WO2004101820A2

WO2004101820A2 - Use of sah gene as a marker for psoriasis

Info

Publication number: WO2004101820A2
Application number: PCT/FR2004/001064
Authority: WO
Inventors: Valérie TRINQUET; Patricia Rossio; Michel Demarchez
Original assignee: Galderma Research & Development, S.N.C.
Priority date: 2003-05-07
Filing date: 2004-05-03
Publication date: 2004-11-25
Also published as: FR2854639A1; WO2004101820A3

Abstract

The invention relates to a probe relating to the SAH gene for characterization of a biological sample. The invention also relates to the use of said probe in order to identify an compound for the treatment of psoriasis, an identification method and a novel search tool.

Description

GENE SAH DU PSORIASIS

The present invention relates to the use of a probe relating to the SAH gene for the characterization of a biological sample.

The invention also relates to the use of this probe for the identification of a compound intended for the treatment of psoriasis, a method of identification as well as a new research tool.

Psoriasis is a frequent dermatological pathology which affects 3 to 5% of the European population. Psoriasis is commonly associated with other pathologies, such as hypertension, arthritis, or AIDS (Farber EM, Nall L, In Roenigk HH, Maibach Hl (Eds.): Psoriasis Third Edition, Revised and Expanded (1998), chapter 9: 107-157; Duvic M, 1990 J Invest Dermatol 1990 Nov; 95 (5): 38S-40S).

Based on the age of onset of symptoms and the association with certain genes of the major histocompatibility complex type I (MHC I), psoriasis has been classified into two subtypes called I and II (Henseler T, Christophers E, In Roenigk HH, Maibach Hl (Eds.): Psoriasis Third Edition, Revised and Expanded (1998), chapter 10: 159-166). Studies have also shown that 30 to 50% of psoriasis patients have a family history with one of the parents of ¹ or 2 ^nd degree. These observations have made it possible to suggest that psoriasis is a genetic disease with an autoimmune component (Bos, Br. J. Dermatol. (1988) 118: 141; Baadsgaard et al., J. Invest. Dermatol. (1990) 95: 328 ).

The main pathophysiological components of psoriasis include hyperproliferation associated with abnormal differentiation of keratinocytes of the epidermis, an inflammation characterized by an inflammatory infiltrate (mainly activated helper T lymphocytes) in the dermis and epidermis and by the release of pro-inflammatory cytokines, and finally an abnormal vascularization of the dermis (Krueger JG, J Am Acad Dermatol (2002) Jan; 46 (1): 1-23; quiz 23-6). The involvement of the inflammatory process in the pathophysiology of psoriasis is supported by the mechanism of action of certain agents having a therapeutic effect beneficial for the disease. For example, cyclosporine, used in the treatment of severe forms of psoriasis, exerts an inhibitory effect on the synthesis of interleukin-2, a cytokine promoting the proliferation of T lymphocytes and the secretion of inflammatory cytokines by these cells. Consequently, the modulators of inflammation make it possible to limit the inflammatory component characterizing in part psoriasis. Clinically, psoriasis is defined by an hyperplasia of the epidermis, as well as by scaling, erythema and inflammation of the skin.

Currently available methods of treatment include topical therapy, phototherapy, photo-chemotherapy and systemic therapy, reserved for the more severe forms of the disease. Nevertheless, these therapies only offer periods of remission to the patients, but the pathology generally resurfaces after the treatment is stopped.

Glucocorticoids administered topically are most commonly prescribed in the initial treatment of psoriasis, and have beneficial anti-inflammatory, antimitotic and antipruritic effects.

Raw coal tar is a complex mixture of thousands of hydrocarbon compounds. Its therapeutic effect is linked to the inhibition of enzymes and to antimitotic properties. Although effective, this treatment is by its unpleasant odor to use for the patient and can induce skin cancer. Phototherapy and photo-chemotherapy based on the administration of methoxsalen, the active photosensitizing ingredient, are only temporarily effective. Treatment should be repeated and offers palliative, but not curative, therapy. In addition, these treatments have long-term side effects, including immunologic alterations and an increased risk of carcinogenesis.

Methotrexate is the most commonly used systemic cytotoxic agent for severe forms of surface-spreading psoriasis important body. There are many contraindications for this treatment and stopping treatment may be accompanied by worsening of symptoms. Hydroxyurea, etretinate, cyclosporine, and AZT are also the systemic drugs used to fight psoriasis but all have serious side effects.

To date, no treatment allows a lasting remission of this pathology without causing significant side effects, because psoriasis is a complex pathology of multifactorial origin. Understanding the pathophysiological mechanism leading to the psoriatic phenotype therefore remains an important challenge in dermatological medical treatment. Indeed, many aspects of the disease remain to be elucidated, such as the heterogeneous response to certain treatments, of patients with similar clinical signs.

The majority of people with hypertension have hypertension, the origin of which remains unknown. However, in most patients there is a significant hereditary tendency. Blood pressure is not regulated by a simple pressure system but by different systems linked together. Blood pressure can be increased in severe cases by 40 to 60%, the blood circulation in the kidneys is halved. The kidneys no longer perform their function of excreting normal levels of salts and water, while blood pressure is increased. This dysfunction is not yet elucidated. Current treatments for this type of hypertension are based on vasodilators and diuretics.

In view of current knowledge of dysregulations of blood pressure and more precisely of hypertension problems, nothing allows a person skilled in the art to deduce any implication thereof in the physiological dysregulation leading to the psoriatic phenotype.

Analysis of gene expression in the skin of psoriatic patients constitutes a way to better understand the pathophysiological mechanism of this disease (Bowcock AM, Shannon W, Du F, et al., Hum Mol Genêt (2001) 10 (17): 1793-805; Oestreicher JL, Walters IB, Kikuchi T, et al., Pharmacogenomics J (2001) 1 (4): 272-87).

The technology of cDNA filters ("arrays") (NGuyen C, Rocha D, Granjeaud S, et al., Genomics (1995)) is one of the technologies allowing to analyze simultaneously and quickly the expression of several hundred mRNAs from a skin biopsy taken from the patient, as opposed to other heavier technologies to implement, such as SAGE (“Sériai Analysis of Gène Expression”) (Velculescu VE, Zhang L, Vogelstein B, Kinzler KW , Science (1995 Oct) 20; 270 (5235): 484-7), or the Differential Display (Liang P, Pardee AB, Science (1992); 257: 967-971; Zhang L, et al., Science: ( 1997); 276: 1268-1272).

The Applicant has identified a new marker gene for psoriasis.

The subject of the present invention is a probe relating to the SAH gene for the characterization of a biological sample, and also a new tool for characterizing a compound intended for the treatment of psoriasis, as well as a new research tool.

In particular, the invention relates to the use of a polynucleotide probe relating to the SAH gene having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the gene SAH (Accession number NM 005622.1) as a psoriasis research tool.

The use of the polynucleotide probe according to the invention allows in particular the characterization of a biological sample by measuring the level of expression of the SAH gene.

According to the present invention, a biological sample corresponds to any sample or sample of living organism, preferably a mammal, in particular a human organism, in an amount sufficient to be characterized. Mention may be made, as a biological sample, by way of nonlimiting example, of a skin, scalp, mucosa, or cell sample.

According to the present invention, the characterization of a biological sample consists in measuring the level of expression of a gene of interest, that is to say more precisely the quantity of messenger RNA or protein corresponding to this gene which is present in the biological sample.

By polynucleotide probe is meant a single or double-stranded oligo- or polynucleotide, of RNA or DNA, the sequence of which is complementary to a target sequence corresponding to part or all of said gene and which can range from 10 to conventionally 500 base pairs and whose sequence is complementary to the target sequence by at least 80% and preferably by at least 90% (percentage of nucleic acid bases paired between two sequences, see the calculation methods proposed by Atschul et al J. Molec. Biol. (1990), 215: 403).

The polynucleotide probes according to the invention may be oligo- or polynucleotides comprising one or more nucleotides modified by substitution, deletion or insertion, these modifications will be such that the sequence of the probe will allow it to specifically recognize the target sequence fragment.

The polynucleotide probes may also have a modified backbone giving it, for example, better stability. For example, the phosphodiester bonds of the natural RNA strands can be modified to include at least one nitrogen or sulfur atom. Furthermore, the polynucleotide probes can be modified so that their phosphodiester bonds are replaced by peptide-like sequences (peptide nucleic acids).

In addition, the polynucleotide probes according to the invention can comprise bases other than the 4 conventional bases. i

The polynucleotide probes according to the invention can be synthesized according to numerous synthesis methods in vivo or in vitro manually or automatically.

The in vitro synthesis methods can be chemical or enzymatic. For example, a single-stranded cRNA probe may be obtained by in vitro transcription of a template of DNA sequence of interest serving as a template, using an RNA polymerase (for example T3, T7 or SP6 RNA polymerase). A single-stranded cDNA probe can be generated by reverse transcription of an mRNA of interest in the presence of a reverse transcriptase (for example M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase isolated from Escherichia coli).

Many methods of in vivo synthesis of double stranded RNA are described in the literature, they can be carried out in various cell types of bacteria or higher organisms. Reference may also be made to the synthesis methods described in patent applications WO01 / 36646 and WO01 / 75164. These double stranded RNAs can then be denatured to serve as a single stranded polynucleotide.

Regarding its environment, the polynucleotide probe can be or be part of a plasmid, a viral genome or another type of vector. It can, in other cases, be part of the genome of a cell, or else of a cell genetically modified to contain this probe in its genome. It can also be an isolated molecule.

As regards the regions framing this probe, it is preferably under the control of regulatory sequences. If the probe is inserted into a vector, said vector preferably comprises all the sequences necessary for the transcription and optionally the translation of the probe. This probe can also be surrounded by flanking regions allowing a step of homologous recombination with another polynucleotide fragment, possibly leading to the insertion of the probe of the invention into the genomic DNA of a target cell.

The invention also relates to the use of a polypeptide probe relating to the expression product of the SAH gene as a tool for the search for psoriasis.

The use of the polypeptide probe according to the invention allows in particular the characterization of a biological sample by measuring the level of expression of the SAH gene.

The term “polypeptide probe relating to the expression product of the SAH gene” means a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a polypeptide. homologous, modified or variant of the wild protein. Said antibody can be obtained according to methods known to those skilled in the art (Catty D (Ed): Antibodies, A Practical Approach, Volume I (1989), chapters 2, 3 and 4; or Harlow E. and Lane D. Antibodies, A laboratory Manual (1988), chapters 6 and 7) using said wild protein or said homologous polypeptide.

They may, for example, be mono- or polyclonal antibodies, or their biologically active fragment capable of recognizing the protein encoded by the gene of interest. These antibodies can be labeled, for example immuno-conjugated with enzymes or labeled using fluorescent compounds, biotin or even radiolabelled.

By homologous polypeptides, is meant polypeptides whose amino acid sequences have at least 80%, and preferably. 90% amino acids in common with the wild protein encoded by the gene of interest.

By variant polypeptide is meant a mutated polypeptide or corresponding to a polymorphism which may exist, in particular in humans and which may exhibit truncation, substitution, deletion and / or addition of at least one amino acid relative to the sequence of the normal wild-type polypeptide.

The term “modified polypeptide” is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis, exhibiting a modification with respect to the normal wild-type sequence. These modifications may in particular relate to the pre-, pro- or mature domains of the polypeptides, to the amino acids at the origin of a spectrum specificity or of activity efficiency, or at the origin of the structural conformation. , charge or hydrophobicity, and the capacity for multimerization and membrane insertion of the polypeptides. It will thus be possible to create polypeptides of equivalent, narrower or wider activity. The modifications may also relate to the sequences involved in the processing, transport and addressing of the polypeptides.

The term “biologically active fragment of a polypeptide” is intended to denote a polypeptide fragment having conserved at least one activity of the polypeptide from which it is derived.

A comparative study of gene expression in psoriatic skin compared to healthy skin, enabled the Applicant to identify a gene,

_* called SAH (Human Hypertension-associated protein SA) Genbank reference (NCBI): NM_005622.1, whose transcriptional activity is induced in diseased skin.

In particular, the present invention relates to the use of a polynucleotide probe relating to the SAH gene as defined above for diagnosing psoriasis or a predisposition to psoriasis of a biological sample by measuring the level of expression of the SAH gene.

The present invention also relates to the use of a probe polynucleotide relating to the SAH gene as defined above for the identification of a compound intended for the treatment of psoriasis.

In a particular embodiment of the invention, the polynucleotide probe is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

In another embodiment of the invention, the polynucleotide probe will be used in the method described below.

The invention thus relates to a method for identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the amount of messenger RNA corresponding to the SAH gene in biological samples using a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (access no. NM 005622.1); d) selection of said compounds for which an inhibition of the transcription of the messenger RNAs of the SAH gene is measured in the sample treated in step b).

In particular, step c) of the method can be implemented with a polynucleotide probe which is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the cDNA probe. single strand of sequence SEQ. ID. # 2 or with the cDNA probe double strand of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

According to the embodiment given in Example 1, the measurement of the expression of the SAH gene in a biological sample can be carried out using the technology of cDNA filters ("arrays") on nylon support, according to which the extracted mRNAs of the biological sample are transcribed into radiolabelled single-stranded cDNAs which are then deposited on cDNA filters.

According to another possible embodiment, the total mRNAs extracted from the biological sample tested can be analyzed according to one of the detection methods known to those skilled in the art (Sambrook et al.: Molecular Cloning, A Laboratory Manual , Volume I (1989) Second Edition, chapter 7) as the technique of hybridization by Northern-Blot using a denatured double stranded cDNA probe.

According to another possible embodiment, the labeling of the probe may comprise a cycle of synthesis of double-stranded cDNA, from the total mRNAs extracted from the biological sample, coupled to an in vitro transcription in the presence of biotinyl ribonucleotides, generating Complementary labeled RNAs, these labeled antisense RNAs serving as a probe during hybridization on a DNA chip (Affymetrix, Gene Expression Monitoring, Technical Note; US 5,716,785, US 5,891,636, US 6,291,170).

In another embodiment, the present invention relates to the use of a polypeptide probe relating to the SAH gene as defined above for diagnosing psoriasis or a predisposition to psoriasis of a biological sample by measuring the level expression of the SAH gene.

The present invention also relates to the use of a polypeptide probe relating to the SAH gene as defined above for the identification of a compound intended for the treatment of psoriasis. The present invention also relates to a method for identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the quantity of the protein produced by the expression of the SAH gene in biological samples using a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild protein resulting from the expression the SAH gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein; d) selection of said compounds for which an inhibition of the expression of said protein is measured in the sample treated in step b).

By inhibition of the transcription of a SAH gene or of the expression of the protein is meant a reduction in the amount of messenger RNA or of the protein corresponding to said gene in a test sample.

In another embodiment, the present invention relates to a research tool comprising a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (access no. NM 005622.1).

In particular, the polynucleotide probe is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

The present invention also relates to a research tool comprising a polypeptide probe, said probe being a monoclonal antibody or polyclonal specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous, modified or variant polypeptide of the wild-type protein.

The research tool may also allow the analysis of the effects of certain treatments on the expression of marker genes for psoriasis, such as HSA, or the analysis of mutations in the sequence of these genes in individuals suffering from psoriasis.

The research tool may also allow the analysis of the expression profile of the SAH gene in the cells constituting the epidermis, in particular the keratinocytes, for example according to their state of proliferation or differentiation. The research tool may also allow constitutive expression, overexpression or inhibition of the expression of the SAH gene, according to in vitro or in vivo tests known to those skilled in the art.

For example, according to a research tool envisaged, the polynucleotide probe according to the invention can be used, for example in a hybridization test by Northern blot, or also in an amplification test by PCR (“Polymerase Chain Reaction” ), to follow the expression of the SAH gene or of a variant, modified or partial homolocal sequence in cells constituting the epidermis, in particular keratinocytes, expressing the SAH gene in vitro, for example as a function of state of differentiation or proliferation of cells. In particular, said expression can be constitutive or even inducible, the SAH gene being able to be placed under the control of a constitutive promoter such as the SV40 or CMV promoter, or of an inducible promoter such as the Tet Off Gene Expression System ( Invitrogen), classical techniques of genetic engineering and transfection are used (Gossen M, Bujard H., Annu Rev Genet. (2002) 36: 153-73).

According to another research tool envisaged, the polynucleotide probe according to the invention can be used, for example in a hybridization test by Northern-blot, or in a PCR amplification test (“Polymerase Chain Reaction”), to follow the expression of the SAH gene or of a homologous sequence, variant, modified, or partial, in the cells constituting the epidermis of a strain of laboratory animals, in particular in keratinocytes, overexpressing said gene or sequence in vivo, for example under the control of a promoter specifically activated in the epidermis, such as the promoters of Keratin K14, K15 or Involucrine, for example depending on the state of differentiation or proliferation of cells.

According to another envisaged research tool, the polynucleotide probe according to the invention can be used for the analysis for example by Southern blotting of an inactivation by deletion of the SAH gene, for example in cells constituting the epidermis, in particularly keratinocytes, cultivated in vitro or even in vivo in the cells of a strain of laboratory animals. This inactivation can be carried out according to conventional techniques of homologous recombination.

According to another research tool envisaged, the polynucleotide probe according to the invention can be used to draw and synthesize siRNAs, according to techniques known to those skilled in the art (TuschI T, Borkhardt A, Mol Interv (2002) Jun ; 2 (3): 158-67). These antisense RNAs can be used for the inhibition of the expression of the SAH gene, for example in a line of constituent cells of the epidermis, in particular keratinocytes, in vitro, or even in vivo, for example in a strain d laboratory animals.

For example, this search tool could be such that the probe or probes that it comprises, are brought into contact with the corresponding target sequence (s) deposited on a support such as a filter in nylon.

The probe can also be used in a hybridization test (for example a mismatch detection test), sequencing, microsequencing. According to other research tools envisaged by the present invention, the probe according to the invention may be associated with another detectable probe, the nature of which may preferably be fluorescent, radioactive or enzymatic. This characteristic can make it possible, among other things, to track the location of the probe, from the extracellular medium to the cell, or from the cytoplasm to the nucleus, or else to specify its interaction with DNA, or RNA or proteins. Those skilled in the art will know which detectable probe is best suited as a function of the characteristic that they want to be able to follow.

According to a research tool envisaged, the polypeptide probe according to the invention can be used for immunolabelling of the target polypeptide as defined above, on biological samples, according to the conventional techniques known to those skilled in the art, for example on section of epidermis of laboratory animals, for example animals exhibiting an overexpression of the SAH protein or of a homologous, variant, modified or active polypeptide polypeptide, or in vitro on cells constituting the epidermis, in particular keratinocytes, which may be the subject of an overexpression of the protein or of said polypeptide, or else of an inhibition of this expression, for example by the use of siRNA as envisaged previously.

According to another research tool envisaged, the polypeptide probe can also be used to purify said protein or said polypeptide, for example, according to immunoprecipitation techniques conventionally known to those skilled in the art.

The techniques conventionally known to those skilled in the art can be the techniques described by Sambrook J, Fritsch EF and Maniatis T in Molecular Cloning, A Laboratory Manual.

The discovery of the overexpression of the SAH gene in psoriatic skin makes it possible on the one hand to diagnose psoriasis, and on the other hand, to develop new therapeutics based on the inhibition of expression and / or SAH function to treat psoriasis.

In a particularly preferable aspect, the present invention provides a method for characterizing skin samples to determine predisposition to psoriasis.

The Applicant has demonstrated that the expression of the SAH gene is induced in psoriatic lesional skin with a plaque of psoriasis, compared to healthy skin (average expression measured in 13 psoriatic subjects and 10 healthy subjects). The results obtained are presented in Table 1.

The measurement of the expression of the SAH gene makes it possible to characterize a lesional psoriatic skin independently of the type and location of the plaque of psoriasis (Tables 2 and 3). Table 2 represents the expression of the SAH gene relative to an average expression in healthy subjects, in patients with predominantly plaques at the level of the trunk and asymmetric plaques. Table 3 represents the expression of the SAH gene relative to an average expression in healthy subjects, in patients with predominantly symmetrical plaques.

In addition, apart from the characterization of a skin having a plaque of psoriasis compared to a healthy skin, the discovery of these new markers of psoriasis also makes it possible to discriminate a skin of psoriatic patient not lesional (clinically healthy) but already committed towards the psoriatic process, of non-lesional skin having the real characteristics of healthy skin.

The Applicant has surprisingly discovered that certain non-lesional skins also overexpress the SAH gene. On the basis of the expression of these genes, the diagnosis of psoriasis is achievable very early since the expression of the SAH gene makes it possible to differentiate at the molecular level non-lesional skin already affected, while the clinical signs do not allow it. The results obtained are presented in Table 4. Thus, the invention also relates to a method for diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of the SAH gene by bringing a polynucleotide probe into contact with the target sequences isolated from said biological sample, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (access no. NM 005622.1).

In particular, the polynucleotide probe may be a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

The invention also relates to a method for diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of the protein produced by the expression of the SAH gene by bringing into contact with a polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous, modified or variant polypeptide of the wild protein.

By overexpression of a gene in a psoriatic sample is meant a higher expression of the messenger RNA or of the protein corresponding to this gene in this sample compared to a healthy reference sample.

Furthermore, the invention also relates to a diagnostic kit comprising a polynucleotide or polypeptide probe relating to the SAH gene. The object of the present invention therefore relates to a diagnostic kit comprising a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1).

In particular, said polynucleotide probe of the diagnostic kit is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

The subject of the present invention also relates to a diagnostic kit comprising a polypeptide probe such as a monoclonal or polyclonal antibody specifically directed against the wild protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein , or against a homologous, modified or variant polypeptide of the wild-type protein.

The observations described above also indicate that SAH is a potential therapeutic target for the development of new therapies aimed at treating chronic vulgar plaque psoriasis, whatever the predominant type of plaque in the patient, and the location of the psoriasis plaque on the body.

Thus, another object of the present invention relates to the use of a polynucleotide probe complementary to the SAH gene to inhibit the expression of the SAH gene.

In particular, the polynucleotide probe may be an antisense RNA or a siRNA which has the role of inhibiting the expression of a specific gene (Fire A., Trends in Genetic (1999); 15: 358-363).

In a particular embodiment of the invention, the polynucleotide probe will be a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the RNA probe of sequence SEQ. ID. # 4.

Finally, the invention also relates to the use of a polynucleotide probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (no. of accession NM 005622.1) for the manufacture of a pharmaceutical composition intended for the treatment of psoriasis.

The polynucleotide probe may more particularly be a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

EXAMPLE 1 - MEASUREMENT OF GENE EXPRESSION IN HEALTHY SKIN AND IN PSORIASIC SKIN

A clinical study carried out in the presence of healthy volunteers and patients suffering from chronic vulgar plaque psoriasis has made it possible to demonstrate the involvement of the SAH gene in psoriasis. Skin biopsies are taken from healthy volunteers, as well as from psoriasis plaques and from non-lesional areas that appear clinically healthy, from patients with psoriasis. The presence or absence of psoriasis on the area of biopsied skin, as well as the severity of the psoriasis plaque, are determined by the evaluation of local clinical parameters (erythema, scaling and plaque elevation scores). psoriasis). The analysis of gene expression is then carried out from each individual biopsy. The comparison of the gene profiles corresponding to healthy skin and psoriatic non-lesional and lesional skin, makes it possible to identify genes whose expression is induced in the disease. The determination of the gene profiles is based on the hybridization of a complex single-stranded cDNA probe (generated by the reverse transcription of the mRNAs from each skin biopsy) on fragments of cDNA of interest, which are deposited on a support such as a nylon filter. The hybridization signals obtained are proportional to the quantity of each labeled cDNA in the probe and reflect the level of expression of each mRNA in the analyzed skin sample (Nguyen C et al. Genomics (1995 Sep) 1; 29 (1): 207-16).

Filters developed by the Applicant, specific to dermatology have been developed. These filters are made up of 474 cDNAs involved in the physiological processes of human skin. In parallel, commercial filters with 4200 human cDNAs (Invitrogen GF211) were used. These cDNA filters will make it possible to determine the expression of genes of interest in healthy skin as well as in non-lesional and lesional skin of patients suffering from chronic vulgar plaque psoriasis.

Method

The measurement of gene expression in healthy skin and in psoriatic skin is carried out using the technology of cDNA filters (“arrays”) on nylon support.

The principle of this technology is based on the hybridization of a radiolabeled single-strand cDNA probe on target cDNA fragments deposited at high density on nylon filters (Nguyen C et al. Genomics (1995 Sep) 1; 29 (1): 207-16). In this study, nylon filters (GF211) sold by the company Invitrogen on which 4200 identified human cDNAs are deposited, as well as filters dedicated to dermatology with 474 human cDNAs, developed by the Applicant, are used. The radiolabelled cDNA probes are generated by reverse transcription of the total RNAs (Invitrogen protocol), isolated from the different biopsies taken from 10 healthy volunteers and 13 subjects suffering from chronic plaques of vulgar psoriasis (RNEasy protocol, Qiagen). The hybridization signals obtained are proportional to the quantity of each labeled cDNA present in the probe, which hybridizes on the cDNA which is complementary to it on the filter and thus reflect the transcriptional activity of the mRNAs in the biopsies analyzed.

Normalization analysis of the results of αenic expression and comparison of geniαues profiles between healthy skin and psoriatic skin

The hybridization signals are measured by phospholuminescence, then quantified using the image analysis software Imagen (Biodiscovery). In order to discriminate the specific hybridization signals of the genes studied, the background noise linked to non-specific hybridization is measured on each filter on virgin areas of cDNA deposition (GF211 filters), or within a surrounding crown. each spot (dedicated filters). After transformation of the data into log a detection threshold allowing to discriminate a measurement pertaining to a real signal or noise, is automatically determined for each filter using the method described previously by Fogel et al, 2002.

In order to compare the results of gene expression between the different biopsies analyzed, the hybridization signals are normalized by the median of the set of signals obtained for each biopsy (filters GF211), or by the expression of an RNA. exogenous messenger, serving as a reference, the expression of which is invariant between the samples analyzed. This exogenous transcript is added in constant quantity to the total RNA preparations from the different samples, before synthesis of the radiolabelled cDNA probe. The target sequence of this exogenous transcript is deposited on each filter.

For each gene, an average reference expression is determined on all the biopsies of healthy subjects, then subtracted from the expression obtained in each biopsy of non-lesional and lesional psoriatic skin (values in log). The ratio between the expression of each gene in psoriatic skin compared to healthy skin is then obtained by taking the base 10 exponential of the value calculated previously (Tables 1 to 4).

The results obtained make it possible to highlight the overexpression of the SAH gene in lesional human skin with a plaque of psoriasis compared to healthy human skin (Table 1).

The results of Tables 2 and 3 indicate that this overexpression is observed whatever the predominant type of plaque expressed in the patient (symmetrical or truncated) and also independently of the location of the psoriasis plaques. The abbreviations mentioned in these tables represent the location of the biopsies taken from the body for each psoriatic subject (SP) and have the following meaning: JG: left leg, JD: right leg, BD: right arm, BG: left arm, T: trunk.

The results in Table 4 show that the expression of these genes is also induced in non-lesional psoriatic skin. The abbreviations have the same meaning as before.

EXAMPLE 2 - MEASUREMENTS OF LOCAL CLINICAL PARAMETERS RELATED TO THE PSORIASIS PLATE: SCORES OF ERYTHHEM, DEQUAMATION AND PLATE ELEVATION

The different local clinical scores used to define the presence or absence of psoriasis on the biopsied skin area, as well as the severity of the psoriasis plaque, are the erythema, scaling and plaque elevation scores. These scores are conventionally measured using the following rankings:

The measurement of erythema is done using the following classification:

0 = none (no erythema)

1 = slight (slight redness present)

2 = moderate (easily detectable redness)

3 = severe (intense redness) 4 = very severe (very intense redness)

The measurement of flaking is done using the following classification: 0 = none (no flaking) 1 = light (dander detected, only one layer of flaking)

2 = moderate (scales of intermediate degree between mild and severe)

3 = severe (Several layers of dander)

4 = very severe (several layers of thick dander)

The plate elevation is measured using the following classification:

0 = none (no thickness elevation)

1 = slight (slight elevation detectable by touch)

2 = moderate (moderate thickness) 3 = severe (strong thickening)

4 = very severe (very thickening)

This study demonstrates that the local clinical parameters obtained with non-lesional psoriatic skin are not different from those obtained for healthy skin. They therefore do not make it possible to differentiate psoriatic non-lesional skin from healthy skin.

On the other hand, the results of table 4 made it possible to highlight at the molecular level, an overexpression of the SAH gene in certain non-lesional psoriatic skins. Measuring the expression of the SAH gene therefore makes it possible to carry out a very early diagnosis of psoriasis, since the expression of these genes makes it possible to differentiate at the molecular level non-lesional skin already affected, while the clinical signs do not allow it. .

Claims

1. Use of a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1) , as a psoriasis research tool.

2. Use of a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1) , to diagnose psoriasis or a predisposition to psoriasis in a biological sample by measuring the level of expression of the SAH gene.

3. Use of a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1) , for the identification of a compound intended for the treatment of psoriasis.

4. Use according to any one of claims 1 to 3, characterized in that said probe is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the cDNA probe single strand of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

5. A method of identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the amount of messenger RNA corresponding to the SAH gene in biological samples using a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1); d) selection of said compounds for which an inhibition of the transcription of the messenger RNAs of the SAH gene is measured in the sample treated in step b).

6. Method according to claim 5, characterized in that said probe of step c) is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the cDNA probe single strand of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the sequence RNA probe

SEQ. ID. # 4.

7. A method of diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of the SAH gene by bringing a polynucleotide probe into contact with the target sequences isolated from said biological sample, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession number NM 005622.1).

8. A diagnostic method according to claim 7, characterized in that said probe is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID No. 2 or with the double-stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ.

ID. # 4.

9. Psoriasis diagnostic kit comprising a polynucleotide probe, said probe having a sequence of 10 to 500 base pairs and being at least 80% complementary with a target sequence corresponding to all or part of the SAH gene (accession no. NM 005622.1).

10. Diagnostic kit according to claim 9, characterized in that said probe is a fragment of 10 to 500 base pairs representing at least 80%, and preferably at least 90%, of homology with the single-stranded cDNA probe of sequence SEQ. ID. n ° 2 or with the double stranded cDNA probe of sequence SEQ. ID. n ° 3 or with the RNA probe of sequence SEQ. ID. # 4.

11. Use of a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous, modified polypeptide or variant of the wild protein as a psoriasis research tool.

12. Use of a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous, modified polypeptide or variant of the wild-type protein for diagnosing psoriasis or a predisposition to psoriasis of a biological sample by measuring the level of expression of the SAH gene.

13. Use of a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous, modified polypeptide or variant of the wild-type protein for the identification of a compound intended for the treatment of psoriasis.

14. A method of identifying a compound intended for the treatment of psoriasis comprising the following steps: a) preparation of at least two biological samples; b) bringing one of the samples into contact with one or more compounds to be tested; c) measuring the quantity of the protein produced by the expression of the SAH gene in biological samples using a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild protein resulting from the expression the SAH gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild-type protein; d) selection of said compounds for which an inhibition of the expression of said protein is measured in the sample treated in step b).

15. A method of diagnosing psoriasis or a predisposition to psoriasis comprising a step of measuring the overexpression in a biological sample of the SAH gene by bringing a polypeptide probe into contact with the target sequences isolated from said biological sample, said probe being a monoclonal or polyclonal antibody specifically directed against the wild protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or against a homologous, modified or variant polypeptide of the wild protein.

16. Diagnostic kit comprising a polypeptide probe, said probe being a monoclonal or polyclonal antibody specifically directed against the wild-type protein resulting from the expression of the SAH gene, or against a biologically active fragment of said protein, or also against a homologous polypeptide, modified or variant of wild protein.