an immunoglobulin chain can be humanized. Humanized antibodies, including humanized immunoglobulin chains, can be generated by replacing sequences of the Fv variable domain which are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Humanized antibodies can be produced by a variety of methods, including CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., US 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141 :4053-4060; Winter US 5,225,539. Still other methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and US 5,585,089, US 5,693,761 and US 5,693,762. hi some implementations, monoclonal, chimeric and humanized antibodies can be modified by, e.g., deleting, adding, or substituting other portions of the antibody, e.g., the constant region. For example, an antibody can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, e.g., a constant region meant to increase half-life, stability or affinity of the antibody, or a constant region from another species or antibody class; or (iii) by modifying one or more amino acids in the constant region to alter, for example, the number of glycosylation sites, agonist cell function, Fc receptor (FcR) binding, complement fixation, among others. Antibody constant regions can be altered. Antibodies with altered function, e.g. altered affinity for an agonist ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, US 5,624,821 and US 5,648,260). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

To identify an antibody that not only binds, but also has a particular function (e.g., inhibits), an antibody can be evaluated in a functional assay. For example, a plurality of antibodies that bind to a target (e.g., Wnt or a Wnt receptor) can be evaluated in this manner.

Nucleic Acid Antagonists

Li certain implementations, nucleic acid antagonists are used to decrease expression of a target protein, e.g., a positively acting component of the Wnt pathway (e.g., Wnt or a Wnt receptor), an IGF protein, or other protein whose expression is upregulated in tissue from scleroderma patients, e.g., a protein listed in Table 2. hi one embodiment, the nucleic acid antagonist is an siRNA that targets mRNA encoding the target protein. Other types of antagonistic nucleic acids can also be used, e.g., a nucleic acid aptamer, a dsRNA, a ribozyme, a triple-helix former, or an antisense nucleic acid. siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens, J. C. et al. (2000) Proc. Natl. Sci. USA 97, 6499-6503; Billy, E. et al. (2001) Proc. Natl. Sci. USA 98, 14428-14433; Elbashir et al. (2001) Nature. 411(6836):494-8; Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 9942-9947, US 2003-0166282, 2003-0143204, 2004-0038278, and 2003-0224432.

Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the

oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA can interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5- propynylcytosine and C5-propynyluracil. Other suitable modified nucleobases include N ⁴ -(C ₁ -C ₁₂ )alkylaminocytosines and N ⁴ ,N ⁴ --(C ₁ -C ₁₂ )dialkylaminocytosines. Modified nucleobases may also include 7-substituted-8-aza-7-deazapurines and 7-substituted-7- deazapurines such as, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines, 7- aminocarbonyl-7-deazapurines. Examples of these include 6-amino-7-iodo-7- deazapurines, 6-amino-7-cyano-7-deazapurines, 6-amino-7-aminocarbonyl-7- deazapurines, 2-amino-6-hydroxy-7-iodo-7-deazapurines, 2-amino-6-hydroxy-7-cyano-7- deazapurines, and 2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore, N ⁶ -(C ₁ -C ₁₂ )alkylaminopurines and N ⁶ ,N ⁶ -(C ₁ -C ₁₂ )dialkylaminopurines, including N ⁶ - methylaminoadenine and N ⁶ ,N ⁶ -dimethylaminoadenine, are also suitable modified nucleobases. Similarly, other 6-substituted purines including, for example, 6-thioguanine may constitute appropriate modified nucleobases. Other suitable nucleobases include 2- thiouracil, 8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine.

Derivatives of any of the aforementioned modified nucleobases are also appropriate. Substituents of any of the preceding compounds may include C ₁ -C ₃₀ alkyl, C ₂ -C ₃₀ alkenyl, C ₂ -C ₃₀ alkynyl, aryl, aralkyl, heteroaryl, halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like. Descriptions of other types of nucleic acid agents are also available. See, e.g.,

US 4,987,071; US 5,116,742; US 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. ( 199 Y) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N Y. Acad. Sci. 660:27-36; and Maher, LJ. (1992) Bioassays 14:807-15.

Artificial Transcription Factors

Artificial transcription factors can also be used to regulate genes whose expression is altered in scleroderma, e.g., to increase the expression of a gene listed in Columns 2, 4, and 6 of Table 1 or to decrease expression of a gene list in Table 2. Artificial transcription factors can also be used to regulate genes that encode components of the Wnt pathway (e.g., to increase expression of negatively acting components (e.g., an sFRP, WIF, or Cerberus) or to decrease expression of positively acting components (e.g., a Wnt or Wnt receptor)). The protein can be designed or selected from a library. For example, the protein can be prepared by selection in vitro (e.g., using phage display, US 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., WO

00/42219 and US 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol 19:656; and Wu et al. (1995) Proc. Nat. Acad. Sci. USA 92:344 for, among other things, methods for creating libraries of varied zinc finger domains. Optionally, the zinc finger protein can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription. The zinc finger protein can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., US 6,534,261. The heterologous nucleic acid that includes a sequence encoding the zinc finger protein can be operably linked to an inducible promoter, e.g., to enable fine

control of the level of the zinc finger protein in the cell. Artificial zinc finger proteins can be administered directly, e.g., using a protein transduction domain, or by delivering a nucleic acid encoding the artificial zinc finger protein, e.g., using a gene therapy vector.

Recombinant Protein Production The nucleic acids encoding proteins that function as agents for the methods described herein may be operably linked to an expression control sequence in a vector in order to produce the protein recombinantly. General methods of expressing recombinant proteins are exemplified in Kaufman, Methods in Enzymology 185, 537-566 (1990), Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3 ^rd Edition, Cold Spring Harbor Laboratory, N. Y. (2001) and Ausubel et al, Current Protocols in Molecular

Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Examples of regulatory sequences that can be used to direct gene expression are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Cells can be, for example prokaryotic (e.g., E. coli), yeast, plant, or mammalian.

Exemplary mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-I cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF3, 32D, FDCP-I, PC12, Mix or C2C12 cells. Transgenic animals (particularly mammals) can also be used to produce the recombinant protein (e.g., in milk).

Treatments and Pharmaceutical Compositions

A therapeutic agent described herein can be provided as a pharmaceutical composition. Exemplary therapeutic agents include an agent that inhibits Wnt signaling an agent that inhibits IGF activity, an agent that decreases activity or expression a gene product listed in Columns 2, 4, and 6 of Table 1, or an agent that increases activity or expression of a gene product listed in Table 2.

The pharmaceutical composition may include a therapeutically effective amount of an agent described herein. A therapeutically effective amount is an amount effective,

at dosages and for periods of time necessary, to achieve the desired therapeutic result or to prevent or delay onset of a disorder. A therapeutically effective amount of the composition may va3y according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective amount preferably modulates a measurable parameter, e.g., an indicia of scleroderma, e.g., to a statistically significant degree. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder, using in vitro assays, e.g., an assay described herein, or using appropriate human trials. A variety of animal models of scleroderma can be used. Examples are described in Clark (2005) Curr Rheumatol Rep. 7(2): 150-5.

Particular effects mediated by an agent may show a difference that is statistically significant (e.g., P value < 0.05 or 0.02). Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. An increase or decrease can cause a qualitative or quantitative difference relative to a reference state, e.g., a statistically significant difference (e.g., P value < 0.05 or 0.02).

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is possible to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. An exemplary, non-limiting range for a therapeutically effective amount of an agent described herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg. Dosage values may

vary with the type and severity of the condition to be alleviated. For any individual subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Accordingly, the dosage ranges set forth herein are only exemplary.

Subjects who can be treated include human and non-human animals, e.g., non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, mice, sheep, dogs, cows, pigs, etc.

An agent described herein may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may contain, in addition to the agent and carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials. Pharmaceutically acceptable carriers are non-toxic materials that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier typically depend on the route of administration.

In practicing the method of treatment or use, a therapeutically effective amount of an agent is administered to a subject, e.g., mammal (e.g., a human). The agent may be administered either alone or in combination with other therapies such as other treatments for fibrotic disorders, e.g., scleroderma. When co-administered with one or more agents, the agent may be administered either simultaneously with the second agent, or sequentially. If administered sequentially, the attending physician can decide on the appropriate sequence of administering the agent described herein with other agents.

Administration of an agent described herein can be carried out in a variety of ways, including, for example, oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection or administration. Topic administration can include direct application to a lesion, e.g., to sclerotic skin.

For oral administration, the agent can be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% of the agent or from about 25 to 90% of the agent. When administered in liquid form, a liquid carrier such as water, petroleum,

oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the agent, e.g., from about 1 to 50% or the agent.

To administer an agent, e.g., by intravenous, cutaneous or subcutaneous injection, the agent can be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. An exemplary pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to the agent an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle. The pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants, or other additive.

The amount of an agent to be delivered can depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone. The attending physician can decide the amount of agonist with which to treat each individual patient. Initially, for example, the attending physician can administer low doses of the agent and observe the patient's response. Larger doses of the agent may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further, or by monitoring one or more symptoms.

In the case of an agent that is an immunoglobulin (e.g., a full length antibody), an exemplary pharmaceutical compositions may contain about 0.1 μg to about 10 mg of the immunoglobulin agent per kg body weight. For example, useful dosages can include between about 10 μg-1 mg, 0.1-5 mg, and 3-50 mg of the agent per kg body weight.

The duration of therapy using the pharmaceutical composition can vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. The duration of each application of the agent can be, e.g., in the range of 12 to 24 hours of continuous intravenous

administration. The attending physician can decide on the appropriate duration of intravenous therapy using a pharmaceutical composition described herein.

With respect to agents that are proteins or nucleic acids, the disease or disorder can also be treated or prevented by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The polynucleotides that encode an agent or that provide a nucleic acid agent activity can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see US 5,328,470), injection (e.g., US 2004-0030250 or 2003-0212022) or stereotactic injection (e.g., Chen et al. Proc. Natl. Acad. Sci. USA 91 :3054-3057,

1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Diagnostics

Information about the expression of one or more genes described herein (e.g., one or more genes listed in Table 1 or 4) can be used to evaluate a subject or a culture. The subject can be evaluated, e.g., to determine risk for scleroderma or other fibrotic disorder, e.g., to predict whether the subject is likely to get the disorder prior to its onset, or to determine whether the subject has the disorder. The subject can be an adult, child, fetus, or gamete. The evaluation can be made by comparing a value indicative of expression (e.g., qualitative or quantitative values) in the subject to a reference, e.g., a reference, e.g., a reference obtained from a control (e.g., a healthy subject) or a reference that is a statistical representation of a cohort (e.g., cohort of diseased or healthy subjects). The subject can be evaluated prior to, during, or after a treatment, e.g., to determine efficacy of the treatment. Information from the evaluation can be used to modify the treatment, e.g., to increase or decrease the dose of an agent on subsequent administrations. Values indicative of expression (e.g., qualitative or quantitative values)

can be compared to a reference, e.g., a reference for the subject obtained prior to an initial treatment, a reference for the subject obtained prior to disease onset, or other reference, e.g., reference obtained from a control (e.g., a healthy subject) or a statistical representation of a cohort (e.g., cohort of diseased or healthy subjects). The evaluation can include evaluate a single gene, e.g., a gene encoding WIFl,

IGFBP, or collagen, e.g., collagen XL The evaluation can also include evaluating expression of multiple genes, e.g., a plurality of genes described herein. Information about the expression of multiple genes can be used to provide a gene expression profile. An exemplary scheme for producing and evaluating profiles is as follows. Nucleic acid is prepared from a sample, e.g., a sample of interest and hybridized to an array, e.g., with multiple addresses. Hybridization of the nucleic acid to the array is detected. The extent of hybridization at an address is represented by a numerical value and stored, e.g., in a vector, a one-dimensional matrix, or one-dimensional array. The vector x {x _a , X _b ...} has a value for each address of the array. For example, a numerical value for the extent of hybridization at a first address is stored in the variable x _a . The numerical value can be adjusted, e.g., for local background levels, sample amount, and

I other variations. Nucleic acid is also prepared from a reference sample and hybridized to an array (e.g., the same or a different array), e.g., with multiple addresses. The vector y is construct identically to vector x. The sample expression profile and the reference profile can be compared, e.g., using a mathematical equation that is a function of the two vectors. The comparison can be evaluated as a scalar value, e.g., a score representing similarity of the two profiles. Either or both vectors can be transformed by a matrix in order to add weighting values to different nucleic acids detected by the array.

The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column corresponds to a nucleic acid being assayed, e.g., an address or an array, and each row corresponds to a sample. A separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.

Nucleic acids that are similarly regulated can be identified by clustering expression data to identify coregulated nucleic acids. Nucleic acids can be clustered using hierarchical clustering (see, e.g., Sokal and Michener (1958) Univ. Kans. ScL Bull. 38: 1409), Bayesian clustering, k-means clustering, and self-organizing maps (see, Tamayo et al. (1999) Proc. Natl. Acad. Sd. USA 96: 2907).

Expression profiles obtained from nucleic acid expression analysis on an array can be used to compare samples and/or cells in a variety of states as described in Golub et al. ((1999) Science 286: 531). For example, multiple expression profiles from different conditions and including replicates or like samples from similar conditions are compared to identify nucleic acids whose expression level is predictive of the sample and/or condition. Each candidate nucleic acid can be given a weighted "voting" factor dependent on the degree of correlation of the nucleic acid's expression and the sample identity. A correlation can be measured using a Euclidean distance or a correlation coefficient, e.g., the Pearson correlation coefficient. The similarity of a sample expression profile to a predictor expression profile

(e.g., a reference expression profile that has associated weighting factors for each nucleic acid) can then be determined, e.g., by comparing the log of the expression level of the sample to the log of the predictor or reference expression value and adjusting the comparison by the weighting factor for all nucleic acids of predictive value in the profile. As described above, information about gene expression can include information obtained by evaluating mRNA levels or by evaluating protein levels.

Fibrotic Disorders

In addition to scleroderma, the methods described herein (particularly the therapeutic methods) may also be generally applicable to other fibrotic disorders. Fibrotic disorders include fibrosis of an internal organ, a dermal fibrosing disorder, and fibrotic conditions of the eye.

Fibrosis of internal organs (e.g., liver, lung, kidney, heart blood vessels, gastrointestinal tract), occurs in disorders such as pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, renal interstitial fibrosis, renal fibrosis in patients receiving

cyclosporin, and HIV associated nephropathy. Dermal fibrosing disorders include, e.g., scleroderma, morphea, keloids, hypertrophic scars, familial cutaneous collagenoma, and connective tissue nevi of the collagen type. Fibrotic conditions of the eye include conditions such as diabetic retinopathy, post-surgical scarring (for example, after glaucoma filtering surgery and after cross-eye surgery), and proliferative vitreoretinopathy.

Additional fibrotic conditions include: rheumatoid arthritis, diseases associated with prolonged joint pain and deteriorated joints; progressive systemic sclerosis, polymyositis, dermatomyositis, eosinophilic fascitis, morphea, Raynaud's syndrome, and nasal polyposis.

EXAMPLE

The following exemplary methods were used to identify changes in gene expression associated with scleroderma.

Patients and Controls

Patients who (i) fulfilled the preliminary classification criteria for scleroderma of the American Rheumatism Association (ARA now the American College of Rheumatology) and (ii) had diffuse cutaneous disease according to the classification of LeRoy et al (J Rheumatol. 1988 Feb;15(2):202-5) were selected. Consecutive outpatients with early (less than 3 years from the onset of the first non-Raynaud's phenomenon scleroderma disease manifestation) and diffuse disease were chosen in an attempt to diminish heterogeneity of disease expression. To be eligible for the study, patients could not have been on doses of prednisone greater or equal to 20 mg in the 4 weeks prior to the biopsy, nor on other immunosuppressive treatment within this time period. Healthy controls were chosen on the basis of lack of Raynaud's phenomenon by history, lack of a diagnosis of a systemic autoimmune disease, and willingness to participate. Control subjects who did not use glucocorticoids or immunosuppressive agents were selected. Characteristics of the subjects are summarized in Table 3, below.

Table 3: Characteristics Of The Study Population That Was Used For Gene Expression Analysis

Two 3 -mm punch biopsies were taken from the same distal forearm (involved skin in the case of the scleroderma patients) of each subject in a side-by-side fashion. One biopsy specimen was immediately placed in RNALater® (Ambion) and stored at 4°C prior to RNA preparation. RNA was prepared within 15 days of tissue harvest. The other biopsy piece was placed in culture.

Fibroblast Culture Conditions Biopsy tissue was rinsed several times with antibiotic-antimycotic solution (Life

Technologies Cat. No. 15240-062). Tissue was then placed in 1 ml of collagenase solution and incubated for 24 hours at 37°C. Collagenase solution contained 0.25% collagenase type I (Sigma) and 0.05% DNaseI (Sigma) in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) (HyClone, Logan, UT). The entire 1 ml was mixed together with 5 ml of media (DMEM 4- 20% FCS), then plated into 25 cm2 flask, and left undisturbed for 48 hours at 37°C in a 5% CO2 atmosphere. The resulting confluent culture was then designated passage- 1 or Pl . Cells were then split 1 :4 to generate 4X25 cm2 flasks (P2). Two flasks at passage 4 were trypsinized, the trypsin was neutralized using soybean trypsin inhibitor, and cell pellets were washed twice with PBS prior to addition of RNAlater® (Ambion). Cell pellets were shipped frozen on dry ice by overnight delivery and stored frozen at -8O ⁰ C prior to RNA preparation. A 3mm biopsy stored in RNAlater® according to manufacturers instructions gave ample material for a labeling and hybridization reaction without amplification. Twenty-eight biopsies were processed. Most biopsies gave yields in excess of 2 μg RNA and were hybridized.

Total RNA Purification

Skin biopsies were removed from RNAlater® solution (Ambion, Austin, TX), placed in a weighing dish containing 1 ml of TRIzol® reagent (Invitrogen, Carlsbad, CA), minced using a razor blade, and poured into a 2 ml tube. RNAlater® was removed from fibroblast pellets, which were then resuspended in 1 ml of TRIzol® reagent.

Fibroblasts and biopsies were homogenized using a PowerGen™ 125 (Fisher Scientific, Hampton, NH) for 2 to 5 minutes at top speed. Total RNA was extracted from TRIzol® according to manufacturer's protocol. Biopsy extraction included a centrifugation step to treat samples with high content of fat and extracellular materials. Total RNA was resuspended in 100 μL of water and further purified using an RNEASY MINI™ column (Qiagen, Valencia, CA) according to manufacturer's protocol.

Probe labeling, hybridization and scanning

Sample labeling, hybridization, and staining were carried out according to the Eukaryotic Target Preparation protocol in the Affymetrix Technical Manual (701021 rev. 4) for GENECHIP® Expression Analysis (Affymetrix, Santa Clara, CA). In summary, 1 to 5 μg of purified total RNA was used in a 20 μL first strand reaction with 200 U Superscript II (Invitrogen) and 0.5 μg (dT)-T7 primer in 1 X first strand buffer (Invitrogen) with a 42°C incubation for 1 hour. Second strand synthesis was carried out by the addition of 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, 10 U E. coli DNA Ligase in IX second strand buffer (Invitrogen) followed by incubation at 16°C for 2 hrs.

The second strand synthesis reaction was purified using the GENECHIP® Sample Cleanup Module according to the manufacturer's protocol (Affymetrix). Purified cDNA was amplified and biotinylated using BIOARRAY HIGHYIELD™ RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY) according to manufacturer's protocol. 15 μg of labeled cRNA was fragmented and resuspended in 300 μL IX hybridization buffer containing 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% TWEEN® 20, 0.5 mg/mL acetylated BSA, 0.1 mg/mL herring sperm DNA, control oligo B2, and eukaryotic control transcripts. The labeled material was applied to a Human Genome 133 A GENECHIP® (Affymetrix). Hybridized arrays were washed and stained

on a GENECHIP® Fluidics Station 450 and visualized using a GENECHIP® Scanner 3000.

Quantitative RTPCR

First-Strand cDNA was synthesized from RNA using SUPERSCRIPT III PLATINUM™ Two-Step qRT-PCR Kit (Invitrogen). For each reaction IuL of RNA containing 100ng-lug was used. RT-PCR reactions were set up in Optical 96-Well Reaction Plates (Applied Biosystems) using TAQMAN™ Gene Expression Assays Protocol for 50 μL Reactions. Concentration of cDNA was determined by spectroscopy using a BIOPHOTOMETER™ (Eppendorf). For all reactions 40 ng of cDNA was used. RT-PCR thermal profile: 3 :00 @ 95°; then 55 cycles of 0: 15 @ 95°, 1 :00 @ 56°. RT- PCR data was collected using an Mx3000P™ PCR machine (Stratagene) and analyzed using Mx3000P™ software (Stratagene).

Array Analysis

Array data in the form of CEL files were imported into BRB array tools. Biopsy data and fibroblast data were imported and normalized independently using RMA algorithm. Datasets normalized using the MAS 5 algorithm implemented in R were used when comparisons between fibroblasts and biopsies were necessary. Class comparisons and class predictions were carried out using the BRB software package (available from Dr. Richard Simon and Amy Peng Lam, National Cancer Institute, Bethesda MD).

Immunohistochemistry

Specimens were analyzed from individuals with similar age and history as those used for the gene array experiments, hnmunohistochemistry was performed according to manufacturer's instructions using diaminobenzidine (DAB) as chromogen.

Comparison of Normal and SSC biopsies Unsupervised (agglomerative) clustering of all the samples demonstrated that the scleroderma phenotype was the dominant influence on expression profile, and was not confounded by patient sex, age, race, or origin of the biopsy. Class comparisons on all 22000 qualifiers between normal and scleroderma biopsies showed 1839 qualifiers

distinguishing normal skin from scleroderma at a p value ≤O.Ol, n = 17 (unpaired T- tests, with random variance model and a false discovery rate < 0.1), of which the 50 most significant by p value are shown below: WIFl, CTGF, NNMT, PRSS23, LBH, SFRP4, CHNl, LUM, SERPINE2, D2S448, THBSl, ABHD6, LOXLl, RAB31, IGFl, COMP, RAB31, IFITM2, FKBPIl, ASPN, IGFBP6, IGFBP3, THYl, CDHI l, THYl, OASl, GARP, SERPINEl, N0X4, HCAl 12, ADCY2, RELB, SGCG, GALNTlO, TNFRSF6B, COL6A3, FNl, TP53I3, COL4A2, ADAM12, CLDN8, CAPN3, IGFBP3, and TNFSF4.

Comparison of normal and SSc fibroblasts and relationship to the biopsies

Class comparisons between normal and scleroderma fibroblasts showed 223 qualifiers distinguishing normal from scleroderma at p < 0.01. Of these, 105 were up- regulated in scleroderma, and 118 down-regulated. The intersection between qualifiers dysregulated in biopsies and those in fibroblasts (26, of which 9 are discordant, 17 are concordant) was far greater than that which would be seen by chance (p < 0.02, Fisher's Exact test of observing an intersection of that many or more by random sampling of gene lists from a pool of 22000 qualifiers.), and included a large proportion of extracellular matrix genes, including collagen VIII, fibulin I, fibrillin 2, and decorin. Interestingly, a subset of these showed inverse regulation in the context of the biopsy and the fibroblasts, notably ephrin B2.

The extent of change in gene expression levels is complicated by the fact that each biopsy contains maintains multiple cell types. A particular change in gene expression may occur in only one of the many cell types in the biopsy. As an approximation, we considered genes that were up-regulated at least five fold in cultured fibroblasts as likely to be of fibroblast origin in the biopsy, and, conversely, genes that were down-regulated at least five-fold in fibroblasts relative to the biopsy were unlikely to be of fibroblast origin in the biopsy. Genes between these two extremes of differential expression were considered indeterminate in source.

In order that genes influenced by the disease process would not be averaged out, class comparisons of scleroderma biopsies to sclerodeπna fibroblasts were made independently of control biopsies versus control fibroblasts, and a union made of the five-fold downregulated (or five-fold upregulated) qualifiers. These lists were intersected

with the class comparison of scleroderma biopsies versus control biopsies. Thus, for example, qualifiers q dysregulated in the fibroblast component were found such that

(((qfϊbrossc^X qwopsyssoj P < -01) OR (qfibronl^X qbiopsy, P < -01) ) AND (qbiopsynl <> qWopsyssc, p < .01)). The top 25 hyp value of "probable fibroblast" and "probable non-fibroblast" genes are shown below.

Nonfibroblast: WIFl, SFRP4, ABHD6, IGFl, COMP, OASl, ADCY2, CLDN8, CAPN3, ClQRl, PECAMl, IGFl, IFI27, ACADL, ZNF204, ClQB, CD14, RALGPSl, CCLl 9, A2M, ILl F7, GPM6A, ALDH5A1, C4A, CD209, and ERBB3.

Fibroblast: CTGF ₅ NNMT, PRSS23, CHNl, SERPINE2, D2S448, THBSl, LOXLl, IGFBP3, THYl, CDHl 1, THYl, GARP, SERPINEl, N0X4, ADAM12,

IGFBP3, D2S448, CDHI l, SERPINEl, DAB2, TIMPl, ANXA6, DAB2, ADAM12, and FNl.

We created an exemplary list of genes that are useful as an expression profile for distinguishing normal from scleroderma fibroblasts and for distinguishing scleroderma biopsies from controls. Biopsies, unlike cultured fibroblasts, may reflect actual expression of cells in a subject as the evaluated cells in a biopsies are not given the opportunity for cellular adjustment that may result during culturing. We performed a class comparison between scleroderma fibroblasts and normal fibroblasts at p < 0.01. The 223 member qualifier list that resulted from this comparison was used to generate a classifier for the biopsies. Leave-one-out cross validation analysis selected a subset of 26 genes, which were successfully distinguished scleroderma from normal biopsies according to multiple models. Fifteen genes could be matched to qualifiers in the HU95A chip. Examples of these genes are: ADSL, ClQA, MX2, COL8A1, ICAMl, C7orfl9, OASl, HS3ST3A1, IFI16, ANGPT2, DAP, NINJ2, SLC16A3, TNIP2, SDC3, FBN2, FZD2, LOC51334, MTCPl, PAQR6, DCN, ABCA6, LOCI 14977, PLPl, EFNB2, and FBLNl.

Changes in the population or activity of immune cells in biopsies can be detected using T and B cell markers. Examples of T cell markers include CD3, CD4, and CD28, the monocyte markers CD 14, CD 163, and CDlIb, the antigen presenting cell co- stimulatory protein B7-2 (CD86). Examples of B cell markers include CD83, CD84, and Ig kappa. CD3 (T cell) counts were not obviously different on immunohistochemistry

between control and scleroderma samples, and CD20 (B cell) stains showed very few cells on all specimens. This suggests that even very small numbers of lymphocytes can be detected by gene expression profiling. The gene expression analysis detected an immune cell signature from B cells, T cells and macrophages, consistent with the abundant auto-antibodies characteristic of scleroderma.

We observed that the expression of choηdrogenesis associated collagens, such as collagen XI and collagen X was increased, as was that of the chondrogenesis associated protein (COMP), the nonfibrillar collagen IV, the network forming collagens (collagens X and VIII), the endothelial basement membrane collagen XV, and BMPl (a collagen and biglycan processing enzyme). Significantly, the expression of collagen XI is increased even more than that of other collagens: ~ 5-fold, relative to ~2-fold for most other collagens. Collagen V forms fibrils with collagen I to control fibril diameter and collagen XI forms fibrils with collagen II in an analogous fashion. Collagen XI is found in association with collagen I in fibrocartilage of the intervertebral disc of normal individuals, as well as in the embryonic tendon. Thus, the fibrotic response resembles the specific developmental program for fibrocartilage. Interestingly, in a study of lung fibroblast responses to TGFjS, collagen IV was the only collagen significantly dysregulated.

The small leucine rich family of proteins (SLRPs) also show some characteristic alterations in scleroderma (Table 4). Decorin is down regulated in scleroderma, and may have TGF/3 antagonistic properties of its own. On the other hand, biglycan, versican and lumican are up regulated, as is asporin, a homolog of decorin. The potential regulatory roles of the SLRPs in matrix assembly are beginning to be dissected. While all of them are associated with collagen fibrils, each may be synthesized by different subsets of cell types. All of these proteins have generally inhibitory effects on collagen fibril diameter and on fibroblast proliferation.

Table 4

Upregulated in Ssc Downregulated in SSc Not regulated

Collagens, particularly 11, 10, Decorin Fibromodulin

5,and 4

Thrombospondin Tenascin X

Tenascin C Laminin

Lumican Fibulin

Fibronectin

Biglycan

Versican

Aggrecan

Fibrillin 2

The TGFjS pathway is activated in cultured scleroderma fibroblasts. The expression of many gene expression targets of TGF/3 was increased in scleroderma biopsies. Many of these targets are no longer differentially expressed in explanted fibroblasts, notably the bulk of the collagens, which suggests that the profibrotic drivers are from cell types which do not persist in fibroblast culture. Transcripts for TGF/3 itself were not increased in scleroderma biopsies, suggesting that increased TGF/3 signal is due to increased activation of latent TGF/3 protein. Thrombospondin I, an activator of latent TGFjS, is significantly upregulated in the scleroderma biopsies. Studies have suggested that the subset of pulmonary fibroblasts which are Thy-1 positive are in fact TGF/3 insensitive due to failure of TGF/3 activation. Expression of Thy-1 was increased in scleroderma skin along with thrombospondin I, suggesting that this relationship does not hold in dermal fibrosis. Changes seen in the Wnt pathway encompass many gene products more likely to be related to cell types other than fibroblasts, including decreased expression of WIFl, frizzled related protein, and frizzled homolog 7, as well as increased expression of secreted frizzled-related protein 4. These changes are consistent with a general increase in Wnt signalling. Interestingly, a Wnt regulatory system has been shown to be active in mesenchymal stem cells in culture. There was a decrease in Dkk and a reciprocal increase in Wnt5A associated with a deceleration in cell growth.

Changes in gene expression of members of the CCN family were also detected. The CCN family includes connective tissue growth factor (CTGF, CCN2), a putative

downstream target of TGFjS and a profibrotic cytokine. CCN2 is up regulated in scleroderma skin and scleroderma fibroblasts (see also). Expression of Cyrόl (CCNl), which provides integrin dependent promigratory stimuli to fibroblasts and vascular smooth muscle cells is also increased in scleroderma, while expression of WISP2 (CCN5) is decreased. Loss of Cyr61 is associated with differentiation of mesenchymal stem cells into any daughter lineage. Thus its up regulation may reflect expansion of an uncommitted fibroblast phenotype. Reciprocal regulation of WISP2 and CTGF has been noted in the fibroblast response to TGF/3.

With the observations above, profiling data from both biopsies and fibroblasts enabled us to make some informed guesses about the tissue type contribution of different genes in scleroderma. For example, increased expression of monocyte/macrophage genes CD 14,TLR 1 and 2, integrins /32, oϋi, and oM appear to be associated with increased expression of IL6, ILl 6, and CXCL3, all of which maybe synthesized by fibroblasts. Similarly, a decrease in expression of IGFBP 5 and 6 and WISP2, likely from a nonfibroblast source, and a reciprocal increase in expression of IGFBP 3 and 4, both presumably in fibroblasts, as well as CTGF, PAI-I, and CIq, the latter from a nonfibroblast source were observed. Accordingly, in scleroderma lesions, there may be a loss of inhibitory signals from a non-fibroblast cells and concomitant increase in profibrotic behavior by fibroblasts. It is possible that the downregulation of some epithelial-derived genes is due to loss of epithelial adnexae in sclerodeπnatous skin.

However, while there was a two fold decrease in expression of keratin 15, associated with early hair follicle "bulge" cells, no other markers of follicles, such as keratin 19 and the hair keratins, were significantly or consistently altered in the scleroderma biopsies.

The comparison of changes to fibroblast cultures in scleroderma to changes in primary biopsies in scleroderma enabled dissecting fibroblast driven from non-fibroblast driven behavior. While there is a robust set of genes whose expression distinguishes scleroderma from normal fibroblasts; there is a reduced number of genes whose expression distinguishes scleroderma biopsies from control at the same level of significance (223 vs. 1839 atp=0.01). For example, CTGF, well known as a fibrosis driver downstream of TGF/3, as well as Thy-1, are upregulated in scleroderma biopsies relative to noπnal controls, but in the cultured fibroblasts expression of these genes does

not clearly correlate with disease. At the same time, matrix targets of CTGF and TGF/3, such as some of the collagens and fibronectin, are upregulated in the scleroderma fibroblasts. Thus, while the fibroblasts show alterations in gene expression that are a signature of the disease, they are likely to act at the end of a chain of events initiated by another cell type. Possible originators of this process may include the endothelial cell or its partner the pericyte, both targets of scleroderma mediated injury. In culture, however, disease-driving cells may be diluted out as fibroblasts proliferate in the culture system, leading to reversion of the fibroblast behavior.

A list of exemplary genes whose expression is significantly altered is provided below:

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

7h3 ABCA12 7h3 ABCA12 7h3 ABHD5

A2M ABCA3 A2M ABCA3 A2M ABHD6

ACP2 ABCA5 ACTG 1 ABCA5 ACTG1 ACAD8

ACTB ABCA6 ADAM 12 ABCA6 ADAMTS6 ACADL ABCB1 /// ABCB1 /// ACTG 1 ABCB4 ADAM 19 ABCB4 ADSL ACSL1

ADAM12 ABHD5 ADAMDEC1 ABHD5 AGTRL1 ADCY2

ADAM 19 ABHD6 ADAMTS2 ABHD6 ALOX5 ADPN

ADAMDEC1 ABLIM1 ADAMTS6 ACAD8 AP2B1 AGXT

ADAMTS2 ACAD8 ADCYAP1 ACADL APOL2 AKAP1 AKR1C1 /// ADAMTS6 ACADL ADORA3 ACADSB APOL6 AKR1C2

ADCYAP1 ACADSB ADRBK2 ACSL1 ARHGAP8 AKR1C2

ADORA3 ACSL1 ADSL ACVR1 B ARHGDIB ALAD

ADRBK2 ACVR1B AGC1 ADCY2 ASPN ALOXE3

ADSL ADCY2 AGTRL1 ADD3 ASRGL1 ANKH

AEBP1 ADD1 ALDH1 B1 ADPN ATP8B2 ANKRA2 ATXN7L1 /// AGC1 ADD3 ALOX5 AGTR1 ATXN7L4 ANKRD27

AGTRL1 ADPN ALOX5AP AGXT B4GALT5 ARHGAP19 BGN /// AIF1 AGTR1 ANGPT2 AHNAK SDCCAG33 ARHGEF10

AKAP2 AGXT AOAH AKAP1 BICC1 ARL10C AKR1C1 /// ALDH1 B1 AHNAK AP2B1 AKR1C2 BM039 ARMCX1

ALOX5 AIM1 APC AKR1C2 C10orf3 ATP5A1

ALOX5AP AKAP1 APOBEC3B ALAD C11orf24 ATP5L AKR1C1 /// ANGPT2 AKR1C2 APOL2 ALDH2 C13orf18 ATP6V0A4

ANGPTL2 AKR1C2 APOL6 ALDH3A2 C16orf30 ATPIF1

ANXA5 ALAD ARHGAP4 ALDH5A1 C19orf22 BBS1

ANXA6 ALDH2 ARHGAP8 ALOX12 C1orf33 BCAN

AOAH ALDH3A2 ARHGDIB ALOXE3 C1QA BCL11 B

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma _, Scleroderma scleroderma Scleroderma scleroderma Scleroderma

AP2B1 ALDH5A1 ARRB2 AMT C1QR1 BMP2

APC ALDH9A1 ARSA ANG C20orf103 BRP44L

APOBEC3B ALOX12 ARSB ANK3 C20orf39 C10orf57

APOL2 ALOXE3 ASPN ANKH C3 C14orf131

APOL6 AMT ASRGL1 ANKMY2 C6orf97 C14orf2

ARF4 AMY2B ATP8B2 ANKRA2 C7orf10 C14orf92

ATXN7L1 ///

ARHGAP4 ANG ATXN7L4 ANKRD27 CALD1 C18orf11

ARHGAP8 ANK3 B4GALT5 ANKRD28 CALM3 C18orf9

ARHGDIB ANKH BASP1 ANXA2 CARHSP1 C1orf35

ARPC1 B ANKMY2 BCL3 APP CASP2 C1QDC1

BGN ///

ARRB2 ANKRA2 SDCCAG33 AQP9 CD209 C20orf111

ARSA ANKRD27 BICC1 AR CD28 C20orf38

ARSB ANKRD28 BICD1 ARHGAP19 CD4 C20orf42

ASPN ANXA2 BM039 ARHGEF10 CD84 C9orf55

ASRGL1 APP BMP1 ARHGEF12 CD86 CAB39L

ATP8B2 APPBP2 BRCA2 ARHGEF9 CDC6 CABC1

ATXN7L1 /// i

ATXN7L4 AQP9 C10orf3 ARHI CECR1 CAT j

B4GALT5 AR C11orf24 ARI H 1 CENTA2 CCNG2 J

BASP1 ARHG AP 19 C13orf18 ARL10C CHD7 CD9

BCL3 ARHGEF10 C16orf30 ARL4A CINP CDC16

ARL6IP /// I

BF ARHGEF12 C19orf10 RPS15A CKAP1 CDC5L I

BGN ARHGEF9 C19orf22 ARMCX1 CKIP-1 CDK5RAP1

BGN ///

SDCCAG33 ARHI C1orf33 ARMCX6 CLN2 CELSR1

BICC1 ARIH1 C1orf38 ASCC3L1 CNN2 CGI-143

BICD1 ARL10C C1QA ASGR1 CNTNAP1 CGI-41

BM039 ARL4A C1QR1 ATP1B3 COL1A1 CHCHD2

ARL6IP ///

BMP1 RPS15A C20orf103 ATP2B4 COL5A1 CHN2

BRCA2 ARMCX1 C20orf39 ATP5A1 COL5A2 CLCA4

BST2 ARMCX6 C3 ATP5H COL6A1 CLDN1

C10orf10 ASCC3L1 C3AR1 ATP5L COL8A1 COX15

C10orf3 ASGR1 C4A ATP6V0A4 COL8A2 COX7C

C11orf24 ATP1B3 C6orf32 ATP7A CORO1B CPE

C13orf18 ATP2B4 C6orf97 ATPIF1 COTL1 CRBN

C16orf30 i ATP5A1 C7orf10 ATRN CRN7 CRYZL1

C19orf10 ATP5H C7orf19 B3GNT6 CSGIcA-T CUL3

C19orf22 ATP5J CALD1 B4GALT3 CTSB CYP39A1

C1orf29 ATP5L CALM3 B4GALT4 CXCL10 CYP3A5

C1orf33 ATP5O CAPNS 1 BAT8 CYLD CYP4F3

C1orf38 ATP6V0A4 CARHS P 1 BBS1 DACT1 DAF

C1QA ATP6V1 H CASP2 BCAN DAPK3 DCN

C1QB ATP7A CCNB2 BCAR3 DEPDC6 DDR1

C1QR1 ATP9A CCND3 BCKDHB DKFZP434B044 DKFZp434C0328

C1R ATPIF1 CD209 BCL11B DKFZP434H132 DKFZp761P211

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma __ Scleroderma scleroderma Scleroderma scleroderma Scleroderma

C1S ATRN CD28 BCL2L2 DKFZP434P211 DLG3

C2 AUH CD37 BDNF DKFZp762C186 DNCH2

C20orf103 B3GNT6 CD4 BEXL1 DKFZp762E1312 DOK4

C20orf39 B4GALT3 CD83 BLCAP DNM3 DSG2

C3 B4GALT4 CD84 BLMH DPYSL2 DVL1

C3AR1 BAT8 CD86 BMP2 DPYSL3 EBP

C4A BBS1 CDC14B BRP44L EFEMP2 ECHDC3

C5orf13 BCAN CDC6 BTBD3 EFHD2 EDD

C6orf32 BCAR3 CDC7 C10orf57 ERP70 EFHC2

C6orf97 BCKDHB CECR1 C12orf22 FAD104 EGF

C7orf10 BCL11B CENTA2 C14orf1 FAP EIF2B1

C7orf19 BCL2L2 CFLAR C14orf124 FARS 1 EIF2C1

CALCRL BDNF CH25H C14orfϊ31 FCGR2A elF3k

CALD1 BEXL1 CHC1 C14orf132 FGF13 EIF3S6IP

CALM3 BLCAP CHD7 C14orf2 FKBP11 ELL3

CALU BLMH CHN1 C14orf92 FLJ10292 ELOVL4

CAPNS1 BMP2 CINP C18orf11 FLJ 10385 EMX2

CARHSP1 BRP44L CKAP1 C18orf9 FLJ 10647 EPB41L4B

CASP2 BTBD3 CKIP-1 C1orf21 FLJ 10815 EPHX2

CCL13 C10orf116 CLN2 C1orf35 FLJ 11259 FAHD2A

CCL18 C10orf57 CMKLR1 C1QDC1 FLJ 12438 FBXO42

CCL19 C12orf22 CNN2 C20orf111 FLJ12439 FBXO9

CCL2 C14orf1 CNTNAP1 C20orf38 FLJ 13725 FGFR1

CCL8 C14orf124 COL10A1 C20orf42 FLJ22175 FH

CCNB2 C14orf131 COL1A1 C21orf107 FLJ23221 FHL1

CCND3 C14orf132 COL4A4 C6orf130 FLJ23556 FLJ10415

CD14 C14orf2 COL5A1 C9orf55 FLJ30656 FLJ10521

CD163 C14orf92 COL5A2 CA11 FLNA FLJ10769

CD209 C18orf11 COL6A1 CAB39L FLRT2 FLJ 10847

CD28 C18orf9 COL8A1 CABC1 FMO3 FLJ 10948

CD37 C1orf21 COL8A2 CAT FOLH1 FLJ 10986

CD4 C1orf35 CORO1A CBX7 FOSL1 FLJ 11036

CD47 C1QDC1 CORO1B CCNG2 G6PC3 FLJ11848

CD53 C20orf111 COTL1 CCNI GANAB FLJ 12270

CD63 C20orf38 CR1 CD34 GJA4 FLJ 12895

CD74 C20orf42 CRN7 CD9 GLT25D1 FLJ13197

CD83 C21orfϊ07 CSF1 CDC16 GPR1 FLJ13646

CD84 C6orf130 CSF1R CDC5L GRB10 FLJ 14297

CD86 C9orf55 CSGIcA-T CDK5RAP1 GREM1 FLJ20010

CDC14B C9orf61 CTSB CDS1 GRP58 FLJ20265

CDC25B CA11 CXCL1 CELSR1 GTSE1 FLJ20315

CDC6 CAB39L CXCL10 Cep152 HAPLN1 FLJ20345

CDC7 CABC1 CXCL9 CES1 HCA112 FLJ20457

CDH11 CAPN3 CXCR4 CFH HDGFRP3 FLJ20489

CEBPD CAT CXorfθ CGI-143 HEM1 FLJ20604

CECR1 CBX7 CYLD CG 1-41 HIP1 FLJ20618

CENTA2 CCNG2 CYP26A1 CHCHD2 HKE2 FLJ20701

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

CETP CCNI DACT1 CHN2 HLA-B FLJ20859

CFLAR CD34 DAPK3 CLASP1 HLA-C FLJ20920

CH25H CD9 DEPDC6 CLASP2 HLA-DPA1 FLJ21069

CHC1 CDC16 DKFZP434B044 CLCA4 HLA-DRA FLJ21511

HLA-DRB1 ///

HLA-DRB3 ///

CHD7 CDC42BPA DKFZP434H132 CLDN1 HLA-DRB4 FLJ23091

CHN1 CDC5L DKFZP434P211 CLDN8 HLA-DRB6 FLJ23186

CHST1 CDH12 DKFZp566C0424 CNKSR1 HNT FLJ23861 cig5 CDK11 DKFZp762C186 COBL HPCAL1 FOXC1

CINP CDK5RAP1 DKFZp762E1312 CORO2B HRH1 FRMD4A

CKAP1 CDS1 DNM3 COX15 HS3ST3A1 FXYD3

CKIP-1 CELSR1 DOK5 COX4I1 HSGP25L2G FYCO1

CLN2 Cep152 DPT COX7C IGH@ /// IGHG1 GAL3ST4

CMKLR1 CES1 DPYSL2 CP IGLJ3 /// IGLC2 GALNT11

CNN2 CFH DPYSL3 CPE IGLL1 GATM

CNTNAP1 CG 1-143 ECGF1 CRBN ILT8 GDPD2

COL10A1 CG 1-41 EFEMP2 CRKL INHBA GHITM

COL11A1 CG 1-51 EFHD2 CRYZL1 ISG20 GLRX

COL15A1 CHCHD2 EMILIN1 CS KCNJ8 GLTSCR2

COL1A1 CHN2 EPHA3 CTDSPL KDELR3 GOLGA7

COL4A1 CHS1 EPHB2 CTNNBIP1 KERA GPM6B

COL4A2 CLASP1 EPIM CTNND1 KIAA0040 GPR48

COL4A4 CLASP2 ERP70 CTSF KIAA0746 GPR87

COL5A1 CLCA4 FAD104 CTSL2 KIAA0792 GRHPR

COL5A2 CLDN1 FAP CUL3 KIAA1644 GSTA3

COL6A1 CLDN8 FARS 1 CYP2J2 LBH GULP1

COL6A3 CNKSR1 FBN2 CYP39A1 LBP H2AFJ

COL8A1 COBL FCGR1A CYP3A5 LGALS3BP HBS1L

COL8A2 CORO2B FCGR2A CYP4F12 LILRA2 HCNGP

COMP COX15 FGF13 CYP4F3 LILRB2 HEBP1

COPS8 COX4I1 FKBP11 D2LIC LMCD1 HERC2

CORO1A COX7C FLJ 10292 DAF LOC146489 HEY2

CORO1 B CP FLJ 10385 DAG 1 LOC254359 HNRPD

COTL1 CPE FLJ 10647 DBI LOC284021 HOXA9

CR1 CRBN FLJ10815 DCN LOC51334 HOXC10

CRN7 CRKL FLJ 11259 DCT LR8 HPS4

CSF1 CRYZL1 FLJ 12438 DDR1 LRCH4 HRASLS

CSF1 R CS FLJ12439 DDX42 MAGEB4 HSD17B7

CSGIcA-T CTDSPL FLJ12443 DGCR2 MAP4K1 IDE

CSPG2 CTNNBIP1 FLJ 13725 DGKA MARCKS IDS

CSRP2 CTNND1 FLJ22175 DHX57 MCM5 IHPK2

CTGF CTSF FLJ23221 DKFZp434C0328 MDS018 IL1F7

CTSB CTSL2 FLJ23556 DKFZp434N2030 MGC16664 IL20RA

MGC27165 ///

IGH@ /// IGHG1

CTSC CUL3 FLJ30656 DKFZp667G2110 /// IGHM IL22RA1

_CTSL. _ . . CYP2J2 FLNA _ DKFZp_761P211_ MGC27165 ///. . . . .JNG4...

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma _^ _ _Sc[eroderma scleroderma Scleroderma

IGHG1

CXCL1 CYP39A1 FLOT2 DLG3 MGC4294 INPP5A

CXCL10 CYP3A5 FLRT2 DNAL4 MGC4368 IRS3L

CXCL9 CYP4F12 FMO3 DNCH2 MICAL2 ITPR1

CXCR4 CYP4F3 FOLH1 DOCK9 MICAL-L2 KCNJ13

CXorfθ D2LIC FOSL1 DOK4 MMP19 KCNK1

CYBA DAF FTHP1 DSG2 MS4A4A KIAA0033

CYBB DAG1 FTL DSTN MS4A6A KIAA0495

CYLD DBI FZD2 DTNA MTRF1L KIAA0794

CYP26A1 DCN G6PC3 DVL1 MTVR1 KIAA1305

CYP7B1 DCT GAA EBP MXD4 KIAA1815

CYR61 DDB1 GADD45B ECHDC3 MYO9B KIF1 B

D2S448 DDR1 GALNT10 EDD NAGA KIT

DAB2 DDX42 GANAB EFHC2 NCBP1 KLHDC2

DACT1 DF GARP EFNB2 NEDD9 LAMC3

DAP DGCR2 GDF15 EFS NF1 LASS6

DAPK3 DGKA GGTLA1 EGF NINJ2 LGTN

DCTD DHX57 GJA4 EGFL5 NOX4 LIN7B

DEPDC6 DKFZp434C0328 GLA EIF2B1 NPR3 LOC254531

DIO2 DKFZP434C212 GLT25D1 EIF2C1 NRG 1 LOC51136

DKFZP434B044 DKFZp434N2030 GNLY elF3k NRI P3 LOC51149

DKFZP434H132 DKFZP586H2123 GPR1 EIF3S3 NRP1 LOC63928

DKFZP434P211 DKFZp667G2110 GPSM3 EIF3S6IP OAS3 LOC81558

DKFZp564l1922 DKFZp761 P211 GPX7 EIF4B OR2B2 LRP16

DKFZP564K0822 DLEU1 GRB10 ELF5 OSBPL10 LRP1B

DKFZp566C0424 DLG3 GREM1 ELL3 P101-PI3K LRRC16

DKFZp762C186 DNAL4 GRP58 ELOVL4 P2RY13 LRRC2

DKFZp762E1312 DNCH2 GTSE1 EMP2 PAPPA LRRFIP2

DNAJC7 DNCM GUCY1A3 EMX2 PARP8 LUC7L

DNM3 DOCK9 HA-1 EPB41L4B PCDH12 MAGEF1

DNMT1 DOK4 HAPLN1 EPHB6 PDGFC MCCC1

DOK5 DSCR1L1 HCA112 EPHX1 PDLIM7 MCCC2

DPT DSG2 HDGFRP3 EPHX2 Pfs2 MCOLN3

DPYSL2 DSTN HEG EPN2 PHTF1 MDH1

DPYSL3 DTNA HEM1 ERBB3 PI4KII MFN1

ECGF1 DVL1 HFE ERCC3 PILRA MGC3123

EFEMP2 EBP HIP1 ERCC5 PITPNC1 MGC5139

EFHD2 ECHDC3 HKE2 ETFDH PLAU MLL

EIF4A1 EDD HLA-B EXTL2 PLSCR3 MMP27

EMILIN1 EFHC2 HLA-C EZH1 PLVAP MOCS2

ENG EFNA4 HLA-DPA1 FAHD2A PLXDC1 MPPE1

EPHA3 EFNB2 HLA-DRA FAM8A1 PP2447 MRPS30

HLA-DRB1 ///

HLA-DRB3 ///

EPHB2 EFS HLA-DRB4 FBLN1 PPM1 F MST4

EPIM EGF HLA-DRB6 FBXO21 PRO1855 MSTP9

ERP70 EGFL5 HNT FBXO42 __PSCp4_ NALPI

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma _ Scleroderma _

FAD104 EIF2B1 HPCAL1 FBXO9 PTGER4 NAV2

FAP EIF2C1 HRH1 FDFT1 RAB15 NCOA1

FARS1 elF3k HS3ST3A1 FGFR1 RAB20 NDRG2

FBN2 EIF3S3 HSGP25L2G FGFR2 RAB35 NDRG3

FCER1G EIF3S6IP HTR2A FH RAMP NEIL1

FCGR1A EIF4B HTR2C FHL1 RCN3 NFS1

FCGR2A ELF5 ICAM1 FLJ 10415 RELA NIP30

FGF13 ELL3 ICAM2 FLJ10521 RFX5 NIT2

FKBP11 ELOVL4 IF FLJ10769 RIN3 NOL3

FLJ 10292 EMP2 IFI35 FLJ 10847 RIOK3 NOTCH2

FLJ 10385 EMX2 IFIT2 FLJ 10948 RNF17 NPY1R

FLJ10647 EPB41 L4B IFIT3 FLJ 10986 RRM2 OCLN

FLJ10815 EPHB6 IFIT5 FLJ 11036 SAA1 /// SAA2 ORC4L

FLJ 11259 EPHX1 IGF1 FLJ 11220 SCAND1 PAQR6

FLJ 12438 EPHX2 IGFBP2 FLJ 11848 SEC61A1 PARD3

FLJ12439 EPN2 IGH@ /// IGHG1 FLJ 12270 SEMA6B PAWR

FLJ12443 ERBB3 IGHM FLJ 12895 SH3TC1 PCSK2

FLJ 13725 ERCC3 IGKV1D-13 FLJ 13197 SHOX2 PDCD4

FLJ22175 ERCC5 IGLC2 FLJ 13646 SLAMF8 PDCD6IP

FLJ23221 ETFA IGLJ3 /// IGLC2 FLJ13910 SLC11A1 PDGFD

FLJ23556 ETFDH IGLL1 FLJ14297 SLC12A8 PDHA1

FLJ30656 EXTL2 IL10RA FLJ20010 SLC15A3 PECI

FLNA EZH1 IL2RG FLJ20265 SLC16A1 PELI2

FLOT1 F10 IL4R FLJ20315 SLC24A6 PERLD1

FLOT2 FAHD2A IL6 FLJ20345 SLC25A22 PGRMC1

SLC2A14 ///

FLRT2 FAM8A1 ILT8 FLJ20457 SLC2A3 PGRMC2

FMO3 FBLN1 INHBA FLJ20489 SLC2A3 PJA1

FN1 FBXO21 IRF1 FLJ20604 SLC43A3 PLP1

FOLH1 FBXO28 IRF7 FLJ20618 SLC4A7 PLXNA3

FOLR2 FBXO42 ISG20 FLJ20701 SLCO1A2 POGK

FOSL1 FBXO9 ITGA5 FLJ20859 SN POLR3E

FTH 1 FDFT1 ITGAM FLJ20920 SNFT POU2F3

FTHP1 FGFR1 ITGAX FLJ21069 SNX11 PPA2

FTL FGFR2 ITGBL1 FLJ21511 SOD2 PPP1CB

FZD2 FH ITIH3 FLJ23091 SPHK1 PPT2

G1 P2 FHL1 K-ALPHA-1 FLJ23186 SPHK2 PREP

G1P3 FLJ 10326 KCNJ 15 FLJ23861 SPTLC2 PSAT1

G6PC3 FLJ10415 KCNJ8 FLJ30092 SSH1 PTD015

GAA FLJ10521 KCNQ1 FLNB STC2 PWP1

GADD45B FLJ 10769 KDELR3 FMO5 SYNJ2 RAB33B

GALNT10 FLJ 10847 KERA FNBP2 TACC3 RAB3-GAP150

RABL2A///

GANAB FLJ 10948 KIAA0040 FOXC1 TAL1 RABL2B

GARP FLJ 10986 KIAA0342 FOXJ3 TAZ RAD54B

GBP1 FLJ 11036 KIAA0508 FRMD4A TBC1D5 RAI2

GDF15 FLJ 11220 KIAA0602 FRZB TBX2 REC14

GEM FLJ11848 KIAA0746 FXYD3 JMEFF1 RGNEF

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

GGTLA1 FLJ 12270 KIAA0792 FYCO1 TMEM2 RHOT1

GJA4 FLJ 12895 KIAA0894 FZD7 TMEM8 RNF128

GLA FLJ 13197 KIAA0963 GABBR1 TMPRSS4 RPL13

GLT25D1 FLJ13646 KIAA1036 GAL3ST4 TNFAIP2 RPL13A

GMFG FLJ13910 KIAA1644 GALNT11 TNFRSF12A RPL17

GNG5 FLJ14297 LAIR1 GALNT3 TNFRSF21 RPL29

GNLY FLJ20010 LAPTM5 GATA3 TNFRSF6B RPL3

TNFSF12-

TNFSF13 ///

GPR1 FLJ20265 LBH GATM TNFSF13 RPL31

GPSM3 FLJ20315 LBP GCHFR TNIP2 RPL7A

GPX1 FLJ20345 LGALS1 GDPD2 TNRC5 RRAGD

GPX7 FLJ20457 LGALS3BP GGPS1 TPMT RTN3

GRB10 FLJ20489 LGALS9 GHITM TRAF3 RYK

GREM1 FLJ20604 LILRA2 GLRX TREM2 SARG

GRP58 FLJ20618 LILRB2 GLS2 TRIAD3 SCAMP1

GTSE1 FLJ20701 LMCD1 GLTSCR2 TXNDC5 SERPINB13

GUCY1A3 FLJ20859 LOC146489 GNPAT UBE2I SERTAD3

HA-1 FLJ20920 LOC254359 GNRH1 WAS SETMAR

HAPLN1 FLJ21069 LOC284021 GOLGA7 WASPIP SFRS 11

HCA112 FLJ21511 LOC51334 GPLD1 YWHAH SGPL1

HCK FLJ23091 LOC56902 GPM6A ZC3HDC1 SLC19A2

HCLS1 FLJ23186 LR8 GPM6B ZDHHC14 SLC25A12

HDGFRP3 FLJ23861 LRCH4 GPNMB ZFP36L2 SLC35F2

HEG FLJ30092 LTB GPR48 SLC6A14

HEM1 FLNB LY64 GPR56 SNCA

HFE FMO5 LY6E GPR87 SP192

HIP1 FNBP2 LY86 GPRASP1 SPATS2

HKE2 FOXC1 M6PRBP1 GREB1 SSB3

HLA-B FOXJ3 MAGEB4 GRHPR SSH3

HLA-C FRMD4A MAN2B1 GSTA3 ST13

HLA-DMA FRZB MAP4K1 GSTA4 STS

HLA-DMB FXYD3 MARCKS GSTT1 STXBP6

HLA-DPA1 FYCO1 MATN3 GULP1 SViL

HLA-DRA FZD7 MCM5 H2AFJ TAF7L HLA-DRB1 /// HLA-DRB3 /// HLA-DRB4 GABARAPL2 MDS018 HARSL TDE1

HLA-DRB3 GABBR1 METTL1 HBP1 TFAP2A

HLA-DRB6 GAL3ST4 MFAP2 HBS1 L TFAP2B

HNT GALNT11 MFNG HCNGP TFB1 M

HPCAL1 GALNT3 MGC16664 HEBP1 TM4SF6 MGC27165 /// IGH@ /// IGHG1

HRH1 GATA3 /// IGHM HERC2 TMOD1 MGC27165 /// HS3ST3A1 GATM IGHG1 HEY2 TNMD

HSGP25L2G GCHFR MGC3047 HLF TPD52L1

HSPG2 GDPD2 MGC4294 HMGCS2 TRIM2

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

HTR2A GGPS1 MGC4368 HNRPD TRIT1

HTR2C GHITM MICAL2 HOP TUFT1

ICAM1 GLCE M1CAL-L2 HOXA9 TULP4

ICAM2 GLRB MICB HOXC10 TXNDC4

IF GLRX MMP11 HOXD4 UBAP2

IFI16 GLS2 MMP14 HPGD Ufd

IFI27 GLTSCR2 MMP19 HPS4 USP21

IFI30 GNPAT MMP3 HRASLS USP34

IFI35 GNRH1 MMP9 HSD11B1 VAV3

IFI44 GOLGA7 MOXD1 HSD17B7 XLKD1

IFIT2 GPLD1 MS4A4A HSPB3 XPNPEP1 J IFIT3 GPM6A MS4A6A IDE ZDHHC11 I IFIT5 GPM6B MT1 F IDS ZDHHC13 I IFITM1 GPNMB MTRF1L IGBP1 ZFD25

IFITM2 GPR48 MTVR1 IGFBP5 ZFP161

IFNGR2 GPR56 MVP IHPK2 ZFYVE21

IGF1 GPR87 MXD4 IL11RA ZNF16

IGF2 GPRASP1 MYO9B IL1F7 ZNF224

IGFBP2 GREB1 NAGA IL20RA ZNF266

IGFBP3 GRHPR NCBP1 IL22RA1 ZNF395 I IGFBP4 GSN NEDD9 ING4 ZNF506

IGFBP7 GSTA3 NF1 INPP5A

IGH@ /// IGHG1 GSTA4 NINJ2 IRS3L

IGHG1 GSTM5 NK4 IRX5

IGHM GSTT1 NKG7 ITPR1

GTF2I ///

IGKC GTF2IP1 NOX4 ITPR2

IGKV1D-13 GULP1 NPR3 JARID1B

IGLC2 H2AFJ NR2F1 JMJD1 B

IGLJ3 /// IGLC2 HARSL NRG1 KAZALD1

IGLL1 HBP1 NRGN KCNJ13

IL10RA HBS1L NRIP3 KCNK1

IL2RG HCNGP NRP1 KIAA0033

IL4R HEBP1 NRP2 KIAA0157

IL6 HERC2 NUDT3 KIAA0174

ILT8 HEY2 OAS 1 KIAA0182

INHBA HLF OAS3 KIAA0232

IRF1 HMGCR OLFML2B KIAA0240

IRF7 HMGCS2 OR2B2 KIAA0265

ISG20 HNRPD OSBPL10 KIAA0368

ITGA5 HOP P101-PI3K KIAA0459

ITGAM HOXA9 P2RX4 KIAA0495

ITGAX HOXC10 P2RY13 KIAA0515

ITGB2 HOXD4 PAFAH 1B2 KIAA0553

ITGBL1 HPGD PAPPA KIAA0582

ITIH3 HPS4 PAPSS2 KIAA0657

K-ALPHA-1 HRASLS PARP8 KIAA0703

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

KCNJ15 HSD11 B1 PARVB KIAA0794

KCNJ8 HSD17B4 PCDH12 KIAA0828

KCNQ1 HSD17B7 PCDH17 KIAA0895

KDELR3 HSPB3 PCSK5 KIAA1007

KERA IDE PDE1A KIAA1018

KIAA0040 IDS PDGFC KIAA1093

KIAA0101 IGBP1 PDLIM7 KIAA1117

KIAA0342 IGFBP5 PDXK KIAA1155

KIAA0508 IGFBP6 PENK KIAA1305

KIAA0602 IHPK2 Pfs2 KIAA1467

KIAA0746 IL11RA PGM3 KIAA1536

KIAA0792 IL1F7 PHTF1 KIAA1815

KIAA0894 IL20RA PI4KII KIF1B

KIAA0963 IL22RA1 PILRA KIT

KIAA0992 ING4 PITPNC1 KLHDC2

KIAA1036 INPP5A PKN1 KLK1

KIAA1199 INSIG2 PLAU KRT15

KIAA1462 IRS3L PLCB2 L3MBTL

KIAA1644 IRX5 PLEK LAMC3

LAI R1 ITPR1 PLSCR3 LANCL1

LAPTM5 ITPR2 PLTP LASS6

LBH JARID1B PLVAP LEPR

LBP JMJD1B PLXDC1 LETMD1

LGALS1 KAZALD1 PML LGTN

LGALS3BP KCNJ 13 POLD1 LIN7B

LGALS9 KCNK1 POLE2 L0C114977

LGMN KIAA0033 POSTN L0C157567

LHFPL2 KIAA0157 PP2447 LOC201725 /// TOMM7

LILRA2 KIAA0174 pp9099 LOC254531

LILRB1 /// LYZ KIAA0182 PPIB LOC51136

LILRB2 KIAA0232 PPM1 F LOC51149

LIPA KIAA0240 PRELP LOC63928

LMCD1 KIAA0251 PRKR LOC81558

LMNB1 KIAA0265 PRO1855 LRP16

LNK KIAA0368 PSCD4 LRP1B

LOC 146489 KIAA0459 PSMB10 LRRC16

LOC254359 KIAA0494 PTGER4 LRRC2

LOC284021 KIAA0495 PTP4A2 LRRFI P2

LOC51334 KIAA0515 PTPN18 LSS

LOC56902 KIAA0553 RAB13 LTA4H

L0XL1 KIAA0582 RAB15 LUC7L

L0XL2 KIAA0657 RAB20 MAGEA11

LR8 KIAA0703 RAB35 MAGEF1

LRCH4 KIAA0794 RAC2 MAN2A2

LTB KIAA0828 RAMP MAP3K4

LTBP2 KIAA0895 RASGRP2 MASK-BP3 /// EIF4EBP3

LTF KIAA1007 RCN3 MATN2

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma .. _§5l£ ^r 2<_. ^e [!Tl ^a . ! ^c J§J5φ ^r JIL ^a ~ Scleroderma _ scleroderma Scleroderma

LUM KIAA1018 RELA MBP

LY64 KIAA1093 RELB MCCC1

LY6E KIAA1117 RFX5 MCCC2

LY86 KIAA1155 RGS 16 MCOLN3

LY96 KIAA1305 RIN3 MDH1

LYN KIAA1467 RIOK3 METTL3

M6PRBP1 KIAA1536 RNF17 MFN1

MAGEB4 KIAA1815 RODH MGC22014

MAN2B1 KIF1 B RRM2 MGC2650

MAP4K1 KIT RUNX1 MGC3123

MAP4K4 KLHDC2 SAA1 /// SAA2 MGC48332

MAPRE1 KLK1 SAMHD1 MGC5139

MARCKS KRT15 SCAND1 MGEA5

MATN3 L3MBTL SCG2 MLL

MCM5 LAMC3 SCO2 MMP27

MDK LANCL1 SDC3 MOAP1

MDS018 LASS6 SEC61A1 MOCS2

METTL1 LEPR SECTM1 MPPE1

MFAP2 LETMD1 SELL MRPS30

MFNG LGTN SEMA6B MRS2L

MGAT1 LIN7B SGCD MSH5

MGC16664 LKAP SH3TC1 MST1

MGC27165 LOC114977 SHOX2 MST4

MGC27165 ///

IGH@ /// IGHG1

/// IGHM LOC157567 SIL MSTP9

MGC27165 /// LOC201725 ///

IGHG1 TOMM7 SIPA1 MTCP1

MGC3047 LOC254531 SLAMF8 MTMR3

MGC4294 LOC51136 SLC11A1 MTM R4

MGC4368 LOC51149 SLC12A8 MUT

MICAL2 LOC63928 SLC15A3 MXH

MICAL-L2 LOC81558 SLC16A1 NAB1

MICB LONP SLC1A3 NAB2

MMP11 LRBA SLC20A1 NACA

MMP14 LRP16 SLC24A6 NAG

MMP19 LRP1B SLC25A22 NALP1

SLC2A14 ///

MMP3 LRRC16 SLC2A3 NAV2

MMP9 LRRC2 SLC2A3 NAV3

MOXD1 LRRFIP2 SLC39A14 NCOA1

MRPS6 LSS SLC43A3 NDRG2

MS4A4A LTA4H SLC4A7 NDRG3

MS4A6A LUC7L SLC7A7 NDUFB6

MSN MAGEA11 SLCO1A2 NDUFC1

MT1 F MAGEF1 SMA4 NEBL

MTHFD2 MAN2A2 SN NEIL1

MTRF1L MAP3K4 SNFT NFATC3

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma ^ScIe I9J^ ^rm _ ^a _ scleroderma _ Scleroderma __

^{' "} MASK-BP37// ^{"" "}

MTVR1 EIF4EBP3 SNX11 NFIB

MVP MATN2 SOD2 NFS1

MX1 MATN4 SPHK1 NFYC

MX2 MBP SPHK2 NIP30

MXD4 MCCC1 SPTLC2 NIT2

MYD88 MCCC2 SSH1 NOL3

MYO5A MCOLN3 STARD8 NOTCH2

MYO9B MDH1 STAT2 NPAS 1

NAGA MECP2 STC2 NPR2

NCBP1 METTL3 STMN1 NPY1 R

NCF4 MFN1 STRN NR3C2

NEDD9 MGC22014 SYNJ2 NRD1

NF1 MGC2650 SYT11 NUMA1

NID2 MGC3123 T1A-2 OACT2

NINJ2 MGC48332 TACC3 OCLN

NK4 MGC5139 TAL1 ODAG

NKG7 MGEA5 TAP2 OGT

NMI MLL TAZ OLFML1

NNMT MMP27 TBC1 D5 0LFML2A

NOX4 MOAP1 TBX2 0RC4L

NPR3 MOCS2 TBXAS 1 0SBPL1A

NR2F1 MPPE1 TGFB1 I1 0SBPL2

NRG1 MRPS30 TIE OSR2

NRGN MRS2L TIMELESS P8

NRIP3 MSH5 TK2 PABPN1

NRP1 MST1 TMEFF1 PAM

NRP2 MST4 TMEM2 PAQR6

NUDT3 MSTP9 TMEM8 PARD3

OAS1 MTCP1 TMPRSS4 PAWR

OAS2 MTMR1 TMSB10 PBP

OAS3 MTMR3 TNFAIP2 PCCA

OLFML2B MTMR4 TNFRSF12A PCDH21

OR2B2 MUT TNFRSF21 PCSK2

OSBPL10 MXH TNFRSF4 PDCD4

OSMR NAB1 TNFRSF6B PDCD6IP TNFSF12- TNFSF13 /// P101-PI3K NAB2 TNFSF13 PDGFD

P2RX4 NACA TNFSF4 PDGFRL

P2RY13 NAG TN1P2 PDHA1

PAFAH 1B2 NALP1 TNRC5 PDZK3

PAPPA NAV2 TOSO PECI

PAPSS2 NAV3 TP53I3 PELI2

PARP8 NCALD TPMT PERLD1

PARVB NCAM 1 TRAF3 PEX1

PCDH12 NCOA1 TREM2 PFAAP5

PCDH17 NDRG2 TRIAD3 PFDN5

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6

Upregulated in Downregulated in Upregulated in Downregulated Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

PCSK5 NDRG3 TRIO PFKM

PDE1A NDUFB6 TRIP13 PGAP1

PDGFA NDUFC1 TXNDC5 PGRMC1

PDGFC NDUFS4 TYMS PGRMC2

PDLIM1 NEBL U2AF1 PHKA2

PDLIM7 NEDD8 UBD PHYH

PDXK NEIL1 UBE2I PIAS3

PECAM 1 NEK9 UCK2 PIGC

PENK NFATC3 UPP1 PIGH

Pfs2 NFIB WAS PIK3C2G

PGM3 NFS1 WASPIP PINK1

PHC2 NFYC WISP1 PJA1

PHTF1 NIP30 YWHAH PLA2G4A

PI4KII NISCH ZC3HDC1 PLCB4

PILRA NIT2 ZDHHC14 PLEKHE1

PITPNC1 NOL3 ZFP36L2 PLP1

PKN1 NOTCH2 ZNF364 PLXNA3

PLAU NPAS1 PMM1

PLAUR NPR2 POGK

PLCB2 NPY1 R P0LR3E

PLEK NR3C2 POU2F3

PLSCR1 NRD1 POU6F1

PLSCR3 NUMA1 PPA2

PLTP NUP133 PPL

PLVAP NY-REN-7 PPM1A

PLXDC1 OACT2 PPOX

PLXND1 OCLN PPP1CB

PML ODAG PPP1CC

POLD1 OGT PPP1R7

POLE2 0LFML1 PPT2

POSTN 0LFML2A PRDM2

PP2447 ORC4L PREP pp9099 OSBPL1A PRKCH

PPIB 0SBPL2 PRPF8

PPM1F OSR2 PRPSAP2

PPT1 P11 PSAT1

PRCP P8 PSMD5

PRDX4 PABPN 1 PTD015

PRELP PAM PTPN21

PRG1 PAQR6 PTPN3

PRKR PARD3 PTPRF

PRO1855 PAWR PUM2

PRPS1 PBP PWP1

PRSS23 PCCA QPCT

PSCD4 PCDH21 RAB11A

PSMB10 PCSK2 RAB33B

_L PSMB8_ _ PDCD4 RAB3-GAP150

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

TPST1 SMARCC2 VPS13D

TRAF3 SNCA VPS45A

TREM2 SNX27 WASF1

TRIAD3 SOD3 WASF3

TRIB2 SOSTDC1 WDR42A

TRIM22 SOX9 WISP2

TRIO SP192 XK

TRIP13 SPAG 1 XLKD1

TXNDC5 SPATS2 XPNPEP1

TYMS SPINK5 XPO7

TYROBP SPTAN1 ZDHHC11

U2AF1 SPTBN1 ZDHHC13

UBD SS18L1 ZDHHC3

UBE2I SSB3 ZFD25

UBE2L6 SSBP2 ZFP161

UBE2S SSH3 ZFYVE21

UCK2 SSPN ZFYVE26

UCP2 ST13 ZNF10

UPP1 STAR ZNF133

VAMP5 STAT6 ZNF16

VCAM1 STS ZNF175

VSIG4 STXBP6 ZNF204

VWF SVIL ZNF211

WARS TACC2 ZNF224

WAS TACSTD2 ZNF263

WASPIP TAF7L ZNF266

WISP1 TBC1 D4 ZNF395

YWHAH TBC1 D8 ZNF506

ZC3HDC1 TCFL5 ZNF516

ZDHHC14 TDE1 ZNF539

ZFP36L2 TDRD3 ZNF6

ZNF364 TFAP2A ZNF647

ZYX TFAP2B ZNF75

TFB 1 M ZNF91

TGFA

THOC1

THRAP1

TIAF1

TIAM 1

TM4SF11

TM4SF3

TM4SF6

TMOD1

TNA

TNKS

TNMD

TNNC2

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

TNNI2

TNXB

TOB1

TPD52L1

TRIM2

TRIT1

TRRAP

TSC1

TTC15

TUFT1

TULP3

TULP4

TUSC4

TXNDC4

TYR

UBAP2

UBE4A

UBE4B

Ufd

UNC13B

UQCRC2

UREB1

USP21

USP34

USP46

USP6NL

UST

VAV3

VIPR1

VPS11

VPS13D

VPS45A

WASF1

WASF3

WDR42A

WIF1

WISP2

XK

XLKD1

XPNPEP1

XPO7

ZDHHC11

ZDHHC13

ZDHHC3

ZFD25

ZFP161

ZFYVE21

TABLE 1

COLUMN 1 COLUMN 2 COLUMN 3 COLUMN 4 COLUMN 5 COLUMN 6 Upregulated in Downregulated in Upregulated in Downregulated in Upregulated in Downregulated in scleroderma Scleroderma scleroderma Scleroderma scleroderma Scleroderma

ZFYVE26

ZNF10

ZNF133

ZNF16

ZNF175

ZNF204

ZNF211

ZNF224

ZNF263

ZNF266

ZNF273

ZNF395

ZNF506

ZNF516

ZNF539

ZNF6

ZNF647

ZNF75

ZNF91

This study has demonstrated that multi-center studies of skin gene profiles in scleroderma can generate meaningful results not confounded by the source of material, and show that the scleroderma transcript profile is very robust. We identified changes in matrix expression, such as a disproportionate increase in expression of collagen XI, consistent with an invocation of a fibrocartilage program in the fϊbrotic process. One scleroderma patient sample, notably younger (29 years) than the others, was an outlier. Thus, gene profiling can also be used to distinguish between different subtypes of scleroderma.

Other embodiments are within the scope of the following claims.