EP1311688A2 - Lipase humaine 50250 et utilisations - Google Patents

Lipase humaine 50250 et utilisations

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Publication number
EP1311688A2
EP1311688A2 EP01937794A EP01937794A EP1311688A2 EP 1311688 A2 EP1311688 A2 EP 1311688A2 EP 01937794 A EP01937794 A EP 01937794A EP 01937794 A EP01937794 A EP 01937794A EP 1311688 A2 EP1311688 A2 EP 1311688A2
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EP
European Patent Office
Prior art keywords
nucleic acid
polypeptide
protein
acid molecule
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01937794A
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German (de)
English (en)
Inventor
Rosana Kapeller-Libermann
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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Publication of EP1311688A2 publication Critical patent/EP1311688A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • Lipid metabolism and storage are important processes not only in normal cellular functioning, but also in such systemic processes as cardiovascular regulation and fat deposition.
  • a family of enzymes that facilitate the hydrolysis of lipids termed the lipase family, has been identified. These enzymes catalyze the hydrolysis of a variety of lipids and lipid-containing molecules into fatty acids and less-substituted lipid molecules.
  • Lipases are serine esterases, and the lipase family is itself a subgroup of the alpha/beta hydrolase superfamily (Ollis et al. (1992) Protein Eng. 5: 197-211). All members of this superfamily share a conserved catalytic triad consisting of a nucleophilic serine, an aspartate and a glutamate residue in a conserved spatial relationship, where the serine is directly involved in the catalytic activity of the enzyme (Ollis et al. (1992), supra).
  • the lipases are typically highly glycosylated; for example, human gastric lipase has been found to have four N-glycosylation sites located throughout the enzyme (Bodmer et al. (1987) Biochim. Biophys. Acta 909: 147-153).
  • Different lipases e.g., human lipases
  • tissue distributions and different specificities which permits them to be separately regulated depending on the needs of the organism.
  • lipases specific to the pancreas, to the stomach, and to tlie liver have been identified, having specificity for triglycerides, a variety of dietary lipids, and phospholipids and triglycerides of plasma lipoproteins, respectively.
  • These enzymes are also adapted to different environmental conditions', for example, the gastric lipases only function at low pH, such as that found in the stomach. While lipases have substrate preferences, most of these enzymes will hydrolyze a wider range of substrates, albeit with varying degrees of efficiency (Gotor (1999) Bioorg. Med. Chem. 7: 2189-2197).
  • lipases have been exploited in the biosynthesis and degradation of biologically important molecules that are otherwise difficult or expensive to synthesize chemically, such as chiral alcohols, lactones, and esters (Gotor (1999), supra). Lipases play an important role in the breakdown of lipids and lipid-based compounds (e.g., triglycerides, phospholipids, and cholesterol esters) and in the production of fatty acids.
  • lipids and lipid-based compounds e.g., triglycerides, phospholipids, and cholesterol esters
  • fatty acids are key substituents in a variety of metabolic pathways, and are necessary for membrane biosynthesis, among many other functions
  • intra- and inter-cellular communication for example, fatty acids and other lipid degradation products serve as second messengers in a number of signal transduction pathways.
  • their activity contributes to the ability of the cell to grow and differentiate, to proliferate, and to communicate and interact with other cells.
  • Lipases are also critical to the functioning of systemic processes, such as cardiovascular regulation, the ability to take up lipids from ingested food (absorption), and the ability to store lipids (deposition) as energy reserves (e.g., adipocyte formation).
  • the present invention is based, at least in part, on the discovery of novel members of the family of lipase molecules, referred to herein as LP nucleic acid and protein molecules.
  • the LP nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, differentiation, and/or migration.
  • this invention provides isolated nucleic acid molecules encoding LP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of LP-encoding nucleic acids.
  • an LP nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:l, 3, 4, or 6 or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or a complement thereof.
  • the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 1 -2 of SEQ ID NO: 1. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 795-2031 of SEQ ID NO: 1. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-47 of SEQ ID NO:4. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO: 6 and nucleotides 795-2031 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6. In another embodiment, the nucleic acid molecule of the present invention includes nucleotides residues 1-62 and/or 928-1049 and/or 1617-1705 and/or 1883-2031 shown in SEQ ID NO: 1.
  • an LP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO: 2 or 5, or an amino acid sequence encoded by the
  • an LP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • an isolated nucleic acid molecule encodes the amino acid sequence of human LP.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • the nucleic acid molecule is at least 50, 100, 150, 177, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 756, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or more nucleotides in length.
  • the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 756, 800, 850, 900, 950, 1000, 1021, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or more nucleotides in length and encodes a protein having an LP activity (as described herein).
  • nucleic acid molecules preferably LP nucleic acid molecules, which specifically detect LP nucleic acid molecules relative to nucleic acid molecules encoding non-LP proteins.
  • a nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, 3, 4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the nucleic acid molecules are at least 15 (e.g., 15 contiguous) nucleotides in length and hybridize under stringent conditions to the nucleotide molecule set forth in SEQ ID NO:l or 4, or a complement thereof.
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 5, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:l, 3, 4, or 6, respectively, under stringent conditions.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an LP nucleic acid molecule, e.g., the coding strand of an LP nucleic acid molecule.
  • Another aspect of the invention provides a vector comprising an LP nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention provides a host cell containing a nucleic acid molecule of the invention.
  • the invention also provides a method for producing a protein, preferably an LP protein, by culturing in a suitable medium, a host cell, e.g. , a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
  • an isolated LP protein includes at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif.
  • an LP protein includes at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • an LP protein includes at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif, and has an LP activity (as described herein).
  • an LP protein includes at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6.
  • the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2 or 5, wherein the fragment comprises at least 15, 30, 50, 100, 150, 200, 250 or 263 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2 or 5, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number .
  • an LP protein has the amino acid sequence of SEQ ID NO: 2 or 5.
  • the invention features an LP protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or a complement thereof.
  • This invention further features an LP protem which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or a complement thereof.
  • the proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-LP polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably LP proteins.
  • the LP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of an LP nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an LP nucleic acid molecule, protein, or polypeptide such that the presence of an LP nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of LP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of LP activity such that the presence of LP activity is detected in the biological sample.
  • the invention provides a method for modulating LP activity comprising contacting a cell capable of expressing LP with an agent that modulates LP activity such that LP activity in the cell is modulated.
  • the agent inhibits LP activity.
  • the agent stimulates LP activity.
  • the agent is an antibody that specifically binds to an LP protein.
  • the agent modulates expression of LP by modulating transcription of an LP gene or translation of an LP mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an LP mRNA or an LP gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted LP protein or nucleic acid expression or activity by administering an agent which is an LP modulator to the subject.
  • the LP modulator is an LP protein.
  • the LP modulator is an LP nucleic acid molecule.
  • the LP modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant or unwanted LP protein or nucleic acid expression is a lipase-associated disorder, e.g., a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or deposition.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an LP protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of an LP protein, wherein a wild- type form of the gene encodes a protein with an LP activity.
  • the invention provides methods for identifying a compound that binds to or modulates the activity of an LP protein, by providing an indicator composition comprising an LP protein having LP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on LP activity in the indicator composition to identify a compound that modulates the activity of an LP protein.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human LP (clone Fbh50250).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2031 of SEQ ID NO:l.
  • the amino acid sequence corresponds to amino acids 1 to 263 of SEQ ID NO:2.
  • the coding region without the 3' or 5' untranslated region of the human LP gene is shown in SEQ ID NO:3.
  • Figure 2 depicts the cDNA sequence and predicted amino acid sequence of a fragment of human LP (clone Fbh50250), LP 48-794 .
  • the nucleotide sequence corresponds to nucleic acids 1 to 2031 of SEQ ID NO:4.
  • the amino acid sequence corresponds to amino acids 1 to 248 of SEQ ID NO:5.
  • the coding region without the 3' untranslated or 5' untranslated region of the human LP 48-794 gene is shown in SEQ ID NO:6.
  • Figure 3 depicts the results of an analysis of the expression pattern of LP in a panel of human tissues as determined by quantitative PCR using the TaqManTM procedure.
  • Figure 4 depicts the results of an analysis of the expression pattern of LP in a panel of breast, ovary and lung tumors as compared to normal breast, ovary and lung samples as determined by quantitative PCR using the TaqManTM procedure.
  • Figure 5 depicts the results of an analysis of the expression pattern of LP in a panel of colon, brain and liver tumors as compared to normal colon, brain and liver samples as determined by quantitative PCR using the TaqManTM procedure.
  • Figure 6 depicts the results of an analysis of the expression pattern of LP in a panel of various normal and diseased liver samples as determined by quantitative PCR using the TaqManTM procedure.
  • Figure 7 depicts the results of an analysis of the expression pattern of LP in a panel of normal and diseased cardiovascular vessels as determined by quantitative PCR using the TaqManTM procedure.
  • lipase or "LP” nucleic acid and protein molecules (e.g., human LP or LP50250), which are novel members of a family of enzymes possessing lipase activity.
  • lipase includes a molecule which is involved in catalysis of lipid hydrolysis.
  • Lipases are involved, for example, in the hydrolysis of triglycerides, phospholipids and cholesterol esters to produce fatty acids, and are therefore involved in cellular metabolic and catabolic processes (including respiration, fatty acid biosynthesis, and purine biosynthesis), and intra- and inter-cellular communication (e.g., by producing molecules, such as fatty acids, which are involved in a number of signal transduction pathways) which contribute to the ability of the cell to grow, proliferate, and differentiate.
  • Lipase molecules are also involved in systemic lipid absorption and deposition (e.g. , digestion of lipids and adipocyte production), and in cardiovascular regulation.
  • the LP molecules of the present invention provide novel diagnostic targets and therapeutic agents to control lipase-associated disorders.
  • a lipase-associated disorder includes a disorder, disease or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation) of lipase activity. Lipase-associated disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, or migration, or systemic responses in an organism, such as cardiovascular function or lipid absorption or deposition.
  • cardiovascular disorders examples include cardiovascular disorders.
  • Cardiovascular system disorders in which the LP molecules of the invention may be directly or indirectly involved include arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, and arrhythmia.
  • Lipase disorders also include cellular proliferation, growth, differentiation, or migration disorders.
  • Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process" is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus.
  • the LP molecules of the present invention are involved in signal transduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes.
  • the LP molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, or migration.
  • disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.
  • LP-associated or related disorders also include disorders of lipid absorption or deposition.
  • LP-associated or related disorders also include disorders affecting tissues in which LP protein is expressed.
  • a “lipase-mediated activity” includes an activity which involves the hydrolysis of lipids to form fatty acids and less-substituted lipid molecules. Lipase-mediated activities include those cellular or systemic activities which require the breakdown of lipids or the production of fatty acids. Such activities include cellular metabolism, modulation or regulation of cellular growth, proliferation, or differentiation, and systemic activities, such as cardiovascular regulation and lipid absorption and deposition.
  • family when referring to the protein and nucleic acid molecules of the invention (e.g., the LP family of proteins and/or nucleic acids) is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin, e.g., mouse or monkey proteins.
  • Members of a family may also have common functional characteristics.
  • the family of LP proteins comprises at least one signal sequence or signal peptide.
  • the prediction of such a signal peptide can be made, for example, utilizing the computer algorithm SignalP (Henrik, et al. (1997) Protein Engineering 10:1-6).
  • SignalP Harik, et al. (1997) Protein Engineering 10:1-6).
  • a "signal sequence” or “signal peptide” includes a peptide containing about 15 or more amino acids which occurs at the N-terminus of secretory and membrane bound proteins and which contains a large number of hydrophobic amino acid residues.
  • a signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, and more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine or Phenylalanine).
  • a signal sequence also referred to in the art as a “signal peptide” serves to direct a protein containing such a sequence to a lipid bilayer, and is cleaved in secreted and membrane bound proteins.
  • an LP molecule of the present invention is identified based on the presence of at least one N-glycosylation site.
  • N- glycosylation site includes an amino acid sequence of about 4 amino acid residues in length which serves as a glycosylation site. More preferably, an N-glycosylation site has the consensus sequence Asn-Xaa-Ser/Thr (where Xaa may be any amino acid).
  • N- glycosylation sites are described in, for example, Prosite PDOC00001 (http://www.expasy.ch cgi-bin/get-prodoc-entry7PDOC00001), the contents of which are inco ⁇ orated herein by reference.
  • Amino acid residues 101-104 of the LP protein (SEQ ID NO:2) comprise an N-glycosylation site.
  • Amino acid residues 89-92 of the LP 48-794 protein (SEQ ID NO:5) comprise an N-glycosylation site. Accordingly, LP proteins having at least one N-glycosylation site are within the scope of the invention.
  • an LP molecule of the present invention is identified based on the presence of a "lipase/acylhydrolase domain with a GDSL-like motif in the protein or corresponding nucleic acid molecule.
  • a "lipase/acylhydrolase domain with a GDSL-like motif includes a protein domain having an amino acid sequence of about 25-125 amino acid residues, and a bit score of at least 10 when compared against a lipase/acylhydrolase domain with a GDSL-like motif Hidden Markov Model (HMM), e.g., PFAM accession number PF00657.
  • HMM Hidden Markov Model
  • a lipase/acylhydrolase domain with a GDSL-like motif includes a protein domain having an amino acid sequence of about 50-100 amino acid residues and a bit score of at least 15.
  • a lipase/acylhydrolase domain with a GDSL-like motif includes a protein domain having an amino acid sequence of about 77-79 amino acid residues and a bit score of 22-42.
  • HMMs Hidden Markov Models
  • the lipase/acylhydrolase domain with a GDSL-like motif has been assigned the PFAM Accession PF00657 (http://genome.wustl.edu/Pfam/html).
  • HMM GDSL-like motif
  • PF00657 http://genome.wustl.edu/Pfam/html
  • a search was performed against the HMM database using the amino acid sequence of human LP (SEQ ID NO:2), resulting in the identification of a lipase/acylhydrolase domain with a GDSL-like motif in the amino acid sequence of human LP (SEQ ID NO:2) at about residues 31-107 of SEQ ID NO:2, having a score of 22.8.
  • an LP protein is identified based on the presence of at least one "LP signature motif in the protein or corresponding nucleic acid molecule.
  • LP signature motif includes an amino acid sequence that contains at least about 5-15 amino acid residues that are conserved among LP family members.
  • an LP signature motif includes an amino acid sequence at least about 7-13 amino acid residues, more preferably about 9-11 amino acid residues, more preferably 10 amino acid residues in length and having the following amino acid sequence: [LIVMFYAG](4)-G-D-S-[LIVM]-X(2), (SEQ ID NO:7), where X indicates any amino acid (see, for example, Upton and Buckley (1995) Trends Biochem.Sci. 20: 178-179).
  • preferred proteins include the conserved amino acid residues of the above-recited LP signature motif. Proteins including at least 5, 6, 7, 8, 9 or more conserved amino acid residues of the above- recited LP signature motif are also considered to be within the scope of the present invention.
  • the LP molecules of the invention include at least one, preferably two or more of the following motifs or domains: a signal peptide, an N- glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif.
  • Isolated proteins of the present invention preferably LP proteins, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2 or 5, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:l, 3, 4, or 6.
  • the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g.
  • amino acid residue which has a similar side chain amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical.
  • amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.
  • an "LP activity”, “biological activity of LP” or “functional activity of LP” refers to an activity exerted by an LP protein, polypeptide or nucleic acid molecule on an LP responsive cell or tissue, or on an LP protein substrate, as determined in vivo, or in vitro, according to standard techniques.
  • an LP activity is a direct activity, such as an association with an LP-target molecule.
  • a "target molecule” or “binding partner” is a molecule with which an LP protein binds or interacts in nature, such that LP-mediated function is achieved.
  • An LP target molecule can be a non-LP molecule (e.g., a cofactor) or an LP protein or polypeptide of the present invention.
  • an LP target molecule is an LP substrate (e.g., a triglyceride, a phospholipid, a cholesterol ester, or structurally related molecule).
  • an LP activity is an indirect activity, such as a metabolic activity mediated by interaction of the LP protein with an LP substrate. The biological activities of LP are described herein.
  • the LP proteins of the present invention have at least one of the following activities: i) interaction with an LP substrate; ii) interaction with a cofactor; and iii) conversion of an LP substrate to product (e.g., catalysis of the conversion of substrate to product).
  • the LP proteins of the present invention have one or more of the following activities: 1) modulate fatty acid production, 2) modulate metabolism and catabolism of important molecules, 3) modulate intra- or inter-cellular communication, 4) modulate cellular growth, proliferation, or differentiation, 5) regulate cardiovascular activity; and 6) modulate lipid absorption or deposition.
  • LP proteins and polypeptides having an LP activity features isolated LP proteins and polypeptides having an LP activity.
  • Other preferred proteins are LP proteins having one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif and, preferably, an LP activity.
  • Additional preferred proteins have at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6.
  • the nucleotide sequence of the isolated human LP cDNA and the predicted amino acid sequence of the human LP polypeptide are shown in Figure 1 and in SEQ ID NOs:l and 2, respectively.
  • the nucleotide sequence of the isolated cDNA and the predicted amino acid sequence of the LP 48-794 fragment of the human LP polypeptide are shown in Figure 2 and in SEQ ID NOs: 4 and 5, respectively.
  • a plasmid containing the nucleotide sequence encoding human LP and LP 48- 94 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-
  • the human LP gene which is approximately 2031 nucleotides in length, encodes a protein having a molecular weight of approximately 28.9 kD and which is approximately 263 amino acid residues in length.
  • nucleic acid molecules that encode LP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify LP-encoding nucleic acid molecules (e.g., LP mRNA) and fragments for use as PCR primers for the amplification or mutation of LP nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (t.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated LP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • LP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number as a hybridization probe, LP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid
  • nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to LP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6.
  • This cDNA may comprise sequences encoding the human LP protein (i.e., "the coding region", from nucleotides 3-794), as well as 5' untranslated sequences (nucleotides 1-2) and 3' untranslated sequences (nucleotides 795-2031) of SEQ ID NO:l.
  • the nucleic acid molecule can comprise only the coding region of SEQ ID ⁇ O:l (e.g., nucleotides 3-794, corresponding to SEQ ID NO:3).
  • this cDNA may comprise sequences encoding the LP 48-794 fragment of the human LP protein (i.e., "the coding region", from nucleotides 48-794), as well as 5' untranslated sequence (nucleotides 1-47) and 3' untranslated sequences (nucleotides 795-2031).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , respectively, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of an LP protein, e.g., a biologically active portion of an LP protein.
  • the nucleotide sequence determined from the cloning of the LP gene allows for the generation of probes and primers designed for use in identifying and/or cloning other LP family members, as well as LP homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number of an anti-sense sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or of a naturally occurring allelic variant or mutant of SEQ ID
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900, 1900-1950, 1950-2000 or more nucleotides in length and hybridizes under
  • Probes based on the LP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an LP protein, such as by measuring a level of an LP-encoding nucleic acid in a sample of cells from a subject e.g., detecting LP mRNA levels or determining whether a genomic LP gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of an LP protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number which encodes a polypeptide having an LP biological activity (the biological activities of the LP proteins are described herein), expressing the encoded portion of the LP protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the LP protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number due to degeneracy of the genetic code and thus encode tlie same LP proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or 5.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the LP proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the LP genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding an LP protein, preferably a mammalian LP protein, and can further include non-coding regulatory sequences, and introns.
  • Allelic variants of human LP include both functional and non-functional LP proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human LP protein that maintain the ability to bind an LP ligand or substrate and/or modulate cell proliferation and/or migration mechanisms.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or 5, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the human LP protein that do not have the ability to either bind an LP ligand and/or modulate any of the LP activities described herein.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2 or 5, or a substitution, insertion or deletion in critical residues or critical regions of the protein.
  • the present invention further provides non-human orthologues of the human LP protein.
  • Orthologues of the human LP protein are proteins that are isolated from non- human organisms and possess the same LP ligand binding and/or modulation of membrane excitability activities of the human LP protein.
  • Orthologues of the human LP protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2 or 5.
  • nucleic acid molecules encoding other LP family members and, thus, which have a nucleotide sequence which differs from the LP sequences of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention.
  • another LP cDNA can be identified based on the nucleotide sequence of human LP.
  • nucleic acid molecules encoding LP proteins from different species and which, thus, have a nucleotide sequence wliich differs from the LP sequences of SEQ ID NO: 1 , 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention.
  • a mouse LP cDNA can be identified based on the nucleotide sequence of a human LP.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the LP cDNAs of the invention can be isolated based on their homology to the LP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the LP cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the LP gene.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or a complement thereof, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • the nucleic acid is at least 50-100, 100-150, 150-200, 200-250, 250- 300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700- 750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100- 1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450- 1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800- 1850, 1850-1900, 1900-1950, 1950-2000 or more nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
  • stringent hybridization conditions includes hybridization in 4X or 6X sodium chloride/sodium citrate (SSC), at about 65- 70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C.
  • SSC sodium chloride/sodium citrate
  • a further preferred, non-limiting example of stringent hybridization conditions includes hybridization at 6X SSC at 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X or 6X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70° C or at 42-50°C are also intended to be encompassed by the present invention.
  • SSPE lxSSPE is 0.15M NaCl, lOmM NaH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.
  • blocking agents e.g., BSA or salmon or herring sperm carrier DNA
  • detergents e.g., SDS
  • chelating agents e.g., EDTA
  • Ficoll e.g., Ficoll, PVP and the like.
  • an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 PO , 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH 2 PO 4 , 1% SDS at 65°C, see e.g. , Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2X SSC, 1% SDS).
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, 3, 4, or 6, and corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the LP sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of LP (e.g., the sequence of SEQ ID NO:2 or 5) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the LP proteins of the present invention e.g., those present in a lipase consensus sequence, are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the LP proteins of the present invention and other members of the LP family are not likely to be amenable to alteration.
  • nucleic acid molecules encoding LP proteins that contain changes in amino acid residues that are not essential for activity. Such LP proteins differ in amino acid sequence from SEQ ID NO:2 or 5, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 or 5.
  • An isolated nucleic acid molecule encoding an LP protein identical to the protein of SEQ ID NO:2 or 5 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • Mutations can be introduced into SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in an LP protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an LP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for LP biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant LP protein can be assayed for the ability to metabolize or catabolize important biochemical molecules, to permit intra- or intercellular signaling, to regulate cardiovascular processes, or to modulate lipid absorption or deposition.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an' antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire LP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an LP.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human LP corresponds to SEQ ID NO:3, and the coding region of the LP 48-794 fragment corresponds to SEQ ID NO:6).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding LP.
  • the term “noncoding region” refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of LP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of LP mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of LP mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i. e. , RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an LP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue etal. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave LP mRNA transcripts to thereby inhibit translation of LP mRNA.
  • a ribozyme having specificity for an LP-encoding nucleic acid can be designed based upon the nucleotide sequence of an LP cDNA disclosed herein (i.e., SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ).
  • SEQ ID NO:l SEQ ID NO:l, 3, 4, or 6
  • nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number a derivative of a Tetrahymena L-19 IVS
  • RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an LP-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • LP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from " a pool of RNA molecules. See, e.g., Barrel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • LP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the LP (e.g., the LP promoter and/or enhancers; e.g., nucleotides 1-59 of SEQ ID NO:l) to form triple helical structures that prevent transcription of the LP gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the LP e.g., the LP promoter and/or enhancers; e.g., nucleotides 1-59 of SEQ ID NO:l
  • the LP nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of tlie molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of LP nucleic acid molecules can be used in therapeutic, and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of LP nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g. , by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
  • PNAs of LP can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of LP nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • modified nucleoside analogs e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, pr hybridization-triggered cleavage agent).
  • an endogenous LP gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous LP gene.
  • a heterologous DNA regulatory element for example, an endogenous LP gene which is normally "transcriptionally silent", i.e., an LP gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous LP gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous LP gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • LP proteins and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-LP antibodies.
  • native LP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • LP proteins are produced by recombinant DNA techniques.
  • an LP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the LP protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of LP protein in wliich the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of LP protein having less than about 30% (by dry weight) of non-LP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-LP protein, still more preferably less than about 10% of non-LP protein, and most preferably less than about 5% non-LP protein.
  • non-LP protein also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of LP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of LP protein having less than about 30% (by dry weight) of chemical precursors or non-LP chemicals, more preferably less than about 20% chemical precursors or non-LP chemicals, still more preferably less than about 10% chemical precursors or non-LP chemicals, and most preferably less than about 5% chemical precursors or non-LP chemicals.
  • a "biologically active portion" of an LP protein includes a fragment of an LP protein which participates in an interaction between an LP molecule and a non-LP molecule.
  • Biologically active portions of an LP protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the LP protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or 5, which include fewer amino acids than the full length LP proteins, and exhibit at least one activity of an LP protein.
  • biologically active portions comprise a domain or motif with at least one activity of the LP protein, e.g., modulating membrane excitability.
  • a biologically active portion of an LP protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300 or more amino acids in length.
  • Biologically active portions of an LP protein can be used as targets for developing agents which modulate an LP mediated activity, e.g. , a proliferative response.
  • a preferred biologically active portion of an LP protein of the present invention may contain at least one or more of the following motifs or domains: a signal peptide, an N-glycosylation site, a lipase/acylhydrolase domain with a GDSL-like motif, and/or an LP signature motif.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native LP protein.
  • the LP protein has an amino acid sequence shown in SEQ ID NO:2 or 5.
  • the LP protein is substantially identical to SEQ ID NO:2 or 5, and retains the functional activity of the protein of SEQ ID NO:2 or 5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the LP protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 or 5.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the LP amino acid sequence of SEQ ID NO:2 or 5 having 248 or 263 amino acid residues, at least 50, preferably at least 100, more preferably at least 150, even more preferably at least 200, and even more preferably at least 225 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the invention also provides LP chimeric or fusion proteins.
  • an LP "chimeric protein" or “fusion protein” comprises an LP polypeptide operatively linked to a non-LP polypeptide.
  • LP polypeptide refers to a polypeptide having an amino acid sequence corresponding to an LP molecule
  • non-LP polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the LP protein, e.g., a protein which is different from the LP protein and which is derived from the same or a different organism.
  • the LP polypeptide can correspond to all or a portion of an LP protein.
  • an LP fusion protein comprises at least one biologically active portion of an LP protein.
  • an LP fusion protein comprises at least two biologically active portions of an LP protein.
  • the term "operatively linked" is intended to indicate that the LP polypeptide and the non-LP polypeptide are fused in-frame to each other.
  • the non-LP polypeptide can be fused to the N-terminus or C-terminus of the LP polypeptide.
  • the fusion protein is a GST-LP fusion protein in which the LP sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant LP.
  • the fusion protein is an LP protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of LP can be increased through use of a heterologous signal sequence.
  • the LP fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the LP fusion proteins can be used to affect the bioavailability of an LP substrate.
  • LP fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding an LP protein; (ii) mis-regulation of the LP gene; and (iii) aberrant post-translational modification of an LP protein.
  • the LP-fusion proteins of the invention can be used as immunogens to produce anti-LP antibodies in a subject, to purify LP ligands and in screening assays to identify molecules which inhibit the interaction of LP with an LP substrate.
  • an LP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An LP- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the LP protein.
  • the present invention also pertains to variants of the LP proteins which function as either LP agonists (mimetics) or as LP antagonists.
  • Variants of the LP proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of an LP protein.
  • An agonist of the LP proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an LP protein.
  • An antagonist of an LP protein can inhibit one or more of the activities of the naturally occurring form of the LP protein by, for example, competitively modulating an LP -mediated activity of an LP protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe LP protein.
  • variants of an LP protein which function as either LP agonists (mimetics) or as LP antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an LP protein for LP protein agonist or antagonist activity.
  • a variegated library of LP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of LP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential LP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of LP sequences therein.
  • a degenerate set of potential LP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of LP sequences therein.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential LP sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
  • libraries of fragments of an LP protein coding sequence can be used to generate a variegated population of LP fragments for screening and subsequent selection of variants of an LP protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an LP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the LP protein.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of LP proteins.
  • the most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis (REM) a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify LP variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3): 327-331).
  • cell based assays can be exploited to analyze a variegated LP library.
  • a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to an LP ligand in a particular LP ligand-dependent manner.
  • the transfected cells are then contacted with an LP ligand and the effect of expression of the mutant on, e.g., membrane excitability of LP can be detected.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the LP ligand, and the individual clones further characterized.
  • An isolated LP protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind LP using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length LP protein can be used or, alternatively, the invention provides antigenic peptide fragments of LP for use as immunogens.
  • the antigenic peptide of LP comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 or 5 and encompasses an epitope of LP such that an antibody raised against the peptide forms a specific immune complex with the LP protein.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of LP that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • AN LP immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed LP protein or a chemically synthesized LP polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic LP preparation induces a polyclonal anti-LP antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as an LP.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind LP molecules.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of LP.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular LP protein with which it immunoreacts.
  • Polyclonal anti-LP antibodies can be prepared as described above by immunizing a suitable subject with an LP immunogen.
  • the anti-LP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized LP.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against LP can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2 or 5927-31; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines: These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind LP, e.g., using a standard ELISA assay.
  • a monoclonal anti-LP antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with LP to thereby isolate immunoglobulin library members that bind LP.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S.
  • recombinant anti-LP antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al.
  • An anti-LP antibody (e.g., monoclonal antibody) can be used to isolate LP by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti- LP antibody can facilitate the purification of natural LP from cells and of recombinantly produced LP expressed in host cells.
  • an anti-LP antibody can be used to detect LP protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the LP protein.
  • Anti-LP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1,
  • vectors preferably expression vectors, containing a nucleic acid encoding an LP protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can dep ⁇ nd on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., LP proteins, mutant forms of LP proteins, fusion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of LP proteins in prokaryotic or eukaryotic cells.
  • LP proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Purified fusion proteins can be utilized in LP activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for LP proteins, for example.
  • an LP fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Srudier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
  • the LP expression vector is a yeast expression vector.
  • yeast S. cerevisiae examples include pYepSecl (Baldari, et al, (1987) Embo J. 6:2 or 529-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • LP proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2 or 5156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31- 39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ - fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to LP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to host cells into which an LP nucleic acid molecule of the invention is introduced, e.g. , an LP nucleic acid molecule within a recombinant expression vector or an LP nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an LP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an LP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an LP protein.
  • the invention further provides methods for producing an LP protein using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding an LP protein has been introduced) in a suitable medium such that an LP protein is produced.
  • the method further comprises isolating an LP protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which LP-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous LP sequences have been introduced into their genome or homologous recombinant animals in which endogenous LP sequences have been altered.
  • Such animals are useful for studying the function and/or activity of an LP and for identifying and/or evaluating modulators of LP activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous LP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal .
  • a transgenic animal of the invention can be created by introducing an LP- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the LP cDNA sequence of SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human LP gene such as a mouse or rat LP gene, can be used as a transgene.
  • an LP gene homologue such as another LP family member, can be isolated based on hybridization to the LP cDNA sequences of SEQ ID NO:l, 3, 4, or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to an LP transgene to direct expression of an LP protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of an LP transgene in its genome and/or expression of LP mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an LP protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of an LP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the LP gene.
  • the LP gene can be a human gene (e.g. , the cDNA of SEQ ID NO:3 or 6), but more preferably, is a non- human homologue of a human LP gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:l or 4).
  • a mouse LP gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous LP gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous LP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous LP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous LP protein).
  • the altered portion of the LP gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the LP gene to allow for homologous recombination to occur between the exogenous LP gene carried by the homologous recombination nucleic acid molecule and an endogenous LP gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking LP nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced LP gene has homologously recombined with the endogenous LP gene are selected (see e.g., Li, E. et ⁇ l.
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Ter ⁇ toc ⁇ rcinom ⁇ s and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • aggregation chimeras see e.g., Bradley, A. in Ter ⁇ toc ⁇ rcinom ⁇ s and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al. ; WO 91/01140 by Smithies et al. ; WO 92/0968 by Zijlstra et al.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions The LP nucleic acid molecules, fragments of LP proteins, and anti-LP antibodies
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of an LP protein or an anti-LP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a fragment of an LP protein or an anti-LP antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound ' and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitliramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine. 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the conjugates of the invention can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • an LP protein of the invention has one or more of the following activities: 1) modulation of fatty acid production, 2) modulation of metabolism and catabolism of important molecules, 3) modulation of intra- or intercellular communication, 4) modulation of cellular growth, proliferation, or differentiation, 5) regulation of cardiovascular activity; and 6) modulation of lipid absorption or deposition and, thus, may be used to: 1) modulate fatty acid production, 2) modulate metabolism and catabolism of important molecules, 3) modulate intra- or inter-cellular communication, 4) modulate cellular growth, proliferation, or differentiation, 5) regulate cardiovascular activity; and 6) modulate lipid absorption or deposition.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express LP protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect LP mRNA (e.g., in a biological sample) or a genetic alteration in an LP gene, and to modulate LP activity, as described further below.
  • LP proteins can be used to treat disorders characterized by insufficient or excessive production of an LP substrate or production of LP inhibitors.
  • the LP proteins can be used to screen for naturally occurring LP substrates, to screen for drags or compounds which modulate LP activity, as well as to treat disorders characterized by insufficient or excessive production of LP protein or production of LP protein forms which have decreased, aberrant or unwanted activity compared to LP wild type protein (e.g., lipase-associated disorders).
  • the CAH molecules of the invention are useful for catalyzing the hydrolysis of lipids and lipid-containing compounds (e.g., triacylglycerols, phospholipids, and cholesterol esters) to less-substituted lipid compounds and fatty acids.
  • these molecules may be employed in small or large-scale synthesis of either lipids or fatty acids, or in chemical processes that require the production of these compounds. Such processes are known in the art (see, e.g., Ullmann et al. (1999) Ullmann's Encyclopedia of Industrial Chemistry, 6 th ed. VCH: Weinheim; Gutcho (1983) Chemicals by Fermentation.
  • lipases may be used in biosynthesis of a variety of other compounds, such as chiral alcohols, esters, carboxylic acids, and lactones through hydrolysis and transesterification reactions (Gotor (1999), supra, and references therein).
  • the isolated nucleic acid molecules of the invention can be used, for example, to express LP protein (e.g. , via a recombinant expression vector in a host cell in gene therapy applications), to detect LP mRNA (e.g., in a biological sample) or a genetic alteration in an LP gene, and to modulate LP activity, as described further below.
  • LP proteins can be used to treat disorders characterized by insufficient or excessive production of an LP substrate or production of LP inhibitors.
  • the LP proteins can be used to screen for naturally occurring LP substrates, to screen for drugs or compounds which modulate LP activity, as well as to treat disorders characterized by insufficient or excessive production of LP protein or production of LP protein forms which have decreased, aberrant or unwanted activity compared to LP wild type protein (e.g., lipase-associated disorders, such as cardiovascular disorders (e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, my
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which interact with or bind to LP proteins, have a stimulatory or inhibitory effect on, for example, LP expression or LP activity, or have a stimulatory or inhibitory effect on, for example, the availability of LP substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which interact with or bind to LP proteins, have a stimulatory or inhibitory effect on, for example, LP expression or LP activity, or have a stimulatory or inhibitory effect on, for example, the availability of LP substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of an LP protein or polypeptide or biologically active portion thereof (e.g., triglycerides, phospholipids and cholesterol esters, or compounds which are structurally related thereto).
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an LP protein or polypeptide or biologically active portion thereof (e.g., cofactors or inhibitory molecules).
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
  • an assay is a cell-based assay in which a cell which expresses an LP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate LP activity is determined. Determining the ability of the test compound to modulate LP activity can be accomplished by monitoring, for example, the production of one or more specific LP substrates or products in a cell which expresses LP (see, e.g., Saada et al. (2000) Biochem Biophys. Res. Commun. 269: 382-386).
  • the cell for example, can be of mammalian origin.
  • the ability of the test compound to modulate LP binding to a substrate can also be determined. Determining the ability of the test compound to modulate LP binding to a substrate can be accomplished, for example, by coupling the LP substrate with a radioisotope or paramagnetic label such that binding of the LP substrate to LP can be determined by detecting the labeled LP substrate in a complex.
  • LP could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate LP binding to an LP substrate in a complex.
  • Determining the ability of the test compound to bind LP can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to LP can be determined by detecting the labeled LP compound in a complex.
  • compounds e.g., LP substrates
  • compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds e.g., LP substrates
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with LP without the labeling of either the compound or the LP. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing an LP target molecule (e.g., an LP substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the LP target molecule. Determining the ability of the test compound to modulate the activity of an LP target molecule can be accomplished, for example, by determining the ability of the LP protein to bind to or interact with the LP target molecule.
  • Determining the ability of the LP protein, or a biologically active fragment thereof, to bind to or interact with an LP target molecule can be accomplished by one of the methods described above for dete ⁇ nining direct binding.
  • determining the ability of the LP protein to bind to or interact with an LP target molecule can be accomplished by determining the activity or availability of the target molecule. For example, a target-regulated cellular activity, such as growth or proliferation, may be monitored.
  • an assay of the present invention is a cell-free assay in which an LP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to associate with, to bind to, or to serve as a substrate for the LP protein or biologically active portion thereof is determined.
  • Preferred biologically active portions of the LP proteins to be used in assays of the present invention include fragments which participate in interactions with non-LP molecules, e.g., fragments with high surface probability scores. Binding of the test compound to the LP protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the LP protein or biologically active portion thereof with a known compound which interacts with LP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an LP protein, wherein determining the ability of the test compound to interact with an LP protein comprises determining the ability of the test compound to preferentially bind to or interact with LP or a biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which an LP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the LP protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of an LP protein can be accomplished, for example, by determining the ability of the LP protein to bind to or associate with an LP target molecule by one of the methods described above for determining direct binding. Determining the ability of the LP protein to bind to an LP target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C.
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g. , BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of an LP protein can be accomplished by determining the ability of the LP protein to further modulate the activity of a downstream effector of an LP target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting an LP protein or biologically active portion thereof with a known compound which binds the LP protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the LP protein, wherein determining the ability of the test compound to interact with the LP protein comprises determining the ability of the LP protein to preferentially hydrolyze a target substrate (e.g., a triglyceride, a phospholipid, a cholesterol ester, or a structurally related compound).
  • a target substrate e.g., a triglyceride, a phospholipid, a cholesterol ester, or a structurally related compound.
  • binding of a test compound to an LP protein, or interaction of an LP protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the LP protein to be bound to a matrix.
  • glutathione-S-transferase/LP fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or LP protein, and the mixture incubated under conditions conducive to complex formation (e.g. , at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of LP binding or activity determined using standard techniques.
  • an LP protein can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated LP protein can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with LP protein but which do not interfere with binding of the LP protein to its target molecule can be derivatized to the wells of the plate, and unbound target or LP protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the LP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the LP protein.
  • modulators of LP expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of LP mRNA or protein in the cell is determined.
  • the level of expression of LP mR ⁇ A or protein in the presence of the candidate compound is compared to the level of expression of LP mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of LP expression based on this comparison. For example, when expression of LP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of LP mRNA or protein expression.
  • the candidate compound when expression of LP mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of LP mRNA or protein expression.
  • the level of LP mRNA or protein expression in the cells can be determined by methods described herein for detecting LP mRNA or protein.
  • the LP proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al (1993) Cell 72:2 or 523-232; Madura et al (1993) J Biol. Chem.
  • LP-binding proteins proteins which bind to or interact with LP
  • LP-6-bp proteins which bind to or interact with LP
  • Such LP-binding proteins are also likely to be involved in the propagation of signals by the LP proteins or LP targets as, for example, downstream elements of an LP-mediated signaling pathway.
  • LP-binding proteins are likely to be LP inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for an LP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g. , LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with the LP protein.
  • a reporter gene e.g. , LacZ
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of an LP protein can be confirmed in vivo, e.g., in an animal such as an animal model for atherosclerosis.
  • the ability of the agent to modulate the activity of an LP protein can be tested in an animal such as an animal model for a cellular proliferation disorder, e.g., tumorigenesis.
  • Animal based models for studying tumorigenesis in vivo are well known in the art (reviewed in Animal Models of Cancer Predisposition Syndromes, Hiai, H. and Hino, O. (eds.) 1999, Progress in Experimental Tumor Research, Vol. 35; Clarke, A.R. (2000) Carcinogenesis 21:435-41) and include, for example, carcinogen- induced tumors (Rithidech, K. et al. (1999) Mutat. Res. 428:33-39; Miller, M . et al. (2000) Environ. Mol.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an LPmodulating agent, an antisense LP nucleic acid molecule, a LP-specific antibody, or an LPbinding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • cell-based systems may be used to identify compounds which may act to ameliorate tumorigenic or proliferative disease symptoms.
  • such cell systems may be exposed to a compound, suspected of exhibiting an ability to ameliorate tumorigenic or proliferative disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of tumorigenic or proliferative disease symptoms in the exposed cells.
  • the cells are examined to determine whether one or more of the tumorigenic or proliferative disease cellular phenotypes has been altered to resemble a more normal or more wild type, non- tumorigenic disease or non-proliferative disease phenotype.
  • Cellular phenotypes that are associated with tumorigenic disease states include aberrant proliferation and migration, angiogenesis, anchorage independent growth, and loss of contact inhibition.
  • animal-based tumorigenic disease systems such as those described herein, may be used to identify compounds capable of ameliorating tumorigenic or proliferative disease symptoms.
  • Such animal models may be used as test substrates for the identification of drags, pharmaceuticals, therapies, and interventions which may be effective in treating tumorigenic or proliferative disease.
  • animal models may be exposed to a compound, suspected of exhibiting an ability to ameliorate tumorigenic or proliferative disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of tumorigenic or apoptotic tumorigenic or proliferative disease symptoms in the exposed animals.
  • the response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with tumorigenic disease, for example, by counting the number of tumors and/or measuring their size before and after treatment.
  • the animals may be monitored by assessing the reversal of disorders associated with tumorigenic disease, for example, reduction in tumor burden, tumor size, and invasive and/or metastatic potential before and after treatment.
  • any treatments which reverse any aspect of tumorigenic or proliferative disease symptoms should be considered as candidates for human tumorigenic or proliferative disease therapeutic intervention.
  • Dosages of test agents may be determined by deriving dose-response curves.
  • Gene expression patterns may be utilized to assess the ability of a compound to ameliorate proliferative or tumorigenic disease symptoms.
  • the expression pattern of one or more genes may form part of a "gene expression profile” or “transcriptional profile” which may be then be used in such an assessment.
  • Gene expression profile or “transcriptional profile”, as used herein, includes the pattern of mRNA expression obtained for a given tissue or cell type under a given set of conditions. Such conditions may include, but are not limited to, the presence of a tumor, e.g., a breast or ovary tumor or any of the other tumors described herein, including any of control or experimental conditions described herein.
  • Gene expression profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT- PCR.
  • LP gene sequences may be used as probes and or PCR primers for the generation and corroboration of such gene expression profiles.
  • Gene expression profiles may be characterized for known states, either tumorigenic or proliferative disease or normal, within the cell- and/or animal-based model systems. Subsequently, these known gene expression profiles may be compared to ascertain the effect a test compound has to modify such gene expression profiles, and to cause the profile to more closely resemble that of a more desirable profile.
  • administration of a compound may cause the gene expression profile of a tumorigenic or proliferative disease model system to more closely resemble the control system.
  • Administration of a compound may, alternatively, cause the gene expression profile of a control system to begin to mimic a tumorigenic or proliferative disease state.
  • Such a compound may, for example, be used in further characterizing the compound of interest, or may be used in the generation of additional animal models.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below. 1. Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping.
  • portions or fragments of the LP nucleotide sequences, described herein, can be used to map the location of the LP genes on a chromosome.
  • the mapping of the LP sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, LP genes can be mapped to chromosomes by preparing PCR primers
  • LP sequences (preferably 15-25 bp in length) from the LP nucleotide sequences.
  • Computer analysis of the LP sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the LP sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D ⁇ ustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the LP nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map an LP sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci.
  • FISH Fluorescence in situ hybridization
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the LP gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the LP sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the LP nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the LP nucleotide sequences of the invention uniquely represent portions of the human genome.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each' yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as that in SEQ ID NO:3, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from LP nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (t.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” t.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO: 1 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the LP nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:l having a length of at least 20 bases, preferably at least 30 bases.
  • the LP nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such LP probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such LP probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., LP primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining LP protein and/or nucleic acid expression as well as LP activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted LP expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with LP protein, nucleic acid expression or activity. For example, mutations in an LP gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with LP protein, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of LP in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of LP protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting LP protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes LP protein such that the presence of LP protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting LP protein or nucleic acid e.g., mRNA, or genomic DNA
  • a preferred agent for detecting LP mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to LP mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, the LP nucleic acid set forth in SEQ ID NO:l, 3, 4, or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to LP mRNA or genomic DNA.
  • suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting LP protein is an antibody capable of binding to LP protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a D ⁇ A probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect LP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of LP mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of LP protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of LP genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of LP protein include introducing into a subject a labeled anti-LP antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting LP protein, mRNA, or genomic DNA, such that the presence of LP protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of LP protein, mRNA or genomic DNA in the control sample with the presence of LP protein, mRNA or genomic D ⁇ A in the test sample.
  • kits for detecting the presence of LP in a biological sample can comprise a labeled compound or agent capable of detecting LP protein or mRNA in a biological sample; means for determining the amount of LP in the sample; and means for comparing the amount of LP in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect LP protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted LP expression or activity.
  • aberrant includes an LP expression or activity which deviates from the wild type LP expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant LP expression or activity is intended to include the cases in which a mutation in the LP gene causes the LP gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional LP protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with an LP substrate, or one which interacts with a non-LP substrate.
  • the term "unwanted” includes an unwanted phenomenon involved in a biological response such as cellular proliferation.
  • unwanted includes an LP expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in LP protein activity or nucleic acid expression, such as a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or deposition.
  • a disorder associated with a misregulation in LP protein activity or nucleic acid expression such as a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or deposition.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in LP protein activity or nucleic acid expression, such as a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or deposition.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted LP expression or activity in which a test sample is obtained from a subject and LP protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of LP protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted LP expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., cerebrospinal fluid or serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted LP expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted LP expression or activity in which a test sample is obtained and LP protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of LP protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted LP expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in an agent for a disorder associated with aberrant or unwanted LP expression or activity in which a test sample is obtained and LP protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of LP protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted LP expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in an agent for a disorder associated with aberrant or unwanted LP expression or activity.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an LP -protein, or the mis-expression of the LP gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an LP gene; 2) an addition of one or more nucleotides to an LP gene; 3) a substitution of one or more nucleotides of an LP gene, 4) a chromosomal rearrangement of an LP gene; 5) an alteration in the level of a messenger RNA transcript of an LP gene, 6) aberrant modification of an LP gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of an LP gene, 8) a non- wild type level of an LP- protein, 9) allelic loss of an LP gene, and 10) inappropriate post-translational modification of an LP-protein.
  • a preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. , U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; andNakazawa et al. (1994) Proc. Natl.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an LP gene under conditions such that hybridization and amplification of the LP gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, (1989) Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et al.
  • mutations in an LP gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in LP can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 1: 244-255; Kozal, MJ. et al. (1996) Nature Medicine 2: 753-759).
  • genetic mutations in LP can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the LP gene and detect mutations by comparing the sequence of the sample LP with the corresponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147- 159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the LP gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type LP sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:2 or 586-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in LP cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an LP sequence e.g., a wild- type LP sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in LP genes.
  • SSCP single strand conformation polymorphism
  • Single-strand conformation polymorphism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2 or 5766, see also Cotton (1993) Mutat Res. 285:125-144; and Hayashi (1992) Genet Anal. Tech. Appl. 9:73-79).
  • Single-stranded DNA fragments of sample and control LP nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2 or 5437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :2 or 538).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an LP gene.
  • any cell type or tissue in which LP is expressed may be utilized in the prognostic assays described herein.
  • Monitoring the influence of agents (e.g., drugs) on the expression or activity of an LP protein can be applied not only in basic drag screening, but also in clinical trials.
  • agents e.g., drugs
  • the effectiveness of an agent determined by a screening assay as described herein to increase LP gene expression, protein levels, or upregulate LP activity can be monitored in clinical trials of subjects exhibiting decreased LP gene expression, protein levels, or downregulated LP activity.
  • the effectiveness of an agent determined by a screening assay to decrease LP gene expression, protem levels, or downregulate LP activity can be monitored in clinical trials of subjects exhibiting increased LP gene expression, protein levels, or upregulated LP activity.
  • an LP gene and preferably, other genes that have been implicated in, for example, an LP-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including LP that are modulated in cells by treatment with an agent (e.g., compound, drag or small molecule) which modulates LP activity (e.g., identified in a screening assay as described herein) can be identified.
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of LP and other genes implicated in the LP-associated disorder, respectively.
  • the levels of gene expression e.g., a gene expression pattern
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of LP or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an LP protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the LP protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the LP protein, mRNA, or genomic DNA in the pre- administration sample with the LP protein, mRNA, or genomic DNA in the post administration sample or samples;
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
  • increased administration of the agent may be desirable to increase the expression or activity of LP to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of LP to lower levels than detected, t ' .e. to decrease the effectiveness of the agent.
  • LP expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted LP expression or activity, e.g., a lipase-associated disorder such as a cell proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, or disorders of lipid absorption or deposition.
  • a lipase-associated disorder such as a cell proliferation, growth, differentiation, or migration disorder
  • a cardiovascular disorder e.g., lipid absorption or deposition.
  • treatment includes the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to a cell or tissue from a subject, who has a diseases or disorder, has a symptom of a disease or disorder, or is at risk of (or susceptible to) a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or disorder, the symptom of the disease or disorder, or the risk of (or susceptibility to) the disease or disorder.
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drags in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drag response phenotype", or “drug response genotype”).
  • a drug e.g., a patient's "drag response phenotype", or “drug response genotype”
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the LP molecules of the present invention or LP modulators according to that individual's drag response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drag-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted LP expression or activity, by administering to the subject an LP or an agent which modulates LP expression or at least ( one LP activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted LP expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the LP aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an LP, LP agonist or LP antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. 2.
  • the modulatory method of the invention involves contacting a cell with an LP or agent that modulates one or more of the activities of LP protein activity associated with the cell.
  • An agent that modulates LP protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an LP protein (e.g., an LP substrate), an LP antibody, an LP agonist or antagonist, a peptidomimetic of an LP agonist or antagonist, or other small molecule.
  • the agent stimulates one or more LP activities.
  • stimulatory agents include active LP protein and a nucleic acid molecule encoding LP that has been introduced into the cell.
  • the agent inhibits one or more LP activities.
  • inhibitory agents include antisense LP nucleic acid molecules, anti-LP antibodies, and LP inhibitors.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) LP expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering an LP protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted LP expression or activity.
  • Stimulation of LP activity is desirable in situations in which LP is abnormally downregulated and/or in which increased LP activity is likely to have a beneficial effect.
  • inhibition of LP activity is desirable in situations in which LP is abnormally upregulated and/or in which decreased LP activity is likely to have a beneficial effect.
  • LP molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on LP activity (e.g., LP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) LP-associated disorders (e.g., cell proliferation, growth, differentiation, or migration disorders, cardiovascular disorders, or disorders of lipid absorption or deposition) associated with aberrant or unwanted LP activity.
  • LP-associated disorders e.g., cell proliferation, growth, differentiation, or migration disorders, cardiovascular disorders, or disorders of lipid absorption or deposition
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an LP molecule or LP modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an LP molecule or LP modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):2 or 554-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drags anti-malarials, sulfonamides, analgesics, nitrofurans
  • consumption of fava beans One pharmacogenomics approach to identifying genes that predict drag response, known as "a genome-wide association", relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g.
  • a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drug response or side effect.
  • a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNPs single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA.
  • a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drag response.
  • a gene that encodes a drag target is known (e.g., an LP protein of the present invention)
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drag response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6- formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • the gene expression of an animal dosed with a drag e.g., an LP molecule or LP modulator of the present invention
  • a drag e.g., an LP molecule or LP modulator of the present invention
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an LP molecule or LP modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • E. Electronic Apparatus Readable Media and Arrays Electronic apparatus readable media comprising LP sequence information is also provided.
  • LP sequence information refers to any nucleotide and/or amino acid sequence information particular to the LP molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like.
  • information "related to" said LP sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g.
  • electronic apparatus readable media refers to any suitable medium for storing, holding, or containing data or information that can be read and accessed directly by an electronic apparatus.
  • Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact discs; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybrids of these categories such as magnetic/optical storage media.
  • the medium is adapted or configured for having recorded thereon LP sequence information of the present invention.
  • the term "electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
  • Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatuses; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • sequence information refers to a process for storing or encoding information on the electronic apparatus readable medium.
  • Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the LP sequence information.
  • a variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium.
  • the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in tlie form of an ASCII file, or stored in a database application, such as DB2, Sybase, . Oracle, or the like, as well as in other forms.
  • Any number of dataprocessor structuring formats may be employed in order to obtain or create a medium having recorded thereon the LP sequence information.
  • sequence information in readable form
  • searching means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • the present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has an LPassociated disease or disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder, wherein the method comprises the steps of determining LP sequence information associated with the subject and based on the LP sequence information, determining whether the subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder, and/or recommending a particular treatment for the disease, disorder, or pre-disease condition.
  • the present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder wherein the method comprises the steps of determining LP sequence information associated with the subject, and based on the LP sequence information, determining whether the subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.
  • the present invention also provides in a network, a method for determining whether a subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder associated with LP, said method comprising the steps of receiving LP sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to LP and/or a cellular proliferation, growth, differentiation, and/or migration disorder, and based on one or more of the phenotypic information, the LP information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/
  • the present invention also provides a business method for determining whether a subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder, said method comprising the steps of receiving information related to LP (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to LP and/or related to a cellular proliferation, growth, differentiation, and/or migration disorder, and based on one or more of the phenotypic information, the LP information, and the acquired information, determining whether the subject has a cellular proliferation, growth, differentiation, and/or migration disorder or a pre-disposition to a cellular proliferation, growth, differentiation, and/or migration disorder.
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the invention also includes an array comprising an LPsequence of the present invention.
  • the array can be used to assay expression of one or more genes in the array.
  • the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be LP. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.
  • the invention allows the quantitation of gene expression.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues.
  • one tissue can be perturbed and the effect on gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression.
  • the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect.
  • undesirable biological effects can be determined at the molecular level.
  • the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
  • the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a cellular proliferation, growth, differentiation, and/or migration disorder, progression of a cellular proliferation, growth, differentiation, and/or migration disorder, and processes, such a cellular transformation associated with the cellular proliferation, growth, differentiation, and/or migration disorder.
  • the array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of LP expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells.
  • This provides a battery of genes (e.g., including LP) that could serve as a molecular target for diagnosis or therapeutic intervention.
  • the invention is based, at least in part, on the discovery of a human gene encoding novel proteins, referred to herein as human LP and human LP 48- 94 .
  • human LP and human LP 48- 94 The entire sequence of human clone Fbh50250 was determined and found to contain several open reading frames termed human "LP", and human "LP 48- 94 ", set forth in Figures 1, and 2, respectively.
  • the amino acid sequences of these human LP expression products are set forth in Figures 1 and 2, respectively.
  • the human LP protein the sequence of which is set forth in SEQ ID NO:2, comprises about 263 amino acids and is shown in Figure 1.
  • the human LP 48-794 protein comprises about 248 amino acids and is shown in Figure 2.
  • the coding regions (open reading frames) of human LP and LP 48-794 are set forth as SEQ ID NOs:3 and 6.
  • Clone Fbh50250 comprising the coding regions of human LP and fragment LP 8- , was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, on , and assigned Accession No.
  • the amino acid sequence of human LP and fragment LP 48-794 were analyzed using the program PSORT (http://www. psort.nibb.ac.jp) to predict the localization of the protein within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence.
  • the results of the analyses show the likelihood of human LP (SEQ ID NO:2) being localized, for example, to the mitochondrion, to the nucleus, to the cell wall or extracellular space, or to the cytoplasm.
  • the results further show the likelihood of fragment LP 8- 9 of human LP (SEQ ID NO:5) being localized, for example, to the cytoplasm, to the mitochondrion, or to the nucleus.
  • This example describes the tissue distribution of human LP mRNA, as determined by Northern analysis, by Polymerase Chain Reaction (PCR) on cDNA libraries using oligonucleotide primers based on the human LP sequence, or by in situ analysis. • Northern blot hybridizations with the various RNA samples are performed under standard conditions and washed under stringent conditions, i.e., 0.2xSSC at 65°C. The DNA probe is radioactively labeled with 32p_dCTP using the Prime-It kit (Stratagene, La Jolla, CA) according to the instructions of the supplier.
  • Prime-It kit Stratagene, La Jolla, CA
  • Filters containing human mRNA are probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.
  • Human LP. expression in normal tissues is assessed by PCR using the Taqman ® system (PE Applied Biosystems) according to the manufacturer's instructions.
  • various tissues e.g. tissues obtained from brain, are first frozen on dry ice. Ten-micrometer-thick sections of the tissues are postfixed with 4% formaldehyde in DEPC treated IX phosphate- buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0).
  • Probes are incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1 % sodium thiosulfate for 18 hours at 55°C.
  • a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1%
  • slides are washed with 2X SSC. Sections are then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM ⁇ aCl, and 1 mM EDTA), for 10 minutes, in T ⁇ E with lO ⁇ g of R ⁇ ase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides are then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM ⁇ aCl, and 1 mM EDTA
  • Sections are then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4°C for 7 days before being developed and counter stained.
  • EXAMPLE 2 EXPRESSION OF RECOMBINANT HUMAN LP PROTEIN IN BACTERIAL CELLS
  • human LP is expressed as a recombinant glutathione-S- transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • GST glutathione-S- transferase
  • human LP is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • Expression of the GST-LP fusion protein in PEB 199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB 199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
  • the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire human LP protein and an HA tag (Wilson et al. (1984) Cell 31:161) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the human LP DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the human LP coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the human LP coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the human LP gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HBl 01, DH5 ⁇ , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are subsequently transfected with the LP-pcDNA/Amp plasmid D ⁇ A using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, ⁇ Y, 1989.
  • the expression of the human LP polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S- cysteine available from ⁇ EN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA-specific, FLAG-specific, or LP-specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35 S- methionine (or 35 S-cysteine).
  • the culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1 % NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA-specific, FLAG-specific, or LP-specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • detergents RIPA buffer, 150 mM NaCl, 1 % NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5.
  • DNA containing the human LP coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the human LP polypeptide is detected by radiolabelling and immunoprecipitation using a human LP-specific monoclonal antibody.
  • Tissue Expression Analysis of LP mRNA Using TaqManTM This example describes the tissue distribution of human LP mRNA in a variety of cells and tissues, as determined using the TaqManTM procedure.
  • the TaqmanTM procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cD ⁇ A was generated from the samples of interest, e.g., tumor samples and normal samples, cell lines and the like,and used as the starting material for PCR amplification.
  • a gene-specific oligonucleotide probe (complementary to the region being amplified) was included in the reaction ( . e. , the TaqmanTM probe).
  • the TaqManTM probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6- carboxyfluorescein), TET (6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy- 4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6- carboxy- ⁇ ,N,N',N'-tetramethylrhodamine) at the 3' end of the probe.
  • a fluorescent reporter dye covalently linked to the 5' end of the probe
  • TET 6-carboxy-4,7,2',7'-tetrachlorofluorescein
  • JOE 6-car
  • the fluorescent signal from the 5' dye is quenched.
  • the 5' to 3' nucleolytic activity of taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal.
  • the PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a way of quantitating the initial template concentration. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites.
  • the 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target.
  • the probe fragments are then displaced from the target, and polymerization of the strand continues.
  • the 3' end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product.
  • Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as ⁇ 2 microglobulin which has been labeled with a different fluor on the 5' end (typically JOE).
  • a primer/probe set was designed using Primer Express software and primary cDNA sequence information.
  • Total RNA was prepared from a series of tissues using an RNeasy kit from Qiagen
  • First strand cDNA was prepared from one ⁇ g total RNA using an oligo dT primer and Superscript II reverse transcriptase (GIBCO-BRL).
  • cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction.
  • Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control gene confirms efficient removal of genomic DNA contamination.
  • An array of human tissues were tested. The results of one such analysis are depicted in Figure 3. Expression was greatest in brain cortex, heart with coronary heart failure (CHF), prostate epithelial cells, and glial cells. Expression was also high in primary osteoblasts, cardiovascular tissues such as the human umbilical vein epithelial cells, aortic and coronary smooth muscle cells (SMC), normal heart, kidney, liver and dermal fibroblasts, as well as nerve tissues, including the hypothalamus and dorsal root ganglions.
  • CHF coronary heart failure
  • SMC coronary smooth muscle cells
  • nerve tissues including the hypothalamus and dorsal root ganglions.
  • LP expression levels were measured in various normal and diseased liver samples by quantitative PCR using the TaqmanTM procedure as described above.
  • the relative levels of LP expression in various samples are depicted in Figure 6.
  • expression of LP in the diseased state was moderately decreased as compared to normal samples.
  • LP expression levels were measured in various tissue samples from normal and diseased cardiovascular vessels, using the TaqmanTM procedure described above.
  • the relative levels of LP expression in various samples are depicted in Figure 7.
  • LP expression was decreased in one diseased saphenous vein sample as compared to 5 non- diseased saphenous vein samples.
  • a moderate decrease in expression was also noted in diseased arteries as compared to normal arteries.

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Abstract

L'invention concerne des molécules d'acide nucléique isolées, désignées sous le nom de molécules d'acide nucléique LP, codant pour de nouvelles molécules de lipase LP. L'invention concerne également des molécules d'acide nucléique anti-sens, des vecteurs d'expression de recombinaison contenant des molécules d'acide nucléique LP, des cellules hôtes dans lesquelles ont été introduits les vecteurs d'expression, et des animaux transgéniques non humains dans lesquels un gène LP a été introduit ou interrompu. L'invention concerne également des protéines LP isolées, des protéines de fusion, des peptides antigéniques et des anticorps anti-LP. L'invention concerne en outre des méthodes de diagnostic dans lesquelles sont utilisées des compositions de l'invention.
EP01937794A 2000-05-26 2001-05-29 Lipase humaine 50250 et utilisations Withdrawn EP1311688A2 (fr)

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AU6949200A (en) * 1999-09-01 2001-03-26 Incyte Genomics, Inc. Human hydrolytic enzymes
EP1572987A4 (fr) * 2000-02-03 2005-11-30 Nuvelo Inc Acides nucleiques et polypeptides

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