WO2002081726A2 - Modulation de l'expression genetique dans des modeles genetiques murins de l'atherosclerose - Google Patents

Modulation de l'expression genetique dans des modeles genetiques murins de l'atherosclerose Download PDF

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WO2002081726A2
WO2002081726A2 PCT/US2001/043741 US0143741W WO02081726A2 WO 2002081726 A2 WO2002081726 A2 WO 2002081726A2 US 0143741 W US0143741 W US 0143741W WO 02081726 A2 WO02081726 A2 WO 02081726A2
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seq
polypeptide
polynucleotide
antibody
atherosclerosis
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PCT/US2001/043741
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WO2002081726A3 (fr
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Amedeo Leonardi
Abraham Sartani
James Glass
J. Gregor Sutcliffe
Karl W. Hasel
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Digital Gene Technologies, Inc.
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Priority to AU2001297768A priority Critical patent/AU2001297768A1/en
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Publication of WO2002081726A3 publication Critical patent/WO2002081726A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Atherosclerosis is a common form of cardiovascular disease and accounts for one- third of all deaths in Western societies.
  • the prevalence of atherosclerosis has led to concentrated efforts to understand the underlying basis of the pathology and to develop effective therapeutics for prevention and treatment.
  • the progression of atherosclerosis in humans often occurs over decades, and can be viewed as a state of chronic inflammation resulting from a cascade of interactions between oxidized or modified lipoproteins, monocyte-derived macrophages, T cells, and the cellular components comprising the arterial wall. This inflammatory process results in the appearance of atherosclerotic lesions, or plaques, that cause thickening of the arteries, loss of vascular elasticity, and thrombus formation.
  • the initiation and development of atherosclerotic lesions in the arterial wall proceeds in a series of well-defined stages.
  • the first stage comprises the growth of fatty streak lesions in the sub-endothelial intimal space. Following the initial fatty streak formation, a fibrous plaque coalesces at the lesion site, harboring an acellular lipid core covered by a cap containing smooth muscle cells and extracellular matrix components.
  • the final stage results in a complex lesion containing a necrotic core of macrophage and smooth muscle cell- derived foam cells along with deposits of extracellular cholesterol (Bennet, Cardiovascular Res, 1999; Davies, #r Heart J 1993).
  • the histological hallmark of emerging lesions is the accumulation of macrophages and smooth muscle cells in the vascular wall intima, with the appearance of cholesteryl ester-rich foam cells (Brown, J. Cell Biol, 1979; Ross, Nature, 1993).
  • Prominent histological features accompanying lesion progression include intimal disorganization due to cell migration or proliferation and changes in the composition of the extracellular matrix.
  • the fibrous cap and the thickening and deformation of the arterial wall characterize the advanced stages of lesion histopathology (Breslow, Science, 1996).
  • atherosclerosis is also associated with altered gene expression that initiates cell proliferation and de-differentiation the intima of the arterial wall.
  • the TOGA method is an improved method for the simultaneous sequence- specific identification of mRNAs in an mRNA population which allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA.
  • the method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions such as atherosclerosis.
  • the present invention associates identified polynucleotides and their encoded polypeptides to atherosclerosis such that the polynucleotides and polypeptides may be useful for diagnosis and treatment of atherosclerosis.
  • One embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as atherosclerosis, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention.
  • a further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention.
  • a preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the antibody. In one preferred embodiment, a method for preventing, treating, modulating or ameliorating atherosclerosis is provided.
  • An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject.
  • the method comprises determining the presence or absence of a mutation in a polynucleotide of the invention.
  • a pathological condition or a susceptibility to a pathological condition, such as atherosclerosis is diagnosed based on the presence or absence of the mutation.
  • Even another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as atherosclerosis, in a subject.
  • a pathological condition such as atherosclerosis
  • Especially preferred embodiments include methods of diagnosing atherosclerosis.
  • the method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a. first biological sample is obtained from a patient suspected of having atherosclerosis and a second sample from a suitable comparable control source is obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amount of the polypeptide in the first and second samples is determined.
  • a patient is diagnosed as having atherosclerosis if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as atherosclerosis
  • the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention to the mammalian subject.
  • a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as atherosclerosis
  • the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition.
  • This can be accomplished by, for example, the use of antisense oligonucleotides, triple helix base pairing methodology or ribozymes.
  • drags or antibodies that bind to and inactivate the polypeptide product can be used.
  • the present invention provides novel polynucleotides and the encoded polypeptides.
  • the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides.
  • One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO.T, SEQ ID NO:2, SEQ ID NO:3 5 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ K NO:22, SEQ D NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, S
  • an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of these isolated nucleic acid molecules and an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of these isolated nucleic acid molecules under stringent conditions. Any one of these isolated nucleic acid molecules can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3' ⁇ terminus. Further provided is a recombinant vector comprising any one of these isolated nucleic acid molecules and a recombinant host cell comprising any one of these isolated nucleic acid molecules. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acids.
  • Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ED NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
  • an isolated nucleic acid molecule encoding any of these polypeptides, an isolated nucleic acid molecule encoding a fragment of any of these polypeptides, an isolated nucleic acid molecule encoding a polypeptide epitope of any of these polypeptides, and an isolated nucleic acid encoding a species homologue of any of these polypeptides.
  • any one of these polypeptides has biological activity.
  • any one of the isolated polypeptides comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
  • a recombinant host cell that expresses any one of these isolated polypeptides.
  • Yet another embodiment of the invention comprises an isolated antibody that binds specifically to an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ DD NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ED NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
  • Yet another embodiment of the invention is a method of identifying an activity of an expressed polypeptide in a biological assay.
  • a polypeptide of the invention is expressed in a cell and isolated.
  • the expressed polypeptide is tested for an activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.
  • Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in atherosclerosis, chosen from the group consisting of the DNA molecules shown in SEQ DD NO: 1, SEQ ED NO:2, SEQ DD NO:3, SEQ DD NO:4, SEQ DD NO:5, SEQ DD NO:6, SEQ DD NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ DD NO: 10, SEQ DD NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ HD NO:15, SEQ ID NO:16, SEQ DD NO:17, SEQ ED NO:18, SEQ DD NO:19, SEQ DD NO:20, SEQ DD NO:21, SEQ DD NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ DD NO:25, SEQ ⁇ NO:26, SEQ DD NO:27, SEQ DD NO:28
  • kits for detecting the presence of a polypeptide of the invention in a mammalian tissue sample comprises a first antibody, which immunoreacts with a mammalian protein encoded by a gene corresponding to the polynucleotide of the invention or with a polypeptide encoded by the polynucleotide in an amount sufficient for at least one assay and suitable packaging material.
  • the kit can further comprise a second antibody that binds to the first antibody.
  • the second antibody can be labeled with enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.
  • kits for detecting the presence of genes encoding a protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.
  • Yet another embodiment of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample. A polynucleotide of the invention or fragment thereof having at least 10 contiguous bases is hybridized with the nucleic acid of the sample. The presence of the hybridization product is detected.
  • Figure 1 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases CCGA (SEQ DD NO:54) and the universal 3' PCR primer (SEQ DD NO:47) showing PCR products produced from mRNA extracted from the aortas of wild-type control mice at 2 months (Panels A and F) and 8 months (Panels B and G), ApoE (-/-) mice at 2 months (Panel C), 4 months (Panel D) and 8 months (Panel E), and LDLR (-/-) mice at 4 months (Panel H) and 8 months (Panel I).
  • the horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA analysis (which corresponds to the relative expression of the molecule of that address).
  • the results of the TOGATM runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699 U.S., and pending PCT Application Serial No. PCT/US00/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and pending U.S. Patent Application Serial No. 09/318,679/U.S. and pending PCT Application Serial No.
  • Figure 2 presents a graphical example of the results obtained when a DST is verified by the Extended TOGA method using a primer generated from a cloned product (as described below).
  • the length of the PCR product corresponding to SEQ DD NO: 17 (DST REC5_7) was cloned and a 5' PCR primer was built from the cloned DST (SEQ DD NO:55).
  • the product obtained from PCR with this primer (SEQ DD NO:55) and the universal 3' PCR primer (SEQ DD NO:47) (as shown in the top panel) was compared to the length of the original PCR product that was produced in the TOGA reaction with mRNA extracted from the 2 month apoE (-/-) sample using a 5' PCR primer with parsing bases CCGA (SEQ DD NO:54) and the universal 3' PCR primer (SEQ DD NO:47) (as shown in the middle panel). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which corresponds to relative expression) is found on the vertical axis.
  • Figure 3 is a graphical representation similar to Fig. 1 of the results of TOGA TM runs using a 5' PCR primer with parsing bases TCTA (SEQ DD NO: 56) and the universal 3' PCR primer (SEQ DD NO:47).
  • the vertical line drawn through the nine panels indicates a PCR product of about 428 b.p. and represents the DST molecule identified as REC4_24 (SEQ DD NO:5).
  • Figure 4 is a graphical representation similar to Fig. 1 of the results of TOGATM runs using a 5' PCR primer with parsing bases AATC (SEQ ED NO: 57) and the universal 3' PCR primer (SEQ DD NO:47).
  • the vertical line drawn through the nine panels indicates a PCR product of about 372 b.p. and represents the DST molecule identified as REC4_3 (SEQ DD NO:7).
  • isolated nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of naturally occurring genomic nucleic acid.
  • the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion
  • isolated polypeptide refers to a polypeptide removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • isolated antibody refers to an antibody removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • Polynucleotide or “polynucleotide of the invention” or “polynucleotide of the present invention” refers to a molecule having a nucleic acid sequence contained in SEQ DD NOs:l-43, 58-62.
  • the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ED NOs:l-43, 58-62 or the complement thereof, or the cDNA.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO (5% w/v non-fat dried milk in phosphate buffered saline (“PBS”), heparin, denatured salmon sperm DNA, and other commercially available proprietary formulations.
  • BLOTTO 5% w/v non-fat dried milk in phosphate buffered saline
  • heparin 5% w/v non-fat dried milk in phosphate buffered saline
  • denatured salmon sperm DNA and other commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA ⁇ sequences (such as any 3' terminal polyA ⁇ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a polyA stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • Polypeptide or “polypeptide of the invention” or “polypeptide of the present invention” refers to a molecule having a translated amino acid sequence generated from the polynucleotide as broadly defined.
  • the translated amino acid sequence beginning with the methionine, is identified although other reading frames can also be easily translated using known molecular biology techniques.
  • the polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques, which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. See references below. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides maybe branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteo lyric processing, phosphorylation, prenylation, racemization, selenoylation, sul
  • a polypeptide has "biological activity" when the polypeptide has structural, regulatory or biochemical functions of a naturally occurring molecule.
  • Biological activity can be measured by several kinds of biological assays, both in vitro (e.g., cell cultures) and in vivo (e.g., behavioral or metabolic assays). In these cases, the potency of the biological activity is measured by its dose-response characteristics; in the case of polypeptides with activity similar to the polypeptide of the present invention, the dose-response dependency will be substantially similar in a given activity as compared to the polypeptide of the present invention.
  • Polypeptides may derive their "biological activity" through binding to specific cellular receptors, which mediate secondary signals to the target cell or tissue.
  • DNA refers to deoxyribonucleic acid.
  • RNA refers to ribonucleic acid.
  • mRNA refers to messenger ribonucleic acid.
  • cDNA refers to a deoxyribonucleic acid that is complementary to an mRNA.
  • Gene refers to a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit encompassing coding DNA sequence, the regions preceding and following the coding region (leader or trailer), noncoding regulatory DNA sequences, and introns.
  • Codon refers to the three-nucleotide sequence of an mRNA molecule that codes for one specific amino acid.
  • Vector refers to a vehicle for transfer of DNA into a recipient cell.
  • Standard mutation or “silent substitution” refers to a mutation that causes no functional change in the gene product.
  • Phenotype refers to the appearance, behavior, or other characteristics of a cell or individual due to actual expression, or pattern of expression, of a specific gene or set of genes. Differences in phenotype may be due to changes in the expression or pattern of expression of a specific gene or set of genes, or to differences in the biological activity of one or more genes. These differences may be a result of polymorphic or allelic differences in the coding region of the specific genes or in their regulatory sequences, or to other genetic variations (e.g., new mutations).
  • Hybridization refers to the time- and temperature-dependent process by which two complementary single-stranded polynucleotides associate to form a double helix.
  • Probe refers to a polynucleotide, often radiolabelled, used to detect complementary sequences, e.g. an mRNA used to locate its gene by a corresponding nucleic acid blotting method.
  • Constant amino acid substitution refers to a substitution between similar amino acids that preserves an essential chemical characteristic of the original polypeptide.
  • Phage refers to a virus that infects bacteria. Many phage have proved useful in the study of molecular biology and as vectors for the transfer of genetic information between cells.
  • “Plasmid” refers to a self-replicating extra-chromosomal element, usually a small segment of duplex DNA that occurs in some bacteria; used as a vector for the introduction of new genes into bacteria.
  • Retrovirus refers to a virus with an RNA genome that may be either an mRNA, (+)-RNA, or its complement, (-)-RNA.
  • Class 1 contains (+)-RNA; class 2, (-)-RNA, which is the template for an RNA-dependent RNA polymerase; class 3, double-stranded RNA, in which (+)-RNA is synthesized by an RNA-dependent RNA polymerase; class 4, retrovirus, in which (+)-RNA is a template for an RNA-dependent DNA polymerase (a reverse transcriptase).
  • a Retrovirus may be used as a vector for the introduction of genes into mammalian cells.
  • Multiple Helix refers to the tertiary structure of collagen that twists three polypeptide chains around themselves; also a triple-stranded DNA structure that involves Hoogstein base pairing between B-DNA and a third DNA strand that occupies the major groove.
  • Antibody refers to an immunoglobulin molecule that reacts specifically with another (usually foreign) molecule, the antigen.
  • mAb Monoclonal antibody
  • mAb refers to an immunoglobulin preparation that is completely homogeneous, due to its formation by daughters of a single progenitor cell that has been programmed for the synthesis and secretion of one specific antibody.
  • Polyclonal antibody refers to a heterogeneous immunoglobulin preparation that contains antibodies directed against one or more determinants on an antigen; the product of daughters of several progenitor cells that have been programmed for immunoglobulin synthesis and secretion.
  • “Complementary” as used in nucleic acid chemistry is descriptive of the relationship between two polynucleotides that can combine in an antiparallel double helix; the bases of each polynucleotide are in a hydrogen-bonded inter-strand pair with a complementary base, A to T (or U) and C to G.
  • a to T or U
  • C to G.
  • protein chemistry the matching of shape and/or charge of a protein to a ligand.
  • C-terminus refers to, in a polypeptide, the end with a free carboxyl group.
  • N-terminus refers to, in a polypeptide, the end with a free amino group.
  • a “secreted” protein refers to those proteins capable of being directed to the endoplasmic reticulum, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention. "Identity" per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo et al., SIAM J Applied Math., 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers,” Martin J. Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra.
  • Epitopes refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human.
  • a preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment.
  • a region of a protein molecule to which an antibody can bind is defined as an
  • an "immunogenic epitope” is defined as a part of a protein that elicits an antibody response. (See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1983)).
  • homologous means corresponding in structure, position, origin or function.
  • a “homologous polynucleotide” refers to a polynucleotide that encodes a homologous polypeptide.
  • a "homologous nucleic acid molecule” refers to a nucleic acid molecule that encodes a homologous polypeptide.
  • a “homologous polypeptide” refers to a polypeptide having any of the following characteristics with respect to the polypeptides of the present invention: similar function, similar amino acid sequence, similar subunit structure and formation of a functional heteropolymer, immuno logical cross-reaction, similar expression profile, similar subcellular location, similar substrate specificity, or similar response to specific inhibitors.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.
  • a “specific binding agent” refers to a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.
  • complex refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction.
  • exemplary complexes are immunoreaction products.
  • label and "indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.
  • package refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene, or polycarbonate), paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody, or monoclonal antibody of the present invention.
  • a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed (i.e., linked) so as to be capable of being immunologically bound by an antibody or antigen, respectively.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • DST refers to a Digital Sequence Tag, i.e., a polynucleotide that is an expressed sequence tag of the 3' end of an mRNA.
  • a number of animal models have been utilized to study the molecular events controlling the development of atherosclerosis and to screen for anti-atherogenic agents.
  • the common feature of all of these models is the requirement for a hypercholesterolemic state to produce atherosclerotic lesions and disease characteristics as seen in humans.
  • the classical models for atherosclerosis research have been those involving diet-induced hypercholesterolemia in primates, guinea pigs and rabbits (Bocan, Current Pharmaceutical Design, 1998).
  • Watanabe rabbit strain has been used a model for genetic predisposition to atherosclerosis disease, as these animals lack functional receptors for low density lipoprotein (LDL) and develop atherosclerotic lesions when fed a high cholesterol diet (Tanzawa, FEBSLett, 1980; Rosenfeld, Arterioscler, 1987).
  • LDL low density lipoprotein
  • these investigations have encountered limitations due to difficulties in conducting large studies over the long time frames that are required for developing atherosclerosis in these animals.
  • the inability in these models to adequately control or manipulate key factors in cholesterol homeostatic pathways has restricted their utility for pinpointing pathways for therapeutic intervention.
  • mice strains with targeted deletions in several key genes controlling plasma lipid levels are highly resistant to atherosclerosis; however, several transgenic strains have been engineered to be prone to fatty lesion formation or, when fed high cholesterol diets, will develop lesions characteristic of those seen in humans (Breslow, Science, 1996; Smith, J Intern Med, 1997).
  • the first transgenic strain to be used as a murine atherosclerosis model was created by gene knockout strategies to produce mice deficient in apolipoprotein E (apoE (-/-)) (Zhang, Science, 1992; Plump, Cell, 1992).
  • ApoE is a surface constituent of lipoprotein particles and a ligand for lipoprotein recognition and clearance.
  • a consequence of apoE deficiency is delayed clearance of lipoprotein and subsequent elevated levels of cholesterol, reaching 400 to 600 mg/dl, as compared to levels in normal mice, which are typically ⁇ lOOmg/dl.
  • apoE null mice fatty lesions containing macrophage-derived foam cells develop by three months after birth (Reddick, Arterioscler Throm Vase Biol, 1994).
  • the fatty streaks and fibrous plaques present at vascular sites are typical of those found in human atherosclerotic lesions. If these same animals are fed a high fat, high cholesterol diet, plasma cholesterol levels climb 3 to 4 times higher compared to control littermates, with lesion development occurring more rapidly and with greater severity than those on normal diets.
  • a second atherosclerosis mouse model is the LDL receptor deficient strain (LDLR (-/- )) (Ishibashi, Proc Nat'lAcad Sci, 1994; Ishibashi, J Clin Invest 1993).
  • the LDL receptors recognize apolipoprotein B (apoB) on LDL and apoE on intermediate density lipoprotein and, following membrane internalization, remove these particles from circulation.
  • apoB apolipoprotein B
  • apoE apolipoprotein B
  • atherosclerotic lesions do not develop normally as seen in the apoE (-/-) mice, but lesions that are morphologically similar can be rapidly induced by a high cholesterol diet (Tangirala, J Lipid Res, 1995).
  • Atherosclerosis- susceptible mouse strains permits further alteration of the mouse genetic background by crossing these animals with other strains of transgenic mice to determine the vulnerability of their offspring to develop lesions.
  • breeding the LDLR (-/-) mice with a strain overexpressing an LDL receptor family type, the scavenger receptor B 1 results in heterozygous offspring with decreased atherosclerosis (Huszar, Arterioscler Thronib Vase Biol, 2000; Arai, JBiol Chem, 1999).
  • Transgenes can be also introduced via viral vector mechanisms into the LDL receptor (-/-) background.
  • the increased plasma levels of apoB resulting from overexpression of an apoB mRNA editing enzyme (apoBecl) led to lower levels of plasma LDL and esterified cholesterol (Teng,
  • oxidized LDL causes a substantial induction of the scavenger receptor CD36 through a PPAR ⁇ mechanism (Tontonoz, Cell, 1998; Chawla, Mol Cell, 2001).
  • the control of cholesterol efflux from macrophage foam cells appears to require the ABC transporter proteins ABCAl and ABCGl, two genes that are highly induced in response to changes in cellular lipid levels (Langmann, Biochem Biophys Res Commun, 1999; Nenkateswaran,
  • the TOGA T method was used to monitor and identify genes whose relative expression changed in a temporal manner compared to age-matched controls in two mouse genetic models of atherosclerosis.
  • Digital sequence tags (DSTs) corresponding to mR ⁇ As were detected as genes whose expression was regulated over the time course of fatty lesion development in the aorta of LDLR (-/-) or apoE (-/-) mice.
  • mice Male and female C57BL/6J control (wild type) mice, C57BL/6J-LDLR (-/-), and C57BL/6J-apoE (-/-) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, US). All groups were maintained in the same conditions. Upon arrival, the animals were kept in groups of 6 animals at 24°C with light cycle of 12 hours. The animals were fed with a standard diet (Mucedola, Italy) with free access to water until the sacrifice time.
  • the mR ⁇ A from the above-described groups was isolated from total aorta at different time points.
  • the mR ⁇ A from control mice (wild type) was isolated at 2 and 8 months of age.
  • the mR ⁇ A from experimental transgenic mice was isolated at 4 and 8 months for LDLR (-/-) mice and at 2, 4, and 8 months for apoE (-/-) mice.
  • Each time point represents a group of 50 animals. Animals were sacrificed by cervical dislocation and the entire aorta from the aortic arch to the femoral fork was isolated and washed briefly with diethyl pyrocarbonate (DEPC)- treated (Sigma, Milano, Italy) PBS and kept on ice.
  • DEPC diethyl pyrocarbonate
  • Aortic mRNAs were prepared from each experimental group following retrieval, wash, and placement in storage in 5 volumes per aorta of RNALater (Ambion, Austin, TX, USA) at -20°C, without jeopardizing the quality or quantity of RNA.
  • RNALater RNALater
  • each aorta was briefly washed in ice cold DEPC-treated PBS and pooled with samples of the same experimental group. Aortas were then shredded using a Polytron Devices, Inc. homogenizer. The nuclei, blood cells, and insoluble extracellular matrix remnants were pelleted by centrifugation.
  • RNA was then precipitated from the aqueous phase with ethanol.
  • the polyA containing mRNA fraction was prepared using standard methods of polyA selection known in the art (Scriber, J Mol Biol, 1980).
  • TOGA TOtal Gene expression Analysis
  • the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art.
  • the TOGA TM method further comprised an additional PCR step performed using four 5' PCR primers in four separate reactions and cDNA templates prepared from a population of antisense cRNAs.
  • a final PCR step that used 256 5' PCR primers in separate reactions produced PCR products that were cDNA fragments that corresponded to the 3 '-region of the starting mRNA population.
  • the produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and isolate the 3' region plus the sequence of the preferably four parsing bases immediately 3' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences. Since the length of the obtained PCR products includes known vector sequences at the 5' and 3' ends of the insert, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.
  • the method yields Digital Sequence Tags, that is, polynucleotides that are expressed sequence tags of the 3' end of mRNAs.
  • DSTs that showed changes in relative levels of expression in experimental transgenic animals compared to wild-type controls were selected for further study.
  • the intensities of the laser-induced fluorescence of the labeled PCR products were compared across samples isolated.
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture or set of all 48 5'-biotinylated anchor primers to initiate reverse transcription.
  • One such suitable set is G-A- A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-N-N-N (SEQ DD NO: 44), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double-stranded cDNA.
  • Each biotinylated double-stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG.
  • the resulting fragments of cDNA corresponding to the 3' region of the starting mRNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin- coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads, and paramagnetic porous glass particles.
  • a preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Great Neck, NY).
  • the cDNA fragment product was released by digestion with Notl, which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • the 3' Mspl-Notl fragments which are of uniform length for each mRNA species, were directionally ligated into Clal- Notl-cleaved plasmid pBC SK+ (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene).
  • the ligation regenerates the Notl site, but not the Mspl site, leaving CGG as the first 3 bases of the 5' end of all PCR products obtained.
  • Each library contained in excess of 5 x 10 recombinants to ensure a high likelihood that the 3' ends of all mRNAs with concentrations of 0.001% or greater were multiply represented. Plasmid preps (Qiagen) were made from the cDNA library of each sample under study.
  • each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3' cDNA inserts and their flanking sequences, including the T3 promoter, intact.
  • the product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.
  • T3 RNA polymerase MEGAscript kit, Ambion
  • each of the cRNA preparations was processed in a three-step fashion, hi step one, 250 ng of cRNA was converted to first-strand cDNA using the 5' RT primer (A-G- G-T-C-G-A-C-G-G-T-A-T-C-G-G, (SEQ ID NO: 45).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 46), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-G-T (SEQ ID NO: 47) to yield four sets of PCR reaction products ("Nl reaction products").
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction.
  • This PCR reaction comprised adding 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 47) conjugated to 6-FAM and 100 ng of the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 48), and using a program that included an annealing step at a temperature X slightly above the Tm of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).
  • N4 reaction products The products (“N4 reaction products”) from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration. Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.
  • ABSI GeneScan software package
  • mRNA samples extracted from aortas of wild-type control mice at 2 months and 4 months of age, aortas of LDLR (-/-) mice at 4 months and 8 months, and aortas of apoE (-/- ) mice at 2, 4, and 8 months as described above were analyzed.
  • a summary of the expression levels for 131 mRNAs identified as modulated in apoE (-/-) mice is shown in Table 1A and for 110 mRNAs identified as modulated in LDLR (-/-) mice in Table IB.
  • the number of differentially regulated mRNAs represents approximately 0.7% of the total number of mRNAs identified by TOGATM as expressed in these tissue samples.
  • these molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment.
  • the 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CC GG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step.
  • the digital address length of the fragment was determined by interpolation on a standard curve and, as such, may vary ⁇ 1-2 b.p. from the actual length as determined by sequencing.
  • the entry in Table 1 A that describes a DNA molecule identified by the digital address Mspl CCGA is further characterized as having a 5' terminus partial nucleotide sequence of CGGCCGA and a digital address length of 310 b.p.
  • the DNA molecule identified as Mspl CCGA310 is further described as being expressed in the aortas of apoE (-/- ) mice at 2, 4 and 8 months of age (see Figure 1).
  • the DNA molecule identified as MspI CCGA310 (REC5 7) is described by its nucleotide sequence, which corresponds with SEQ ID NO: 17.
  • the other DNA molecules identified in Table 1 A by their Mspl digital addresses are further characterized by: 1) the level of gene expression in the aortas of wild- type control mice at 2 months, 2) the level of gene expression in the aortas of wild-type control mice at 8 months, 3) the level of gene expression in the aortas of apoE (-/-) mice at 2 months, 4) the level of gene expression in the aortas of apoE (-/-) mice at 4 months, 5) the level of gene expression in the aortas of apoE (-/-) mice at 8 months.
  • the other DNA molecules identified in Table IB by their Mspl digital addresses are further characterized by: 1) the level of gene expression in the aortas of wild-type control mice at 2 months, 2) the level of gene expression in the aortas of wild-type control mice at 8 months, 3) the level of gene expression in the aortas of LDLR (-/-) mice at 4 months, 4) the level of gene expression in the aortas of apoE (-/-) mice at 8 months.
  • DSTs were further characterized as shown in Tables 2, 3, 4, and 5.
  • the ligation of the sequence into a vector does not regenerate the Mspl site; the experimentally determined sequence reported herein has C-G-G as the first bases of the 5' end.
  • the data shown in Figures 1, 3, and 4 were generated with the 5' -PCR primers
  • Figures 1, 3 and 4 are graphical representations of the results of TOGA using 5' PCR primers with parsing bases CCGA (SEQ ID NO:54), TCTA (SEQ DD NO:56), and AATC (SEQ HD NO:57) combined with the universal 3' PCR primer (SEQ DD NO:47) showing PCR products produced from mRNA extracted from the aortas of wild-type control mice at 2 months (Panels A and F) and 8 months (Panels B and G), apoE (-/-) mice at 2 months (Panel C), 4 months (Panel D) and 8 months (Panel E), and LDLR (-/-) mice at 4 months (Panel H) and 8 months (Panel I).
  • the vertical index line in each panel indicates a PCR product of about 310 b.p. that is expressed to a greater level in the apoE (-/-) 2 month and 4 month samples compared to the 2 month or 8 month wild-type controls, with no differences detected between any of the LDLR (-/-) samples and control samples.
  • the vertical index line in each panel indicates a PCR product of about 428 b.p. that is expressed to a greater level in the LDLR (-/-) 4 month and 8 month samples compared to the 2 or 8 month controls, with no differences detected between any of the apoE (-/-) samples and control samples.
  • the vertical index line in each panel indicates a PCR product of about 372 b.p.
  • the TOGA PCR product was sequenced using a modification of a direct sequencing methodology (frinis et al., Proc. Nat'l. Acad. Sci., 85: 9436-9440 (1988)).
  • PCR products corresponding to DSTs were gel purified and PCR amplified again to incorporate sequencing primers at 5' and 3' ends.
  • the sequence addition was accomplished through 5' and 3' ds-primers containing M13 sequencing primer sequences (M13 forward and Ml 3 reverse respectively) at their 5' ends, followed by a linker sequence and a sequence complementary to the DST ends.
  • a master mix containing all components except the gel purified PCR product template was prepared, which contained sterile H 2 0, 10X PCR H buffer, lOmM dNTP, 25 mM MgCl 2 , AmpliTaq/ Antibody mix (1.1 ⁇ g/ ⁇ l Taq antibody, 5 U/ ⁇ l AmpliTaq), 100 ng/ ⁇ l of 5' ds- primer (5' TCC CAG TCA CGA CGT TGT AAA ACG ACG GCT CAT ATG AAT TAG GTG ACC GAC GGT ATC GG 3', SEQ DD NO: 49), and 100 ng/ ⁇ l of 3' ds-primer (5' CAG CGG ATA ACA ATT TCA CAC AGG GAG CTC CAC CGC GGT GGC GGC C 3', SEQ DD NO: 50).
  • PCR was performed using the following program: 94°C, 4 minutes and 25 cycles of 94°C, 20 seconds; 65°C, 20 seconds; 72°C, 20 seconds; and 72°C 4 minutes.
  • the resulting amplified adapted PCR product was gel purified.
  • the purified PCR product was sequenced using a standard protocol for ABI 3700 sequencing. Briefly, triplicate reactions in forward and reverse orientation (6 total reactions) were prepared, each reaction containing 5 ⁇ l of gel purified PCR product as template. In addition, the sequencing reactions contained 2 ⁇ l 2.5X sequencing buffer, 2 ⁇ l Big Dye
  • the 3' sequencing primer was the sequence 5' GGT GGC GGC CGC AGG AAT TTT TTT TTT TTT TT 3 ', (SEQ DD NO: 53). PCR was performed using the following thermal cycling program: 96°C, 2 minutes and 29 cycles of 96°C, 15 seconds; 50°C, 15 seconds; 60°C, 4 minutes.
  • Table 2 contains the database matches for the sequences determined by this method.
  • DST REC5_2 SEQ DD NO: 28
  • the DNA molecule identified by Mspl was one such direct sequenced product.
  • the extended TOGA assay was performed for each DST (see below).
  • Extended TOGATM assay Verification Using the Extended TOGA Method hi order to verify that the TOGA M peak of interest corresponds to the identified DST, an extended TOGATM assay was performed for each DST as described below.
  • PCR primers ("Extended TOGA primers") were designed from sequence determined using one of three methods: (1) in suitable cases, the PCR product was isolated, cloned into a TOPO vector
  • the TOGA PCR product was sequenced using a modification of a direct sequencing methodology (Innis et al., Proc. NatT. Acad. Sci., 85: 9436-9440 (1988)) or (3) in other cases, the sequences listed for the TOGATM PCR products were derived from candidate matches to sequences present in available GenBank, EST, or proprietary databases.
  • PCR was performed using the Extended TOGA primers and the Nl PCR reaction products as a substrate. Oligonucleotides were synthesized with the sequence G-A-T-C-G-A- A-T-C extended at the 3' end with a partial Mspl site (C-G-G), and an additional 18 adjacent nucleotides from the determined sequence of the DST.
  • the 5' PCR primer was G-A-T-C-G-A-A-T-C-C-G-G-C-C-G-A-G-G-A-T-C-G-A-G-T-C-T-T-A (SEQ ID NO:55).
  • This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ DD NO:47) in a PCR reaction using the PCR Nl reaction product as substrate.
  • the length of the PCR product generated with the Extended TOGA p ⁇ mer was compared to the length of the original PCR product that was produced in the TOGA reaction.
  • the results for SEQ DD NO:17, for example, are shown in Figure 2.
  • the length of the PCR product corresponding to SEQ DD NO: 17 (REC5_7) was cloned and a 5' PCR primer was built from the cloned DST (SEQ DD NO:55).
  • the product obtained from PCR with this primer (SEQ DD NO:55) and the universal 3' PCR primer (SEQ DD NO:47) (as shown in the top panel) was compared to the length of the original PCR product that was produced in the TOGA reaction with mRNA extracted from the apoE (-/-) 2 month sample using a 5' PCR primer with parsing bases CCGA (SEQ DD NO:54) and the universal 3' PCR primer (SEQ DD NO:47) (as shown in the middle panel). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which corresponds to relative expression) is found on the vertical axis.
  • sequences listed for the TOGA PCR products were derived from candidate matches to sequences present in available Genbank, EST, or proprietary databases. Table 3 lists the candidate matches for each by accession number of the Genbank entry or by the accession numbers of a set of computer-assembled ESTs used to create a consensus sequence. Extended TOGATM primers were designed based on these sequences (as mentioned previously), and Extended TOGATM was run to determine if the database sequences were the DSTs amplified in TOGATM.
  • sequence includes nucleotide and amino acid sequences.
  • the query sequence can be either protein or nucleic acid or any combination therein.
  • BLAST is a statistically driven search method that finds regions of similarity between a query and database sequences. These are called segment pairs, and consist of gap less alignments of any part of two sequences. Within these aligned regions, the sum of the scoring matrix values of their constituent symbol pairs is higher than a level expected to occur by chance alone.
  • the scores obtained in a BLAST search can be interpreted by the experienced investigator to determine real relationships versus random similarities.
  • the BLAST program supports four different search mechanisms:
  • DST sequences of the present invention were also searched against a database containing computer-assembled consensus sequences derived from mouse, rat and human EST databases.
  • the assemblies were derived using the Paracel Clustering Program (Paracel, Pasadena, CA).
  • the cluster database was searched using BLAST and the resulting matches are indicated in Table 4.
  • the data from Table 1 A and B identify a group of genes that are modulated in response to the genetic loss of apoE and LDLR in transgenic animals.
  • the mRNAs detected in Table 1 whose relative levels of expression were determined by TOGA as differentially expressed and whose sequence identification was completed in Tables 2 and 3 were further analyzed to categorize the patterns of regulation detected in the apoE (-/-) and LDLR (-/-) mice compared to age-matched controls.
  • Genes were organized into seven distinct categories, namely, those 1) up-regulated in aorta of apoE (-/-) mice ( Figure 1), 2) up-regulated in aorta of LDLR (-/-) mice ( Figure 3), 3) up-regulated in aorta of both apoE (-/-) and LDLR (-/-) mice ( Figure 4), 4) down-regulated in aorta of apoE (-/-) mice, 5) down-regulated in aorta of LDLR (-/-) mice, 6) down-regulated in aorta of both apoE (-/-) and LDLR (-/-) mice, and 7) genes that show opposite regulation in aorta of apoE (-/-) mice compared to LDLR (-/-) mice.
  • genes were identified that had no known matches to homologs in sequence databases or whose predicted gene product encoded a protein of unknown biological function. The difference in the consequence and severity of the genetic deficit in these transgenic models is reflected in the patterns of co- expression of these regulated genes. Of the 43 DSTs evaluated, only 6 were co-expressed in a similar manner (either up or down-regulated in both models), suggesting alteration of LDL levels in these animals can result in complex effects on physiology.
  • the differentially expressed nucleotide sequences or their corresponding polypeptides can be used as a diagnostic fingerprint indicative of the status of LDL or apoE levels, or of disease progression in atherosclerosis. Such a diagnostic use can be performed with TOGA or other simultaneous gene expression monitoring techniques, including hybridization-based cDNA or oligonucleotide arrays and sequence tag-based methods.
  • Tables 1 and 5 are useful not only in predicting which genes and pathways (see below) are subject to regulation during changes in disease state, lipid, cholesterol, or lipoprotein levels, but also provide an indication of when to therapeutically intervene in order to deter or reverse the progression of atherosclerosis disease.
  • a complete understanding of diseases, such as atherosclerosis, and the development of new pharmaceutical intervention strategies to prevent or ameliorate disease progression will require an ability to pinpoint the cellular and molecular mechanisms responsible for the underlying pathology. Identifying the genes and regulatory pathways involved in atherosclerosis will provide a foundation for a molecular description of the disease process.
  • the TOGA process revealed a large set of differentially expressed genes in aortic tissues of apoE (-/-) and LDLR (-/-) mouse models of atherosclerosis.
  • the cloning and sequencing of the differentially expressed PCR products generated by TOGATM, use of the extended TOGA assay, and performance of sequence database searches for each DST provided gene identities for 43 of these molecules, as indicated in Tables 2 and 3.
  • the biological function for 26 of the encoded protein products are known and were previously established in the literature or deduced by homology to proteins of known function in other species. The function of each these 26 DSTs is listed in Table 5.
  • Control of lipoprotein metabolism was identified by the altered expression of the mRNAs encoding an LDLR- family member, LRU, in LDLR deficient mice, apolipoprotein D in both apoE (-/-) and LDLR (-/-) animals, and Apobec2 and apolipoprotein Cl in apoE (-/-) tissues.
  • cholesterol metabolic control was implicated by identification of increased expression of cypl l in apoE (-/-) mice.
  • TOGA For each of these genes, the changes in expression shown by TOGA would serve to counter the atherogenic effect of increased plasma LDL levels caused by the loss of function of apoE or LDLR, as indicated in Table 5. Of particular importance, TOGA demonstrates that these expression changes occur in cell types present in the aorta, and suggests that therapeutic benefit might derive from intervention at sites in the aortic vessel wall undergoing fatty lesion development.
  • a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as atherosclerosis
  • the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition.
  • This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention (or a set of polynucleotides and polypeptides including those of the invention) to the mammalian subject.
  • a polynucleotide of the invention can be administered alone or with other polynucleotides to a mammalian subject by a recombinant expression vector comprising the polynucleotide.
  • a mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat, rabbit, guinea pig, rat or mouse.
  • the recombinant vector comprises a polynucleotide shown in SEQ DD NOs: 1-43, 58-62 or a polynucleotide which is at least 98% identical to a nucleic acid sequence shown in SEQ DD NOs: 1-43, 58-62.
  • the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ⁇ NOs: 1-43, 58-62.
  • a polynucleotide or recombinant expression vector of the invention can be used to express a polynucleotide in said subject for the treatment of, for example, atherosclerosis.
  • Expression of a polynucleotide in target cells including but not limited to monocytes, macrophages, smooth muscle cells, and endothelial cells would effect greater production of the encoded polypeptide.
  • target cells including but not limited to monocytes, macrophages, smooth muscle cells, and endothelial cells would effect greater production of the encoded polypeptide.
  • the regulation of other genes may be secondarily up- or down-regulated.
  • a naked polynucleotide can be administered to target cells.
  • Polynucleotides and recombinant expression vectors of the invention can be administered as a pharmaceutical composition.
  • Such a composition comprises an effective amount of a polynucleotide or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration.
  • Suitable formulation materials preferably are 5 non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • the pharmaceutically active compounds i.e., a polynucleotide or a vector
  • the pharmaceutical composition comprising a polynucleotide or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the dosage regimen for treating a disease with a composition comprising a polynucleotide or expression vector is based on a variety of factors, including the type or severity of the atherosclerosis, the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A typical dosage may
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • 25 may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely __________________r ⁇ , j ⁇ h ⁇ £ ⁇ j ⁇ Q ⁇ ⁇
  • the cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro.
  • Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo maybe accomplished using any of a variety of techniques well known to those skilled in the art.
  • U.S. Patent No. 5,672,344 describes an in vivo viral- mediated gene transfer system involving a recombinant neuro trophic HSV-1 vector.
  • compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray.
  • parenteral' '.as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • Another delivery system for polynucleotides of the invention is a "non- viral" delivery system.
  • Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110):926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R., (1993) Science, 260 (5110):926-32).
  • a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as atherosclerosis
  • the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition.
  • This can be accomplished by, for example, the use of antisense oligonucleotides, triple helix base pairing methodology or ribozymes.
  • drugs or antibodies that bind to and inactivate the polypeptide product can be used.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxyrnethyl esters, carbonates, and phosphate triesters.
  • Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a polynucleotide.
  • Antisense oligonucleotides that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide of the invention. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol., 10:152-158; Uhlmann et al., (1990) Chem. Rev., 90:543-584; Uhlmann et al., (1987) Tetrahedron. Lett, 215:3539-3542.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) Ann. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct.
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a polynucleotide of the invention can be used to generate ribozymes that will specifically bind to mRNA transcribed from the polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al., EP 321,201).
  • Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • nucleotide sequences shown in SEQ ED NOs: 1-43, 58-62 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Pathological conditions or susceptibility to pathological conditions can be diagnosed using methods of the invention. Testing for expression of a polynucleotide of the invention or for the presence of the polynucleot de product can correlate with the severity of the condition and can also indicate appropriate treatment. For example, the presence or absence of a mutation in a polynucleotide of the invention can be determined through sequencing techniques known to those skilled in the art and a pathological condition or a susceptibility to a pathological condition is diagnosed based on the presence or absence of the mutation.
  • an alteration in expression of a polypeptide encoded by a polynucleotide of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a first biological sample from a patient suspected of having a pathological condition is obtained along with a second sample from a suitable comparable control source.
  • a biological sample can comprise saliva, blood, cerebrospinal fluid, amniotic fluid, urine, feces, or tissue, such as gastrointestinal tissue.
  • a suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition.
  • the average concentrations and distribution of a polynucleotide or polypeptide of the invention can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amounts of the polypeptide in the first and second samples are compared.
  • a patient is diagnosed as having a pathological condition if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • the amount of polypeptide in the first sample falls within the range of samples taken from a representative group of patients with the pathological condition.
  • the method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
  • the present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such as brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.
  • a body sample such as brain tissue, cell suspensions or tissue sections
  • a body fluid sample such as CSF, blood, plasma or serum
  • a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene corresponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell.
  • the nucleic acid molecule probes can be of a variety of lengths from at least about 10, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length.
  • the probe can be used to detect the polynucleotide through hybridization methods, which are extremely well known in the art and will not be described further here.
  • PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected.
  • the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention.
  • Particularly preferred PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions that are not conserved in other cellular proteins.
  • PCR primer pairs useful for detecting the genes corresponding to the polynucleotides of the present invention and expression of these genes are described in the TOGA Process Section above and in the Tables. Nucleotide primers from the corresponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the corresponding gene in any of a variety of tissues.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample.
  • Such diagnostic kit would be useful for monitoring the fate of a therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention.
  • the system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent.
  • a diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.
  • Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
  • Such labels are themselves well- known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • the labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immuno fluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC),
  • the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
  • An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3- ethyl-benzthiazoline-G-sulfonic acid) (ABTS). Radioactive elements are also useful labeling agents and are used illustratively herein.
  • Elements which themselves emit gamma rays such as I, I, I, I and Cr represent one class of gamma ray emission-producing radioactive element indicating groups.
  • Another group of useful labeling means are those elements such as ⁇ C, 18 F, 15 O and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ⁇ ⁇ indium or 3 H.
  • antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al., Meth. EnzymoL, 73:3-46 (1981)).
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, e.g., Aurameas, et al, Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al.,
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
  • the specific binding agent binds the reagent species when that species is present as part of a complex.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
  • the diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample.
  • ELISA ELISA-activated immunosorbent assay
  • a polypeptide of the present invention an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
  • a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein. Useful solid matrices are also well known in the art.
  • Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose, polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, EL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchlori.de.
  • SEPHADEX Pharmacia Fine Chemicals
  • agarose agarose
  • polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, EL)
  • polyvinyl chloride polystyrene
  • the reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophihzed form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • the present invention also relates to the genes corresponding to SEQ DD NOs: 1-43, 58-62 and translations of SEQ DD NOs:l-43, 58-62.
  • the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
  • homologues including paralogous genes and orthologous genes. Homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. See, e.g., Curr. Prot. Mol. Bio., Chapter 16.
  • the polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below).
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified by the one-step method described in Smith & Johnson (Gene, 67:31-40, 1988).
  • Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods, which are well known in the art.
  • the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty.
  • the present invention provides secreted polypeptides having a sequence corresponding to the translations of SEQ DD NOs: 1-43, 58-62 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • SEQ DD NOs: 1-43, 58-62 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • polypeptides and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
  • Polynucleotide or polypeptide variants differ from the polynucleotides or polypeptides of the present invention, but retain essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention. Further embodiments of the present invention include polynucleotides having at least
  • the above polypeptides should exhibit at least one biological activity of the protein.
  • polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95 %> similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ED NO: 1-43, 58-62.
  • Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and Bestf ⁇ t program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) which uses the local homology algorithm of Smith and Waterman (Adv. in App.Math., 2:482-489 (1981)).
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)).
  • sequence includes nucleotide and amino acid sequences.
  • sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is presented in terms of percent identity.
  • a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ DD NOs: 1-43, 58-62 means that the polynucleotide is identical to a sequence contained in SEQ ED NOs: 1-43, 58-62 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide stretch).
  • a polynucleotide having a nucleotide sequence at least 95% identical to SEQ DD NOs: 1-43, 58-62 up to 5% of the nucleotides in the sequence contained in SEQ ED NOs: 1-43, 58-62 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide means that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA maybe changed to those preferred by a bacterial host, such as E. col ⁇ ).
  • the variants may be allelic variants.
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, ⁇ d., Genes II, John Wiley & Sons, New York (1985)).
  • allelic variants can vary at either the polynucleotide and/or polypeptide level.
  • non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. See, e.g., Curr. Prot. Mol. Bio., Chapter 8.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation.
  • aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al., Clin. Exp. Immunol 2:331-340 (1967); Robbins et al., Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap. Drug Carrier Sys., 10:307-377 (1993)).
  • interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J Biotechnology, 7:199-216 (1988)).
  • the invention further includes polypeptide variants that show substantial biological activity.
  • variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rales known in the art so as have little effect on activity.
  • guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions that have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions that are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function.
  • site-directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al., Science, 244: 1081-1085 (1989)).
  • the resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp; and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol);
  • polypeptide (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • additional amino acids such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ED NOs: 1-43, 58-62.
  • the short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ED NOs: 1-43, 58-62.
  • These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and greater than 150 nucleotides) are preferred.
  • polynucleotide fragments of the invention include, for example, fragments having a sequence from about nucleotide number 1-50, 51- 100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of SEQ DD NOs: 1-43, 58-62.
  • “about” includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt) at either terminus or at both termini.
  • these fragments encode a polypeptide that has biological activity.
  • polypeptide fragment refers to a short amino acid sequence contained in the translations of SEQ HD NOs: 1-43, 58-62. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length. In this context "about” includes the particularly recited ranges, larger or smaller by several amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha- helix-forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Polypeptide fragments of the translations of SEQ DD NOs: 1-43, 58-62 falling within conserved domains are specifically contemplated by the present invention.
  • polynucleotide fragments encoding these domains are also contemplated.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl Acad. Sci. USA, 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211.
  • antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies that specifically bind the epitope. (See, e.g., Wilson et al., Cell, 31:161-11 (1984); Sutcliffe et al, Science, 219:660-666 (1983)).
  • immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, e.g., Sutcliffe et al, (1983) Supra; Wilson et al., (1984) Supra; Chow et al., Proc. Natl. Acad. Sci., USA, 82:910-914; and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985)).
  • a preferred immunogenic epitope includes the secreted protein.
  • the immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse).
  • the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids).
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med., 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and human and humanized antibodies.
  • the antibodies maybe chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. Co et al. (Nature, 351:501-2, 1991).
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • One method for producing a human antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-43, 58-62, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-43, 58-62 are generated in said animal.
  • Procedures have been developed for generating human antibodies in non- human animals.
  • the antibodies may be partially human, or preferably completely human.
  • mice have been prepared in which one or more endogenous immunoglobulin genes are inactivated by various means and human immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes.
  • transgenic mice may be genetically altered in a variety of ways.
  • the genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
  • Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-43, 58-62 are provided herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule.
  • the spleen cells may be fused with myeloma cells to produce hybridomas by conventional procedures. Examples of such techniques are described in U.S. Patent No. 4, 196,265, which is incorporated by reference herein.
  • a method for producing a hybridoma cell line comprises immunizing such a transgenic animal with an immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1- 43, 58-62; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-43, 58-62.
  • hybridoma cell lines and monoclonal antibodies produced therefrom, are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques. Examples of such techniques are described in U.S. Patent No. 4,469,630 and U.S. Patent No. 4,361,549.
  • Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-43, 58-62. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1- 43, 58-62.
  • antibody blockade of the LDL lipoproteins to receptors at sites of endothelium injury may inhibit the formation of atherosclerotic plaques.
  • antibody blockade of macrophage or platelet adhesion to endothelial lesions may block the initiation of plaque formation.
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ED NOs: 1-43, 58-62.
  • detectable or therapeutic agent attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ED NOs: 1-43, 58-62.
  • agents include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs. See, e.g., Thrush et. al (Annu.Rev. Immunol, 14:49-71, 1996, p. 41).
  • the conjugates find use in in vitro or in vivo procedures.
  • any polypeptide of the present invention can be used to generate fusion proteins.
  • the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
  • domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides of the present invention including fragments and, . specifically, epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • EP A 0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pha ⁇ nacokinetic properties (see, e.g., EP A 0 232 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of ML- 5 (See, Bennett et al., J Mol. Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem., 270:9459-9471 (1995)).
  • the polypeptides of the present invention can be fused to marker sequences, such as a peptide that facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821 -824 (1989)).
  • HA hemagglutinin protein
  • fusion proteins may use the ability of the polypeptides of the present invention to target the delivery of a biologically active peptide. This might include focused delivery of a toxin to tumor cells, or a growth factor to stem cells.
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6.
  • the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. See, e.g., Curr. Prot. Mol. Bio., Chapters 9.9, 16.15.
  • the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16A, PNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector.
  • a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • Polypeptides of the present invention can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
  • the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ D NOs: 1-43, 58-62. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ED NOs: 1-43, 58-62 will yield an amplified fragment.
  • somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments.
  • Other gene-mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • FISH fluorescence in situ hybridization
  • the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Prefe ⁇ ed polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross-hybridization during chromosomal mapping.
  • the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease.
  • polynucleotide and the co ⁇ esponding gene between affected and unaffected individuals can be examined.
  • the polynucleotides of SEQ DD NOs: 1-43, 58-62 can be used for this analysis of individuals. These may be indirect, through associations with risk factors such as diabetes, or direct, through genetic defects in lipid or cholesterol metabolism. These can be used as markers to identify individuals with susceptibility to atherosclerosis.
  • a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al., Nuc. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al, Science, 251:1360 (1991) for discussion of triple helix formation) or to the mRNA itself (see, Okano, J. Neurochem, 56:560 (1991); and
  • Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) for a discussion of antisense technique).
  • Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.
  • a polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
  • protein expression in tissues can be studied with classical immunohisto logical methods (Jalkanen, et a , J Cell. Biol, 101 :976-985 (1985); Jalkanen, et al., J. Cell. Biol, 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RJA). See, e.g., Curr.
  • Suitable antibody assay labels include enzyme labels, such as glucose oxidase; and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( m In), and technetium ( 99m Tc); fluorescent labels, such as fluorescein and rhodamine; and biotin.
  • proteins can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR), or electron spin resonance (ESR).
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a protein-specific antibody or antibody fragment that has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., 131 I, ⁇ ⁇ In, 99m Tc), a radio- opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal.
  • a radioisotope e.g., 131 I, ⁇ ⁇ In, 99m Tc
  • a radio- opaque substance e.g., a radio- opaque substance, or a material detectable by NMR
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells that contain the specific protein.
  • In vivo tumor imaging is described in Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982)).
  • the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
  • a diagnostic method of a disorder which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
  • macrophages activated in atherosclerotic lesions may show specific changes in gene expression. If these changes are also found in macrophages circulating in peripheral blood, this may be detected in blood samples from patients.
  • polypeptides of the present invention can be used to treat disease.
  • patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide; to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.g., by binding to a receptor); to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble tumor necrosis factor (TNF) receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth).
  • a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide; to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.
  • antibodies directed to a polypeptide of the present invention can also be used to treat disease.
  • administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • Polypeptides can be used as antigens to trigger immune responses.
  • activated macrophages that are filled with lipid in atherosclerotic lesions may express genes unique to this activated state. Immunization against these markers may stimulate antibody responses or cellular immune responses that could eliminate the lipid-laden macrophages, and eliminate the atherosclerotic lesions.
  • Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. See, e.g., Curr. Prot. Mol. Bio., Chapter 11.15. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
  • polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of neuroblasts, stem cells, or glial cells. Also, a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the mechanisms of synaptic transmission, synthesis, metabolism and inactivation of neural transmitters, neuromodulators and trophic factors, and by activating or inhibiting the expression and incorporation of enzymes, structural proteins, membrane channels, and receptors in neurons and glial cells.
  • the etiology of these deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorder), acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular nervous system disease or disorder.
  • the disorder or disease can be any of Alzheimer's disease, Pick's disease, Binswanger's disease, other senile dementia, Parkinson's disease, parkinsonism, obsessive compulsive disorders, epilepsy, encephaolopathy, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder.
  • the polypeptide or polynucleotide of the present invention can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus.- Additionally, particularly with reference to mRNA species occurring in particular structures within the central nervous system, the polypeptide or polynucleotide of the present invention can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • the etiology of these immune deficiencies or disorders maybe genetic, somatic (such as cancer or some autoimmune disorders) acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
  • a polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells.
  • a polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
  • immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g., agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Di George's Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCDDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
  • blood protein disorders e.g., agammaglobulinemia, dysgammaglobulinemia
  • ataxia telangiectasia common variable immunodeficiency
  • common variable immunodeficiency e.g., Di George's Syndrome
  • HIV infection e.g., HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte
  • a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (bleeding cessation) or thrombolytic activity (clot formation).
  • a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
  • a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting.
  • a polynucleotide or polypeptide of the present invention may also be useful in the treatment or detection of autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, or in some way results in the induction of tolerance, may be an effective therapy in preventing autoimmune disorders.
  • autoimmune disorders examples include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospho lipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention.
  • these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
  • Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
  • an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells maybe an effective therapy in preventing organ rejection or GVHD.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate inflammation.
  • the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with atherosclerosis, infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over roduction of cytokines (e.g., TNF or IL-1.)
  • cytokines e.g., TNF or IL-1.
  • a polypeptide or polynucleotide can be used to treat or detect hyperproliferative disorders, including neoplasms.
  • a polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.
  • a polypeptide or polynucleotide of the present invention may proliferate other cells that can inhibit the hyperproliferative disorder.
  • hyperproliferative disorders can be treated.
  • This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • decreasing an immune response may also be a method of treating hyperproliferative disorders, such as by administering the polypeptide or polynucleotide as a chemotherapeutic agent.
  • other hyperproliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention.
  • hyperproliferative disorders include, but are not limited to hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases maybe treated.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention.
  • viruses include, but are not limited to, the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis),
  • Herpesviridae such as Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentiviras), and Togaviridae (e.g., Rubiviras).
  • Mononegavirus e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae
  • Orthomyxoviridae e.g., Influenza
  • Papovaviridae e.g., Parvoviridae
  • Picornaviridae Pi
  • Virases falling within these families can cause a variety of diseases or symptoms, including, but not limited to, arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., ADDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemo ⁇ hagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's warts), and viremia.
  • arthritis bronchiollitis, encephalitis
  • eye infections e.g., conjunctivitis, keratitis
  • chronic fatigue syndrome e.g., conjunctivitis, keratitis
  • hepatitis A, B,
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to, the following Gram-Negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Bo ⁇ elia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsielia, Salmonella, Serratia, Yersinia), Erysipelothr
  • bacteremia e.g., endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., ADDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections (such as whooping Cough or empyema), sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually-transmitted diseases, skin diseases (e.g., cell
  • parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,
  • Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas can cause a variety of diseases or symptoms, including, but not limited to, Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., ADDS related), Malaria, pregnancy complications, and toxoplasmosis.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy).
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
  • a polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues (see, Science, 276:59-87 (1997)).
  • the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery (including cosmetic plastic surgery), fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, ligament) tissue.
  • organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • nervous hematopoietic
  • skeletal bone, cartilage, tendon, ligament
  • Regeneration also may include angiogenesis.
  • atherosclerosis improper healing of vascular endothelium lesions may be the primary trigger of atherosclerotic plaque formation. Molecules that may induce more efficient wound healing may prevent plaque formation.
  • a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon ligament regeneration would quicken recovery time after damage.
  • a polynucleotide or polypeptide of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.
  • tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present invention to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke).
  • diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) could all be treated using the polynucleotide or polypeptide of the present invention.
  • a polynucleotide or polypeptide of the present invention may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, f ⁇ broblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • a polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells.
  • chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body.
  • chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location.
  • Chemotactic molecules of the present invention can also attract f ⁇ broblasts, which can be used to treat wounds.
  • a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. Such molecules could also be used to treat a variety of disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis. Blockade of this step may prevent development of atherosclerotic plaques.
  • Atherosclerotic plaque develops over several decades and involves inflammatory cell infiltration, smooth muscle cell proliferation, accumulation of extracellular matrix, fibrous cap formation, and angiogenesis.
  • Chemotaxis is involved in the early development of atherosclerosis. Cell populations migrate toward the inner part of the vascular wall and originate the neointima, which leads to the formation of an atherosclerotic plaque. For example, monocyte chemotaxis is induced by monocyte chemoattractant protein 1 (MCP-1), which is expressed early in the development of atherosclerosis in the injured arterial wall.
  • MCP-1 monocyte chemoattractant protein 1
  • IGF insulin-like growth factors
  • vascular endothelial growth factor has been shown to be a critical regulator of angiogenesis that stimulates proliferation, migration and proteolytic activity of endothelial cells.
  • VEGF vascular endothelial growth factor
  • VEGF is able to stimulate chemotaxis in monocytes and can enhance matrix metalloproteinase expression and accelerate smooth muscle cell migration.
  • Blockade of one or more of these chemotaxic activities may prevent development of atherosclerotic plaques.
  • a polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic (see, Coligan et al., Current Protocols in Immunology 1(2), Chapter 5 (1991)).
  • the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site). In either case, the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells that express the polypeptide, either as a secreted protein or on the cell membrane.
  • Prefe ⁇ ed cells include cells from mammals, yeast, Drosophila, or E. coli.
  • Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
  • the assay can be carried out using cell- free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
  • the assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
  • an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
  • the antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
  • All of these above assays can be used as diagnostic or prognostic markers.
  • the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule.
  • the assays can discover agents that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
  • many of the diagnostic tools are only able to identify risk factors for atherosclerosis (e.g., hyperlipidemia), and do not indicate the presence of actively developing atherosclerotic plaques.
  • New assays using markers generated from materials of the present invention may provide some specific indicators of active disease.
  • the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occu ⁇ ed.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.
  • a polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells from a lineage other than the above-described hemopoietic lineage.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).
  • a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
  • a polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, the response to opiates and opioids, tolerance to opiates and opioids, withdrawal from opiates and opioids, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
  • a polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors, or other nutritional components.
  • Bocan TMA Animal models of atherosclerosis and interpretation of drug intervention studies. Current Pharmaceutical Design 1998;4:37-52.
  • HBP High-density lipoprotein- binding protein
  • Sobue K, Hayashi K, Nishida W Expressional regulation of smooth muscle cell- specific genes in association with phenotypic modulation. Mol Cell Biochem 1999; 190(1 - 2):105-18. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine models: co ⁇ elation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res 1995;36(11):2320-2328.
  • TOGA gene expression data are shown for mRNAs identified by their digital address. RNA samples were analyzed in duplicate (labeled 1 and 2 in the column headings for each condition) and the relative fluorescent units detected for the peak at each digital address are presented. TOGA expression data associated with a respective SEQ ID NO and DST ID are given in the Table.
  • Table 5 The pattern of gene expression changes in LDLR (-/-) and apoE (-/-) mice for each identified DST listed in Table 2 and 3 was determined and placed into one of seven distinct categories. For each condition, changes in relative expression levels of the individual DSTs were compared to age-matched, wild-type controls. Alterations in expression were denoted as UP (greater than 1.8 fold versus control), DOWN (less than 1.8 fold versus control), or ND (no significant difference detected). The corresponding gene identity and biological function of the encoded protein is shown for each DST. The atherogenic effect of the gene expression change, where known, is indicated in the rightmost column.

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Abstract

L'invention concerne des polynucléotides, des polypeptides, des trousses et des méthodes associées à des gènes régulés caractéristiques de l'athérosclérose.
PCT/US2001/043741 2000-11-15 2001-11-15 Modulation de l'expression genetique dans des modeles genetiques murins de l'atherosclerose WO2002081726A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5871995A (en) * 1989-08-15 1999-02-16 Shiseido Company, Ltd. Purified enzymes participating in C-terminal amidation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5871995A (en) * 1989-08-15 1999-02-16 Shiseido Company, Ltd. Purified enzymes participating in C-terminal amidation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] MARRA ET AL.: 'The WashU-NCI EST project 1999', XP002959566 Retrieved from NCBI Database accession no. (AI591507) *
OGONOWSKI ET AL.: 'Antiinflammatory and analgesic activity of an inhibitor of neuropeptide amidation' THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS vol. 280, no. 2, 1997, pages 846 - 853, XP002959564 *

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