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WO2003040393A2 - Acides nucleiques codant des proteases - Google Patents

Acides nucleiques codant des proteases Download PDF

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Publication number
WO2003040393A2
WO2003040393A2 PCT/IB2002/004615 IB0204615W WO03040393A2 WO 2003040393 A2 WO2003040393 A2 WO 2003040393A2 IB 0204615 W IB0204615 W IB 0204615W WO 03040393 A2 WO03040393 A2 WO 03040393A2
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nucleic acid
polypeptide
agent
protease gene
protease
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WO2003040393A3 (fr
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Roger Alfonso Moraga Martinez
Gunnar Thor Sigurdsson
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Decode Genetics ehf
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Decode Genetics ehf
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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

Definitions

  • a “protease” or “protease enzyme” is a protein which has proteolytic activity. These enzymes have important applications in industry, biology and medicine.
  • proteases include food processing (e.g., serine proteases are used to produce dairy products and protein-rich concentrates from fish and livestock), brewing, and alcohol production, and are common additives in laundry detergents (e.g., the serine protease subtilisin is used to remove protein- based stains). They are also used in research, e.g., to degrade undesirable proteins during purification processes.
  • proteases are involved in a wide array of biological pathways, in regulatory capacities, as treatments, or in pathological capacities.
  • Tissue plasminogen activator (t-PA) and thrombin are commonly used in pharmaceutical settings, while improper regulation of elastase can result in lung problems such as emphysema.
  • Proteases have also been implicated in the occurance of many diseases, including Alzheimer's disease, cystic fibrosis, muscular dystrophy, arthritis, emphysema and other respiratory ailments, gastrointestinal diseases, hypertension, degenerative skin disorders, tumor invasion and metastasis and also viral-associated diseases, where they are important in viral maturation.
  • the aspartic acid proteases pepsin and gastricsin are secreted into the stomach for food digestion, and are diagnostic indicators for stomach ulcer and cancer.
  • Collagen and elastase degrade, respectively, the structural proteins collagen and elastin.
  • Many elastase degrade, respectively, the structural proteins collagen and elastin.
  • Many human pathogens e.g., Haemophil s influenza, Neisseria gonorrhoeae, Streptococcus sanguis, Streptococcus pneumonia, Neisseria meningitides
  • Proteases are also involved in cellular responses to starvation, heat-shock, and other stresses in which cells might find it advantageous to break down proteins to salvage component amino acids.
  • the present invention relates to human protease genes, particularly nucleic acids comprising protease genes, and the amino acids encoded by such nucleic acids. These sequences are shown in Appendix I.
  • each protease entry lists the University of California at Santa Cruz contig designation from which the sequence was analyzed (e.g., "ctgchrllq_l"), the name (e.g., "MOOSE13873”), the exon locations (e.g., "7240429 . .7240464, . . .”), following by the amino acid sequence and the nucleic acid sequence.
  • Sub-family information on the sequences is shown in Appendix H
  • the name e.g., "MOOSE13873”
  • the University of California at Santa Cruz contig designation from which the sequence was analyzed e.g., "ctgchrl lq_ l”
  • the assignments were made on the basis of the best E- value with which the sequence aligned.
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-47, as shown in Appendix I, and the complements thereof.
  • the invention further relates to a nucleic acid molecule which hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-47, as shown in Appendix I, and the complements thereof.
  • the invention additionally relates to isolated nucleic acid molecules (e.g., cDNA molecules) encoding a protease polypeptide (e.g., encoding a polypeptide selected from the group consisting of SEQ ID NOs:48-94, as shown in Appendix I).
  • the invention further provides a method for assaying a sample for the presence of a nucleic acid molecule encoding all or a portion of a protease in a sample, comprising contacting said sample with a second nucleic acid molecule comprising a nucleotide sequence encoding a protease polypeptide (e.g. , one of SEQ ' ID NOs:l-47, as shown in Appendix I, or the complement of one of SEQ LD NOs:l- 47; a nucleotide sequence encoding one of SEQ ID NOs:48-94, as shown in Appendix 1), or a fragment or derivative thereof, under conditions appropriate for selective hybridization.
  • a protease polypeptide e.g. , one of SEQ ' ID NOs:l-47, as shown in Appendix I, or the complement of one of SEQ LD NOs:l- 47; a nucleotide sequence encoding one of SEQ ID NOs:48
  • the invention additionally provides a method for assaying a sample for the level of expression of a protease polypeptide, or fragment or derivative thereof, comprising detecting (directly or indirectly) the level of expression of the protease polypeptide, fragment or derivative thereof.
  • the invention also relates to a vector comprising an isolated nucleic acid molecule of the invention operatively linked to a regulatory sequence, as well as to a recombinant host cell comprising the vector.
  • the invention also provides a method for preparing a polypeptide encoded by an isolated nucleic acid molecule described herein (a protease polypeptide), comprising culturing a recombinant host cell of the invention under conditions suitable for expression of said nucleic acid molecule.
  • the invention further provides an isolated polypeptide encoded by isolated nucleic acid molecules of the invention (e.g., protease polypeptide), as well as fragments or derivatives thereof.
  • the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:48-94, as shown in Appendix I.
  • the invention also relates to an isolated polypeptide comprising an amino acid sequence which is greater than about 90 percent identical to an amino acid sequence selected from the group consisting of SEQ LD NOs:48-94, preferably about 95 percent identical.
  • the invention also relates to an antibody, or an antigen-binding fragment thereof, which selectively binds to a polypeptide of the invention, as well as to a method for assaying the presence of a polypeptide encoded by an isolated nucleic acid molecule of the invention in a sample, comprising contacting said sample with an antibody which specifically binds to the encoded polypeptide.
  • the invention further relates to methods of diagnosing a predisposition to a condition mediated by a protease.
  • the methods of diagnosing such a predisposition in an individual include detecting the presence of a mutation in a protease, as well as detecting alterations in expression of a protease polypeptide, such as the presence of different splicing variants of protease polypeptides.
  • the alterations in expression can be quantitative, qualitative, or both quantitative and qualitative.
  • the invention additionally relates to an assay for identifying agents which alter (e.g. , enhance or inhibit) the activity or expression of one or more protease polypeptides.
  • a cell, cellular fraction, or solution containing a protease polypeptide or a fragment or derivative thereof can be contacted with an agent to be tested, and the level of protease polypeptide expression or activity can be assessed.
  • the activity or expression of more than one protease polypeptide can be assessed concurrently (e.g., the cell, cellular fraction, or solution can contain more than one type of protease polypeptide, such as different splicing variants, and the levels of the different polypeptides or splicing valiants can be assessed).
  • the invention relates to assays to identify polypeptides which interact with one or more protease polypeptides.
  • a first vector is used which includes a nucleic acid encoding a DNA binding domain and also a protease polypeptide, splicing variant, or fragment or derivative thereof
  • a second vector is used which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the protease polypeptide, splicing variant, or fragment or derivative thereof (e.g. , a protease polypeptide binding agent or receptor).
  • Incubation of yeast containing both the first vector and the second vector under appropriate conditions allows identification of polypeptides which interact with the protease polypeptide or fragment or derivative thereof, and thus can be agents which alter the activity of expression of a protease polypeptide.
  • Agents that enhance or inhibit protease polypeptide expression or activity are also included in the current invention, as are methods of altering (enhancing or inhibiting) protease polypeptide expression or activity by contacting a cell containing protease and/or polypeptide, or by contacting the protease polypeptide, with an agent that enhances or inhibits expression or activity of protease or polypeptide.
  • the invention pertains to pharmaceutical compositions comprising the nucleic acids of the invention, the polypeptides of the invention, and/or the agents that alter activity of protease polypeptide.
  • the invention further pertains to methods of treating conditions mediated by proteases, by administering protease therapeutic agents, such as nucleic acids of the invention, polypeptides of the invention, the agents that alter activity of protease polypeptide, or compositions comprising the nucleic acids, polypeptides, and/or the agents that alter activity of protease polypeptide.
  • the present invention relates to nucleic acids comprising proteases, and the protease amino acids encoded by those nucleic acids.
  • proteases are enzymes having proteolytic activity, and so comprise an extremely large class of enzymes having a wide array of functions.
  • the proteolytic activity of proteases can be used in many different industrial processes, including food and beverage processing (e.g., beer brewing, manufacture of dairy products, protein processing, etc.), and in detergent formulation (e.g. , for removal of proteinaceous stains).
  • food and beverage processing e.g., beer brewing, manufacture of dairy products, protein processing, etc.
  • detergent formulation e.g. , for removal of proteinaceous stains.
  • New versions of such proteases possessing increased levels of activity or binding would therefore be of increased utility in such industrial settings.
  • those proteases with pharmaceutical application, e.g., t-PA and thrombin would also be more useful if versions were found possessing increased activity and/or more specific activity.
  • proteases are also involved in many cellular functions, in both humans and other organisms, of which some of the functions can lead to disease conditions. Where proteases are associated with pathogenesis, it is therefore desirable to interfere with the activity of such enzymes. For instance, where a disease or undesirable condition is due at least in part to the action of or mediation by one or more protease enzymes (e.g., emphysema, cystic fibrosis, hypertension, etc.), a potential method of prophylaxis would be to prevent the protease associated with the disease from acting at all, or from acting at the time and/or with the ligand necessary to cause the disease or condition. For instance, for those pathogens which infect humans, e.g., by cleaving the hinge region of human IgAl, pathogenesis could be prevented by inhibiting the action of that protease in the pathogenic organism.
  • protease enzymes e.g., emphysema, cystic fibrosis,
  • HMMs Hidden Markov Models
  • DiAlign 2 Merge-Met al. (1994) was built from multiple sequence alignments of the different protein families obtained with DiAlign 2 (Morgenstern, Bioinformatics 15(3):211-8(1999)). DiAlign works based on segment-to-segment comparisons instead of arbitrary thresholds for gap opening and extension, which makes it ideally suited for building models that represent an entire, full-length sequence, since the alignments built this way have more match states that would be assigned as insertion states when using other alignment algorithms. The models were built using the standard HMMer package.
  • a classification was obtained in which the sequences are grouped by length and similarity. Each one of these groups was then used to build a HMM profile representing this group of sequences.
  • This approach aims to have models that can represent the full length of the encoded proteins for a whole range of proteins, without being too specific for any one of them or being too general, as would be a HMM built for large groups of sequences.
  • This classification was based either on existing expert-supervised classifications, or by retrieval of sequences and classification based on pairwise alignment distances. These models were then searched against the August 2001 fixed release of the Santa Cruz contigs using the Paracel GeneMatcherX Hardware Accelerator with the GeneWise algorithm. The sequences were chopped at 100 Kb with an overlap of 1 Kb.
  • the results represent the coding regions of the complete final protein as it is found in the organism.
  • the cross-validation of the results was done in two steps. First, all of the hits o with an E-value lower than 10 " that do not overlap with one another were selected, and in the event of overlapping, the one with lowest E-value was selected. After selecting all of those matches, the DNA sequences were compared against the RefSeq database (Pruitt et al, Trends Genet. 16(l):44-47 (2000)) using BLAST (Altschul et al. Nucleic Acids Res. 25:3389-3402 (1997)).
  • a number of the genes were found to be linked with markers known to be associated with human diseases genes. These are shown in Appendix in.
  • the diseases were linked to the HMM genes in the following manner: (1) the HMM gene models were compared to the consensus of the human genome sequence, located and the results kept in a relational database; (2) all possible markers (Sequence Tagged Sites (STS's)) (public or deCODE genetics) are also located in the same consensus using ePCR or BLAT and results kept in a relational database; and (3) LOD scores for diseases are linked to markers. A span of one LOD drop around the marker was also given. A computer program takes each LOD peak and links it to the consensus through the markerhit in the database. The database is then queried for all HMM genes within the span of one LOD drop or a minimum of 15 Mb in each direction from the marker. The output is the name of the peak marker and its distance to the HMM gene.
  • protease refers to an isolated nucleic acid molecule selected from the group shown in Appendix I, and consisting of SEQ LD NOs: 1-47, and also to a portion or fragment of the isolated nucleic acid molecule (e.g. , cDNA or the gene) that encodes protease polypeptide (e.g., a polypeptide selected from the group shown in Appendix I, and consisting of SEQ JD NOs:48-94).
  • protease polypeptide e.g., a polypeptide selected from the group shown in Appendix I, and consisting of SEQ JD NOs:48-94.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-47 or the complement of such a nucleic acid molecule.
  • the isolated nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA.
  • DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense, strand or the non-coding, or antisense, strand.
  • the nucleic acid molecule can include all or a portion of the coding sequence of the gene and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example). Additionally, the nucleic acid molecule can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide. Such sequences include, but are not limited to, those which encode a glutathione-S-transferase (GST) fusion protein and those which encode a hemagglutinin A (HA) polypeptide marker from influenza.
  • GST glutathione-S-transferase
  • HA hemagglutinin A
  • an "isolated" nucleic acid molecule is one that is separated from nucleic acids which normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material will fonn part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • genomic DNA the term “isolated” also can refer to nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence which is synthesized chemically or by recombinant means. Therefore, recombinant DNA contained in a vector are included in the definition of "isolated" -li ⁇
  • isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleotide sequences are also encompassed by "isolated" nucleotide sequences.
  • isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis.
  • the present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a protease polypeptide (e.g., a polypeptide having an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ LD NOs:48-94), or another splicing variant of a protease polypeptide or polymorphic variant thereof.
  • a protease polypeptide e.g., a polypeptide having an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ LD NOs:48-94
  • DNA molecules which comprise a sequence that is different from the naturally- occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode a protease polypeptide of the present invention are also the subject of this invention.
  • the invention also encompasses nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a protease polypeptide.
  • variants can be naturally-occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
  • Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides which can result in conservative or non-conservative amino acid changes, including additions and deletions.
  • nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a protease polypeptide.
  • nucleotide sequences are fragments that comprise one or more polymorphic microsatellite markers.
  • nucleotide sequences are fragments that comprise one or more single nucleotide polymorphisms in a protease gene.
  • nucleic acid molecules of the invention can include, for example, labeling, methylation, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates
  • charged linkages e.g., phosphorothioates, phosphorodithioates
  • pendent moieties e.g., polypeptides
  • intercalators e.g., acridine, ps
  • synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequences via hydrogen bonding and other chemical interactions.
  • Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the invention also pertains to nucleic acid molecules which hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g. , nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide).
  • the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ JD NOs: 1-47.
  • the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g.
  • nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions).
  • Specific hybridization refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed).
  • “Stringency conditions” for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%o, 95%). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
  • conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another.
  • hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.
  • washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each °C by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by l%o in the maximum extent of mismatching among the sequences that hybridize.
  • the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
  • a low stringency wash can comprise washing in a solution containing 0.2X SSC/0, 1% SDS for 10 minutes at room temperature;
  • a moderate stringency wash can comprise washing in a prewarmed solution (42°C) solution containing 0.2X SSC/0.1% SDS for 15 minutes at 42°C;
  • a high stringency wash can comprise washing in prewarmed (68°C) solution containing 0.1X SSC/0.1%>SDS for 15 minutes at 68°C.
  • washes can be performed repeatedly or sequentially to obtain a desired result as known in the art.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
  • the percent homo logy or identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment).
  • % identity # of identical positions/total # of positions x 100.
  • the length of a sequence aligned for comparison purposes is at least 30%, for example, at least 40%, in certain embodiments at least 60%), and in other embodiments at least 70%, 80%, 90% or 95% of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm.
  • a preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al, Nucleic Acids Res. 25:389-3402 (1997).
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4(1):11-17 (1988). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 , and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl Biosci. 10:3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl Acad. Sci. USA 85:2444-8 (1988).
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK) using either a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using a gap weight of 50 and a length weight of 3.
  • the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-47, or the complement of such a sequence, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence encoding an amino acid sequence selected SEQ ID NOs:48-94, or polymorphic variant thereof.
  • the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies as described below.
  • nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein.
  • Probes or “primers” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules.
  • probes and primers include polypeptide nucleic acids, as described in Nielsen et al, Science 254:1497-1500 (1991).
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and more typically about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-47, or the complement of such a sequence, or a sequence encoding an amino acid sequence selected from SEQ ID NOs:48-94, or polymorphic variant thereof.
  • a probe or primer comprises 100 or fewer nucleotides, preferably from 6 to 50 nucleotides, preferably from 12 to 30 nucleotides.
  • the probe or primer is at least 70%o identical to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • the nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein.
  • nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-47, or the complement of such a sequence, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein.
  • synthetic oligonucleotide primers designed based on one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-47, or the complement of such a sequence, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein.
  • the nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothennal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • the amplified DNA can be labelled, for example radiolabelled, and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZLPLOX or other suitable vector.
  • Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight.
  • the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). Additionally, fluorescence methods are also available for analyzing nucleic acids (Chen et al, Genome Res. 9, 492 (1999)) and polypeptides. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
  • Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of one or more of SEQ ID NOs: 1-47 and/or the complement of one or more of SEQ ID NOs: 1-47, and/or a portion of one or more of SEQ LD NOs: 1-47, or the complement of one or more of SEQ ID NOs: 1-47 and/or a sequence encoding the amino acid sequences of one or more of SEQ ID NOs:48-94, or encoding a portion of one or more of SEQ ID NOs:48-94, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid molecule e.g., an antisense oligonucleotide
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occuning nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
  • the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers which are labeled to map related gene positions.
  • the nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify one or more of the disorders described above, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample.
  • the nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses.
  • Portions or fragments of the nucleotide sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
  • nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as markers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states.
  • the nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
  • nucleic acid constructs containing a nucleic acid molecule selected from the group consisting of SEQ LD NOs: 1-47 and the complements thereof (or a portion thereof).
  • nucleic acid constructs containing a nucleic acid molecule encoding an amino acid sequence of SEQ ID NOs:48-94 or polymorphic variant thereof.
  • the constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., Replication defective retroviruses, adenovirases and adeno-associated viruses) that serve equivalent functions.
  • viral vectors e.g., Replication defective retroviruses, adenovirases and adeno-associated viruses
  • Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell.
  • the recombinant expression vectors include - one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • "operably linked” or "operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, “Gene Expression Technology", Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g. , tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells e.g., E. coli
  • insect cells e.g., yeast or mammalian cells
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e. , express) a polypeptide of the invention.
  • the invention further provides methods for producing a polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • a host cell of the invention can also be used to produce nonhuman trans genie animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule of the invention has been introduced (e.g., an exogenous protease gene, or an exogenous nucleic acid encoding a protease polypeptide).
  • a nucleic acid molecule of the invention e.g., an exogenous protease gene, or an exogenous nucleic acid encoding a protease polypeptide.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a trans gene.
  • rodent such as a rat or mouse
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous.
  • DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al, Nature 385:810-813 (1997) and PCT Publication Nos. WO 97/07668 and WO 97/07669.
  • the present invention also pertains to isolated polypeptides encoded by proteases ("protease polypeptides”) and fragments and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants).
  • proteases proteases
  • polypeptides encoded by nucleotide sequences described herein e.g., other splicing variants.
  • polypeptide refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g. , in a "fusion protein") and still be “isolated” or “purified.”
  • polypeptides of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.
  • the language "substantially free of cellular material” includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10%) other proteins, or less than about 5% other proteins.
  • a polypeptide When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.
  • the language "substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% ⁇ chemical precursors or other chemicals, less than about 10%o chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • a polypeptide of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-47, or the complement of such a nucleic acid, or portions thereof, e.g., SEQ LD NO:48-94, or a portion or polymorphic variant thereof.
  • the polypeptides of the invention also encompass fragment and sequence variants. Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants.
  • Variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ JD NOs: 1-47, or a complement of such a sequence, or portions thereof, or having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of nucleotide sequences encoding SEQ ID NOs:48-94, or polymorphic variants thereof.
  • Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism, i.e., an ortholog.
  • Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis.
  • Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods
  • two polypeptides are substantially homologous or identical when the amino acid sequences are at least about 45-55%), in certain embodiments at least about 70-75%, in other embodiments at least about 80-85%), and in other embodiments greater than about 90%o or more homologous or identical.
  • a substantially homologous amino acid sequence will be encoded by a nucleic acid molecule hybridizing to one or more of SEQ JD NOs: 1-47, or portion thereof, under stringent conditions as more particularly described above, or will be encoded by a nucleic acid molecule hybridizing to a nucleic acid sequence encoding one of SEQ ID NOs:48- 94, a portion thereof or polymorphic variant thereof, under stringent conditions as more particularly described thereof.
  • the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • variant polypeptides can be fully functional or can lack function in one or more activities.
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Functional variants can also contain substitution of similar - amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non- functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro proliferative activity. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffmity labeling (Smith et al, J. Mol Biol 224:899-904 (1992); de Vos et al, Science 255:306-312 (1992)).
  • the invention also includes polypeptide fragments of the polypeptides of the invention. Fragments can be derived from a polypeptide encoded by a nucleic acid molecule comprising one of SEQ ID NOs: 1-47, or a complement of such a nucleic acid (e.g., SEQ JD NOs:48-94, or other variants). However, the invention also encompasses fragments of the variants of the polypeptides described herein. As used herein, a fragment comprises at least 6 contiguous amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide as well as fragments that can be used as an immunogen to generate polypeptide-specific antibodies.
  • Biologically active fragments can comprise a domain, segment, or motif that has been identified by analysis of the polypeptide sequence using well-known methods, e.g., signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites. Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl tenninus of the fragment.
  • the invention thus provides chimeric or fusion polypeptides.
  • These comprise a polypeptide of the invention operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide.
  • "Operatively linked” indicates that the polypeptide and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide.
  • the fusion polypeptide does not affect function of the polypeptide per se.
  • the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences.
  • fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example ⁇ -galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions.
  • enzymatic fusion polypeptides for example ⁇ -galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions.
  • Such fusion polypeptides particularly poly-His fusions, can facilitate the purification of recombinant polypeptide.
  • expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
  • EP A 0 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions.
  • the Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP A 0 232 262).
  • human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists. Bennett et al, Journal of Molecular Recognition, 5:52-58 (1995) and Johanson et al, The Journal of Biological Chemistry, 270,16:9459-9471 (1995).
  • this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
  • a chimeric or fusion polypeptide can be produced by standard recombinant
  • DNA techniques For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of nucleic acid fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • a nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.
  • the isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the polypeptide expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • polypeptides of the present invention can be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods.
  • the polypeptides of the present invention can be used to raise antibodies or to elicit an immune response.
  • the polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g., a ligand) in biological fluids.
  • polypeptides can also be used as markers for cells or tissues in which the corresponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state.
  • the polypeptides can be used to isolate a corresponding binding agent, e.g., ligand, such as, for example, in an interaction trap assay, and to screen for peptide or small molecule antagonists or agonists of the binding interaction.
  • Polyclonal and/or monoclonal antibodies that specifically bind one form of the gene product but not to the other form of the gene product are also provided.
  • Antibodies are also provided that bind a portion of either the variant or the reference gene product that contains the polymorphic site or sites.
  • the invention provides antibodies to the polypeptides and polypeptide fragments of the invention, e.g., having an amino acid sequence of one of SEQ JD NOs:48-94 or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ ID NOs: 1-47, or a complement or another variant or portion . thereof.
  • antibody refers to immunoglobulin molecules and immuno logically active portions of immunoglobulin molecules, i. e. , molecules that contain an antigen binding site that specifically binds an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g. , from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard teclmiques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al, Immunol Today 4:72 (1983)), the EBV-hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp. 77-96) or trioma techniques.
  • standard teclmiques such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al, Immunol Today 4:72 (1983)), the EBV-hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp. 77-96) or triom
  • hybridomas The technology for producing hybridomas is well l ⁇ iown (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • lymphocytes typically splenocytes
  • a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurJZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al, Bio/Technology 9:1370-1372 (1991); Hay et al, Hum.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention e.g., a monoclonal antibody
  • a polypeptide-specif ⁇ c antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells, Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, dete ⁇ nine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include I, I, S or H.
  • the present invention also pertains to a method of diagnosing or aiding in the diagnosis of a disease or condition associated with a protease gene or gene product in an individual.
  • Diagnostic assays can be designed for assessing protease gene expression, or for assessing activity of protease polypeptides of the invention.
  • the assays are used in the context of a biological sample (e.g. , blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or condition associated with a protease, or a defect in a protease.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is susceptible to a disease or condition associated with a protease.
  • mutations in the gene can be assayed in a biological sample.
  • Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of symptoms associated with a susceptibility to a disease or condition associated with a protease.
  • Another aspect of the invention pertains to assays for monitoring the influence of agents (e.g., drugs, compounds or other agents) on the gene expression or activity of polypeptides of the invention, as well as to assays for identifying agents which bind to a polypeptides.
  • nucleic acids, probes, primers, polypeptides and antibodies described herein can be used in methods of diagnosis of a susceptibility to a disease or condition associated with a protease, as well as in kits useful for diagnosis of a susceptibility to a disease or condition associated with a protease.
  • diagnosis of a susceptibility to a disease or condition associated with a protease is made by detecting a polymorphism in a protease as described herein.
  • the polymorphism can be a mutation in a protease, such as the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift mutation; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene; duplication of all or a part of the gene; transposition of all or a part of the gene; or rear
  • More than one such mutation may be present in a single gene.
  • sequence changes cause a mutation in the polypeptide encoded by a protease gene.
  • the mutation is a frame shift mutation
  • the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
  • a polymorphism associated with a susceptibility to a disease or condition associated with a protease can be a synonymous alteration in one or more nucleotides (i.e., an alteration that does not result in a change in the polypeptide encoded by a protease gene).
  • Such a polymorphism may alter splicing sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the gene.
  • a protease gene that has any of the alterations described above is referred to herein as a "altered gene.”
  • hybridization methods such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, including all supplements through 2001).
  • a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or carrying a defect for, a susceptibility to a disease or condition associated with a protease (the "test individual").
  • the individual can be an adult, child, or fetus.
  • the test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • genomic DNA such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • a test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.
  • the DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism in a protease is present, and/or to determine which splicing variant(s) encoded by the protease is present.
  • nucleic acid probe can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism in a protease or contains a nucleic acid encoding a particular splicing variant of a protease.
  • the probe can be any of the nucleic acid molecules described above (e.g., the gene, a fragment, a vector comprising the gene, a probe or primer, etc.).
  • a hybridization sample is formed by contacting the test sample containing a protease, with at least one nucleic acid probe.
  • a preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • the nucleic acid probe can be all or a portion of one of SEQ ID NOs: 1-47, or the complement thereof, or a portion thereof; or can be a nucleic acid encoding a portion of one of SEQ ID NOs:48-94.
  • Other suitable probes for use in the diagnostic assays of the invention are described above (see e.g., probes and primers discussed under the heading, "Nucleic Acids of the Invention").
  • the hybridization sample is maintained under conditions which are sufficient to allow specific hybridization of the nucleic acid probe to a protease.
  • Specific hybridization indicates exact hybridization (e.g. , with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency. Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the protease in the test sample, then the protease has the polymorphism, or is the splicing variant, that is present in the nucleic acid probe.
  • More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphism in the protease, or of the presence of a particular splicing variant encoding the protease and is therefore diagnostic for a disease or a susceptibility to a disease or condition associated with a protease. hi Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F.
  • RNA from the individual is obtained from the individual by appropriate means.
  • Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymorphism in a protease, or of the presence of a particular splicing variant encoded by a protease, and is therefore diagnostic for a disease or a susceptibility to a disease or condition associated with a protease.
  • nucleic acid probes For representative examples of use of nucleic acid probes, see, for example, U.S. Patents No. 5,288,611 and 4,851,330.
  • PNA peptide nucleic acid
  • a nucleic acid probe can be used instead of a nucleic acid probe in the hybridization methods described above.
  • PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P.E. et al, Bioconjugate Chemistry 5, American Chemical Society, p. 1 (1994).
  • the PNA probe can be designed to specifically hybridize to a gene having a polymorphism associated with a disease or a susceptibility to a disease or condition associated with a protease. Hybridization of the PNA probe to a protease is diagnostic for a disease or a susceptibility to a disease or condition associated with a protease.
  • mutation analysis by restriction digestion can be used to detect a mutant gene, or genes containing a polymorphism(s), if the mutation or polymorphism in the gene results in the creation or elimination of a restriction site.
  • a test sample containing genomic DNA is obtained from the individual.
  • Polymerase chain reaction (PCR) can be used to amplify a protease (and, if necessary, the flanking sequences) in the test sample of genomic DNA from the test individual.
  • RFLP analysis is conducted as described (see Current Protocols in Molecular Biology, supra).
  • the digestion pattern of the relevant DNA fragment indicates the presence or absence of the mutation or polymorphism in the protease, and therefore indicates the presence or absence of this disease or a susceptibility to a disease or condition associated with a protease.
  • Sequence analysis can also be used to detect specific polymorphisms in a protease.
  • a test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the gene, and/or its flanking sequences, if desired.
  • the sequence of a protease, or a fragment of the gene, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods.
  • the sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the gene, cDNA (e.g., one or more of SEQ JD NOs: 1-47, or a complement thereof, or a nucleic acid sequence encoding one of SEQ ID NOs:48- 94 or a fragment thereof) or mRNA, as appropriate.
  • cDNA e.g., one or more of SEQ JD NOs: 1-47, or a complement thereof, or a nucleic acid sequence encoding one of SEQ ID NOs:48- 94 or a fragment thereof
  • the presence of a polymorphism in the protease indicates that the individual has a disease or a susceptibility to a disease or condition associated with a protease.
  • Allele-specific oligonucleotides can also be used to detect the presence of a polymorphism in a protease, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al, Nature 324:163-166 (1986)).
  • ASO allele-specific oligonucleotide
  • an “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to a protease, and that contains a polymorphism associated with a disease or a susceptibility to a disease or condition associated with a protease.
  • An allele-specific oligonucleotide probe that is specific for particular polymorphisms in a protease can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra).
  • a test sample of DNA is obtained from the individual.
  • PCR can be used to amplify all or a fragment of a protease, and its flanldng sequences.
  • the DNA containing the amplified protease (or fragment of the gene) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified protease is then detected.
  • Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymorphism in the protease, and is therefore indicative of a disease or a susceptibility to a disease or condition associated with a protease.
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer, which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product, which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual can be used to identify polymorphisms in a protease.
  • an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations.
  • oligonucleotide arrays also described as “GenechipsTM,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.
  • These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al, Science 251:161-111 (1991), Phrung et al, U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al, PCT Publication No. WO 92/10092 and U.S. Pat.
  • a nucleic acid of interest is hybridized with the array and scanned for polymorphisms.
  • Hybridization and scanning are generally carried out by methods described herein and also in, e.g., published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein.
  • a target nucleic acid sequence which includes one or more previously identified polymorphic markers is amplified by well known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream from the polymorphism.
  • Asymmetric PCR techniques may also be used.
  • Amplified target generally incorporating a label, is then hybridized with the array under appropriate conditions.
  • the array Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.
  • the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.
  • arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymorphisms.
  • detection blocks maybe grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the arcay, For example, it may often be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.
  • nucleic acid analysis can be used to detect polymorphisms in a protease or variants encoding by a protease.
  • Representative methods include direct manual sequencing (Church and Gilbert, Proc. Natl Acad. Sci. USA 81:1991- 1995 (1988); Sanger, F. et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977); Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single- stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V.C.
  • diagnosis of a disease or condition associated with a nucleic acid or a susceptibility to a disease or condition associated with a nucleic acid can also be made by expression analysis by quantitative PCR (kinetic thermal cycling).
  • quantitative PCR kinetic thermal cycling
  • This technique utilizing TaqMan® can be used to allow the identification of polymorphisms and whether a patient is homozygous or heterozygous. The technique can assess the presence of an alteration in the expression or composition of the polypeptide encoded by the nucleic acid or splicing variants encoded by the nucleic acid. Further, the expression of the variants can be quantified as physically or functionally different.
  • diagnosis of a disease or a susceptibility to a disease or condition associated with a protease can also be made by examining expression and/or composition of a protease polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • Western blots Western blots
  • immunoprecipitations immunofluorescence.
  • a test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a protease, or for the presence of a particular variant encoded by a protease.
  • An alteration in expression of a polypeptide encoded by a protease can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by a protease is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant protease polypeptide or of a different splicing variant), hi a preferred embodiment, diagnosis of a disease or a susceptibility to a disease or condition associated with a protease is made by detecting a particular splicing variant encoded by that protease, or a particular pattern of splicing variants.
  • An "alteration" in the polypeptide expression or composition refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by a protease in a control sample.
  • a control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by a susceptibility to a disease or condition associated with a protease.
  • An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample is indicative of a disease or a susceptibility to a disease or condition associated with a protease.
  • the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, is indicative of a disease or a susceptibility to a disease or condition associated with a protease.
  • Various means of examining expression or composition of the polypeptide encoded by a protease can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al, U.S. Pat. No. 4,376,110) such as immunoblotting (see also Current Protocols in Molecular Biology, particularly Chapter 10).
  • an antibody capable of binding to the polypeptide e.g., as described above
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or a fragment thereof e.g., Fab or F(ab') 2
  • the term "labeled", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • Western blotting analysis using an antibody as described above that specifically binds to a polypeptide encoded by a mutant protease, or an antibody that specifically binds to a polypeptide encoded by a non-mutant gene, or an antibody that specifically binds to a particular splicing variant encoded by a protease, can be used to identify the presence in a test sample of a particular splicing variant or of a polypeptide encoded by a polymorphic or mutant protease, or the absence in a test sample of a particular splicing variant or of a polypeptide encoded by a non- polymorphic or non-mutant gene.
  • the presence of a polypeptide encoded by a polymorphic or mutant gene, or the absence of a polypeptide encoded by a non- polymorphic or non-mutant gene, is diagnostic for a disease or a susceptibility to a disease or condition associated with a protease, as is the presence (or absence) of particular splicing variants encoded by the protease gene.
  • the level or amount of polypeptide encoded by a protease in a test sample is compared with the level or amount of the polypeptide encoded by the protease in a control sample.
  • a level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the protease, and is diagnostic for a disease or a susceptibility to a disease or condition associated with that protease.
  • the composition of the polypeptide encoded by a protease in a test sample is compared with the composition of the polypeptide encoded by the protease in a control sample (e.g., the presence of different splicing variants).
  • a difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample, is diagnostic for a disease or a susceptibility to a disease or condition associated with that protease, hi another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.
  • a difference in the amount or level of the polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition is indicative of a disease or a susceptibility to a disease or condition associated with that protease.
  • the invention further pertains to a method for the diagnosis and identification of susceptibility to disease in an individual, by identifying an at-risk haplotype in a protease gene, i one embodiment, the at-risk haplotype is one which confers a significant risk of a disease associated with a protease.
  • significance associated with a haplotype is measured by an odds ratio, i a further embodiment, the significance is measured by a percentage.
  • a significant risk is measured as an odds ratio of at least about 2.2, including by not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • an odds ratio of at least 1.2 is significant.
  • an odds ratio of at least about 1.5 is significant.
  • a significant increase in risk is at least about 1.7 is significant.
  • a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 98%.
  • a significant increase in risk is at least about 50%). It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.
  • the invention also pertains to methods of diagnosing a disease or a susceptibility to a disease associated with a protease in an individual, comprising screening for an at-risk haplotype in the nucleic acid that is more frequently present in an individual susceptible to a protease associated disease (affected), compared to the frequency of its presence in a healthy individual (control), wherein the presence of the haplotype is indicative of a disease or a susceptibility to a protease associated disease.
  • Standard techniques for genotyping for the presence of SNPs and/or micro satellite markers that are associated with a protease gene can be used, such as fluorescent based techniques (Chen, et al, Genome Res.
  • the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in the nucleic acid that are associated with a protease associated disease, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual has a protease associated disease or is susceptible to a protease associated disease.
  • Kits useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as decribed herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies which bind to altered or to non- altered (native) protease polypeptide, means for amplification of nucleic acids comprising a protease, or means for analyzing the nucleic acid sequence of a protease or for analyzing the amino acid sequence of a protease polypeptide, etc.
  • hybridization probes or primers as decribed herein e.g., labeled probes or primers
  • restriction enzymes e.g., for RFLP analysis
  • allele-specific oligonucleotides e.g., antibodies which bind to altered or to non- altered (native) prote
  • a kit for diagnosing a protease associated disease or susceptibility to a protease associated disease can comprise primers for nucleic acid amplification of a region in a nucleic acid comprising an at-risk haplotype that is more frequently present in an individual having a protease associated disease or is susceptible to a protease associated disease.
  • the primers can be designed using protion of the nucleic acids flanking SNPs that are indicative of a protease associated disease.
  • the invention provides methods (also referred to herein as "screening assays") for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention, hi one embodiment, the presence (or absence) of a nucleic acid molecule of interest (e.g., a nucleic acid that has significant homology with a nucleic acid of the invention) in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of one of SEQ ID NOs: 1-47, or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of one of SEQ LD NOs:48-94, or a fragment or variant of such nucleic acids), under stringent conditions as described above, and then assessing the sample for the presence (or absence) of hybridization, hi
  • a sample containing the nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleotide sequence (e.g., a primer or a probe as described above) that is at least partially complementary to a part of the nucleic acid molecule of interest (e.g., a protease nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization.
  • a nucleic acid containing a contiguous nucleotide sequence is completely complementary to a part of the nucleic acid molecule of interest.
  • the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically hybridizes to the polypeptide of interest (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.
  • an antibody that specifically hybridizes to the polypeptide of interest e.g., an antibody such as those described above
  • the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein.
  • agents e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein.
  • such agents can be agents which bind to polypeptides described herein (e.g., protease binding agents); which have a stimulatory or inhibitory effect on, for example, activity of polypeptides of the invention; or which change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with protease binding agents (e.g., receptors or other binding agents); or which alter posttranslational processing of the protease polypeptide (e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.
  • protease binding agents e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.
  • the invention provides assays for screening candidate or test agents that bind to or modulate the activity of polypeptides described herein (or biologically active portion(s) thereof), as well as agents identifiable by the assays.
  • Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S., Anticancer Drug Des. 12:145 (1997)).
  • a cell, cell lysate, or solution containing or expressing a protease polypeptide e.g. , one of SEQ LD NOs:48-94, or another splicing variant encoded by a protease), or a fragment or derivative thereof (as described above)
  • a protease polypeptide e.g. , one of SEQ LD NOs:48-94, or another splicing variant encoded by a protease
  • a fragment or derivative thereof as described above
  • the level (amount) of protease activity is assessed (e.g., the level (amount) of protease activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the protease polypeptide or active fragment or derivative thereof in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of a protease polypeptide. An increase in the level of protease activity relative to a control, indicates that the agent is an agent that enhances (is an agonist of) protease activity.
  • a decrease in the level of protease activity relative to a control indicates that the agent is an agent that inhibits (is an antagonist of) protease activity.
  • the level of activity of a protease polypeptide or derivative or fragment thereof in the presence of the agent to be tested is compared with a control level that has previously been established. A level of the activity in the presence of the agent that differs from the control level by an amount that is statistically significant indicates that the agent alters protease activity.
  • the present invention also relates to an assay for identifying agents which alter the expression of a protease gene (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the gene or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays.
  • a solution containing a nucleic acid encoding a protease polypeptide e.g. , a protease gene
  • an agent to be tested e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
  • the solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution which comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired.
  • the level and/or pattern of protease expression e.g. , the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants
  • a control i.e., the level and/or pattern of the protease expression in the absence of the agent to be tested.
  • the agent is an agent that alters the expression of a protease.
  • Enhancement of protease expression indicates that the agent is an agonist of protease activity.
  • inhibition of protease expression indicates that the agent is an antagonist of protease activity, hi another embodiment, the level and/or pattern of protease polypeptide(s) (e.g. , different splicing variants) in the presence of the agent to be tested, is compared with a control level and/or pattern that has previously been established.
  • a level and/or pattern in the presence of the agent that differs from the • control level and/or pattern by an amount or in a manner that is statistically significant indicates that the agent alters protease expression.
  • Such methods can be used to identify compounds that inhibit proteases involved in infection by pathogens.
  • agents which alter the expression of a protease gene or which otherwise interact with the nucleic acids described herein can be identified using a bell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the protease gene operably linked to a reporter gene.
  • the level of expression of the reporter gene (e.g., the level of mRNA or of protein expressed) is assessed, and is compared with the level of expression in a control (i.e., the level of the expression of the reporter gene in the absence of the agent to be tested). If the level in the presence of the agent differs, by an amount or in a manner that is statistically significant, from the level in the absence of the agent, then the agent is an agent that alters the expression of the protease, as indicated by its ability to alter expression of a gene that is operably linked to the protease gene promoter. Enhancement of the expression of the reporter indicates that the agent is an agonist of protease activity.
  • inhibition of the expression of the reporter indicates that the agent is an antagonist of protease activity.
  • the level of expression of the reporter in the presence of the agent to be tested is compared with a control level that has previously been established. A level in the presence of the agent that differs from the control level by an amount or in a manner that is statistically significant indicates that the agent alters expression.
  • Agents which alter the amounts of different splicing variants encoded by a protease e.g., an agent which enhances activity of a first splicing variant, and which inhibits activity of a second splicing variant
  • agents which are agonists of activity of a first splicing variant and antagonists of activity of a second splicing variant can easily be identified using these methods described above.
  • assays can be used to assess the impact of a test agent on the activity of a polypeptide in relation to a protease binding agent.
  • a cell that expresses a compound that interacts with a protease (herein referred to as a "protease binding agent", which can be a polypeptide or other molecule that interacts with a protease, such as a receptor) is contacted with a protease in the presence of a test agent, and the ability of the test agent to alter the interaction between the protease and the protease binding agent is determined.
  • a cell lysate or a solution containing the protease binding agent can be used.
  • test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a test agent to interact with the polypeptide without the labeling of any of the interactants.
  • a microphysiometer can be used to detect the interaction of a test agent with a protease or a protease binding agent without the labeling of either the test agent, protease, or the protease binding agent. McConnell, H.M. et al, Science 257:1906-1912 (1992).
  • a "microphysiometer” e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.
  • these receptors can be used to screen for compounds that are agonists for use in treating a susceptibility to a disease or condition associated with a protease or antagonists for studying a susceptibility to a disease or condition associated with a protease.
  • Drugs could be designed to regulate protease activation which in turn can be used to regulate signaling pathways and transcription events of genes downstream.
  • assays can be used to identify polypeptides that interact with one or more protease polypeptides, as described herein.
  • a yeast two-hybrid system such as that described by Fields and Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to identify polypeptides that interact with one or more protease polypeptides.
  • vectors are constructed based on the flexibility of a transcription factor which has two functional domains (a DNA binding domain and a transcription activation domain).
  • transcriptional activation can be achieved, and transcription of specific markers (e.g. , nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation.
  • specific markers e.g. , nutritional markers such as His and Ade, or color markers such as lacZ
  • a first vector which includes a nucleic acid encoding a DNA binding domain and also a protease polypeptide, splicing variant, or fragment or derivative thereof
  • a second vector which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the protease polypeptide, splicing variant, or fragment or derivative thereof (e.g., a protease polypeptide binding agent or receptor).
  • yeast containing the first vector and the second vector under appropriate conditions (e.g., mating conditions such as used in the MatchmakerTM system from Clontech (Palo Alto, California, USA)) allows identification of colonies which express the markers of interest. These colonies can be examined to identify the polypeptide(s) which interact with the protease polypeptide or fragment or derivative thereof. Such polypeptides may be useful as agents which alter the activity of expression of a protease polypeptide, as described above.
  • binding of a test agent to the polypeptide, or interaction of the polypeptide with a binding agent in the presence and absence of a test agent can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein e.g., a glutathione-S-transferase fusion protein
  • a fusion protein e.g., a glutathione-S-transferase fusion protein
  • modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, or solution containing a nucleic acid encoding a protease is contacted with a test agent and the expression of appropriate mRNA or polypeptide (e.g., splicing variant(s)) in the cell, cell lysate, or solution, is determined.
  • appropriate mRNA or polypeptide e.g., splicing variant(s)
  • the level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent.
  • the test agent can then be identified as a modulator of expression based on this comparison.
  • the test agent when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test agent when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a test agent that is a modulating agent, an antisense nucleic acid molecule, a specific antibody, or a polypeptide-binding agent
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • an agent identified as described herein can be used to alter activity of a polypeptide encoded by a protease, or to alter expression of a protease, by contacting the polypeptide or the gene (or contacting a cell comprising the polypeptide or the gene) with the agent identified as described herein.
  • the present invention also pertains to pharmaceutical compositions comprising nucleic acids described herein, particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NOs:48-94); and/or comprising other splicing variants encoded by a protease; and/or an agent that alters (e.g., enhances or inhibits) protease gene expression or protease polypeptide activity as described herein.
  • nucleic acids described herein particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NOs:48-94); and/or comprising other splicing variants encoded by a protease; and/or an agent that alters (e.g., enhances or inhibits) protease gene expression or protease polypeptide activity as described herein.
  • a polypeptide, protein e.g., a protease receptor
  • an agent that alters protease gene expression or a protease binding agent or binding partner, fragment, fusion protein or prodrug thereof, or a nucleotide or nucleic acid construct (vector) comprising a nucleotide of the present invention, or an agent that alters protease polypeptide activity
  • a physiologically acceptable carrier or excipient can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release fonnulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as
  • compositions of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal.
  • Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • nonsprayable forms viscous to semi-solid or solid fonns comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, 'aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the agent may be incorporated into a cosmetic formulation.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • Agents described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the agents are administered in a therapeutically effective amount.
  • the amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of a susceptibility to a disease or condition associated with a protease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration.
  • the pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g. , separately, sequentially or concurrently), or the like.
  • the pack or kit may also include means for reminding the patient to take the therapy.
  • the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages.
  • the agents can be separated, mixed together in any combination, present in a single vial or tablet.
  • Agents assembled in a blister pack or other dispensing means is preferred.
  • unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
  • the present invention also pertains to methods of treatment (prophylactic and/or therapeutic) for a disease or condition associated with a protease or susceptibility to a disease or condition associated with a protease, using a protease therapeutic agent.
  • a "protease therapeutic agent” is an agent that alters (e.g., enhances or inhibits) protease polypeptide activity and/or protease gene expression, as described herein (e.g., a protease agonist or antagonist).
  • Protease therapeutic agents can alter protease polypeptide activity or gene expression by a variety of means, such as, for example, by providing additional protease polypeptide or by upregulating the transcription or translation of the protease gene; by altering posttranslational processing of the protease polypeptide; by altering transcription of protease splicing variants; or by interfering with protease polypeptide activity (e.g., by binding to a protease polypeptide), or by downregulating the transcription or translation of a protease gene.
  • protease therapeutic agents include the following: nucleic acids or fragments or derivatives thereof described herein, particularly nucleotides encoding the polypeptides described herein and vectors comprising such nucleic acids (e.g., a gene, cDNA, and/or mRNA, such as a nucleic acid encoding a protease polypeptide or active fragment or derivative thereof, or an oligonucleotide; for example, one of SEQ ID NOs: 1-47, or a complement thereof, or a nucleic acid encoding one of SEQ JD NOs:48-94, or fragments or derivatives thereof); polypeptides described herein (e.g., one or more of SEQ JD NOs:48-94, and/or other splicing variants encoded by a protease, or fragments or derivatives thereof); other polypeptides (e.g., protease receptors); protease binding agents; peptidomimetics; fusion proteins or
  • More than one protease therapeutic agent can be used concurrently, if desired.
  • a protease therapeutic agent that is a nucleic acid is used in the treatment of a susceptibility to a disease or condition associated with a protease.
  • treatment refers not only to ameliorating symptoms associated with the disease, but also preventing or delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease.
  • the therapy is designed to- alter (e.g., inliibit or enhance), replace or supplement activity of a protease polypeptide in an individual.
  • a protease therapeutic agent can be administered in order to upregulate or increase the expression or availability of the protease gene or of specific splicing variants of protease, or, conversely, to downregulate or decrease the expression or availability of the protease gene or specific splicing variants of the protease.
  • Upregulation or increasing expression or availability of a native protease gene or of a particular splicing variant could interfere with or compensate for the expression or activity of a defective gene or another splicing variant; downregulation or decreasing expression or availability of a native protease gene or of a particular splicing variant could minimize the expression or activity of a defective gene or the particular splicing variant and thereby minimize the impact of the defective gene or the particular splicing variant.
  • the protease therapeutic agent(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease).
  • a therapeutically effective amount i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease.
  • the amount which will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard, clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a nucleic acid of the invention e.g., a nucleic acid encoding a protease polypeptide, such as one of SEQ JD NOs: 1-47, or a complement thereof; or another nucleic acid that encodes a protease polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding one of SEQ ID NOs:48-94
  • a nucleic acid of the invention can be used, either alone or in a pharmaceutical composition as described above.
  • a protease or a cDNA encoding a protease polypeptide can be introduced into cells (either in vitro or in vivo) such that the cells produce native protease polypeptide. If necessary, cells that have been transformed with the gene or cDNA or a vector comprising the gene or cDNA can be introduced (or re-introduced) into an individual affected with the disease.
  • cells which, in nature, lack native protease expression and activity, or have mutant protease expression and activity, or have expression of a disease-associated protease splicing variant, can be engineered to express the protease polypeptide or an active fragment of the protease polypeptide (or a different variant of the protease polypeptide).
  • nucleic acid encoding a protease polypeptide, or an active fragment or derivative thereof can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal.
  • Other gene transfer systems including viral and nonviral transfer systems, can be used.
  • nonviral gene transfer methods such as calcium phosphate coprecipitation, mechanical techniques (e.g., microinjection); membrane fusion-mediated transfer via liposomes; or direct DNA uptake, can also be used.
  • a nucleic acid of the invention in another embodiment, can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of a protease is administered or generated in situ.
  • a nucleic acid e.g., an oligonucleotide
  • the antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the protease polypeptide, e.g. , by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above.
  • the plasmid When the plasmid is transcribed in the cell, it produces RNA which is complementary to a portion of the mRNA and/or DNA which encodes the protease polypeptide.
  • the antisense construct can be an oligonucleotide probe which is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of the protease.
  • the oligonucleotide probes are modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo.
  • exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
  • oligodeoxyribonucleotides derived from the translation initiation site are preferred.
  • oligonucleotides mRNA, cDNA or DNA
  • the antisense oligonucleotides bind to protease mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA indicates that a sequence has sufficient complementarity to . be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation maybe assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
  • the oligonucleotides used in antisense therapy can be DNA, RNA, or - chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotides can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci.
  • the oligonucleotide may be conjugated to another molecule (e.g., apeptide, hybridization triggered cross-linldng agent, transport agent, hybridization-triggered cleavage agent).
  • another molecule e.g., apeptide, hybridization triggered cross-linldng agent, transport agent, hybridization-triggered cleavage agent.
  • the antisense molecules are delivered to cells which express protease in vivo.
  • a number of methods can be used for delivering antisense DNA or RNA to cells; e.g. , antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
  • a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g. , pol LU or pol LI).
  • RNAs that will fonn complementary base pairs with the endogenous protease transcripts and thereby prevent translation of the protease mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above.
  • a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site.
  • viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
  • Endogenous protease expression can also be reduced by inactivating or "knocking out” protease or its promoter using targeted homologous recombination (e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51 :503-512 (1987); Thompson et al, Cell 5:313-321 (1989)).
  • targeted homologous recombination e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51 :503-512 (1987); Thompson et al, Cell 5:313-321 (1989)).
  • a mutant, non-functional protease flanked by DNA homologous to the endogenous protease (either the coding regions or regulatory regions of protease) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the protease in vivo. hisertion of the DNA construct, via targeted homologous recombination, results in inactivation of the protease.
  • the recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above.
  • targeted homologous recombination can be used to insert a DNA construct comprising a non-mutant, functional protease, e.g., a gene having one of SEQ JD NOs: 1-47, or the complement thereof, or a portion thereof, in place of a mutant protease in the cell, as described above.
  • targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid that encodes a protease polypeptide variant that ' differs from that present in the cell.
  • endogenous protease expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of a protease (i.e., the protease promoter and/or enhancers) to fonn triple helical structures that prevent transcription of the protease in target cells in the body.
  • a protease i.e., the protease promoter and/or enhancers
  • the antisense constructs described herein by antagonizing the normal biological activity of one of the protease proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
  • the anti-sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a protease RNA or gene sequence
  • Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
  • other protease therapeutic agents as described herein can also be used in the treatment or prevention of a susceptibility to a disease or condition associated with a protease.
  • the therapeutic agents can be delivered in a composition, as described above, or by themselves.
  • the therapeutic agents can be administered systemically, or can be targeted to a particular tissue.
  • the therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade et al.), for example, and can be isolated using standard means such as those described herein.
  • a combination of any of the above methods of treatment e.g., administration of non-mutant protease polypeptide in conjunction with antisense therapy targeting mutant protease mRNA; administration of a first splicing variant encoded by a protease in conjunction with antisense therapy targeting a second splicing encoded by a protease
  • administration of non-mutant protease polypeptide in conjunction with antisense therapy targeting mutant protease mRNA can also be used.
  • APVA (SEQ JD NO: 48) ctggtagatgaacagcccctggagaactacctggatatggagtacttcggcactatcggcatcggaactcctgcccagga tttcaccgtcgtctttgacaccggctcctccaacctgtgggtgccctcagtctactgctccagtcttgcctgcACCaacca caaccgcttcaaccctgaggattcttccacctaccagtccaccagcgagacagtctccatcacctacggcaccggcagca
  • SWEELVRDSFGGYRNASAYCLMY ⁇ NDKA (SEQ LD NO: 59) tatgatagaaaaagacaggacaaagcfcccgttgggctaaagaatgttggcaatacttgttggtttagtgctgttattcagtc attatttaatcttttggaatttagaagattagttctgaattacaagcctccatcaaatgcttaccccgaaaccaaaaag gctttttcttttcacagcaagatgtgagtgagtttacacacaaattattagattggttagaagatgccttccaaatgaaagctg aagaggagACGgttggcaaggatgtggagaaattgaacccttgtgcagttggtgaggatatatattgagg
  • MOOSE13873 _ctgchrllq_l Al Pepsin MOOSE13874 _ctgchrllq_l Al Pepsin MOOSE13895 _ctgl0008 Cl Papain MOOSE13908 _ctgChr_13ctg3 C12 Ubiquitin C-terminal hydrolase family 1 MOOSE13930 _ctgl5ctg27 C15 Pyroglutamyl-peptidase I MOOSE13940 _ctgChr_Xctg264 C19 Ubiquitin C-terminal hydrolase family 2 MOOSE13941 .
  • NLDDM NLDDM (NON-LNSULLN DEP. DIABETES)

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Abstract

L'invention concerne des acides nucléiques codant des protases et des procédés permettant de les utiliser.
PCT/IB2002/004615 2001-11-06 2002-11-04 Acides nucleiques codant des proteases Ceased WO2003040393A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347048A4 (fr) * 2000-12-25 2004-07-21 Yamanouchi Pharma Co Ltd Nouvelle protease
US6855532B2 (en) 1999-11-19 2005-02-15 Solvay Pharmaceuticals B.V. Human enzymes of the metalloprotease family

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EP0848062A3 (fr) * 1996-12-14 2000-07-05 Smithkline Beecham Corporation Protéase aspartique ASP1
CA2374610A1 (fr) * 1999-06-28 2001-01-04 Jordan J. N. Tang Memapsine recombinante catalytiquement active et procedes d'utilisation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6855532B2 (en) 1999-11-19 2005-02-15 Solvay Pharmaceuticals B.V. Human enzymes of the metalloprotease family
EP1347048A4 (fr) * 2000-12-25 2004-07-21 Yamanouchi Pharma Co Ltd Nouvelle protease

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