WO2000070033A1 - Htra2 murin - Google Patents

Htra2 murin Download PDF

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Publication number
WO2000070033A1
WO2000070033A1 PCT/US2000/014037 US0014037W WO0070033A1 WO 2000070033 A1 WO2000070033 A1 WO 2000070033A1 US 0014037 W US0014037 W US 0014037W WO 0070033 A1 WO0070033 A1 WO 0070033A1
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Prior art keywords
polypeptide
seq
sequence
polynucleotide
htra2
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Caretha Lee Creasy
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides. to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • the drug discovery process is currently undergoing a fundamental revolution as it embraces 'functional genomics', that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on 'positional cloning'. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
  • E colt htrA gene product (high temperature requirement) encodes a se ⁇ ne protease required for growth at elevated temperatures [Seol et al , Biochem. Biophys. Res. Com. 176, 730-736 (1991)] Null mutants in E. colt htrA show decreased ability to degrade abnormal pe ⁇ plasmic proteins and the protein is required for the degradation of mis-folded proteins formed at high temperatures [Strauch, et al , J. Bacte ⁇ ol. 171, 2689-2696(1989); Laskoska, et al, Mol. Micro. 22, 555-571 (1996), Kolmar et al., J Bacte ⁇ ol. 178, 5925-5929 (1996)].
  • the present invention relates to Mus musculus htra2, in particular Mus musculus htra2 polypeptides and Mus musculus htra2 polynucleotides, recombinant materials and methods for their production
  • the invention relates to methods for identifying agonists and antagonists/inhibitors of the Mus musculus htra2 gene
  • This invention further relates to the generation of in vitro and in vivo compa ⁇ son data relating to the polynucleotides and polypeptides in order to predict oral absorption and pharmacokmetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides.
  • Such a compa ⁇ son of data will enable the selection of drugs with optimal pharmacokmetics m man, i.e., good oral bioavailabi ty, blood-bram bar ⁇ er penetration, plasma half life, and minimum drug interaction.
  • the present invention further relates to methods for creating transgemc animals, which overexpress or underexpress or have regulatable expression of a htra2 gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a htra2 gene. Furthermore, this invention relates to transgemc and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods.
  • IBD Intra-deficiency disease
  • pso ⁇ asis pso ⁇ asis, dermatitis, asthma, allergies
  • infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2
  • HIV-assoc ⁇ ated cachexia and other immunodeficiency disorders HIV-assoc ⁇ ated cachexia and other immunodeficiency disorders
  • septic shock pain; injury; cancers; anorexia; bulimia
  • Parkinson's disease cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; uimary retention; angina pecto ⁇ s; ulcers; benign prostatic hypertrophy
  • psychotic and neurological disorders including anxiety, schizophrenia, manic depression, de ⁇ um, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, hereinafter referred to as "the
  • the present invention relates to Mus musculus htra2 polypeptides.
  • Such polypeptides include isolated polypeptides comprising an amino acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ LD NO:2 over the entire length of SEQ ID NO:2.
  • Such polypeptides include those comp ⁇ s g the ammo acid of SEQ ID NO:2.
  • an isolated polypeptide comprising a polypeptide sequence having at least a 95%, 97%, 98%, or 99%o identity to the polypeptide sequence of SEQ LD NO:2;
  • polypeptide sequence of SEQ ID NO:2 (e) the polypeptide sequence of SEQ ID NO:2; and (f) variants and fragments thereof; and portions of such polypeptides in (a) to (e) that generally contain at least 30 amino acids, more preferably at least 50 ammo acids, thereof.
  • Polypeptides of the present invention are believed to be members of the se ⁇ ne protease family of polypeptides. They are, therefore, of interest, because agonists and antagonists of se ⁇ ne proteases have proven to be useful therapeutics. Furthermore, the polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokmetics in man and thus enhance compound and formulation design, among others These properties, either alone or in the aggregate, are hereinafter referred to as "Mus musculus htra2 activity" or "Mus musculus htra2 polypeptide activity” or "biological activity of htra2.” Preferably, a polypeptide of the present invention exhibits at least one biological activity of Mus musculus htra2.
  • Polypeptides of the present invention also includes va ⁇ ants of the aforementioned polypeptides, including alleles and splice va ⁇ ants.
  • Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative.
  • Particularly preferred va ⁇ ants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination.
  • Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO:2, or an isolated polypeptide comp ⁇ sing an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the ammo acid sequence of SEQ ID NO:2.
  • biologically active fragments which are those fragments that mediate activities of htra2, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or lmmunogenic in an animal, especially in a human. Particularly preferred are fragments comp ⁇ sing receptors or domains of enzymes that confer a function essential for viability of Mus musculus or the ability to initiate, or maintain cause the Diseases in an individual, particularly a human. Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • polypeptides of the present invention may be in the form of a "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid m pu ⁇ fication, for instance, multiple histidme residues, or an additional sequence for stability during recombinant production
  • the present invention also includes va ⁇ ants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics.
  • Typical substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • Particularly preferred are va ⁇ ants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • Polypeptides of the present invention can be prepared in any suitable manner.
  • polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepa ⁇ ng such polypeptides are well understood in the art.
  • the present invention relates to Mus musculus htra2 polynucleotides.
  • Such polynucleotides include isolated polynucleotides comp ⁇ smg a nucleotide sequence encodmg a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO.2.
  • polypeptides which have at least a 97% identity are highly preferred, while those with at least a 98-99%> identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comp ⁇ sing the nucleotide sequence contained m SEQ ID NO: 1 encodmg the polypeptide of SEQ ID
  • polynucleotides of the present mvention include isolated polynucleotides comp ⁇ sing a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encodmg a polypeptide of SEQ ID NO:2, over the entire coding region.
  • polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
  • polynucleotides of the present mvention include isolated polynucleotides comp ⁇ sing a nucleotide sequence having at least a 95%> identity, to SEQ ID NO 1 over the entire length of SEQ ID NO: 1.
  • polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99%) identify are more highly preferred, and those with at least a 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comp ⁇ smg the polynucleotide of SEQ ID NO: 1 , as well as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the nucleotide sequence of SEQ ID NOJ shows homology with PSP-1 (GPS accession number,V2953; patent number EP-828003-A2).
  • the nucleotide sequence of SEQ ED NO: 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotides 1 to 1374) encodmg a polypeptide of 458 ammo acids, the polypeptide of SEQ ID NO:2.
  • the nucleotide sequence encoding the polypeptide of SEQ LD NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ LD NO.1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO.2.
  • the polypeptide of SEQ LD NO:2 is structurally related to other proteins of the se ⁇ ne protease family, having homology and/or structural simila ⁇ ty with PSP-1 (GPS accession number, W56769; patent number EP-828003-
  • Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one htra2 activity.
  • Polynucleotides of the present mvention may be obtamed, using standard cloning and screening techniques, from a cDNA library de ⁇ ved from rnRNA in cells of Mus musculus adult bram, using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252: 1651-1656; Adams, M.D. et al , Nature (1992) 355.632-634, Adams, M.D., et al , Nature (1995) 377 Supp.: 3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized using well known and commercially available techniques.
  • EST expressed sequence tag
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions.
  • a marker sequence that facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz, et al , Proc Natl Acad Sci USA ( 1989) 86: 821 -824, or is an HA tag.
  • the polynucleotide may also comp ⁇ se non-coding 5' and 3' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encodmg polypeptide va ⁇ ants that comp ⁇ se the amino acid sequence of SEQ LD NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 1 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ LD NO: 1.
  • a preferred embodiment of the invention is a polynucleotide of consistmg of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1377 set forth m SEQ ID NOJ, which encodes a htra2 polypeptide.
  • the mvention also includes a polynucleotide consisting of or comp ⁇ smg a polynucleotide of the formula: X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R ⁇ and
  • R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from SEQ LD NO 1 or a modified nucleic acid sequence thereof.
  • R 2 is o ⁇ ented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the ⁇ ght, bound to R3.
  • any stretch of nucleic acid residues denoted by either Rj and/or R 2 , where m and/or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double- stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ LD NO: 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encodmg polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (mcludmg genes encoding homologs and orthologs from species other than Mus musculus) that have a high sequence identity to SEQ LD NO.1 Typically these nucleotide sequences are 95% identical to that of the referent.
  • PCR nucleic acid amplification
  • Preferred probes or p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 nucleotides. Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from a species other than Mus musculus may be obtained by a process comp ⁇ smg the steps of screening an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ LD NO 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comp ⁇ sing said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to the skilled artisan Preferred stringent hyb ⁇ dization conditions include overnight incubation at 42°C m a solution comp ⁇ sing: 50%> formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in OJx SSC at about 65°C.
  • 5xSSC 150mM NaCl, 15mM t ⁇ sodium citrate
  • 50 mM sodium phosphate pH7.6
  • 5x Denhardt's solution 10 % dextran sulfate
  • 20 microgram/ml denatured, sheared salmon sperm DNA followed by washing the filters in OJx SSC at about 65°C.
  • the present mvention also mcludes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screenmg an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ LD NOJ or a fragment thereof, preferably of at least 15 nucleotides.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template du ⁇ ng the polyme ⁇ zation reaction), failing to complete a DNA copy of the mRNA template du ⁇ ng 1st strand cDNA synthesis.
  • the PCR reaction is then repeated using 'nested' p ⁇ mers, that is, p ⁇ mers designed to anneal within the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific p ⁇ mer that anneals further 5' m the known gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existmg cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' p ⁇ mer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comp ⁇ sing expression systems. Accordingly, m a further aspect, the present invention relates to expression systems comp ⁇ sing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides to host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis, et al, BASIC METHODS LN MOLECULAR BIOLOGY (1986) and Sambrook, et al., MOLECULAR CLONLNG: A LABORATORY MANUAL, 2nd Ed., Cold Spring
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • Representative examples of approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E.
  • coli Streptomyces and Bacillus subtdis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells.
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenovrruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids.
  • the expression systems may comp ⁇ se control regions that regulate as well as engender expression.
  • any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used
  • the approp ⁇ ate nucleotide sequence may be mse ⁇ ed mto an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook. et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra)
  • a polypeptide of the present invention is to be expressed for use m screenmg assays, it is generally preferred that the polypeptide be produced at the surface of the cell.
  • the cells may be harvested p ⁇ or to use in the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and/or pu ⁇ fication.
  • Mus musculus htra2 gene products can be expressed in transgemc animals.
  • Animals of any species including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human p ⁇ mates, e g , baboons, monkeys, chimpanzees, may be used to generate htra2 transgemc animals.
  • This invention further relates to a method of producing transgemc animals, preferably Mus musculus, over-expressing htra2, which method may comp ⁇ se the introduction of several copies of a segment compns g at least the polynucleotide sequence encoding SEQ LD NO.2 with a suitable promoter into the cells of a Mus musculus embryo, or the cells of another species, at an early stage
  • This mvention further relates to a method of producing transgemc animals, preferably Mus musculus, under-expressing or regulatably expressing htra2, which method may comp ⁇ se the introduction of a weak promoter or a regulatable promoter (e g , an mducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ LD NOJ into the cells of a Mus musculus embryo at an early stage
  • a weak promoter or a regulatable promoter e g , an mducible or repressible promoter
  • This invention also relates to transgemc animals, characte ⁇ zed in that they are obtained by a method, as defined above
  • Any technique known in the art may be used to introduce a Mus musculus htra2 transgene mto animals to produce a founder line of animals
  • Such techniques include, but are not limited to: pronuclear microinjection (U.S. Patent No 4,873,191); retrovm ⁇ s mediated gene transfer into germ lines (Van der Putten, et al , Proc Natl Acad Set , USA 82: 6148-6152 (1985); gene targeting m embryonic stem cells (Thompson, et al , Cell 56 313-321 (1989); electropolation of embryos (Lo, Mol Cell Biol.
  • a further aspect of the present invention involves gene targeting by homologous recombination in embryonic stem cells to produce a transgemc animal with a mutation in a htra2 gene ("knock-out" mutation).
  • knock-out animals
  • there is mactivation of the htra2 gene or altered gene expression such that the animals are useful to study the function of the htra2 gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or irradiation mutagenesis.
  • Another aspect of the present invention involves the generation of so-called "knock-in” animals m which a portion of a wild-type gene is fused to the cDNA of a heterologous gene.
  • This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressmg htra2
  • a Mus musculus htra2 cDNA SEQ LD NO: 1
  • SEQ LD NO: 1 a Mus musculus htra2 cDNA
  • the method used to create a knockout mouse is characte ⁇ zed m that- a suitable mutation is produced m the polynucleotide sequence of the mu ⁇ ne htra2 genomic clone, which inhibits the expression of a gene encoding mu ⁇ ne htra2, or inhibits the activity of the gene product; said modified mu ⁇ ne htra2 polynucleotide is introduced mto a homologous segment of mu ⁇ ne genomic DNA, combined with an approp ⁇ ate marker, so as to obtain a labeled sequence comp ⁇ sing said modified mu ⁇ ne genomic DNA, said modified mu ⁇ ne genomic DNA comp ⁇ smg the modified polynucleotide is transfected mto embryonic stem cells and correctly targeted events selected in vitro; then said stem cells are remjected mto a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through
  • a mutation is generated in a mu ⁇ ne htra2 allele by the introduction of a DNA construct comp ⁇ sing DNA of a gene encodmg mu ⁇ ne htra2, which mu ⁇ ne gene contains the mutation.
  • the mutation is targeted to the allele by way of the DNA construct.
  • the DNA of the gene encoding mu ⁇ ne htra2 comp ⁇ sed in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct comp ⁇ sed of synthetic or natural genetic components, or a mixture of these.
  • the mutation may constitute an insertion, deletion, substitution, or combination thereof
  • the DNA construct can be introduced mto cells by, for example, calcium-phosphate DNA co-precipitation. It is preferred that a mutation be introduced into cells using electroporation, microinjection, virus infection, hgand-DNA conjugation, virus-hgand-DNA conjugation, or hposomes.
  • mice obtained by a method of producing recombinant mice as defined above, among others.
  • Another aspect of this invention provides for in vitro htra2 "knock-outs", i.e , tissue cultures.
  • Animals of any species including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human p ⁇ mates, e.g , baboons, monkeys, chimpanzees, may be used to generate in vitro htra2 "knock-outs".
  • Methods for "knocking out" genes in vitro are descnbed in Gal - Tahadoros, et al , Journal of ' Immunological Methods 181 : 1-15 (1995).
  • Transgemc "knock-in”, and “knock-out” animals, as defined above, are a particularly advantageous model, from a physiological point of view, for studying se ⁇ ne protease. Such animals will be valuable tools to study the functions of a htra2 gene. Moreover, such animal models are expected to provide information about potential toxicological effects m humans of any compounds discovered by an aforementioned screening method, among others.
  • a Mus musculus htra2 gene functions in these animal models is expected to provide an insight mto treating and preventing human diseases including, but not limited to: Alzheimer's Disease; bone loss mcludmg osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arth ⁇ tis, Osteoarth ⁇ tis, Inflammatory Bowel Disease (LBD), pso ⁇ asis, dermatitis, asthma, allergies; infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HLV-1 or HLV-2; HIV-associated cachexia and other immunodeficiency disorders; septic shock; pain; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; u ⁇ nary retention; angina pecto ⁇ s; ulcers; benign prostatic hypertrophy;
  • the present mvention provides for a method of screening compounds to identify those that stimulate or inhibit the function of the polypeptide.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned.
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
  • Such agonists and antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional numerics thereof (see Co gan, et al , CURRENT PROTOCOLS LN IMMUNOLOGY 1(2): Chapter 5 (1991)).
  • the screening method may simply measure the bmding of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e.g , agonist or antagonist). Further, screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems approp ⁇ ate to cells bea ⁇ ng the polypeptide.
  • Antagonists are generally assayed m the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed Further, screenmg methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide of the present invention, to form a mixture, measuring Mus musculus htra2 activity in the mixture, and compa ⁇ ng a Mus musculus htra2 activity of the mixture to a control mixture which contains no candidate compound.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also m high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method desc ⁇ bed by Schullek, et al , Anal Biochem., 246, 20-29, (1997).
  • Fusion proteins such as those made from Fc portion and Mus musculus htra2 polypeptide, as herein desc ⁇ bed, can also be used for high-throughput screenmg assays to identify antagonists of antagonists of the polypeptide of the present invention (see D. Bennett, et al , J. Mol Recognition, 8:52-58 (1995); and K. Johanson, et al , J Biol Chem., 270(16):9459-9471 (1995)).
  • Another aspect of the invention is a method for assaying for the presence of a substance that modulates htra2 activity by direct binding to htra2 protein. Examples of modulators include, but are not limited to, peptides and small organic molecules including peptidomimetics.
  • Modulator candidates are synthesized on a solid support by techniques such as those disclosed m Lam et al , Nature 354, 82 (1991) or Burbaum et al, Proc. Natl. Acad. Sci. USA 92, 6027 (1995) to provide solid support-associated modulator candidates.
  • a labeled htra2 protein is provided having the ammo acid sequence of htra2 (SEQ LD NO. 2) or a functional de ⁇ vative thereof.
  • Exemplary labels include directly attached fluorescent or colored dyes, biotin, radioisotopes or epitope tags, which are detectable by a suitable antibody.
  • a mixture of solid support-associated modulator candidates and labeled htra2 protein is incubated under conditions which can permit the formation of a htra2 protein/modulator candidate complex.
  • the solid support is separated from free soluble labeled htra2 protein.
  • An assay is performed for the presence of solid support-associated labeled protein.
  • Solid supports complexed with labeled protein are isolated and the identity of the modulator candidate determined by techniques well known to those skilled in the art, such as the TOF-SLMS method in Brummel et al, Science 264, 399-402(1994).
  • polypeptide antagonists include antibodies or, m some cases, o gopeptides or proteins that are closely related to hgands, substrates, receptors, enzymes, etc , as the case may be, of a htra2 polypeptide, e.g., a fragment of a hgand, substrate, receptor, enzyme, etc.; or small molecules which bind to a hrra2 polypeptide but do not elicit a response, so that an activity of a htra2 polypeptide is prevented.
  • a htra2 polypeptide e.g., a fragment of a hgand, substrate, receptor, enzyme, etc.
  • small molecules which bind to a hrra2 polypeptide but do not elicit a response, so that an activity of a htra2 polypeptide is prevented.
  • the present invention relates to a screenmg kit for identifying agonists, antagonists, inhibitors, hgands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds comp ⁇ se a member selected from the group consisting of: (a) a polypeptide of the present invention;
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; which polypeptide is preferably that of SEQ LD NO:2
  • a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by: (a) determining m the first instance the three-dimensional structure of the polypeptide;
  • the present invention relates to the use of Mus musculus htra2 polypeptides, polynucleotides, and recombinant materials thereof m selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Mus musculus htra2.
  • the data from such a selection screen is expected to provide in vitro and in vivo compa ⁇ sons and to predict oral absorption, pharmacokmetics in humans.
  • the ability to make such a compa ⁇ son of data will enhance formulation design through the identification of compounds with optimal development characteristics, i e , high oral bioavailabihty, ULD (once a day) dosmg, reduced drug interactions, reduced va ⁇ abihty, and reduced food effects, among others.
  • “Allele” refers to one or more alternative forms of a gene occurnng at a given locus in the genome
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide
  • “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ LD NO: 1
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comp ⁇ sing va ⁇ ous portions of constant region of immunoglobulm molecules together with another human protein or part thereof.
  • employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262]
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similanty between the two sequences as hereinbefore defined Falling withm this gene ⁇ c term are the terms, "ortholog”, and “paralog” "Ortholog” refers to polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have common descent based on structural and functional considerations "Paralog” refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated va ⁇ ants withm a genome.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng the sequences.
  • identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared
  • a "% identity” may be determined
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences This may include inserting "gaps" either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length
  • “Similanty” is a further, more sophisticated measure of the relationship between two polypeptide sequences
  • “similanty” means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking mto account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% similanty" of the two sequences can then be determined. Methods for comparing the identity and simila ⁇ ty of two or more sequences are well known m the art.
  • programs available in the Wisconsin Sequence Analysis Package, version 9J may be used to determine the % identity between two polynucleotides and the % identity and the % similanty between two polypeptide sequences.
  • BESTFIT uses the "local homology" algo ⁇ thm of
  • BESTFIT is more suited to compa ⁇ ng two polynucleotide or two polypeptide sequences that are dissimilar m length, the program assuming that the shorter sequence represents a portion of the longer In compa ⁇ son, GAP aligns two sequences, finding a "maximum similanty", according to the algo ⁇ thm of Neddleman and Wunsch (J Mo.l Biol , 48, 443-453, 1970). GAP is more suited to compa ⁇ ng sequences that are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight” and "Length Weight” used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • %> identities and simila ⁇ ties are determined when the two sequences being compared are optimally aligned.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • FASTA Pearson W R and Lipman D.J., Proc Nat Acad Sci USA, 85: 2444-2448 (1988) (available as part of the Wisconsin Sequence Analysis Package).
  • BLOSUM62 amino acid substitution matnx Hemkoff S. and Hemkoff J.G.
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore desc ⁇ bed.
  • a polynucleotide sequence having, for example, at least 95%) identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence. Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • point mutations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups withm the reference sequence.
  • a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence up to 5% of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • % identities such as 96%, 97%, 98%, 99%> and 100%).
  • a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence.
  • Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion
  • These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence.
  • a sequence polypeptide sequence having at least 95%> identity to a reference polypeptide sequence up to 5% of the amino acids of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • % identities such as 96%, 97%, 98%>, 99%, and 100%.
  • Polynucleotide embodiments further include an isolated polynucleotide comp ⁇ sing a polynucleotide sequence having at least a 95, 97 or 100%o identity to the reference sequence of SEQ LD NO- 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ LD
  • nucleotide NO: 1 may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ED NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ED NOJ, or n n ⁇ x n - (x n • y),
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides SEQ ED
  • y is 0.95 for 95%, 0.97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer pnor to subtracting it from x n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ
  • ED NO:2 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Polypeptide embodiments further include an isolated polypeptide compnsing a polypeptide having at least a 95, 97 or 100%> identity to a polypeptide reference sequence of SEQ LD
  • polypeptide sequence may be identical to the reference sequence of SEQ ED NO:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ED NO.2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids m SEQ ED NO:2, or:
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ED
  • y is 0.95 for 95%, 0 97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer pnor to subtracting it from x a .
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs m nature, it has been changed or removed from its o ⁇ ginal environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated", as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or non-living
  • Knock- refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene
  • Knock-out refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell.
  • the "knock-out” can be affected by targeted deletion of the whole or part of a gene encoding a protein, in an embryonic stem cell. As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal in which it is normally expressed.
  • RNA molecules produced from RNA molecules initially transcnbed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • Alternative RNA splicing occurs when a pnmary RNA transc ⁇ pt undergoes splicing, generally for the removal of introns, which results m the production of more than one mRNA molecule each of that may encode different ammo acid sequences.
  • the term splice va ⁇ ant also refers to the proteins encoded by the above cDNA molecules.
  • Transgemc animal refers to an animal to which exogenous DNA has been introduced while the animal is still m its embryonic stage
  • the transgemc approach aims at specific modifications of the genome, e g , by introducing whole transcnptional units into the genome, or by up- or down-regulatmg pre-existing cellular genes.
  • the targeted character of certain of these procedures sets transgemc technologies apart from expe ⁇ mental methods in which random mutations are conferred to the germlme, such as administration of chemical mutagens or treatment with ionizing solution.
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybnd molecules comp ⁇ sing DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to t ⁇ ple-stranded regions comp ⁇ sing RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs comp ⁇ sing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, t ⁇ tylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characte ⁇ stic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any peptide or protein comp ⁇ sing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i e , peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may comp ⁇ se amino acids other than the 20 gene-encoded ammo acids "Polypeptides" include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art. Such modifications are well desc ⁇ bed m basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini.
  • polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as argmylation, and ubiquitination (see, for instance
  • a typical va ⁇ ant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes m the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical va ⁇ ant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, in many regions, identical.
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a va ⁇ ant of a polynucleotide or polypeptide may be a naturally occumng such as an allelic va ⁇ ant, or it may be a vanant that is not known to occur naturally.
  • Non- naturally occumng vanants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • the PSP-1 N4F1 heterozygous animals display no overt phenotype.
  • N4F1 homozygous animals are noticeably smaller than their litter-mates.
  • the homozygous animals display an abnormal shifting gait and tend to clench their paws and appear unstable on their feet.
  • this instability is more pronounced and have an impaired ⁇ ghting reflex. The impairments are so severe that the animals must be culled.
  • Macroscopic organ lesions were limited to moderate thymic atrophy present in one male homozygous animal.

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Abstract

L'invention concerne des polypeptides et des polynucléotides htra2 Mus musculus et des méthodes de production desdits polypeptides par des techniques recombinantes. L'invention concerne également des méthodes de criblage pour des composés agonistes ou antagonistes du htra2 Mus musculus. Ces composés sont utiles pour le traitement de maladies humaines, y compris mais pas exclusivement: la maladie d'Alzheimer; la perte osseuse comme l'ostéoporose; les maladies inflammatoires telles que le syndrome de l'insuffisance respiratoire chez l'adulte (ARDS), l'arthrite rhumatoïde, l'ostéoarthrite, la maladie intestinale inflammatoire, le psoriasis, la dermatite, l'asthme ou les allergies; les infections bactériennes, fongiques ou d'origine protozoaire ou virale, notamment les infections causées par le VIH-1 ou le VIH-2, la cachexie associée au VIH ou autres troubles de l'immunodéficience; le choc septique; la douleur; les blessures; les cancers; l'anorexie; la boulimie; la maladie de Parkinson; les maladies cardiovasculaires notamment la resténose, l'athérosclérose; l'insuffisance cardiaque aiguë, l'infarctus du myocarde; l'hypotension; l'hypertension; la rétention urinaire; l'angine de poitrine; les ulcères; l'hypertrophie prostatique bénigne; et les troubles psychotiques ou neurologiques, notamment l'anxiété, la schizophrénie, la manie-dépression, le délire, la démence, le retard mental grave, et les dyskinésies comme la chorée de Huntington ou la maladie de Gilles de la Tourette.
PCT/US2000/014037 1999-05-19 2000-05-19 Htra2 murin Ceased WO2000070033A1 (fr)

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WO2005071107A1 (fr) * 2004-01-27 2005-08-04 Eberhard-Karls-Universität Tübingen Mutation en a141s et g399s dans la proteine omi/htra2 dans la maladie de parkinson
WO2008112798A1 (fr) * 2007-03-12 2008-09-18 Mayo Foundation For Medical Education And Research Traitement de l'éclampsisme et des maladies cardiovasculaires
WO2014109617A1 (fr) * 2013-01-14 2014-07-17 가톨릭대학교 산학협력단 Composition pour la prévention ou le traitement de maladies auto-immunes, contenant htra2 comme principe actif

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DATABASE EST IN GENBANK, 14 April 1997 (1997-04-14), MARRA ET AL.: "The washingotn U-HHMI mouse EST project" *
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HU ET AL.: "Human HtrA, an evolutionarily conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage", JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 273, no. 51, December 1998 (1998-12-01), pages 34406 - 34412, XP002930913 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071107A1 (fr) * 2004-01-27 2005-08-04 Eberhard-Karls-Universität Tübingen Mutation en a141s et g399s dans la proteine omi/htra2 dans la maladie de parkinson
WO2008112798A1 (fr) * 2007-03-12 2008-09-18 Mayo Foundation For Medical Education And Research Traitement de l'éclampsisme et des maladies cardiovasculaires
WO2014109617A1 (fr) * 2013-01-14 2014-07-17 가톨릭대학교 산학협력단 Composition pour la prévention ou le traitement de maladies auto-immunes, contenant htra2 comme principe actif
KR101463325B1 (ko) 2013-01-14 2014-11-20 가톨릭대학교 산학협력단 HtrA2 단백질을 유효성분으로 포함하는 자가면역질환의 예방 또는 치료용 조성물

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