HK1068797B - Use of long pentraxin ptx3 in the manufacture of a medicament for increasing reproductive ability in a female - Google Patents

Description

Use of long pentaxin PTX3 for the manufacture of a medicament for increasing the reproductive capacity of a female animal

Background

The present invention relates to the need for PTX3 activity for female fertility. Genetic mutations that reduce PTX3 activity result in female sterility.

Pentraxin is a superfamily of proteins characterized by cyclic multimeric structures [1 ]. The classical short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP) are acute phase proteins in humans and mice, respectively, that are produced in the liver in response to inflammatory mediators; in particular, they are directly induced by interleukin-6 [2-3 ].

Long pentraxin shares similarities with classical short pentraxin, but differs by the presence of an unrelated long N-terminal domain coupled to the C-terminal pentraxin domain, as well as ligands for genomic organization, chromosomal localization, cellular origin, induction of stimulation and recognition. Long pentraxin 3(PTX3) is the first long pentraxin identified as a gene [4] inducible by interleukin-1 (IL-1) in endothelial cells and a gene [5] inducible by tumor necrosis factor-alpha (TNF α) in fibroblasts. PTX3 is also produced by macrophages and other cell types and tissues when stimulated by the major inflammatory mediators (LPS, IL-1, TNF α) [6-8 ]. PTX3 consists of a C-terminal 203 amino acid pentraxin-like domain and an N-terminal 178 amino acid unrelated domain. When secreted, glycosylated PTX3 protomer (45kDa) assembled to form a 10-20 multimer [9 ]. PTX3 does not bind to classical pentraxin ligands such as phosphoethanolamine, phosphocholine, high pyruvate agarose, collagen IV, fibronectin or gelatin. In contrast, PTX3 specifically binds C1q with high affinity via the pentraxin domain [9 ]. PTX3 plasma levels are very low (. ltoreq.2 ng/ml) under normal conditions but increase in several pathological conditions (10-100ng/ml) including infection [10 ].

Other long pentraxins cloned after PTX3 include guinea pig apexin [11, 12], which is expressed in the sperm acrosome; XL-PXN1[13] from Xenopus laevis; rat neuron pentaxin 1(NP1) [14 ]; human NP1 and NP2[15, 16 ]; mouse NP1 and NP2[15], Narp [17], and neuronal pentraxin receptors (NRPs), a putative integral membrane pentraxin [18-9 ]. The in vivo function of long pentraxin is not yet clear.

PTX3 consists of two domains: an N-terminal domain not associated with any known molecule and a C-terminal domain similar to a short pentraxin such as C-reactive protein (Brevia et al, J.biol. chem., 267: 22190-22197, 1992) substantial similarity was found between human PTX3(hPTX3) and mouse PTX3(mPTX 3). The degree of identity between the human and murine PTX3 genes was 82%, 90% if conservative substitutions were considered (Introna et al, Blood, 87: 1862-1872, 1996). The genes are located in the collinear chromosomal location. The high similarity between the sequences of hPTX3 and mPTX3 is an indication that pentraxin is highly conserved during evolution (Pepys & Baltz, adv. Immunol., 34: 141-212, 1983). A review of pentraxin was made by Gewurz et al (curr. opin. immunol., 7: 54-64, 1995).

WO 99/32516 describes the use of PTX3 for the therapeutic treatment of cancer, inflammation and infectious diseases.

US patent 5,767,252 describes growth factors for neuronal cells belonging to the pentraxin family.

WO 02/36151 describes the use of PTX3 for the preparation of a medicament for the prevention and treatment of autoimmune pathologies.

In contrast to the foregoing, studies of mice genetically modified at their PTX3 genetic locus (which were generated by homologous recombination in embryonic stem cells) and their effects revealed that PTX3 activity is involved in female fertility.

It is an object of the present invention to manipulate PTX3 activity and thereby regulate female fertility. The effects of female sterility can be ameliorated, reproductive capacity can be increased or decreased as desired, female fertility can be increased, or a combination thereof. Other treatments such as in vitro fertilization require invasive procedures and complex techniques. Thereby addressing the need for therapies that affect female fertility. Additional advantages and modifications are discussed below or are apparent from the disclosure herein.

Pharmaceutical compositions, methods of their use and preparation, and other objects are described below.

Summary of The Invention

It is an object of the present invention to provide a pharmaceutical composition comprising an active agent that alters the activity of PTX3 in an amount sufficient to affect the reproductive ability of a female organism. The finding that PTX3 activity is required may be used as a treatment for female patients or animals with reproductive defects or for diagnosing their reproductive capacity.

Examples of such agents include polynucleotides corresponding to the PTX3 gene, polypeptides corresponding to the PTX3 protein encoded thereby and other substances that increase or decrease expression of the PTX3 gene. This includes the nucleotide and amino acid sequences listed herein, analogs thereof, those containing mutations or polymorphisms, and other variants (e.g., partial length oligonucleotides and oligopeptides). Hybrids between at least one PTX3 moiety and a heterologous moiety (polynucleotide or polypeptide) are considered chimeric genes or fusion protein variants, respectively. The genetic vector may be used to shuttle at least one PTX3 moiety into a host or to express at least one PTX3 moiety by transcription and/or translation in a host or using at least partially purified components. Activators (e.g., interleukin-6, NF-. kappa.B, receptor agonists) or inhibitors (e.g., antibodies, I.kappa.B, receptor antagonists) may also be used as agents that modulate the activity of PTX 3. The active agent can be derived from a human or non-human animal (e.g., a mammal).

The subject may be a female patient or a female animal. The composition may be suitable for systemic administration or for local administration (i.e. within or around the reproductive organs of the female organism). The composition can be used for treating infertility or as contraceptive.

It is another object of the invention to provide a method of administering a pharmaceutical composition to a subject in need of treatment for female infertility or in need of female contraception in an amount sufficient to increase or decrease, respectively, reproductive capacity in the subject.

It is another object of the present invention to detect PTX3 in a female biological subject and correlate that amount with its reproductive capacity. Mutations in the human PTX3 genetic locus may map to chromosome 3q24-q 28; mutations in the interacting genes can be mapped outside the PTX3 locus. The function of a PTX3 variant can be determined by comparison with the known PTX3 sequence or other pentraxin sequences; folding, glycosylation, secretion, or formation of multimers; receptor binding or signal transduction; effects on reproductive capacity, fertility or infertility; or a combination thereof.

It is another object of the present invention to screen at least one active agent that alters PTX3 activity and thereby affects the reproductive capacity of a female organism, and to obtain an active agent by this method. Several examples of such active agents are disclosed.

It is another object of the present invention to provide mammalian cells and non-human mammals genetically mutated to reduce PTX3 activity. They provide in vitro and in vivo models for reproductive deficiencies (e.g., infertility). They can be used to screen for or test potential therapeutic agents.

Other aspects of the invention will be apparent to those skilled in the art from the following description and claims, and generalizations thereof.

Brief description of the drawings and sequence listing

FIGS. 1A-1F illustrate abnormal morphology of cumulus ovatus from PTX 3-/-mice. Cumulus was recovered 14-16 hours after hCG treatment. Shown are after collection (a and B) or 4 hours (C and D). In PTX3+/+ mice (a and C), granulocytes form dense and stable cumulus around oocytes (arrows da meters). In PTX 3-/-mice (B and D), they were loosely bound to oocytes and cumulus disappeared completely within 4 hours. Histological examination of the ovaries of PTX3+/+ (E) and PTX3-/- (F) mice revealed normal antral follicles (antral folliculle).

FIGS. 2A-2D show PTX3mRNA and protein expression in ovarian tissue. (A) The kinetics of PTX3 expression in the ovaries after hormone-induced superovulation (PMS treatment, 48 hours followed by hCG treatment) are shown at the mRNA level. Ovaries were collected 0, 6, 16, 24 or 48 hours after PMS treatment and then 2, 6, 16, 24 or 48 hours after hCG treatment. Mu.g of total RNA was loaded per lane. The gel was stained with ethidium bromide, shown below. (B) In situ hybridization of ovaries: granulosa cells express PTX3mRNA only in mature follicles. (C) PTX3 expression by cumulus (c.o.), cumulus cells (c.o. cells) and oocytes was detected by western blot. Cumulus was recovered from 4 PTX3+/+ and PTX 3-/-superovulated females; cumulus cells and oocytes were obtained from 7 and 14 PTX3+/+ superovulated females, respectively. (D) Phase contrast (right panel) and immunofluorescence analysis (left panel) of cumulus from PTX3-/- (lower panel) and PTX3+/+ (upper panel) mice are shown.

The sequences of human cDNA and its translated open reading frame (SEQ ID NOS: 1-2, respectively), mouse cDNA and its translated open reading frame (SEQ ID NOS: 3-4, respectively), human and mouse upstream regulatory regions (SEQ ID NOS: 5-6, respectively), and PCR primers (SEQ ID NOS: 7-10) are shown in the sequence Listing. Alignment of the human and mouse amino acid sequences showed 312 of 381 residues to be identical (82%) and 351 residues to be at least similar (92%). Both genes contain 3 exons: the first encodes 43 amino acid residues; the second encodes 135 amino acid residues, which do not have high similarity to known sequence motifs; and the third one encodes 203 amino acid residues, with similarity to pentraxin. A pentraxin-like domain includes the 2 Cys residues at positions 162 and 254 and the consensus "pentraxin-like" sequence His-Xaa-Cys-Xaa-Ser/Thr-Trp-Xaa-Ser (SEQ ID NO: 11).

Description of specific embodiments of the invention

Polynucleotides, including mutants and other variants thereof, corresponding to all or part of a PTX3 nucleic acid (e.g., transcript or gene) may be used to increase PTX3 activity (e.g., in vivo or in vitro expression of a PTX3 polypeptide), complement or correct genetic defects (e.g., transfection, infection), decrease PTX3 activity (e.g., antisense, ribozyme, siRNA), or detect complementary polynucleotides. Similarly, polypeptides corresponding to the PTX3 protein, including mutants and other variants thereof, may be used directly to provide PTX3 activity (if functional); production of inhibitory antibodies, agonists and antagonists; and identifying, isolating or detecting interacting proteins (e.g., antibodies, receptor agonists or antagonists) by binding assays.

Native PTX3 is glycosylated (potential N-linked glycosylation site at position 203). The multimeric PTX3 complex eluted in the gel filtration had a relative molecular weight of about 900 kDa. It migrated as a prominent band of about 440kDa (e.g., the 9-or 10-mer of the approximately 45kDa protomer) in gel electrophoresis under non-denaturing and non-reducing conditions, with 2 minor bands in the 540-600kDa range. Circular dichroism analysis showed that PTX3 contains mainly β -sheet structures and has some α -helical structures. PTX3 polypeptides or complexes thereof may be identified, isolated or detected indirectly by binding molecules (e.g., antibodies, natural or non-natural peptidomimetics) to the PTX3 gene product.

Candidate compounds for affecting reproductive performance may interact with representative PTX3 polynucleotides or polypeptides and be screened for their ability to provide diagnostic or therapeutic methods. These products can be used in assays (e.g., diagnostics) or for therapy; conveniently, they are packaged in an assay kit or pharmaceutical form. Binding to C1q is specific and saturable (a PTX3 pro-gen binds to a C1q receptor), K_dIs 7.4X 10^-8And M. K is obtained by kinetic analysis and calculation_onIs 2.6X 10⁵M^-1s^-1And K_offIs 4 x 10^-4s^-1. The ligand to which C1q binds is the pentraxin-like domain of PTX3, multimerization being required for binding (possibly via an intramolecular cysteine bond). Other receptors for PTX3 can be characterized.

Another aspect of the invention is a hybrid PTX3 polynucleotide or polypeptide: such as transcription chimeras or translation fusions. In transcription chimeras, at least the transcriptional regulatory region of a heterologous gene is linked to a PTX3 polynucleotide or the transcriptional regulatory region of a PTX3 gene is linked to at least one heterologous polynucleotide. For translational fusions, the reading frame of the PTX3 polypeptide and at least one heterologous amino acid domain are aligned by an ordered linkage. If a reporter or selectable marker is used as the heterologous region or domain, the effect of the mutated PTX3 nucleotide or amino acid sequence on the function of PTX3 can be readily detected. In particular, the transcription chimeras can be used to localize the regulatory promoter of the PTX3 gene, while the translational fusions can be used to localize the PTX3 protein in the cell. For example, a transcriptional regulatory region, ligand binding domain, or multimerization domain from PTX3 may be included in the hybrid molecule.

"PTX 3" refers to human and mouse genes and proteins, mutants and polymorphisms found in nature, and variant forms thereof (e.g., mutants and analogs not found in nature), and analogs thereof. The chemical structure of PTX3 can be a polymer of natural or unnatural nucleotides that are linked by natural or unnatural covalent bonds (i.e., polynucleotides), or a polymer of natural or unnatural amino acids that are linked by natural or unnatural covalent bonds (i.e., polypeptides). For a non-limiting list of natural and non-natural nucleotides and amino acids, see tables 1-4 of WIPOStandard ST.25 (1998).

"mutants" are PTX3 polynucleotides and polypeptides having at least one more active or less active function, an existing function that is altered or deleted, a new function that does not occur naturally, or a combination thereof. A "polymorphism" is a PTX3 polynucleotide and polypeptide that has been genetically altered, but the alteration does not necessarily have a functional consequence. "analogs" are PTX3 polynucleotides and polypeptides that have a different chemical structure but substantially equivalent function compared to the native gene or protein. PTX3 function is described in detail herein. Mutants, polymorphisms and analogs can be made by genetic engineering or chemical synthesis, but the latter are preferred for non-natural nucleotides, amino acids or linkages.

"oligonucleotides" and "oligopeptides" are short polynucleotides and polypeptides (e.g., less than 30, 60, 90, or 180 nucleotides or amino acids). They may be fragments of the PTX3 nucleotide or amino acid sequence described herein. Typically, they are prepared by chemical synthesis, but methods of cleaving longer polynucleotides or polypeptides may also be employed. Electrophoresis and/or reverse phase High Performance Liquid Chromatography (HPLC) are suitable biochemical techniques for purifying short products.

The PTX3 gene can be identified using stringent hybridization: for example, for oligonucleotides, 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50 ℃ or 70 ℃; for polynucleotides of 50 bases or longer, 500mM NaHPO₄pH 7.2, 7% Sodium Dodecyl Sulfate (SDS), 1% Bovine Serum Albumin (BSA), 1mMEDTA, 45 ℃ or 65 ℃. PTX3 protein can be identified using stringent binding using antibodies or other binding proteins as probes: for example, 50mM Tris-HCl pH 7.4, 500mM NaCl, 0.05% TWEEN 20 surfactant, 1% BSA, room temperature. The wash conditions can be varied by adjusting the salt concentration and temperature so that the signal-to-noise ratio is sufficient for specific hybridization or binding. This isolation method can be used to identify unknown PTX 3-related nucleic acids or proteins using probes that detect known PTX3 nucleic acids or proteins, respectively. For example, a mixture of nucleic acids or proteins may be isolated by one or more physical, chemical and/or biological properties, and the presence or absence of PTX3 nucleic acid or protein may then be detected by specific binding of the probe. The probes may also be used to detect the presence or absence of a known PTX3 gene or protein, or to identify a previously unknown PTX3 gene or protein. The blocking and washing conditions can be varied to obtain a nucleic acid hybridization or protein binding signal that is target specific and/or reduces background.

An "isolated" product is at least partially purified from the cell (e.g., human, other mammalian, bacterial, yeast) or production source from which it is derived. For example, compared to a lysate of the source cell, the isolated product is at least 50%, 75%, 90%, 95%, or 98% purified from other chemically similar solutes (e.g., total nucleic acid for polynucleotides, or total protein for polypeptides). For chemically synthesized nucleotide or amino acid polymers, purity is determined by comparison with products that are prematurely terminated or blocked, and can be considered isolated without purification. Purification can be achieved by biochemical techniques such as cell fractionation, centrifugation, chromatography, electrophoresis, precipitation, specific binding, or combinations thereof. Typically, solvents (e.g., water) and functionally inert chemicals (e.g., salts and buffers) are disregarded when determining purity. Cloning is often used to isolate the desired product. Thus, the pharmaceutical composition may include an active agent responsible for most, if not all, PTX3 activity.

The meaning of "heterologous" depends on the context. For example, the joining of heterologous nucleotide regions to form a chimera means that these regions are not found colinear in nature (e.g., a human-derived PTX3 polynucleotide is joined to a human non-PTX 3 transcriptional regulatory region). Another example is the fusion of amino acid domains that are not found colinear in humans (e.g., a PTX3 polypeptide derived from a human linked to a human non-PTX 3 multimerization domain). The linkage of nucleotide regions or the linkage of amino acid domains, one from human and the other from animal, is heterologous in that they are derived from different species. In another example, transfection of a vector or expression construct into a heterologous host cell or gene transfer (transgenesis) of a heterologous non-human organism means that the vector or expression construct is not found in the genome of that cell or organism in nature. The "recombinant" product is the result of the ligation of a heterologous region (for a recombinant nucleotide) or fusion of a heterologous domain (for a recombinant polypeptide). Recombination can be genetically engineered, in vitro with purified enzymes, or in vivo in cultured cells.

According to one aspect of the invention, polynucleotides (e.g. DNA or RNA, single or double stranded) that specifically hybridize to the PTX3 gene and its transcripts may be used as probes or primers. Such polynucleotides may be full-length, covering the entire gene or transcribed information (e.g., recombinant clones in phagemids, plasmids, phage, cosmids, yeast artificial chromosomes or YACs, bacterial artificial chromosomes or BACs, or other vectors), N-terminal "PTX 3-unique" or C-terminal "pentraxin-like" domains, exons or specific coding regions, or shorter length sequences that are unique to PTX3 gene or its transcript but contain only a portion thereof. The probe will stably bind to its target to generate a hybridization signal specific for the PTX3 polynucleotide or polypeptide, whereas the primer may bind less stably to its target because repeated cycles of polymerization or ligation reactions will also generate a specific amplification signal. The polynucleotide may be at least 15, 30, 45, 60, 90, 120, 240, 360, 480, 600, 720, 1200, 2400, 5000, 10K, 20K, 40K, 100K, 250K, or 500K nucleotides in length (including intermediate ranges thereof).

Typically, the sequence of SEQ ID NO: 1 or 3, except for any deletions or insertions that may be present, the nucleotide sequences may exhibit as little as 85% sequence identity, more preferably at least 90% sequence identity, while still being considered related. And SEQ ID NO: amino acid sequences having as little as 90% sequence identity compared to 2 or 4 are considered related. But preferably 95% or greater, and more preferably 98% or greater.

If the sequences can be aligned without introducing many gaps, complex mathematical algorithms need not be used. Such algorithms are known in the art and are implemented in commercial software packages using default parameters. See Doolittle, Of URFS and ORFS, University scienceBooks, 1986; gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991; and references cited herein. The percent identity between a pair of sequences can be calculated by an algorithm implemented in the BESTFIT computer program (Smith and Water-man, J.mol.biol., 147: 195-. Another algorithm for calculating sequence differences is suitable for rapid database searches and is implemented in BLAST computer programs (Altschul et al, Nucl. acids Res., 25: 3389-.

Conservative amino acid substitutions (e.g., Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys or Gln/Asn pairs) may also be considered when making comparisons, as chemical similarity of these pairs of amino acid residues is expected to result in functional equivalence in many cases. Amino acid substitutions that would preserve the biological function of the polypeptide are expected to preserve the chemical properties of the amino acid residue being substituted, such as hydrophobicity, hydrophilicity, side chain charge, or size. Functional equivalence or conservation of biological function can be assessed by the methods of structural determination and bioassay described herein. Thus, amino acid sequences having as little as 90% sequence similarity between two polypeptides are considered related; but preferably 95% or greater, and more preferably 98% or greater.

The codons used in the native nucleotide sequence may be adapted for translation in a heterologous host by taking the codon bias of the host. This will accommodate the translation machinery of the heterologous host without substantially altering the chemical structure of the polypeptide.

PTX3 polypeptides and variants thereof (i.e., deletions, domain shuffling or duplications, insertions, substitutions or combinations thereof) are also useful for determining structure-function relationships (e.g., alanine scanning, conservative or non-conservative amino acid substitutions). For example, folding and processing of PTX3 protein, secretion and multimer formation of PTX3 protomer, ligand binding to receptor, signal transduction, or a combination thereof. See Wells (Bio/Technology, 13: 647-651, 1995) and U.S. Pat. No. 5,534,617. Directed evolution by random mutagenesis or gene shuffling using PTX3 can be used to gain new and improved functions based on selection criteria. Mutant, polymorphic and analogue PTX3 polypeptides are encoded by suitable mutant, polymorphic and analogue PTX3 polynucleotides. The structure-activity relationship of PTX3 can be studied using variant polypeptides produced from expression constructs transfected into host cells with or without endogenous PTX3 (i.e., SAR studies). Mutations in discrete domains of PTX3 polypeptides may therefore be associated with a decrease in protein function or even an increase in activity.

The PTX3 nucleotide sequence can be used to generate a fusion polypeptide having at least one heterologous peptide domain (e.g., an affinity tag or epitope tag). Oligopeptides may be used for the generation of specific antibodies and for epitope mapping of antibodies specific for PTX 3. The polypeptide may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids long (including intermediate ranges thereof). The oligopeptide may be bound to an affinity tag of a specific binding pair (e.g. antibody-digoxigenin/hapten/peptide, biotin-avidin/streptavidin, glutathione S-transferase-glutathione, maltose binding protein-maltose, protein a or G/immunoglobulin, poly-histidine-nickel). Full-length PTX3 polypeptides (e.g., SEQ ID NOs: 2 or 4) or shorter fragments (e.g., N-terminal or C-terminal domains) can be prepared; optionally including a heterologous peptide domain. PTX3 polypeptides may be synthesized chemically, purified from natural sources, synthesized in transfected host cells, or a combination thereof.

The PTX3 nucleotide sequence or a portion thereof may be used to monitor PTX3 expression, determine PTX3 sequence, and/or detect PTX3 variants. The invention also provides hybridization probes and amplification primers (e.g., polymerase chain reaction, ligation chain reaction, other isothermal amplification reactions). One pair of such primers can be used in an RT-PCR assay to quantify PTX3 transcript abundance in cells. The amplification primers may be 15-30 nucleotides long (preferably about 25 nucleotides), anneal to the sense or antisense strand (preferably the primer pair will be complementary to each strand), and are set forth in SEQ ID NOS: 1, 3 and 5-6 or complements thereof terminate at the 3' end. Thus, the present invention will be useful for developing and using PTX3 primers and other oligonucleotides to quantify homologous RNA and DNA in cells.

The binding of the polynucleotide or polypeptide may occur in solution or on a substrate. The assay format may or may not require separation of bound from unbound. The detectable signal can be direct or indirect, attached to any portion of the bound complex, a competitive assay, amplification, or a combination thereof. Blocking or washing steps may be inserted to increase sensitivity and/or specificity. Attaching a polynucleotide or polypeptide, interacting protein or binding molecule to a substrate before, after or during binding results in capture of the unattached species. This fixation will be stably attached to the substrate under the washing conditions. See US patents 5,143,854 and 5,412,087.

Changes in gene expression can be expressed in a cell by affecting transcription initiation, transcript stability, translation of the transcript into a protein product, protein stability, glycoprotein processing, folding, or secretion rate, or a combination thereof. Genes, transcripts or polypeptides may also be assayed by techniques such as in vitro transcription, in vitro translation, Northern hybridization, nucleic acid hybridization, reverse transcription-polymerase chain reaction (RT-PCR), concatenated transcription, Southern hybridization, metabolic protein labeling, antibody binding, Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), fluorescent labeling or histochemical staining, microscopy and digital image analysis, and fluorescence activated cell analysis or sorting.

Reporter or selectable marker genes whose products are readily detectable can be used for convenient detection. Reporter genes include, for example, alkaline phosphatase, beta-galactosidase (LacZ), Chloramphenicol Acetyltransferase (CAT), beta-Glucuronidase (GUS), Luciferase (LUC), green and red fluorescent proteins (GFP and RFP, respectively), horseradish peroxidase (HRP), beta-lactamase and its derivatives (e.g., blue EBFP, blue-green ECFP, yellow-green EYFP, destabilized GFP variants, stabilized GFP variants, or fusion variants sold by Clontech as LIVINGCOLORS fluorescent protein). The reporter gene will use a cognate substrate, preferably as determined by a chromogen, fluorescence or luminescence signal. Alternatively, the test product may be supplemented with heterologous epitopes (e.g. FLAG, MYC, SV40T antigen, glutathione transferase, polyhistidine, maltose binding protein) for which cognate antibodies or affinity resins can be derived. Examples of drugs where there is a selectable marker gene conferring resistance are ampicillin, geneticin/kanamycin/neomycin, hygromycin, puromycin and tetracycline. Metabolic enzymes (e.g., dihydrofolate reductase, HSV-1 thymidine kinase) can be used as selectable markers in sensitive host cells or auxotrophs. For example, methotrexate may increase the copy number of the polynucleotide associated with the DHFR selectable marker, or ganciclovir may negatively select for a viral thymidine kinase selectable marker.

The polynucleotide may be linked to a linker oligonucleotide or bind to a member of a specific binding pair (e.g., antibody-digoxigenin/hapten/peptide epitope, biotin-avidin/streptavidin, glutathione S transferase or GST-glutathione, lectin-sugar, maltose-binding protein-maltose, polyhistidine-nickel, protein a/G-immunoglobulin). The polynucleotides may be bound by ligation of nucleotide sequences encoding binding members. The polypeptide may be linked to one member of a specific binding pair by generating a fusion encoded by such linked or conjugated polynucleotides, or by chemically crosslinking a reactive moiety that is directly chemically bonded to the binding member. Such polynucleotides and polypeptides can be used as affinity reagents to identify, isolate and detect interactions involving specific binding of the transcript or protein product of an expression vector. A member attached to a polynucleotide or polypeptide can bind its cognate binding member before or after affinity binding of the transcript or protein product. This can be done in solution to produce a complex or immobilized on a support. Protease recognition sites (e.g., for enterokinase, factor Xa, ICE, secretase, thrombin) can be included between adjacent domains to allow site-specific proteolysis to occur which separates those domains and/or inactivates protein activity.

Probes and primers can be used to identify the PTX3 gene or variants thereof. For example, probes or primers specific for the human PTX3 gene identified herein can be used to detect the presence or absence of the gene, thereby inferring the presence or absence, respectively, of the source of the gene. Genetic polymorphisms and mutations in the PTX3 gene can be specifically detected by: the potential mismatched base is placed in the middle of the probe or 3' of the primer to stabilize or destabilize binding of the probe or primer to its target, depending on whether the target sequence at that position is complementary to that base, respectively.

Genetic polymorphisms and mutations can also be detected by the following methods: restriction Fragments (RFLP), nuclease protected fragments (e.g., S1 nuclease, DNase I, RNase A, H or T1) or a change in the length of the amplification product. For complex genetic fingerprints, it may not be necessary to identify each component, since differences (e.g., RAPD) can be easily detected by comparison of observations in parallel next to each other. Differences can also be detected by: changes in Molecular Weight (MW) or isoelectric point (pI) of PTX3 protein were visualized by gel electrophoresis or isoelectric focusing, respectively.

The presence of PTX3 protein can be used as an indicator of PTX3 activity in human or animal fluids or tissues. The liquid may be blood, a blood product (e.g. plasma, serum), lavage liquid, sputum or the like. Exemplary tissues are those of epithelial (e.g., lung) or mucosal (e.g., oral, vaginal) tissues, although infections may be systemic and involve other tissue types. For solid tissues, the signal can be detected in situ; on dispersed or homogenized tissue, in solution (e.g. diluted or undiluted body fluids, washing fluids), or on cell smears or contact preparations. Oocytes that can be fertilized can be selected by expression of PTX 3.

Construction of shuttle or expression vectors

The shuttle or expression vector is a recombinant polynucleotide in the chemical form of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). The physical form of the vector may be single-stranded or double-stranded; the topology may be linear or circular. The vector is preferably a double-stranded deoxyribonucleic acid (dsDNA) or is converted to dsDNA upon introduction into a cell (e.g., insertion of a retrovirus into a host genome as a provirus). The vector may comprise one or more regions from mammalian, insect, plant or fungal genes or viruses (e.g., adenovirus, adeno-associated virus, cytomegalovirus, fowlpox virus, herpes simplex virus, lentivirus, Moloney leukemia virus, mouse mammary tumor virus, rous sarcoma virus, SV40 virus, poxviruses), as well as regions suitable for genetic manipulation (e.g., selectable markers, linkers with multiple recognition sites for restriction endonucleases, promoters for in vitro transcription, primer annealing sites for in vitro replication). The vector may be combined with proteins and other nucleic acids in a carrier (e.g., packaged in a viral particle) or agglomerated with chemicals (e.g., cationic polymers) to target into cells or tissues. The selection of vector polynucleotides and methods of introducing them into the reproductive system of a female organism (e.g., endometrium, ovary) are within the skill of the art.

The expression vector may further comprise regulatory regions for gene expression (e.g., promoters, enhancers, silencers, splice donor or acceptor sites, polyadenylation signals, cellular localization sequences). Using an agent, and a regulatory system responsive to the agent (e.g., tetracycline/tetR or FK506/FKBP), different levels of transcription can be achieved. The vector may also include one or more splice donor and acceptor sites within the expression region; kozak consensus sequence upstream of the expression region for translation initiation; and downstream of the expression region, multiple forward reading frames (forward) triple (in the three) stop codons to ensure termination of translation, one or more mRNA degradation signals, termination of transcription signals, polyadenylation signals, and 3' cleavage signals. For expression regions that do not contain introns (e.g., coding regions from cDNA), a pair of splice donor and acceptor sites may or may not be preferred. However, if it is desired to express one or more downstream regions only under inducing conditions, it may be useful to include an mRNA degradation signal.

The shuttle vector may further comprise an origin of replication (ARS) which allows the vector integrated into the host genome to replicate or to replicate as an autonomously replicating episome. Centromere and telomere sequences may also be included for chromosome segregation and chromosome end protection purposes, respectively. Random or targeted integration into the host genome is more likely to ensure maintenance of the vector, but the episome can be maintained by selection pressure, or for applications where the vector is only transiently present is preferred.

The vector may be a shuttle vector or an expression vector.

The expression region may be derived from any gene of interest and provided in any orientation relative to the promoter. Expression regions in antisense orientation will be used to prepare antisense polynucleotides or sirnas. The gene may be derived from a host cell or organism, from the same species as it is, or redesigned. Fusions to domains of genes that may share common functions with PTX3 can be assayed to define domains that confer this function or to provide multifunctional fusion proteins. Fusions may also be made with epitope tags (e.g., GFP, GST, HA, MYC). Some genes produce alternative transcripts, encoding subunits that assemble into homo-or heteromultimers, or produce propeptides that are activated by protease cleavage. The expression region can encode a translational fusion; the open reading frames of the regions encoding the polypeptide and the at least one heterologous domain may be aligned in an ordered linkage. If a reporter or selectable marker is used as the heterologous domain, expression of the fusion protein can be readily determined or located. The heterologous domain may be an affinity tag or epitope tag.

Screening candidate Compounds

Other aspects of the invention are chemical or genetic compounds, derivatives thereof and compositions comprising them, which are effective in the treatment of infertility or contraception. The amount administered to a subject in need of treatment, its formulation and the time and route of administration are effective to reduce fertility, increase or decrease reproductive capacity or enhance fertility. Determination of such amounts, formulations, and timing and routes of administration are within the skill of the art.

Screening methods may include administering the candidate compound to an organism or culturing the candidate compound with a cell and then determining whether gene expression is modulated. This modulation may be an increase or decrease in activity that partially or completely compensates for or may cause an alteration in fertility or sterility associated with fertility or sterility. Gene expression can be increased or decreased at the level of the rate of transcription initiation or extension, the stability of the transcript, the rate of translation initiation or extension, the stability of the protein, the rate of protein processing, folding or secretion, the proportion of protein in an active conformation, the formation of multimers, binding to receptors, or a combination thereof. See, for example, US patents 5,071,773 and 5,262,300. Efficient screening assays are possible (e.g., by using parallel processing and/or robotics).

The screening method may comprise culturing the candidate compound with a cell containing a reporter construct comprising a transcriptional regulatory region of PTX3 covalently linked in cis configuration to a downstream gene encoding the assayable product; and determining the production of the detectable product. Chimeras with the upstream region of the PTX3 gene or translational fusions in frame with the initiation ATG codon can be used to provide transcriptional regulatory regions. For example, SEQ ID NO: 5 or 6. Candidate compounds that increase production of a detectable product will be identified as agents that activate gene expression, while candidate compounds that decrease production of a detectable product will be identified as agents that inhibit gene expression. See, for example, US patents 5,849,493 and 5,863,733.

The regulation of PTX3 transcription (e.g., transcriptional regulatory regions and associated transcription factors) has been characterized for mouse and human genes (Altmeye et al, J.biol. chem., 270: 25584-25590, 1995; Basile et al, J.biol. chem., 272: 8172-8178, 1997). PTX3 transcription is similarly specific for certain cells. The responsiveness of PTX3 transcription to cytokine stimulation appears to be mediated through interactions with NF κ B and I κ B transcription factors as well as cell specific factors.

The screening method can include assaying for in vitro transcription from a reporter construct (the reporter construct comprising a transcriptional regulatory region) in the presence or absence of the candidate compound, and then determining whether transcription is altered by the presence of the candidate compound. In vitro transcription can be determined using cell-free extracts, partially purified cell fractions, purified transcription factors or RNA polymerase or a combination thereof. See, for example, US patent 5,453,362; 5,534,410, respectively; 5,563,036, respectively; 5,637,686, respectively; 5,708,158, respectively; and 5,710,025.

Techniques for determining transcription or translation activity in vivo are known in the art. For example, nuclear conjugation assays can be used to determine transcription of reporter genes. Translation of the reporter gene can be determined by measuring the activity of the translation product. The activity of the reporter gene can be determined by measuring one or more of transcription of the polynucleotide product (e.g., RT-PCR or transcript), translation of the polypeptide product (e.g., immunoassay for the protein), and the biological activity of the reporter protein itself.

Compounds that increase or decrease PTX3 gene expression or protein activity can then be assayed for their effect on fertility, reducing fertility, or enhancing fertility.

Epitope-tagged PTX3 protein or an antibody specific for PTX3 protein can be used for affinity purification of multimers or other PTX 3-containing complexes. Candidate compounds can be screened for their ability to decrease abundance (i.e., steady state level of the complex), assemble secretion rate, or biological activity of the complex. For example, compounds that enhance or inhibit binding between the PTX3 protein and its receptor can be identified. The PTX3 protein may be attached to a substrate as described above. The candidate compound is incubated with the immobilized PTX3 protein in the presence of at least one other component in at least partially purified form or as a complex of a crude mixture. Furthermore, one or more components of the complex may be attached to a substrate, and the candidate compound may be incubated with the immobilized components in the presence or absence of additional complex components in at least partially purified form or as a crude mixture, in the presence of PTX3 protein. Examples of the binding conditions are shown below. After incubation, any unbound components may be washed away, leaving one or more compomer components bound to the substrate. Complex formation including PTX3 protein may also occur in solution, and the PTX 3-containing complex may or may not then be immobilized. The reduction is a reversible reaction that untangles the PTX3 multimer. The amount of each component of the complex can be quantified after washing and separating the complex from other proteins (e.g., heterogeneous assay) or from other proteins (e.g., homogeneous assay). For example, the assay may be performed using an immunological assay such as ELISA, RIA or Western blot. Complex formation can be determined by binding of the antibody to an epitope that is either formation dependent or masked after formation. The complex may be immobilized before or after formation by combining at least one component of the complex with a substrate. Binding of the complex to the substrate can be determined without isolation by proximity detection such as SPA or BiaCore. The amount of one or more bound components of the complex is determined with and without the candidate compound. Desirable compounds are those that increase or decrease PTX3 abundance, assembly, secretion, multimer formation, biological activity, or a combination thereof.

Genetic compounds for therapy

Activation can be achieved by: expression vectors comprising expression regions encoding proteins with PTX3 activity or up-regulating PTX3 activity (e.g. the full-length coding region or a functional part of the PTX3 gene; a super allelic mutant, homologue, ortholog (ortholog) or paralog (paralog) thereof; acute phase inducers; positive transcription factors acting on the PTX3 gene) or encoding proteins which release the inhibition of PTX3 activity (e.g. at least partially inhibiting the expression of a negative regulator of the PTX3 gene) are induced. Overexpression of transcription or translation, as well as overexpression of protein function, is a more direct route to gene activation. Alternatively, the downstream expression region may be targeted for homologous recombination into a locus in the genome, thereby replacing the endogenous transcriptional regulatory region of the gene with an expression cassette or a particular gene mutation.

The expression vector can be introduced into a host cell or non-human animal by transfection or gene transfer techniques using, for example, one or more chemicals (e.g., calcium phosphate, DEAE-dextran, lipids, polymers), biolistics, electroporation, naked DNA techniques, microinjection, or viral infection. The introduced expression vector may be integrated into the host genome of the cell or animal, or maintained as an episome. Many neutral and charged lipids, sterols, and other phospholipids are known for use in preparing lipid carriers. For example, neutral lipids are Dioleoylphosphatidylcholine (DOPC) and Dioleoylphosphatidylethanolamine (DOPE); the anionic lipid is dioleoyl phosphatidylserine (DOPS); the cationic lipids are dioleoyl trimethylammonium propane (DOTAP), dioctadecyl diamido-glycyl spermine (DOGS), dioleoyl trimethylammonium (DOTMA), and 1, 3-dioleoyloxy-2- (6-carboxyspermyl) -propionamidotetraacetic acid (DOSPER). Dipalmitoylphosphatidylcholine (DPPC) may be incorporated to improve the efficacy and/or stability of delivery. FUGENE 6, LIPOFECTAMINE, LIPOFECTIN, DMRIE-C, TRANSFECTAM, CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX-4, PFX-5, PFX-6, PFX-7, PFX-8, TRANSFAST, TFX-10, TFX-20, TFX-50, and lipotaxaxi lipids are proprietary formulations. The polymer may be a cationic dendrimer, polyamide, polyamidoamine, polyethylene or polypropylene glycol (PEG), Polyethylenimine (PEI), polylysine, or a combination thereof; or the polymeric material may be formed into nanoparticles or microparticles. In naked DNA technology, a vector (usually as a plasmid) is delivered to a cell or tissue where it may or may not integrate into the host genome without the use of chemical transfection agents (e.g., lipids, polymers) to aggregate the vector prior to its introduction into the cell or tissue.

Animal, insect, fungal or bacterial cells may be transfected; the gene transfer can be performed using a non-human animal. Homologous regions from the gene may be used for targeted integration into a particular genetic locus in the host genome, thereby regulating expression of the gene at that locus (e.g., a promoter-free reporter or selectable marker for homologous recombination at the PTX3 genetic locus) or an ectopic copy of the PTX3 gene may be inserted. The polypeptides may also be produced in vitro using cell extracts or in vivo using genetically manipulated cells.

Expression vectors can be used to replace functions of genes that are down-regulated or completely defective, to supplement functions of partially defective genes, or to compete with the activity of genes. Thus, the cognate gene activity of the host can be neoallelic, subtenomic, superallelic or normal. Functional substitution or supplementation may be accomplished by the methods discussed above, and genetically manipulated cells or organisms may be subjected to selection for high or low expression in the downstream region (e.g., assessing the amount of products of transcription or translation or the biological function of either product). Competition between the downstream region of expression and the neoallelic, superallelic or normal gene can be achieved because of the combined interactions that exist in the multimeric protein complex. Alternatively, the downstream region of the expression vector may encode a negative regulator of intracellular inhibitory function or a single chain antibody. Thus, at least partial inhibition of PTX3 activity may be achieved by antisense, ribozyme or RNA interference techniques in which the expression vector comprises a downstream region of an siRNA molecule corresponding to an unmodified antisense molecule, ribozyme or to a portion of the PTX3 nucleotide sequence.

Compounds that increase or decrease PTX3 gene expression or protein activity can then be assayed for their effect on fertility, reducing fertility or enhancing fertility.

Antisense polynucleotides may function by hybridizing to mRNA transcripts or degrading such gene transcripts, thereby directly blocking translation. Antisense molecules can be prepared recombinantly using at least a functional portion of the antisense-directed gene as a region downstream of a promoter in an expression vector. Chemically modified bases or linkages can be used to stabilize antisense polynucleotides by reducing degradation or extending half-life in vivo (e.g., methylphosphonate, phosphorothioate, peptide nucleic acids). The sequence of the antisense molecule can be complementary to the translation initiation site (e.g., between-10 and +10 of the target nucleotide sequence).

Ribozymes catalyze the specific cleavage of an RNA transcript or genome. The mechanism of action involves sequence-specific hybridization with complementary cellular or viral RNA followed by endonuclease cleavage. Inhibition may or may not be dependent on ribonuclease H activity. Ribozymes include one or more sequences complementary to a target RNA and a catalytic sequence responsible for RNA cleavage (e.g., hammerhead, hairpin, ax motifs). For example, potential ribozyme cleavage sites within the subject RNA were initially identified as: scanning a target RNA for ribozyme cleavage sites, comprising the following three nucleotide sequences: GUA, GUU and GUC. Once identified, oligonucleotides of about 15 to about 20 ribonucleotides corresponding to the region of the subject RNA containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that may render the candidate oligonucleotide sequence unsuitable. The suitability of the candidate sequence can then be assessed by its ability to hybridize to and cleave the target RNA. Ribozymes can be recombinantly produced or chemically synthesized.

siRNA refers to double-stranded RNA of at least 20-25 base pairs that mediates RNA interference (RNAi). Duplex sirnas corresponding to target RNAs can be formed by the following method: transcription of each strand separately, coupled transcription from a pair of promoters with opposite polarity, or annealing of single-stranded RNA with at least partially self-complementary sequences. Alternatively, double-stranded oligoribonucleotides of at least about 21 to about 23 base pairs can be chemically synthesized (e.g., a 21 ribonucleotide double helix with a 2 ribonucleotide 3' overhang) and some substitution by modified bases can be tolerated. However, mismatches in the center of the siRNA sequence can eliminate interference. The region targeted for RNA interference should be transcribed, preferably as the coding region of a gene. Interference appears to be dependent on cytokines (e.g., ribonuclease III) that cleave the target RNA at sites 21-23 bases apart; the position of the cleavage site appears to be defined by the 5 'end of the guide siRNA rather than its 3' end. Priming by a small amount of siRNA can trigger interference after amplification by RNA-dependent RNA polymerase.

Antibodies specific for PTX3 may be used for inhibition or detection. Polyclonal or monoclonal antibodies can be prepared by immunizing an animal (e.g., chicken, hamster, mouse, rat, rabbit, goat, horse) with an antigen, optionally affinity purified against the same or a related antigen. The antigen may be a native protein, a fragment prepared by proteolysis or genetic engineering, a fusion protein, or a protein translated in vitro or synthesized, which includes at least one or more epitopes bound by the antibody. Antibody fragments can be prepared by proteolytic cleavage or genetic engineering; humanized antibodies and single chain antibodies can be prepared by grafting sequences from the antigen-binding region of the antibody to a framework molecule. Other binding molecules (e.g., agonists or antagonists of ligand-receptor binding) can be prepared by screening combinatorial libraries for members that specifically bind antigen (e.g., phage display libraries). The antigen may be a full-length protein encoded by a gene or a fragment thereof. The antibody may be specific for PTX3 or it may cross-react with other pentraxin, depending on how well the epitope recognized by the antibody is conserved among different species. See, e.g., US patents 5,403,484; 5,723,286; 5,733,743, respectively; 5,747,334, respectively; and 5,871,974.

PTX 3-specific binding agents (e.g., polynucleotides, polypeptides) are diagnostically useful for detecting PTX3 nucleic acids or proteins, or for treatment to inhibit PTX3 activity (e.g., transcription, translation, processing, secretion, receptor binding). In particular, agents that affect PTX3 transcription and PTX3 binding to the receptor are desirable.

The compounds of the invention or derivatives thereof may be used as medicaments or for formulating pharmaceutical compositions having one or more of the uses disclosed herein.

Accordingly, an object of the present invention is the use of recombinant human PTX3 for the manufacture of a medicament for increasing the reproductive capacity of a female subject.

Another object of the present invention is the use of a viral or plasmid vector containing the human PTX3 cDNA for the treatment of a female subject in need of increased reproductive capacity.

Another object of the invention is the use of the PTX3 protein as a diagnostic marker of reproductive capacity in human females.

Another object of the present invention is the use of PTX3 as a target protein for screening pharmaceutical compounds to assess their ability to affect the reproductive ability of a female subject.

The compounds of the invention may be administered to cultured cells in vitro, to cells in vivo, or to cells ex vivo that are outside of a subject and then may be returned to the same subject or another subject. The subject is a female of reproductive age; which is intended to be pregnant or is at risk of pregnancy.

The compounds or derivatives thereof may be used in the preparation of medicaments or other pharmaceutical compositions. The use of compositions that also include a pharmaceutically acceptable carrier and compositions that also include ingredients for delivering the compositions to a subject is known in the art. The addition of such carriers and other ingredients to the compositions of the present invention is within the skill of the art.

The pharmaceutical compositions may be administered as a preparation suitable for direct application to the reproductive system of a female organism (e.g. endometrium, ovary) or for passage through the intestinal tract or circulation. Alternatively, the pharmaceutical composition may be added to the culture medium. In addition to the active compound, such compositions may contain pharmaceutically acceptable carriers and other ingredients known to aid in administration and/or to enhance absorption. The composition may be administered in a single dose or in multiple doses administered at different times.

The pharmaceutical compositions may be administered by any known route. By way of example, the compositions may be administered by mucosal, pulmonary, topical or other localized or systemic routes (e.g., enteral and parenteral). In particular, it may be desirable to achieve an effective amount of PTX3 activity in or around the reproductive system. This may include topical application, implantation around reproductive organs, or application of pessaries. The term "parenteral" includes, without limitation, subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intrathecal and other injection or infusion techniques.

Appropriate choices in the amount and timing of the dose, formulation, and route of administration may be made with the goal of achieving a favorable response (i.e., efficacy) in the subject and avoiding undue toxicity or other harm to the subject (i.e., safety). Thus, "effective" refers to the selection which involves conventional manipulation of conditions to achieve the desired effect: for example, affecting reproductive ability, enhancing fertility, or reducing fertility.

Bolus formulations administered once a day to female subjects are a convenient dosing regimen. Alternatively, an effective dose may be administered every other day, once a week, or once a month. Dosage levels of the active ingredient in the pharmaceutical composition can also be varied to achieve temporary or permanent concentrations of the compound or derivative thereof in the subject to be treated and to produce the desired therapeutic response. It is within the skill of the art to start doses from levels below those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The timing of administration can be set relative to the reproductive cycle (e.g., menstruation) of the female biological subject. In practice, body temperature or hormone levels may be used as a surrogate for reproductive events such as ovulation and menstruation.

The amount of compound administered depends on factors such as: such as the biological activity and bioavailability (e.g., half-life, stability and metabolism in vivo) of the compound; chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); the route and regimen of administration; and the like. It will also be understood that the specific dose level to be achieved for any particular subject may depend upon a variety of factors including the age, health, medical history, body weight, combination with one or more other drugs, and the severity of the disease

The term "treatment" refers to the reduction or amelioration of one or more symptoms of infertility in a subject suffering from the disease. For a given subject, the improvement in symptoms, their worsening, regression or progression can be determined by objective or subjective indices. Treatment may also include combination with other existing treatment modalities and agents (e.g., superovulation). Thus, combination therapy may be administered.

Examples

Heterozygous female and male mice genetically modified for the PTX3 gene were normal and fertile. In vivo breeding in populations yielded a predicted number of homozygous nude mice at mendelian frequency. But reproduction between homozygous females and males (PTX3-/-) is completely infertile. Reproductive results indicate that homozygous males are normally fertile when intercourse with either wild-type (PTX3+/+) or heterozygous (PTX3+/-) females, whereas PTX 3-/-females are always non-fertile, independent of male genotype. Mating trials showed no difference in the frequency of spontaneous post-coital plugs (copulation plugs) during the 4 day period or after superovulation between PTX 3-/-and PTX3+/+ females (table 1). The number of spontaneously ovulating eggs (table 1) (average 7/mouse, n ═ 4 in PTX3 +/-and 7.8/mouse, n ═ 8 in PTX 3-/-mice) or the number of hormone-induced ovulating eggs (average 35/mouse, n ═ 9 in PTX3 +/-and 27/mouse, n ═ 18 in PTX 3-/-mice) was comparable in +/+ and-/-mice. Data are from a representative one of 4 trials performed. Morphology of oocytes and zona pellucida was normal, and first polar bodies were observed in about 50% of oocytes obtained from PTX3+/+ and PTX 3-/-mice 16 hours after human chorionic gonadotropin (hCG) treatment (table 1). These data indicate that ovulation and oocyte maturation are normal and not responsible for infertility. In contrast, morphological abnormalities of cumulus collected from the oviduct of PTX 3-/-mice were continuously observed (fig. 1B and 1D) because granulocytes loosely bind to oocytes and do not form corona radiata. The cumulus derived from PTX 3-/-is unstable in vitro and after collection (14-16 hours after hCG, or 0.5 days after natural mating) granulosa cells spontaneously dissociate from oocytes within a short time (15-60 minutes in PTX 3-/-versus hours in PTX3+/+ cumulus) rapidly leading to denudation of oocytes.

Table 1 Normal mating frequency and ovulation in PTX 3-/-mice

	PTX3+/+	PTX3-/-	P value
				Mating frequency spontaneous (a) after superovulation the second day of the first day and the third day	4/92/52/34/4	2/102/82/58/8	NSNSNSNS
Ovulation spontaneous (b): following superovulation of ovulated mouse ova/mouse: ovulated mouse ovum/mouse	4/475/537.8	5/57.86/633.3	NS-#NS-
				The presence of polar bodies in the discharged ovum (c)	53/98(54％)	54/109(49％)	NS-

(a) Females were housed with males for 4 days and checked daily for the presence of sexual plugs.

(b) Ovulation was analyzed in sexually-embolic females.

(c) The presence of the first polar body was assessed in oocytes recovered 15 hours after HCG treatment.

NS, no significant difference from control PTX3+/+ mice by Fischer's exact test (p < 0.05). # Numbers refer to pooled samples from PTX3+/+ or PTX 3-/-mice. Similar lack differences were observed in 4 trials on 5-7 mice.

To understand if and when pregnancy is interrupted, zygotes and embryos are collected at various time points after spontaneous or hormone-induced post-ovulatory mating. No development of oocytes to the 2-cell stage (1.5 days) in vivo (Table 2) or oocytes with two pronuclei was observed, even though viable sperm were found in the oviduct of defective mice. To further identify the cause of infertility, PTX3+/+ blastocysts were transferred to PTX 3-/-pseudopregnant females, but normal pregnancy and labour were observed. This eliminates defects in the implantation and subsequent procedures.

TABLE 2 fertilization in PTX 3-/-mice

Fertilization of	PTX3+/+	PTX3-/-	P value
				Fertilized in vivo eggs spontaneously ovulate relative to total (a): after superovulation: in vitro after removal of zona pellucida (b) use of intact cumulus (c)	17/28(60％)81/162(50％)21/27(77％)79/189(41.8％)	0/39(0％)0/192(0％)21/31(68％)68/169(40％)	＜0.0001#＜0.0001NS+NS

(a) Embryos were collected at 1.5 days post-coital, at the 2-cell stage.

(b) Fusion was assessed 4 hours after insemination by dye transfer techniques.

(c) 2 cell embryos were counted on the day after insemination.

# Fischer's exact test

NS, no significant difference from control PTX3+/+ mice (p < 0.05)

To assess whether PTX 3-/-oocytes could be fertilized, In Vitro Fertilization (IVF) was performed using wild-type sperm from adult males to fertilize PTX3+/+ or PTX 3-/-oocytes (table 2). IVF was first performed using oocytes with zona pellucida removed and stained with the DNA-specific fluorescent dye Hoechst 33258 to visualize fusion. Under these conditions, normal sperm were observed to bind to the plasma membrane of PTX 3-/-oocytes and comparable fusion capacity of PTX3+/+ (77%) and PTX3-/- (68%) oocytes with sperm (Table 2). These results suggest that sperm-egg binding and fusion can occur in the absence of PTX 3. Intact cumulus collected 13-15 hours after hCG treatment was fertilized and fertilization and progression to the 2-cell stage of PTX 3-/-oocytes was observed with a frequency comparable to PTX3+/+ oocytes (table 2). These data confirm that oocyte quality is normal in PTX3 deficient mice. Since cumulus plays a key role for insemination in vivo, but not in vitro, these results suggest that abnormalities of cumulus underlie PTX 3-/-female sterility.

Expression of PTX3mRNA in ovarian tissue was studied by northern blot and in situ hybridization. Following hormone-induced superovulation, PTX3mRNA expression (assessed by northern blot in whole tissues) started 2 hours after hCG treatment and continued up to 12-14 hours (see figure 2A), corresponding to pre-ovulatory distension up to hours post-ovulation [20 ]. Granulosa cells obtained by hyaluronidase treatment of cumulus and isolation from oocytes express PTX3 transcripts.

Expression in the absence of superovulation under normal conditions was studied by in situ hybridization. In situ hybridization of organs from untreated females (fig. 2B) demonstrated expression of PTX3mRNA in the ovary, restricted to granulosa cells of mature follicles, with no evidence of transcription in oocytes.

The expression of PTX3 protein in ovarian tissue was then analyzed. Western blot indicates that PTX3 is associated with PTX3+/+ cumulus (in particular extracellular matrix) because hyaluronidase treatment that isolates cumulus cells from oocytes abrogates immunoreactivity (fig. 2C). Immunofluorescence analysis of PTX3+/+ and-/-cumulus collected after hormone-induced superovulation (13-15 hours after hCG) confirmed that PTX3 was associated with the cumulus intercellular matrix (fig. 2D).

These data suggest that sterility due to PTX3 deficiency is due to lack of oocyte fertilization, as PTX3 deficiency does not affect other steps of reproduction (from mating to ovulation, implantation and pregnancy). The PTX3 transcript is exclusively expressed in normal ovaries by granulosa cells of mature follicles as well as isolated granulosa cells, but not by oocytes. PTX3mRNA expression was induced throughout ovarian tissue following hormone-induced superovulation. Finally, PTX3 protein was identified in the extracellular matrix of isolated cumulus, presumably produced by granulosa cells. Analysis of PTX 3-/-mice identified abnormal cumulus as a determinant of infertility. Cumulus from PTX 3-/-females showed morphological abnormalities. They lack a well-defined corona radiata and, when cultured in vitro, dissociate rapidly from oocytes. The "fragility" of PTX 3-deficient cumulus may reflect changes in the structural role of PTX3 in this particular matrix or the regulatory mechanisms of matrix dissolution. These results identify PTX3 as a novel cumulus extracellular matrix component that plays a key role in fertility. Although not essential in vitro, the cumulus plays a key role for in vivo fertilization. Thus, abnormalities of cumulus may be related to sterility in PTX 3-/-female mice.

Materials and methods

Generation of PTX 3-/-mice

A8.5 kb genomic DNA fragment containing exons 1-2 of the mouse PTX3 gene was used to integrate the IRES-LacZ cassette, followed by the PGK-neomycin resistance gene from the pWH9 plasmid 71bp downstream of the first coding ATG in exon 1. The method of culturing, selecting and identifying ES cells was performed as described [20 ]. 5 independently targeted Rl ES cell clones were identified by southern blot hybridization using probe A (EcoRI/EcoRV 750bp fragment in the second intron). No evidence of random integration was detected with probe B (from the neomycin resistance gene). 2 ES cell clones were injected into C57B1/6 blastocysts. For mouse genotyping, DNA from tail biopsies was amplified by polymerase chain reaction using 2 primer pairs (primer pair 1: 5'-AGCAATGCACCTCCCTGCGAT-3', SEQ ID NO: 7; 5'-TCCTCGGTGGGATGAAGTCCA-3' SEQ ID NO: 8; primer pair 2: 5'-CTGCTCTTTACTGAAGGCTC-3', SEQ ID NO: 9; 5 ' -TCCTCGGTGGGATGAAGT CCA-3; SEQ ID NO: 10) that detected the wild-type or targeted allele, respectively. Phenotypic analysis of 2 cell lines derived from independent clones confirmed the results in a genetic background of 129Sv-C57BI/6 mixed and 129Sv inbred. The PTX3+/+ mice were 129Sv-C57B1/6PTX 3-/-littermates, or 129Sv or C57B1/6 mice from Charles river, Calco, Italy.

The procedures involved in animal and its Care comply with the academy guidelines, both in accordance with the national (4D.L.N.116, G.U., Suppl.40, 18-2-1992) and international laws and policies (EEC Council Directive86/609, OJ L358, 1, 12-12-1987; NIH Guide for the Care and Use of laboratory Animals, U.S. national Research Council, 1996). All efforts have been made to minimize the number of animals used and their suffering.

PTX3mRNA and protein

RNA was extracted from cells using TRIZOL reagent (GIBCO BRL) and purified. Northern blot, probe labeling and hybridization (binding and washing) conditions were performed according to the [21 ].

In situ hybridization: cryostat sections (13 μm) recovered from wild type and PTX 3-/-ovaries, fixed with 4% paraformaldehyde and frozen in liquid nitrogen were used for in situ hybridization according to the [22 ]. Briefly, slides permeabilized with proteinase K and 0.2N HCl were incubated overnight at 65 ℃ with radiolabeled riboprobe prepared from a vector containing PTX3 cDNA (pBluescript) using the Stratagene RNA transcription kit. Subsequently, the specimens were rinsed with formamide-containing buffer, air-dried, soaked in photographic emulsion and incubated in a dark box at 4 ℃ for at least 10 days. After development, the slides were counterstained with a solution of Hoechst 33258 dye at 2. mu.g/ml. For western blot analysis, whole cell extracts obtained from whole cumulus, cumulus cells or oocytes collected from superovulated females were separated by SDS-polyacrylamide gel electrophoresis (Page), electroblotted onto nitrocellulose filters (Hybond ECL, Amersham) and labeled with purified biotinylated anti-mouse PTX3 polyclonal hamster serum (1 μ g/ml) and then labeled with streptavidin-HRP (BIOSPA, Italy). The labeled proteins were detected by enhanced chemiluminescence (ECL, Amersham).

Oocyte and embryo collection, in vitro fertilization and embryo transfer

Cumulus, zygote and embryo are recovered from the oviduct or uterus of untreated females after natural mating [20 ]. Superovulation was induced by treatment with 5 units of pregnant mare serum (PMS, Folligon, Intervet) and 48 hours later with 5 units of human chorionic gonadotropin (hCG, Corulon, Intervet). Cumulus were collected at different times after mating or 13-15 hours after hCG treatment. Cumulus cells and oocytes were isolated by hyaluronidase treatment [20 ].

In Vitro Fertilization (IVF) of ova obtained from superovulated females was performed on the basis of the zona pellucida-free ova [20] stained with intact cumulus [20] or with 1. mu.g/ml hoechst dye (Sigma) [23] in M16 medium and sperm from BDF males as described. Fertilization and sperm-egg fusion were evaluated as follows: embryos at the 2-cell stage were counted by day after fertilization of the intact cumulus, and ova with fluorescent fertilized sperm were counted by 4 hours after fertilization of the ova without zona pellucida.

Embryo transfer was performed according to the [20] using 3.5 day PTX3+/+ blastocysts implanted into the uterus of a 2.5 day pseudopregnant PTX 3-/-female.

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8.Introna et al.，Cloning of mouse PTX3，a new member of thepentraxin gene family expressed at extrahepatic sites.Blood，1996.87：1862-1872.

9.Bottazzi et al.，Multimer formation and ligand recognitionby the long pentraxin PTX3-Similarities and differences with theshort pentraxins C-reactiVe protein and serum amyloid P component.J.Biol.Chem.，1997.272：32817-32823.

10.Muller et al.，Circulating levels of the long pentraxin PTX3correlate with severity of infection in critically ill patients.Crit.CareMed.2001.29：1404-1407.

11.Noland et al.，The sperm acrosomal matrix contains a novelmember of the pentaxin family of calcium-dependent binding proteins.J.Biol.Chem.，1994.269：32607-32614.

12.Reid & Blobel，Apexin，an acrosomal pentaxin.J.Biol.Chem.，1994.269：32615-32620.

13.Seery et al.，Identification of a novel member of thepentraxin family in Xenopus laevis.Proc.R.Soc.Lond.B.Biol.Sci.，1993.253：263-270.

14.Schlimgen et al.，Neuronal pentraxin，a secreted protein withhomology to acute phase proteins of the immune system.Neuron，1995.14：519-526.

15.Omeis et al.，Mouse and human neuronal pentraxin 1(NPTX1)：Cohservation，genomic structure，and chromosomallocalization.Genomics，1996.36：543-545.

16.Hsu & Perin，Human neuronal pentraxin II(NPTX2)：Conservation，genomic structure，and chromosomal localization.Genomics，1995.28：220-227.

17.Tsui et al.，Narp，a novel member of the pentraxin family，promotes neurite outgrowth and is dinamically regulated by neuronalactivity.J.Neurosci.，1996.15：2463-2478.

18.Dodds et al.，Neuronal pentraxin receptor，a novel putativeintegral membrane pentraxin that interacts with neuronal pentraxin 1and 2 and taipoxin-associated calcium-binding protein 49.J.Biol.Chem.，1997.272：21488-21494.

19.Kirkpatrick et al.，Biochemical interactions of the neuronalpentraxins.Neuronal pentraxin(NP)receptor binds to taipoxin andtaipoxin-associated calcium-binding protein 49 via NP1 and NP2.J.Biol.Chem..2000.275：17786-17792.

20.Hogan et al.，Manipulating the Mouse Embryo.A laboratorymanual.2nd Ed.，1994：Cold Spring Harbor Laboratory Press.

21.Introna et al.，Treatment of murine peritoneal macrophageswith bacterial lipopolysaccharide alters expression of c-fos and c-myconcogenes.J.Immunol.，1986.137：2711-2715.

22.Biffo & Tolosano，The use of radioactively labelledriboprobes for in situ hybridization：Background and examples ofapplication.Liver，1992.12：230-237.

23.Conover & Gwatkin，Pre-loading of mouse oocytes withDNA-specific fluorochrome(Hoechst 33342)permits rapid detectionof sperm-oocyte fusion.J.Reprod.Fertil.，1988.82：681-690.

All changes and substitutions that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Claims using the conversion "comprising" may include other elements within the scope of the claims; the invention is also described by such claims using the phrases "consisting essentially of" (i.e., can include other elements within the scope of the claims if they do not materially affect the operation of the invention) and "consisting of" (i.e., can only allow for the elements listed in the claims except for impurities or unreasonable activities ordinarily associated with the invention) in place of the terms "comprising". Any of these three conversions may be used to claim the invention.

It should be understood that the elements described in this specification should not be construed as limitations on the claimed invention unless explicitly recited in the claims. Accordingly, the claims are the basis for determining the scope of legal protection granted instead of a limitation from the specification.

In contrast, the prior art is expressly excluded from the invention to the extent that the claimed invention or specific embodiments that undermine novelty would be expected. In certain embodiments, the generic concept of a polynucleotide or polypeptide (genus) may be recited in the claims, provided that a native nucleic acid or protein (e.g., having a nucleotide or amino acid sequence not given in the sequence listing) is excluded. For example, the degeneracy of the genetic code can be used to provide a polypeptide having the sequence encoding SEQ ID NO: 2 but not the nucleotide sequence of SEQ ID NO: 1. Similarly, by altering SEQ ID NO: 2, may provide a PTX3 polypeptide that is functionally equivalent but not identical (e.g., at least 90% identical) to a mouse and/or human protein.

It will be apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are to be considered in all respects only as illustrative and not restrictive, since the scope of legal protection afforded the invention will be indicated by the appended claims rather than by the description herein.

Sequence listing

<110>Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.

<120> Long Pentaxin PTX3 and female sterility

<130>012-ST-01-US

<140>

<141>

<150>US 60/309,472

<151>2001-08-03

<160>11

<170>PatentIn version 3.1

<210>1

<211>1837

<212>DNA

<213> Intelligent (Homo sapiens)

<300>

<301>Breviario et al.

<302> Interleukin-1 inducible Gene in endothelial cells

<303>Journal of Biological Chemistry

<304>267

<305>31

<306>22190-22197

<307>1992-11-05

<308>X636613

<309>1993-07-29

<400>1

ctcaaactca gctcacttga gagtctcctc ccgccagctg tggaaagaac tttgcgtctc 60

tccagcaatg catctccttg cgattctgtt ttgtgctctc tggtctgcag tgttggccga 120

gaactcggat gattatgatc tcatgtatgt gaatttggac aacgaaatag acaatggact 180

ccatcccact gaggacccca cgccgtgcga ctgcggtcag gagcactcgg aatgggacaa 240

gctcttcatc atgctggaga actcgcagat gagagagcgc atgctgctgc aagccacgga 300

cgacgtcctg cggggcgagc tgcagaggct gcgggaggag ctgggccggc tcgcggaaag 360

cctggcgagg ccgtgcgcgc cgggggctcc cgcagaggcc aggctgacca gtgctctgga 420

cgagctgctg caggcgaccc gcgacgcggg ccgcaggctg gcgcgtatgg agggcgcgga 480

ggcgcagcgc ccagaggagg cggggcgcgc cctggccgcg gtgctagagg agctgcggca 540

gacgcgagcc gacctgcacg cggtgcaggg ctgggctgcc cggagctggc tgccggcagg 600

ttgtgaaaca gctattttat tcccaatgcg ttccaagaag atttttggaa gcgtgcatcc 660

agtgagacca atgaggcttg agtcttttag tgcctgcatt tgggtcaaag ccacagatgt 720

attaaacaaa accatcctgt tttcctatgg cacaaagagg aatccatatg aaatccagct 780

gtatctcagc taccaatcca tagtgtttgt ggtgggtgga gaggagaaca aactggttgc 840

tgaagccatg gtttccctgg gaaggtggac ccacctgtgc ggcacctgga attcagagga 900

agggctcaca tccttgtggg taaatggtga actggcggct accactgttg agatggccac 960

aggtcacatt gttcctgagg gaggaatcct gcagattggc caagaaaaga atggctgctg 1020

tgtgggtggt ggctttgatg aaacattagc cttctctggg agactcacag gcttcaatat 1080

ctgggatagt gttcttagca atgaagagat aagagagacc ggaggagcag agtcttgtca 1140

catccggggg aatattgttg ggtggggagt cacagagatc cagccacatg gaggagctca 1200

gtatgtttca taaatgttgt gaaactccac ttgaagccaa agaaagaaac tcacacttaa 1260

aacacatgcc agttgggaag gtctgaaaac tcagtgcata ataggaacac ttgagactaa 1320

tgaaagagag agttgagacc aatctttatt tgtactggcc aaatactgaa taaacagttg 1380

aaggaaagac attggaaaaa gcttttgagg ataatgttac tagactttat gccatggtgg 1440

tgttgaacag agggacaatt gttttacttt tctttggtta attttgtttt ggccagagat 1560

gaattttaca ttggaagaat aacaaaataa gatttgttgt ccattgttca ttgttattgg 1620

tatgtacctt attacaaaaa aaatgatgaa aacatattta tactacaagg tgacttaaca 1680

actataaatg tagtttatgt gttataatcg aatgtcacgt ttttgagaag atagtcatat 1740

aagttatatt gcaaaaggga tttgtattaa tttaagacta tttttgtaaa gctctactgt 1800

aaataaaata ttttataaaa ctaaaaaaaa aaaaaaa 1837

<210>2

<211>381

<212>PRT

<213> Intelligent people

<220>

<221>SIGNAL PEPTIDE

<222>(1)..(17)

<223>

<220>

<221>MAT_PEPTIDE

<222>(18)..(381)

<300>

<301>Breviario et al.

<302> Interleukin-1 inducible Gene in endothelial cells

<303>Journal of Biological Chemistry

<304>267

<305>31

<306>22190-22197

<307>1992-11-05

<308>CAA45158

<309>1993-07-29

<400>2

Met His Leu Leu Ala Ile Leu Phe Cys Ala Leu Trp Ser Ala Val Leu

-15 -10 -5

Ala Glu Asn Ser Asp Asp Tyr Asp Leu Met Tyr Val Asn Leu Asp Asn

-1 1 5 10 15

Glu Ile Asp Asn Gly Leu His Pro Thr Glu Asp Pro Thr Pro Cys Asp

20 25 30

Cys Gly Gln Glu His Ser Glu Trp Asp Lys Leu Phe Ile Met Leu Glu

35 40 45

Asn Ser Gln Met Arg Glu Arg Met Leu Leu Gln Ala Thr Asp Asp Val

50 55 60

Leu Arg Gly Glu Leu Gln Arg Leu Arg Glu Glu Leu Gly Arg Leu Ala

65 70 75

Glu Ser Leu Ala Arg Pro Cys Ala Pro Gly Ala Pro Ala Glu Ala Arg

80 85 90 95

Leu Thr Ser Ala Leu Asp Glu Leu Leu Gln Ala Thr Arg Asp Ala Gly

100 105 110

Arg Arg Leu Ala Arg Met Glu Gly Ala Glu Ala Gln Arg Pro Glu Glu

115 120 125

Ala Gly Arg Ala Leu Ala Ala Val Leu Glu Glu Leu Arg Gln Thr Arg

130 135 140

Ala Asp Leu His Ala Val Gln Gly Trp Ala Ala Arg Ser Trp Leu Pro

145 150 155

Ala Gly Cys Glu Thr Ala Ile Leu Phe Pro Met Arg Ser Lys Lys Ile

160 165 170 175

Phe Gly Ser Val His Pro Val Arg Pro Met Arg Leu Glu Ser Phe Ser

180 185 190

Ala Cys Ile Trp Val Lys Ala Thr Asp Val Leu Asn Lys Thr Ile Leu

195 200 205

Phe Ser Tyr Gly Thr Lys Arg Asn Pro Tyr Glu Ile Gln Leu Tyr Leu

210 215 220

Ser Tyr Gln Ser Ile Val Phe Val Val Gly Gly Glu Glu Asn Lys Leu

225 230 235

Val Ala Glu Ala Met Val Ser Leu Gly Arg Trp Thr His Leu Cys Gly

240 245 250 255

Thr Trp Asn Ser Glu Glu Gly Leu Thr Ser Leu Trp Val Asn Gly Glu

260 265 270

Leu Ala Ala Thr Thr Val Glu Met Ala Thr Gly His Ile Val Pro Glu

275 280 285

Gly Gly Ile Leu Gln Ile Gly Gln Glu Lys Asn Gly Cys Cys Val Gly

290 295 300

Gly Gly Phe Asp Glu Thr Leu Ala Phe Ser Gly Arg Leu Thr Gly Phe

305 310 315

Asn Ile Trp Asp Ser Val Leu Ser Asn Glu Glu Ile Arg Glu Thr Gly

320 325 330 335

Gly Ala Glu Ser Cys His Ile Arg Gly Asn Ile Val Gly Trp Gly Val

340 345 350

Thr Glu Ile Gln Pro His Gly Gly Ala Gln Tyr Val Ser

355 360

<210>3

<211>1841

<212>DNA

<213> mouse (Mus musculus)

<300>

<301>Introna et al.

<302> cloning of mouse PTX3

<303> blood

<304>87

<305>5

<306>1862-1872

<307>1996-03-01

<308>X83601

<309>1996-01-10

<400>3

actcctgcct cacactatct ctcccgggct caaactcgga tcactgtaga gtctcgcttc 60

ttcccctgcg gctgcgaacg aaatttcgcc tctccagcaa tgcacctccc tgcgatcctg 120

ctttgtgctc tctggtctgc agtagtggct gagacctcgg atgactacga gctcatgtat 180

gtgaatttgg acaacgaaat agacaatgga cttcatccca ccgaggaccc cacgccatgc 240

gactgccgcc aggagcactc ggagtgggac aagctgttca tcatgctgga gaactcgcag 300

atgcgggagg gcatgctgtt gcaggccacc gacgacgtcc tccgtggaga gctgcagcgg 360

ctgcgggcag agctggggcg gctggcgggc ggcatggcga ggccgtgcgc agccggtggc 420

cccgcagacg ccaggctggt gcgggcgctg gagccgctgc tgcaggagag ccgtgacgcg 480

agcctcaggc tggcgcgcct ggaggacgcg gaggcgcggc gacccgaggc gacagtgcct 540

ggcctaggcg ctgtgctgga ggaactgcgg cggacgcgcg ccgacctgag cgccgtgcag 600

agctgggtcg cccgccactg gctgcccgca ggttgtgaaa cagcaatttt cttcccaatg 660

cgttcgaaga agatttttgg aagcgtgcat cctgtgagac caatgaagct tgaatctttt 720

agtacttgca tttgggtcaa agccacagat gtattaaaca aaaccatcct gttttcttat 780

ggcacaaagt ggaaccccta tgagattcag ctgtacctca gttcccagtc cctagtgttg 840

gtggtgggtg gaaaggagaa caagctggct gcagacactg tggtgtccct ggggaggtgg 900

tcccacctgt gtggcacctg gagttcagag caggggagca tgtccctgtg ggcaaacggg 960

gagctggtgg ctaccactgt agagatggcc aaaagtcact ctgttcctga gggtggactc 1020

ctacagattg gccaagaaaa gaatggttgc tgtgtaggtg ggggctttga cgaatcatta 1080

gcattttctg gaagaatcac aggcttcaat atctgggatc gggttctcag cgaggaggag 1140

atacgggcca gtggaggagt cgaatcctgt cacatccggg gaaatgtcgt cgggtgggga 1200

gtcacagaga ttcaggcgca cggaggagcc cagtatgttt cttaagtgtt gtgaaaatct 1260

acttgaagcc aaaggagact cacattttaa atatgccagt tggaaaagtc tgaaaacttc 1320

ggtgcgtaat agacgaatga aggagagact tgagattgtc tttgtttatc ttggcaaaat 1380

actgaataca cagttgaagg gaaggcttga gagagggctc cgggatgttg ttactaagcc 1440

ttatactgtg gtgctttcag attaatgtct gcctctgtca gataaaccct cagataacta 1500

aacatgactg gactctgaac agagggacga ttgtgtgact tttttttttt tttattttgg 1560

ttaattttat tttggccaga gacattttta tattggaaga ataacaaaac aagctctgtt 1620

gcccattgtt cattctttct ggtgtgtatt ttgtgacaaa agagatgatg agaaaaccat 1680

aattatacca caaagtgact tattaacgaa cataaatgta gcttacgtgt tataatccaa 1740

tccatttggg agaaggtagt tgtgtaattt atattgtgaa atgtaattgt attaatttta 1800

tttttgtaaa agtctactgt aaataaattg ttttataaag c 1841

<210>4

<211>381

<212>PRT

<213> mice

<220>

<221>SIGNAL PEPTIDE

<222>(1)..(17)

<223>

<220>

<221>MAT_PEPTIDE

<222>(18)..(381)

<223>

<300>

<301>Introna et al.

<302> cloning of mouse PTX3

<303> blood

<304>87

<305>5

<306>1862-1872

<307>1996-03-01

<308>CAA58580

<309>1996-01-10

<400>4

Met His Leu Pro Ala Ile Leu Leu Cys Ala Leu Trp Ser Ala Val Val

-15 -10 -5

Ala Glu Thr Ser Asp Asp Tyr Glu Leu Met Tyr Val Asn Leu Asp Asn

-1 1 5 10 15

Glu Ile Asp Asn Gly Leu His Pro Thr Glu Asp Pro Thr Pro Cys Asp

20 25 30

Cys Arg Gln Glu His Ser Glu Trp Asp Lys Leu Phe Ile Met Leu Glu

35 40 45

Asn Ser Gln Met Arg Glu Gly Met Leu Leu Gln Ala Thr Asp Asp Val

50 55 60

Leu Arg Gly Glu Leu Gln Arg Leu Arg Ala Glu Leu Gly Arg Leu Ala

65 70 75

Gly Gly Met Ala Arg Pro Cys Ala Ala Gly Gly Pro Ala Asp Ala Arg

80 85 90 95

Leu Val Arg Ala Leu Glu Pro Leu Leu Gln Glu Ser Arg Asp Ala Ser

100 105 110

Leu Arg Leu Ala Arg Leu Glu Asp Ala Glu Ala Arg Arg Pro Glu Ala

115 120 125

Thr Val Pro Gly Leu Gly Ala Val Leu Glu Glu Leu Arg Arg Thr Arg

130 135 140

Ala Asp Leu Ser Ala Val Gln Ser Trp Val Ala Arg His Trp Leu Pro

145 150 155

Ala Gly Cys Glu Thr Ala Ile Phe Phe Pro Met Arg Ser Lys Lys Ile

160 165 170 175

Phe Gly Ser Val His Pro Val Arg Pro Met Lys Leu Glu Ser Phe Ser

180 185 190

Thr Cys Ile Trp Val Lys Ala Thr Asp Val Leu Asn Lys Thr Ile Leu

195 200 205

Phe Ser Tyr Gly Thr Lys Trp Asn Pro Tyr Glu Ile Gln Leu Tyr Leu

210 215 220

Ser Ser Gln Ser Leu Val Leu Val Val Gly Gly Lys Glu Asn Lys Leu

225 230 235

Ala Ala Asp Thr Val Val Ser Leu Gly Arg Trp Ser His Leu Cys Gly

240 245 250 255

Thr Trp Ser Ser Glu Gln Gly Ser Met Ser Leu Trp Ala Asn Gly Glu

260 265 270

Leu Val Ala Thr Thr Val Glu Met Ala Lys Ser His Ser Val Pro Glu

275 280 285

Gly Gly Leu Leu Gln Ile Gly Gln Glu Lys Asn Gly Cys Cys Val Gly

290 295 300

Gly Gly Phe Asp Glu Ser Leu Ala Phe Ser Gly Arg Ile Thr Gly Phe

305 310 315

Asn Ile Trp Asp Arg Val Leu Ser Glu Glu Glu Ile Arg Ala Ser Gly

320 325 330 335

GIy Val Glu Ser Cys His Ile Arg Gly Asn Val Val Gly Trp Gly Val

340 345 350

Thr Glu Ile Gln Ala His Gly Gly Ala Gln Tyr Val Ser

355 360

<210>5

<211>1531

<212>DNA

<213> Intelligent people

<220>

<221>PROMOTER

<222>(1)..(1317)

<223>

<220>

<221>PROTEIN_BIND

<222>(1222)..(1231)

<223>NF-kB

<300>

<301>Basile et al.

<302> characterization of the promoter of human long Pentaxin PTX3

<303>Journal of Biological Chemistry

<304>272

<305>13

<306>8172-8178

<307>1997-03-28

<308>X97748

<309>1997-11-15

<400>5

gaattccccg gatctccctt ctaactctcc acctttggcc taagctttgc ttccacatgg 60

tcatcaacat ttggtggtta tagaactaat aacccctatc tcacttcact cctatgccag 120

aggggcccta gcatcagctc atgggattgt tgtttttgct ttcctctcta tctttggctc 180

cgggattttc cccttacttt aatgggagct catctgtacc ttttaagttt ttattaatat 240

catgtgaaca cagacctgta tatattgtta gaagcagaaa tctctaagtt tacttttaaa 300

acatgatcct tgcctcgaaa ccttgtagaa taatataatg tccacataat accaagttat 360

gaaaagaaac atacctaaat aactaaataa gtatattcct tttttccccc agcttttttt 420

ccccattcta ggttacccag ttgtactgtg ttgtttgtca taggccgggt gaggtggctc 480

acgtctgtaa tcctagcaat ttgggaggcg aaggcgggtg gatcgcctga ggtcaggagt 540

tcgagaccag cctggctaac atggtgaaac cctgtctcta ctaaaaatac aaaaattaac 600

tgggtgtggt ggcgggtgcc tgtaattcca gctacttggg aagctgaggt aggagaatcg 660

cttgaaccca ggatgcggag gttgcagtga gccgagatca caccattgca ctccagcctg 720

ggcaacaaga gcgaaattca gtctcaaaaa aaaaaattat ctataaaagt ataggtgcaa 780

ctcctcaagt attaaagaca agatagctcg gattggactt gactttcaga gccataacta 840

ttcttaatat gttggtttat cttggaatca gaccattttc agtttcaacc tgtaaaacag 900

tgtacaaagg aaacatggaa agttttctat atataaaggg ttgtgaaata ataacagctc 960

acagaaaatg ctgaaatgat gatttgcttc agtaccctct gaaatttctc ccctaccacc 1020

cctccttcat ccccattgct atcaattcaa attacaacag ctaattctca ggagaacagt 1080

agaagcccag tttctctcct ctttcccctc tgaccctcct ccaattaatc tgactgcagc 1140

gtaaaccttt gcggtttaat attgtgcaac ttccacattt ccctcgctct cccacccagc 1200

cccctccccc accaaattca ggggaactcc cgttaccgca gtgccaccag cattactcat 1260

tcatccccat tcaggctttc ctcagcattt attaaggact ctctgctcca gcctctcact 1320

ctcactctcc tccgctcaaa ctcagctcac ttgagagtct cctcccgcca gctgtggaaa 1380

gaactttgcg tctctccagc aatgcatctc cttgcgattc tgttttgtgc tctctggtct 1440

gcagtgttgg ccgagaactc ggatcattat catctcatgt atgtgaattt ggacaacgaa 1500

atagacaatg gactccatcc cactgaggac c 1531

<210>6

<211>2708

<212>DNA

<213> mice

<220>

<221>PROMOTER

<222>(1)..(1373)

<223>

<300>

<301>Altmeyer et al.

<302> promoter structure and transcriptional activation.

<303>Journal of Biological Chemistry

<304>270

<305>43

<306>25584-15590

<307>1995-10-27

<308>U33842

<309>1995-10-27

<400>6

atcccagagg ctctctgtac tggcattagg acctcacagc accacatcag gtttcttaat 60

gtggacteta gaaactgaac tcgagcccac agccttagga gaaaagcacc ttacaaagct 120

gtggctccac actgcccttt aaacaatatc gtattgtctc atattgccat cgctttctga 180

tggctttaac ggtttcaaac ataccctgtc tttagccgtg atctcaaata agtgaagctc 240

ttgagcaggg gcctgatgcc ttttgacttt gtgttgattc atgcttatga tgccctgttc 300

cctccgtgtc tagctatgtt taactgtgga ttcaattttt attggtgggt ggattggtac 360

atgcatgtgc attccagatg cgtgagggca ctcaggccag gaaagccact catgagtctc 420

tgtcaggagc agaggaattt acctatggaa atccaagagc agccttctga gaggcctggc 480

ctgagggtag tacccctccc atcatgatca ggatgtgact ggtaaccctc cccctccatc 540

tcctttgtat attggagact tgtatcagct caggggtatc ctctgggagt ggttccctct 600

agatctgtgt agttttttag atcttgcttt atttggagtt tattctcatg ttttaatttt 660

ttatcactat tattatgact tatcaacacc tatctaggta cttttcactg ggggaggggg 720

caggttttac acacacacac acacacacac acacacacac acacacacac acagtcacta 780

atgtaaaatt taaaacaggg accttgatag gatatgtcca agaataccca agcaccctaa 840

agccactata ttcccgccct cactttcctg ttttactggg ttttgaccca gccatactgt 900

gttttttagt tgctccacca gaggagtcaa gactagttag tcaagattga cttctagagt 960

cataaaaatt cttaatgggt tattttggag tcacggaatc attttctata gcttggtctt 1020

gagaaagtat ccaaaggaaa agtgaaaaaa aaaagttttc cataacttca ggggttgtgg 1080

agtaatgaaa gctcacacca aatgccaaaa tgataattcg ccctgtacct ctgtgctcct 1140

caccccccaa agcgctagca cttcaggtta cagcaactaa tcctcagggg caccagaaaa 1200

gtccagcttc cctccccttc tccccctgac tcgcctctaa ttaatctgcc tgcagtgtgg 1260

acctcggtgg tttaacattg tgcaacctct tcagctccct tgccctccca cccaaccccc 1320

tcccccaaat ccaggggaac tccctcgcgc tgtgccaccg acattagtca ttcatccgct 1380

catgctttgg agcgtttatt aagggcttca ctcctgcctc acactatctc tcccgggctc 1440

aaactcggat cactgtagag tctcgcttct tcccctgcgg gtgcgaagca aatttcggct 1500

ctccagcaat gcacctccct gcgatcctgc tttgtgctct ctggtctgca gtagtggctg 1560

agacctcgga tgactacgag ctcatgtatg tgaatttgga caacgaaata gacaatggac 1620

ttcatcccac cgaggaccgt aagttcattt ttaactctct cagcgtatca aaactacata 1680

actcacttct gggggggcgc gattaacata attaacatag atagccaatg aagcaagcta 1740

aaattatact ttatttgtga aagcaaggac tgggggaaaa aaggaaagca aggaaatatc 1800

tgagaaaagc cagaggtttt aaattatttt tgtaacattt atgatgagtt aagttatacg 1860

aaatctttaa ctgtttttag ctatattaat ggcattttct cagttagttt aacatgtcta 1920

taaagaatag tctgtgtcat ctttgagttt acacgcacgc tgttttcaga gctatcctta 1980

gaaggagagc gttgctgggg acaggctgaa acttggagtc accaagagtg caacccatgg 2040

ccacccagga caagctgata acacttgtgt gtgtcctgcg ttctagccac gccatgcgac 2100

tgcgcccagg agcactcgga gtgggacaag ctgttcatca tgctggagaa ctcgcagatg 2160

cgggagggca tgctgttgca ggccaccgac gacgtcctcc gtggagagct gcagcggctg 2220

cggtcagagc tgggccggct ggcgggcggc atggcgaggc cgtgcgcagc cggtggcccc 2280

gcagacgcca ggctggtgcg ggcgctggag ccgctgctgc aggagagccg tgacgcgagc 2340

ctcaggctgg cgcgcctgga ggacgcggag gcgcggcgac ccgaggcgac agtgcctggc 2400

ctaggcgctg tgctggagga actgcggcgg acgcgctccg acctgagcgc cgtgcagagc 2460

tgggtcgccc accactggct gcccgcaggt aagcccacgg tcggctctgt ccctagaggc 2520

aagcttttgt gggaccctca cactcagagc cccagtactt ttcataggca cactcacaga 2580

gctcacacca cgccaggcag ctcattgcct tttaaaagta tttccaagcc cgaggaaccc 2640

aaaagaaaaa aacgaggatt taaaccatca gtctggaagt tgacgtcaga ggttcctgat 2700

accggatc 2708

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>7

agcaatgcac ctccctgcga t 21

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>8

tcctcggtgg gatgaagtcc a 21

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>9

ctgctcttta ctgaaggctc 20

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>10

tcctcggtgg gatgaagtcc a 21

<210>11

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> consensus "pentraxin-like" sequences

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> any amino acid

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> any amino acid

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Ser or Thr

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> any amino acid

<400>11

His Xaa Cys Xaa Xaa Trp Xaa Ser

1 5

Claims (8)

1. Use of recombinant human PTX3 for the manufacture of a medicament for increasing reproductive capacity in a female in need of such increase.

2. Use of a viral or plasmid vector comprising human PTX3 cDNA for the preparation of a medicament for the treatment of a female subject in need of increased reproductive capacity.

3. The use of claim 1 or 2, wherein the medicament is administered systemically.

4. The use of claim 1 or 2, wherein the medicament is administered topically.

Use of PTX3 as a marker in an in vitro assay for determining the reproductive capacity of a human female, such assay comprising determining the amount of PTX3 present in a biological fluid or tissue sample of a human female.

Use of PTX3 as a target for screening pharmaceutical compounds that affect the reproductive ability of a female.

7. Use of a substance that alters the activity of long pentraxin PTX3 in the manufacture of a medicament for increasing or decreasing reproductive capacity in a female subject in need of such treatment.

8. Use of a substance increasing or decreasing the activity of long pentraxin PTX3 for the manufacture of a medicament for the treatment of infertility or as a contraceptive, respectively.