WO2003102209A2 - Alternatively spliced isoforms of human hmg co-a reductase - Google Patents

Abstract

The present invention features nucleic acids and polypeptides encoding novel splice variant isoforms of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). The polynucleotide sequences of HMGCRsv1, HMGCRsv2, HMGCRsv3, and HMGCRsv4 are provided by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, and SEQ ID NO 7, respectively. The amino acid sequences for HMGCRsv1, HMGCRsv2, HMGCRsv3, and HMGCRsv4 are provided by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, and SEQ ID NO 8, respectively. The present invention also provides methods for using HMGCRsv1, HMGCRsv2, HMGCRsv3, and HMGCRsv4 polynucleotides and proteins to screen for compounds that bind respectively to HMGCRsv1, HMGCRsv2, HMGCRsv3, and HMGCRsv4.

Description

TITLE OF THE INVENTION

ALTERNATIVELY SPLICED ISOFORMS OF HUMAN HMG CO-A REDUCTASE

This application claims priority to U.S. Provisional Patent Application Serial No. 06/385,188 filed on May 30, 2002, and U.S. Provisional Patent Application Serial No. 60/407,155 filed on August 29, 2002, each of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The references cited herein are not admitted to be prior art to the claimed invention.

Mevalonic acid is the precursor of isoprenoids, a class of compounds involved in diverse cellular functions such as sterol synthesis and growth control. Products of the mevalonate pathway include cholesterol, heme and farnesyl-pyrophosphate. Within cells, the concentration of mevalonate is tightly controlled through the activity of 3-hydroxy-3- methylglutaryl-CoA reductase (HMGCR), the enzyme that catalyzes the four-electron reduction of HMG-CoAto mevalonate. The reaction is catalyzed by HMGCR in a reaction that proceeds as follows:

(S)-HMG-CoA + 2NADPH + 2H⁺ → (R)-mevalonate + 2NADP⁺ + CoASH

where NADP⁺ is the oxidized form of nicotinamide adenine dinucelotide, NADPH is the reduced form of NADP⁺, and CoASH is the reduced form of CoA.

Elevated cholesterol levels are a primary risk factor for coronary artery disease. This disease is a major problem in developed countries and currently affects 13 to 14 million adults in the United States alone. Dietary changes and drug therapy reduce serum cholesterol levels and dramatically decrease the risk of stroke and overall mortality (Eisenberg, 1998 Am. J. Med., 104.2S-5S). As shown in large-scale clinical trials, inhibition of HMGCR significantly decreases cholesterol levels and reduces the risks of stroke by 29% and the overall mortality by 22% (Hebert et al., 1997 J. Am. Med. Assoc, 278:313-21).

Inhibitors of HMGCR, commonly referred to as statins, are effective and safe drugs that are widely prescribed in cholesterol-lowering therapy. Statins curtail cholesterol biosynthesis by inhibiting the committed step in the biosynthesis of isoprenoids and sterols (Corsini, et al., 1995 Pharmacol. Res., 31:9-27). Many different statin compounds have been described each with K values in the nanomolar range (Endo, 1985 J. Med. Chem., 28:401-5). In general, statins are very effective in lowering serum cholesterol levels and are prescribed widely in treatment of hypercholesterolemia (Gotto, 1997 Am. J. Cardiol., 79, 1663-6. Many statins share an HMG moiety and are thought to bind competitively in the active site of HMGCR, but do not have an effect on NADPH binding (Endo, et al., 1976 FEBS Lett., 72:323-6). ϊ In addition to lowering cholesterol, statins appear to have a number of additional effects, such as the nitric oxide-mediated promotion of new bloodvessel growth (Kureishi, et al., 2000 Nature Med., 6:1004-10), stimulation of bone formation (Mundy, et al., 1999 Science, 286:1946-9), protection against oxidative modification of low-density lipoprotein, anti-inflammatory effects and a reduction in C-reactive protein levels (Davignon

) and Laaksonen, 1999 Curr. Opin. LipidoL, 10:543-59). In addition, inhibition of HMGCR also induces growth arrest and cell death in several cancer cell types, presumably through the reduction of non-sterol, mevalonate-derived products (Bennis et al., 1993 Int. J. Cancer, 55:640-5; Hawk et al., 1996 Cancer Lett., 109:217-22; Lee et al, 1998 J. Biol. Chem., 273:10618-23; Caruso et al., 1999 Anticancer Res., 19:451-4). Recently a new and ϊ unexpected role for HMGCR was demonstrated in studies on Drosophila melanogaster development, where it was shown that a gene that is required to guide primordial germ cells, encodes HMGCR (Van Doren et al., 1998 Nature, 396:466-9).

HMGCR is among the most highly regulated enzymes known (Goldstein and Brown, 1990 Nature, 343:425-30). Transcription and translation of HMGCR increase when

) the concentrations of products of the mevalonate pathway are low. Conversely, when sterol concentrations are high, the intracellular HMGCR concentration decreases rapidly (Nakanishi et al., 1988 J. Biol. Chem., 263:8929-37). A third level of regulation is achieved by phosphorylation of S872 (human enzyme) by AMP-activated protein kinase, which decreases the enzyme's activity (Omkumar et al., 1994 J. Biol. Chem., 269:6810-4). i Because of the importance of HMGCR as a drug target and its myriad of roles in metabolism, there is a need in the art for compounds that selectively bind to isoforms of human HMGCR. The present invention is directed towards novel HMGCR isoforms (HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4) and uses thereof.

) SUMMARY OF THE INVENTION

Genomic tiling microarrays and RT-PCR have been used to identify and confirm the presence of human splice variants of HMGCR mRNA and resulting HMGCR protein isoforms. More specifically, the present invention features polynucleotides encoding HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4, and the corresponding

> polypeptides: HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4, respectively. The cDNA sequence encoding HMGCRsvl is provided by SEQ ID NO 1. The amino acid sequence for HMGCRsvl is provided by SEQ ID NO 2. The cDNA sequence encoding HMGCRsv2 is provided by SEQ ID NO 3. The amino acid sequence for HMGCRsv2 is provided by SEQ ID NO 4. The cDNA sequence encoding HMGCRsv3 is provided by SEQ ID NO 5. The amino acid sequence for HMGCRsv3 is provided by SEQ ID NO 6. The cDNA sequence encoding HMGCRsv4 is provided by SEQ ID NO 7. The amino acid sequence for HMGCRsv4 is provided by SEQ ID NO 8.

Thus, a first aspect of the present invention describes a purified HMGCR isoform encoding nucleic acid selected from the group consisting of HMGCRsvl, HMGCRsv2, HMGCRsv3, and HMGCRsv4. The HMGCRsvl encoding nucleic acid comprises SEQ ID NO 1 or the complement thereof. The HMGCRsv2 encoding nucleic acid comprises SEQ ID NO 3 or the complement thereof. The HMGCRsv3 encoding nucleic acid comprises SEQ ID NO 5 or the complement thereof. The HMGCRsv4 encoding nucleic acid comprises SEQ ID NO 7 or the complement thereof. Reference to the presence of one region does not indicate that another region is not present. For example, in different embodiments, the nucleic acids of the present invention can comprise, consist, or consist essentially of a nucleic acids encoding for SEQ ID NO 1, or alternatively, can comprise, consist, or consist essentially of the nucleic acid sequence of SEQ ID NO 3, or alternatively, can comprise, consist, or consist essentially of the nucleic acid sequence of SEQ ID NO 5, or alternatively, can comprise, consist, or consist essentially of the nucleic acid sequence of SEQ ID NO 7.

Another aspect of the present invention describes a purified polypeptide selected from a group consisting of HMGCRsvl, HMGCRsv2, HMGCRsv3, and HMGCRsv4. Thus in one embodiment, the HMGCRsvl polypeptide can comprise, consist, or consist essentially of SEQ ID NO 2. In another embodiment, the HMGCRsv2 polypeptide can comprise, consist, or consist essentially of SEQ ID NO 4. In another embodiment, the HMGCRsv3 polypeptide can comprise, consist, or consist essentially of SEQ ID NO 6. In yet another embodiment, the HMGCRsv4 polypeptide can comprise, consist, or consist essentially of the amino acid sequence of SEQ ID NO 8.

Another aspect of the present invention describes HMGCR expression vectors. In one embodiment of the invention, the inventive expression vector comprises a nucleotide sequence encoding polypeptide comprising, consisting, or consisting essentially of SEQ ID NO 2, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter. In another embodiment, the inventive expression vector comprises a nucleotide sequence encoding polypeptide comprising, consisting, or consisting essentially of SEQ ID NO 4, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter. In another embodiment, the inventive expression vector comprises a nucleotide sequence encoding polypeptide comprising, consisting, or consisting essentially of SEQ ID NO 6, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter. In yet another embodiment, the inventive expression vector comprises a nucleotide sequence encoding polypeptide comprising, consisting, or consisting essentially of SEQ ID NO 8, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter.

Another aspect of the present invention describes a recombinant cell comprising an expression vector comprising, consisting, or consisting essentially of the above-described sequences and an RNA polymerase present recognizes the promoters in the cell.

Another aspect of the present invention, describes a recombinant cell made by a process comprising the step of introducing into the cell an expression vector comprising a nucleotide sequence comprising, consisting, or consisting essentially of either SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, or a nucleotide sequence encoding a polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of either SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, respectively, wherein the nucleotide sequence is transcriptionally coupled to an exogenous promoter. The inventive expression vectors can be used to insert recombinant nucleic acids into the host genome or can exist as autonomous pieces of nucleic acid.

Another aspect of the present invention describes a method of producing HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides comprising SEQ ID NO 2 or SEQ ID NO 4 or SEQ ID NO 6 or SEQ ID NO 8, respectively. The method involves the step of growing a recombinant cell containing an inventive expression vector under conditions wherein the selected HMGCRsv polypeptide is expressed from the expression vector.

Another aspect of the present invention provides a method of screening for a compound that binds to a HMGCR isoform polypeptide or to a fragment thereof, said HMGCR isoform polypeptide is selected from the group consisting of HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4. In one embodiment the method comprises the steps of: (a) expressing a polypeptide comprising SEQ ID NO 2 from a recombinant nucleic acid; providing to said polypeptide a test preparation comprising one or more compounds; and measuring the ability of said test preparation to bind said polypeptide. In another aspect of the invention, the above method is performed using polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 4 or SEQ ID NO 6 or SEQ ID NO 8.

In another embodiment of the method, a compound is identified that binds selectively to HMGCRsvl polypeptide as compared to HMGCR polypeptide or an isoform thereof, that is not HMGCRsvl. This method comprises the steps of: providing HMGCRsvl polypeptide comprising SEQ ID NO 2; providing HMGCR isoform polypeptide that is not HMGCRsvl; contacting said HMGCRsvl polypeptide and said HMGCR isoform polypeptide that is not HMGCRsvl with a test preparation comprising one or more test compounds; and determining the binding of said test preparation to said HMGCRsvl polypeptide and said HMGCR isoform polypeptide that is not HMGCRsvl, wherein a compound that binds said HMGCRsvl polypeptide but does not bind said HMGCR isoform polypeptide that is not HMGCRsvl is a compound that selectively binds HMGCRsvl polypeptide. Alternatively, the same method can be performed using HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, respectively.

In another aspect of the invention, a method is provided for screening for a compound able to bind to or interact with a HMGCRsvl protein or a fragment thereof comprising the steps of: expressing a HMGCRsvl polypeptide comprising SEQ ID NO 2 or a fragment thereof from a recombinant nucleic acid; providing to said polypeptide a labeled HMGCR ligand that binds to said polypeptide and a test preparation comprising one or more compounds; and measuring the effect of said test preparation on binding of said labeled HMGCR ligand to said polypeptide, wherein a test preparation that alters the binding of said labeled HMGCR ligand to said polypeptide contains a compound that binds to or interacts with said polypeptide. In alternative embodiments, the above method is performed using HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, or fragments thereof.

Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A presents the results of reverse transcription, polymerase chain reaction (RT-PCT) assays using polyA mRNA obtained from 79 human tissue samples and one polyA mRNA sample obtained from monkey brain. Figure IB presents a complete list of the polyA mRNA samples, with cross reference numbers correlating the mRNA sample to the RT-PCR results depicted in FIG. 1 A.

Figure 2A presents the results of reverse transcription, polymerase chain reaction (RT-PCT) assays using polyA mRNA obtained from three relevant human tissue samples. The three samples were chosen as a subset from the 80 sample-screen described for Figure 1A. The subset was selected to highlight the identification of an HMGCRsv2 splice variant mRNA. Figure 2B presents the results of reverse transcription, polymerase chain reaction (RT-PCT) assays using polyA mRNA obtained from four relevant human tissue samples. The four samples represent a subset from the 80 sample-screen as described for Figure 1 A. This subset was selected to highlight the presence of two HMGCR splice variant mRNA species, designated as HMGCRsv3 and HMGCRsv4.

Figures 3 A, 4A, 5 A and 6A illustrate the exon structures of HMGCR mRNA corresponding to the known reference form of HMGCR mRNA, labeled NMJ300859. Each box in the gray bars represents an exon in the HMGCR mRNA. Exons affected by splice variation are shown as darkened shaded boxes. Figures 3B, 4B, 5B and 6B illustrate the inventive HMGCR splice variant mRNA structures, labeled HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4, respectively. The small horizontal arrows above some of the HMGCR exons show the positions of the oligonucleotide primers used to perform RT-PCR assays to confirm the exon structure of the various HMGCR mRNAs in 80 human and monkey tissue samples (see Figures 1 A and IB). The nucleotide sequences shown in boxes below the exon bars of the reference HMGCR and splice variant HMGCR mRNAs depict the nucleotide sequences of the exon junctions resulting from the splicing of various exons in the HMGCR mRNAs. In Figures 3 through 6, nucleotides shown in italics represent 20 nucleotides at the 3' side of the respective splice junctions and nucleotides shown in underline represent 20 nucleotides at the 5' side of the respective splice junctions. In Figures 3 A, 4 A, 5 A and 6 A, nucleotides in boldface are associated with either the 5' or the 3' sides of dropped exon splice junctions.

DEFECTIONS

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, "HMGCR" refers to 3-hydroxy-3-methylglutaryl-CoA reductase having an amino sequence as set forth in reference HMGCR protein NP_000850. The term "HMGCR" refers to the polynucleotide sequence encoding portion of NM_00859. In contrast, reference to an HMGCR isoform, includes NP_000850 and other polypeptide isoform variants.

As used herein, "HMGCRsvl", which lacks exon 13, refers to an isoform of HMGCR protein having an amino acid sequence set forth in SEQ ID NO 2. "HMGCRsv2", which lacks exon 8, refers to an isoform of HMGCR protein having an amino acid sequence set forth in SEQ ID NO 4. "HMGCRsv3", which lacks exon 18, refers to an isoform of HMGCR protein having an amino acid sequence set forth in SEQ ID NO 6. "HMGCRsv4", which lacks exons 17 and 18, refers to an isoform of HMGCR protein having an amino acid sequence set forth in SEQ ID NO 8.

As used herein, an "isolated nucleic acid" is a nucleic acid molecule that exists in a physical form that is nonidentical to any nucleic acid molecule of identical sequence as found in nature; "isolated" does not require, although it does not prohibit, that the nucleic acid so described has itself been physically removed from its native environment. For example, a nucleic acid can be said to be "isolated" when it includes nucleotides and/or intemucleoside bonds not found in nature. When instead composed of natural nucleosides in phosphodiester linkage, a nucleic acid can be said to be "isolated" when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequence, with respect to the presence of proteins, with respect to the presence of lipids, or with respect the presence of any other component of a biological cell, or when the nucleic acid lacks sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses sequence not identically present in nature. As so defined, "isolated nucleic acid" includes nucleic acids integrated into a host cell chromosome at a heterologous site, recombinant fusions of a native fragment to a heterologous sequence, recombinant vectors present as episomes or as integrated into a host cell chromosome.

A "purified nucleic acid" represents at least 10% of the total nucleic acid present in a sample or preparation. In preferred embodiments, the purified nucleic acid represents at least about 50%, at least about 75%, or at least about 95% of the total nucleic acid in a isolated nucleic acid sample or preparation. Reference to "purified nucleic acid" does not require that the nucleic acid has undergone any purification and may include, for example, chemically synthesized nucleic acid that has not been purified.

The phrases "isolated protein", "isolated polypeptide", "isolated peptide" and "isolated oligopeptide" refer to a protein (or respectively to a polypeptide, peptide, or oligopeptide) that is nonidentical to any protein molecule of identical amino acid sequence as found in nature; "isolated" does not require, although it does not prohibit, that the protein so described has itself been physically removed from its native environment. For example, a protein can be said to be "isolated" when it includes amino acid analogues or derivatives not found in nature, or includes linkages other than standard peptide bonds. When instead composed entirely of natural amino acids linked by peptide bonds, a protein can be said to be "isolated" when it exists at a purity not found in nature — where purity can be adjudged with respect to the presence of proteins of other sequence, with respect to the presence of non-protein compounds, such as nucleic acids, lipids, or other components of a biological cell, or when it exists in a composition not found in nature, such as in a host cell that does not naturally express that protein.

As used herein, a "purified polypeptide" (equally, a purified protein, peptide, or oligopeptide) represents at least 10% of the total protein present in a sample or preparation, as measured on a weight basis with respect to total protein in a composition. In preferred embodiments, the purified polypeptide represents at least about 50%, at least about 75%, or at least about 95% of the total protein in a sample or preparation. A "substantially purified protein" (equally, a substantially purified polypeptide, peptide, or oligopeptide) is an isolated protein, as above described, present at a concentration of at least 70%, as measured on a weight basis with respect to total protein in a composition. Reference to "purified polypeptide" does not require that the polypeptide has undergone any purification and may include, for example, chemically synthesized polypeptide that has not been purified.

As used herein, the term "antibody" refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives. Fragments within the scope of the term "antibody" include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation, and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab', Fv, F(ab)'₂, and single chain Fv (scFv) fragments. Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Marasco (ed.), Intracellular Antibodies: Research and Disease Applications, Springer- Verlag New York, Inc. (1998) (ISBN: 3540641513). As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems, and phage display.

As used herein, a "purified antibody preparation" is a preparation where at least 10% of the antibodies present bind to the target ligand. In preferred embodiments, antibodies binding to the target ligand represent at least about 50%, at least about 75%, or at least about 95% of the total antibodies present. Reference to "purified antibody preparation" does not require that the antibodies in the preparation have undergone any purification.

As used herein, "specific binding" refers to the ability of two molecular species concurrently present in a heterogeneous (inhomogeneous) sample to bind to one another in preference to binding to other molecular species in the sample. Typically, a specific binding interaction will discriminate over adventitious binding interactions in the reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold; when used to detect analyte, specific binding is sufficiently discriminatory when determinative of the presence of the analyte in a heterogeneous (inhomogeneous) sample. Typically, the affinity or avidity of a specific binding reaction is least about 10^"7 M, with specific binding reactions of greater specificity typically having affinity or avidity of at least 10^" M to at least about 10^"9 M.

The term "antisense", as used herein, refers to a nucleic acid molecule sufficiently complementary in sequence, and sufficiently long in that complementary sequence, as to hybridize under intracellular conditions to (i) a target mRNA transcript or (ii) the genomic DNA strand complementary to that transcribed to produce the target mRNA transcript.

The term "subject", as used herein refers to an organism and to cells or tissues derived therefrom. For example the organism may be an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is usually a mammal, and most commonly human.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the nucleic acid sequences encoding isoform variants of human HMGCR. In particular, HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4 polynucleotides encode polypeptides that are splice variant isoforms of HMGCR. This invention also includes HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4 proteins. SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 and SEQ ID NO 7 present polynucleotide sequences representing the full open reading frames that encode HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4, respectively. Similarly, SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6 and SEQ ID NO 8 show amino acid sequences representing the polypeptide sequences of HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4, respectively.

HDAC3svl, HDAC3sv2, HDAC3sv3 and HDAC3sv4 polynucleotide sequences encoding HDAC3svl, HDAC3sv2, HDAC3sv3 and HDAC3sv4 proteins, respectively, as exemplified and enabled herein include a number of specific, substantial and credible utilities. For example, HMGCRsvl, HMGCRsv2, HMGCRsv3 and HMGCRsv4 encoding nucleic acids were identified in mRNA samples obtained from human sources (see Example 1-3). Such nucleic acids can be used as hybridization probes to distinguish between cells that produce HMGCRsvl, HMGCRsv2, HMGCRsv3 or HMGCRsv4 transcripts from human or non-human cells (including bacteria) that do not produce such transcripts. Similarly, antibodies specific for HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be used to distinguish between cells that express HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 proteins, respectively, from human or non-human cells (including bacteria) that do not express HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 proteins.

HMGCR is an important drug target for the management of cholesterol levels. Statins are a class of small molecule compounds that inhibit HMGCR enzyme activity. Given the importance of HMGCR activity to the therapeutic management of high cholesterol levels it is important to identify HMGCR isoforms and to identify HMGCR-ligand compounds that are isoform-specific as well as compounds that are effective ligands for two or more HMGCR isoforms. In particular, it may be very important to identify compounds that are effective inhibitors of HMGCR activity, yet do not bind to all HMGCR isoforms. Compounds that bind to all HMGCR isoforms may require higher drug doses to saturate all HMGCR isoform binding sites, and thereby result in a greater likelihood of secondary non- therapeutic side effects. For the foregoing reasons, HMGCRsvl, HMGCRsv2, HMGCRsv3, and HMGCRsv4 proteins represent important compound-binding target and have utility in the identification of new HMGCR compounds having greater specificity and efficacy.

In some embodiments, one or more activities of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 protein are modulated by a ligand compound to achieve one or more of the following: prevent or reduce the risk of occurrence, or recurrence where the potential exist, of vascular disease (in particular, atherosclerosis), lipid storage diseases, obesity, diabetes, hypercholesterolemia, cancer, and osteoporosis. Compounds that treat hypercholesterolemia are particularly important because of the cause-and-effect relationship between hypercholesterolemia and morbidity and mortality from coronary artery disease (CAD) (For a review, Mahley, R.W. and Bersot, T.P., In, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw-Hill, New York, 1996, Ch. 36, pp. 984-995).

Compounds modulating HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 protein activities include agonists, antagonists, and allosteric modulators. Generally, but not always, HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 antagonists and allosteric modulators negatively affecting either HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 activities, respectively, will be used to inhibit HMG Co-A reductase activity thereby decreasing LDL cholesterol levels and increasing the levels of HDL. The evidence linking elevated serum cholesterol to coronary heart disease is overwhelming. For example, atherosclerosis is a slowly progressive disease characterized by the accumulation of cholesterol within the arterial wall. Compelling evidence supports the concept that lipids deposited in atherosclerotic lesions are derived primarily from plasma LDL; thus, LDLs have popularly become known as the "bad" cholesterol. In contrast, HDL serum levels correlate inversely with coronary heart disease — indeed, high serum levels of HDL are regarded as a negative risk factor. It is hypothesized that high levels of plasma HDL are not only protective against coronary artery disease, but may actually induce regression of atherosclerotic plaques (e.g., see Badimon, et al., 1992 Circulation 86 (Suppl. III):86-94). Thus, HDL has popularly become known as "good" cholesterol. Inhibitors of HMGCR achieve clinical efficacy by a number of effects, including primarily inhibition of hepatic cholesterol biosynthesis, depletion of critical intracellular pools of sterols, and increased transcription of LDL receptors leading to enhanced removal from plasma of LDL and LDL precursors.

HMGCR isoform activities can also be affected by modulating the cellular abundance of transcripts encoding HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4. Compounds modulating the abundance of HMGCR isoform transcripts include cloned polynucleotides comprising HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 coding sequences that can be used to express HMGCRsvl, HDACsv2, HMGCRsv3, or HMGCRsv4, respectively, in vivo, antisense nucleic acids targeted to HMGCRsvl, HDACsv2, HMGCRsv3, or HMGCRsv4 transcripts, and inhibitory ribonucleic acids, such as ribozymes and RNAi, targeted to HMGCRsvl, HDACsv2, HMGCRsv3, HMGCRsv4 transcripts.

In some embodiments, HMGCR isoform polypeptide activities are modulated to achieve a therapeutic effect upon diseases in which cholesterol metabolism is in need of adjustment in a subject. For example, atherosclerosis can be treated by modulating one or more HMGCR isoform protein activities to achieve, for instance, increased levels of HDL. In other embodiments, the risk of developing atherosclerosis is reduced by modulating one or more HMGCR isoform protein activities to achieve, for example, increased levels of HDL.

HMGCR ISOFORM NUCLEIC ACIDS HMGCRsvl nucleic acids contain regions that encode for polypeptides comprising or consisting of SEQ ID NO 2. HMGCRsv2 nucleic acids contain regions that encode for polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 4. HMGCRsv3 nucleic acids contain regions that encode for polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 6. HMGCRsv4 nucleic acids contain regions that encode for polypeptides comprising, consisting, or consisting essentially of SEQ ID NO 8. HMGCR isoform nucleic acids have a variety of uses, such as being used as a hybridization probe or PCR primer to identify the presence of HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4 nucleic acids; being used as hybridization probes or PCR primers to identify nucleic acid encoding for proteins related to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4; and or being used for recombinant expression of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides. In particular, HMGCRsvl polynucleotides do not have a polynucleotide region that comprises exon 13 of the HMGCR gene, NM_000859 (hereafter HMGCR reference gene)(see FIGS. 3A and 3B). HMGCRsv2 polynucleotides do not have a polynucleotide region that comprises exon 8 of the HMGCR reference gene (see FIGS. 4A and 4B). HMGCRsv3 polynucleotides do not have a polynucleotide region that comprises exon 18 of the HMGCR reference gene (see FIGS. 5 A and 5B). Similarly, HMGCRsv4 polynucleotides do not have a polynucleotide region that comprises exons 17 and exons 18 of the HMGCR reference gene (see FIGS. 6A, and 6B).

Regions in HMGCRsvl, HMGCRsv2, HMGCRsv3 or, HMGCRsv4 nucleic acids that do not encode for HMGCRsvl, or HMGCRsv2, or HMGCRsv3, or HMGCRsv4 amino acids, respectively, or are not found in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, respectively, if present, are preferably chosen to achieve a particular purpose. Examples of additional regions that can be used to achieve a particular purpose include capture regions that can be used as part of a sandwich assay, reporter regions that can be probed to indicate the presence of the nucleic acids, expression vector regions, and regions encoding for other polypeptides.

The guidance provided in the present application can be used to obtain nucleic acid sequence encoding for HMGCR isoforms HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 -related proteins from different sources. Obtaining nucleic acids encoding HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4-related proteins from different sources is facilitated by using sets of degenerative probes and primers and the proper selection of hybridization conditions. Sets of degenerative probes and primers are produced taking into account the degeneracy of the genetic code. Adjusting hybridization conditions is useful for controlling probe or primer specificity to allow for hybridization to nucleic acids having similar sequences.

Techniques employed for hybridization detection and PCR cloning are well known in the art. Nucleic acid detection techniques are described, for example, in Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989. PCR cloning techniques are described, for example, in White, Methods in Molecular Cloning, volume 67, Humana Press, 1997.

HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polynucleotide probes and primers can be used to screen nucleic acid libraries containing, for example, cDNA. Such libraries are commercially available, and can be produced using techniques such as those described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998.

Starting with a particular amino acid sequence and the known degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost all amino acids are encoded for by different combinations of nucleotide triplets or "codons". The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990). Amino acids are encoded for by codons as follows:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteine: codons UGC, UGU

D=Asp=Aspartic acid: codons GAC, GAU

E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, UUU

G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His=Histidine: codons CAC, CAU

I=Ile=Isoleucine: codons AUA, AUC, AUU

K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

M=Met=Methionine: codon AUG

N=Asn=Asparagine: codons AAC, AAU p=Pro=Proline: codons CCA, CCC, CCG, CCU

Q=Gln=Glutamine: codons CAA, CAG

R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

T=Thr=Threonine: codons ACA, ACC, ACG, ACU

V=Val=Valine: codons GUA, GUC, GUG, GUU

W=Trp=Tryptophan: codon UGG γ=Tyr=Tyrosine: codons UAC, UAU

Nucleic acid having a desired sequence can be synthesized using chemical and biochemical techniques. Examples of chemical techniques are described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989. In addition, long polynucleotides of a specified nucleotide sequence can be purchased from commercial vendors, such as Blue Heron Biotechnology, Inc. (Bothell, WA). Biochemical synthesis techniques involve the use of a nucleic acid template and appropriate enzymes such as DNA and/or RNA polymerases. Examples of such techniques include in vitro amplification techniques such as PCR and transcription based amplification, and in vivo nucleic acid replication. Examples of suitable techniques are provided by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989, and U.S. 5,480,784.

HMGCR Isoform Probes

HMGCR isoform probes of the present invention contain regions that can specifically hybridize to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 target nucleic acids, respectively, under appropriate hybridization conditions and can distinguish HMGCRBsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 nucleic acids from non-target nucleic acids. Probes complementary and hybridizable to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can also contain nucleic acid regions that are not complementary to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 nucleic acids, respectively.

In embodiments where, for example, one or more HMGCR isoform polynucleotide probes are used in a hybridization assay to specifically detect the presence of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polynucleotides, respectively, in a sample, the HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polynucleotide probe comprises at least 20 nucleotides of a sequence that corresponds to a novel exon junction polynucleotide region of the respective HMGCR isoform mRNA. In other embodiments, the inventive HMGCR isoform probe comprises at least 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides of a sequence that corresponds to the novel exon junction polynucleotide region of the respective HMGCR isoform mRNA.

In one embodiment of a probe for detection of an HMGCRsvl polynucleotide, the probe comprises at least 20 nucleotides of HMGCRsvl sequence that corresponds to an exon junction polynucleotide region created by alternative splicing of exon 12 to exon 14 of a transcript of the HMGCR gene (see FIGS. 3A and 3B). For example, the polynucleotide sequence: 5' -GGGAΓΓAΓAAΓΓACΓCCTTGCTTGGTGGAGGTGCCAGCAG- 3' [SEQ ID

NO 9] represents one possible embodiment of such an inventive HMGCRsvl polynucleotide probe, wherein a first 20 nucleotides region (in italics) is complementary and hybridizable to the 3' end of exon 12 of the HMGCR gene and a second contiguous 20 nucleotide region (underscored) is complementary and hybridizable to the 5' end of exon 14 of the HMGCR gene (see Figure 3B). In another embodiment, an inventive HMGCRsvl polynucleotide probe comprises at least 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of HMGCR sequence that is complementary and hybridizable to an exon junction polynucleotide region created by alternative splicing of exon 12 to exon 14 of a transcript of the HMGCR gene. In most embodiments, the HMGCRsvl polynucleotide probe is selected to comprise a first continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 3' end of exon 12, and a second continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 5' end of exon 14 in HMGCR.

In one embodiment of a probe for detection of an HMGCRsv2 polynucleotide, the probe comprises at least 20 nucleotides of HMGCRsv2 sequence that corresponds to an exon junction polynucleotide region created by alternative splicing of exon 7 to exon 9 of a transcript of the HMGCR gene (see FIGS. 4A and 4B). For example, the polynucleotide sequence: 5 ' -GΓGΓGΓCC7TGGΓA7TAGAGTCTCTAGGCTTGGTTCTTGT-3 ' [SEQ ID NO 10] represents one embodiment of such an inventive HMGCRsv2 polynucleotide probe, wherein a first 20 nucleotides region (italicized) is complementary and hybridizable to the 3' end of exon 7 of the HMGCR gene and a second contiguous 20 nucleotide region (underscored) is complementary and hybridizable to the 5' end of exon 9 of the HMGCR gene (see Figure 4B).

In another embodiment, an inventive HMGCRBsv2 polynucleotide probe comprises at least 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of HMGCR sequence that is complementary and hybridizable to an exon junction polynucleotide region created by alternative splicing of exon 7 to exon 9 of a transcript of the HMGCR gene. In most embodiments, the HMGCRsv2 polynucleotide probe is selected to comprise a first continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 3' end of exon 7, and a second continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 5' end of exon 9 in the HMGCR gene.

In one embodiment of a probe for detection of an HMGCRsv3 polynucleotide, the probe comprises at least 20 nucleotides of HMGCRsv3 sequence that corresponds to an exon junction polynucleotide region created by alternative splicing of exon 17 to exon 19 of a primary transcript of the HMGCR gene (see FIGS. 5A and 5B). For example, the polynucleotide sequence: 5' rCTACA-ZTGCCrGEGGACAGATGCTAGGTGTTCAAG GAGC-3' [SEQ ID NO 11] represents one embodiment of such an inventive HMGCRsv3 polynucleotide probe, wherein a first 20 nucleotides region (italicized) is complementary and hybridizable to the 3' end of exon 17 of the HMGCR gene and a second 20 nucleotide region (underscored) is complementary and hybridizable to the 5' end of exon 19 of the HMGCR gene (see Figure 5B). In another embodiment, an inventive HMGCRBsv3 polynucleotide probe comprises at least 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of HMGCR sequence that is complementary and hybridizable to an exon junction polynucleotide region created by alternative splicing of exon 17 to exon 19 of a transcript of the HMGCR gene. In most embodiments, the HMGCRsv3 polynucleotide probe is selected to comprise a first continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 3' end of exon 17, and a second continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 5' end of exon 19 in the HMGCR gene.

In one embodiment of a probe for detection of an HMGCRsv4 polynucleotide, the probe comprises at least 20 nucleotides of HMGCRsv4 sequence that corresponds to an exon junction polynucleotide region created by alternative splicing of exon 16 to exon 19 of a primary transcript of the HMGCR gene (see FIGS. 6 A and 6B). For example, the polynucleotide sequence: 5' CAGCCAAGG3TGECAGAGAAATGCTAGGTGTTCAAG GAGC-3' [SEQ ID NO 12] represents one embodiment of such an inventive HMGCRsv4 polynucleotide probe, wherein a first 20 nucleotides region (italicized) is complementary and hybridizable to the 3' end of exon 16 of the HMGCR gene and a second 20 nucleotide region (underscored) is complementary and hybridizable to the 5' end of exon 19 of the HMGCR gene (see Figure 6B).

In another embodiment, an inventive HMGCRBsv4 polynucleotide probe comprises at least 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of HMGCR sequence that is complementary and hybridizable to an exon junction polynucleotide region created by alternative splicing of exon 16 to exon 19 of a transcript of the HMGCR gene. In most embodiments, the HMGCRsv4 polynucleotide probe is selected to comprise a first continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 3' end of exon 16, and a second continuous region of at least 5 to 20 nucleotides that is complementary and hybridizable to the 5' end of exon 19 of the HMGCR gene.

As will be apparent to a person of skill in the art, a large number of different HMGCR isoform-specific probes may be selected based on the detection need which will, under appropriate hybridization conditions, have the capacity to detectably hybridize to either HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polynucleotides, respectively, and yet will hybridize to a much less extent to an HMGCR isoform polynucleotides that is not the target HMGCR isoform polynucleotide.

Preferably, non-complementary nucleic acid that is present has a particular purpose such as being a reporter sequence or being a capture sequence. However, additional nucleic acid need not have a particular purpose as long as the additional nucleic acid sequence does not prevent the HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 probe nucleic acids from distinguishing between the target HMGCR isoform polynucleotide and non-target polynucleotides. For example, additional nucleic sequences to a HMGCRsvl probe should not prevent the probe from distinguishing in a hybridization assay between a HMGCRsvl polynucleotide and a non-target polynucleotide, including, but not limited to, a HMGCR isoform polynucleotide not comprising the exon 12 to exon 14 splice junction found in HMGCRsvl.

Hybridization occurs through complementary nucleotide bases. Hybridization conditions determine whether two molecules, or regions, have sufficiently strong interactions with each other to form a stable hybrid.

The degree of interaction between two molecules that hybridize together is reflected by the Tm of the produced hybrid. The higher the Tm the stronger the interactions and the more stable the hybrid. Tm is effected by different factors well known in the art such as the degree of complementarity, the type of complementary bases present (e.g., A-T hybridization versus G-C hybridization), the presence of modified nucleic acid, and solution components (e.g., Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989).

Stable hybrids are formed when the Tm of a hybrid is greater than the temperature employed under a particular set of hybridization assay conditions. The degree of specificity of a probe can be varied by adjusting the hybridization stringency conditions. Detecting probe hybridization is facilitated through the use of a detectable label. Examples of detectable labels include luminescent, enzymatic, and radioactive labels.

Examples of stringency conditions are provided in Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989. An example of high stringency conditions is as follows: Prehybridization of filters containing DNA is carried out for 2 hours to overnight at 65 °C in buffer composed of 6 X SSC (sodium saline citrate), 5 X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hours at 65 °C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2 X SSC, 0.1% SDS. This is followed by a wash in 0.1 X SSC, 0.1% SDS at 50°C for 45 minutes before autoradiography. Other procedures using conditions of high stringency would include, for example, either a hybridization step carried out in 5 X SSC, 5 X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2 X SSPE (sodium saline phophate-EDTA), 0.2% SDS at 65°C for 30 to 60 minutes. Recombinant Expression

HMGCR splice variant polynucleotides, such as those comprising SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, respectively, can be used to make HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides. In particular, HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides can be expressed from recombinant nucleic acids in a suitable host or in a test tube using a coupled transcription- translation system. Recombinantly expressed HMGCRsvl or HMGCRsv2 or HMGCRsv3 or HMGCRsv4 polypeptides can be used, for example, in assays to screen for compounds that bind to or interact with HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides, respectively. Alternatively, HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides can also be used to screen for compounds that bind to or interact with HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4, respectively, but do not bind to or interact with other protein isoforms of HMGCR.

In some embodiments, expression is achieved in a host cell using an expression vector. An expression vector contains recombinant nucleic acid encoding for a polypeptide along with regulatory elements for proper transcription and processing. The regulatory elements that may be present include those naturally associated with the recombinant nucleic acid and exogenous regulatory elements not naturally associated with the recombinant nucleic acid. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing recombinant nucleic acid in a particular host.

Generally, the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal providing for processing in eukaryotic cells. Preferably, an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.

Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. Mammalian expression vectors well known in the art include, but are not restricted to, pcDNA3 (Invitrogen, Carlsbad CA), pSecTag2 (Invitrogen), pMClneo (Stratagene, La Jolla CA), pXTl (Stratagene), pSG5 (Stratagene), pCMVLacl (Stratagene), pCI-neo (Promega), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146) and pUCTag (ATCC 37460), and. Bacterial expression vectors well known in the art include pETlla (Novagen), pBluescript SK (Stratagene, La Jolla), pQE-9 (Qiagen Inc., Valencia), lambda gtll (Invitrogen), pcDNAII (Invitrogen), and pKK223-3 (Pharmacia). Fungal cell expression vectors well known in the art include pPICZ (Invitrogen) and pYES2 (Invitrogen), Pichia expression vector (Invitrogen). Insect cell expression vectors well known in the art include Blue Bac III (Invitrogen), pBacPAK8 (CLONTECH, Inc., Palo Alto) and PfastBacHT (Invitrogen, Carlsbad).

Recombinant host cells may be prokaryotic or eukaryotic. Examples of recombinant host cells include the following: bacteria such as E. coli; fungal cells such as yeast; mammalian cells such as human, bovine, porcine, monkey and rodent; and insect cells such as Drosophila and silkworm derived cell lines. Commercially available mammalian cell lines include L cells L-M(TK^") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS- 7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

To enhance expression in a particular host it may be useful to modify the sequence provided in one or more of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, to take into account codon usage of the host. Codon usages of different organisms are well known in the art (see, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C).

Expression vectors may be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation.

Nucleic acid encoding for a polypeptide can be expressed in a cell without the use of an expression vector employing, for example, synthetic mRNA or native mRNA. Additionally, mRNA can be translated in various cell-free systems such as wheat germ extracts and reticulocyte extracts, as well as in cell based systems, such as frog oocytes. Introduction of mRNA into cell based systems can be achieved, for example, by microinjection.

HMGCR SPLICE VARIANT ISOFORM POLYPEPTIDES HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides contain amino acid sequences comprising, consisting or consisting essentially of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, respectively. HMGCR splice variant isoform polypeptides have a variety of uses, such as, for example, providing a marker for the presence of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides, respectively; being used as immunogens to produce antibodies binding to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4, respectively; being used as target polypeptides to identify compounds binding selectively to one or more HMGCRsvl, HMGCRsv2, HMGCRsv3, Or HMGCRsv4 polypeptides, respectively; or being used in an assay to identify compounds that bind to or interact with other isoforms of HMGCR, but do not bind to or interact with one or more HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides.

In chimeric polypeptides containing one or more regions from a splice variant isoform of HMGCR and one or more regions not from HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4, respectively, the region(s) not from HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be used, for example, to achieve a particular purpose or to produce polypeptides that can substitute for HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4, or fragments thereof. Particular purposes that can be achieved using a chimeric HMGCR splice variant isoform polypeptides include, providing a marker for HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 activities, respectively, enhancing an immune response, and modulating HDL cholesterol level in a subject.

Polypeptides can be produced using standard techniques including those involving chemical synthesis and those involving biochemical synthesis. Techniques for chemical synthesis of polypeptides are well known in the art (see e.g., Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990).

Biochemical synthesis techniques for polypeptides are also well known in the art. Such techniques employ a nucleic acid template for polypeptide synthesis. The genetic code providing the sequences of nucleic acid triplets coding for particular amino acids is well known in the art (see, e.g., Lewin GENES TV, p. 119, Oxford University Press, 1990). Examples of techniques for introducing nucleic acid into a cell and expressing the nucleic acid to produce protein are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989.

Functional HMGCR Splice Variant Isoforms

Functional HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 splice variants are proteins that are isoforms of HMGCR. The identification of the amino acid and nucleic acid sequences of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 provide tools for obtaining functional proteins related to each respective HMGCR splice variant isoform from other sources; for producing chimeric HMGCR splice variant isoform proteins; and for producing other functional derivatives of HMGCR splice variant isoform proteins: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, and SEQ ID NO 8. HMGCR splice variant isoform proteins of the invention can be readily identified and obtained based on their sequence similarity to SEQ ID NO 2 (HMGCRsvl), SEQ ID NO 4 (HMGCRsv2), SEQ ID NO 6 (HMGCRsv3), or SEQ ID NO 8 (HMGCRsv4), respectively. In particular, HMGCRsvl polypeptides lack the amino acids encoded by exon 13 of the HMGCR gene; HMGCRsv2 polypeptides lack the amino acids encoded by exon 8 of the HMGCR gene; HMGCRsv3 polypeptides lack the amino acids encoded by exon 18 of the HMGCR gene; and HMGCRsv4 polypeptides lack the amino acids encoded by exon 17 and exon 18 of the HMGCR gene. Both the nucleic acid and amino acid sequences of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be used to help identify and obtain HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides, respectively. For example, SEQ ID NO 1 can be used to produce degenerative nucleic acid probes or primers for identifying and cloning polynucleotides encoding for an HMGCRsvl polypeptide. A similar strategy can be used to clone and identify nucleic acids encoding HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides.

In addition, polynucleotides comprising, consisting, or consisting essentially of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, or fragments thereof can also be used under conditions of moderate stringency to identify and clone nucleic acid encoding the respective HMGCR splice variant isoform polypeptide from a variety of different organisms.

The use of degenerative probes and moderate stringency conditions for cloning is well known in the art. Examples of such techniques are described by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989.

Starting with HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 proteins, obtained from a particular source, derivatives can be produced. Such derivatives include polypeptides with amino acid substitutions, additions and deletions. Changes to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides to produce derivatives having essentially the same properties should be made in a manner without altering the tertiary structure of the respective HMGCR splice variant isoform protein.

Differences in naturally occurring amino acids are due to different R groups. An R group affects different properties of the amino acid such as physical size, charge, and hydrophobicity. Amino acids are can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tryosine, cysteine, asparagine, and glutamine); basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid).

Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing large changes in polypeptide functioning.

Changes outside of different amino acid groups can also be made. Preferably, such changes are made taking into account the position of the amino acid to be substituted in the polypeptide. For example, arginine can substitute more freely for nonpolar amino acids in the interior of a polypeptide then glutamate because of its long aliphatic side chain (See, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C).

HMGCR Splice Variant Isoform Antibodies

Antibodies recognizing HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be produced using a polypeptide comprising SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, respectively, or fragments thereof as immunogens. Preferably, an HMGCRsvl polypeptide used as an immunogen consists of a polypeptide derived from SEQ ID NO 2 or fragments thereof of having at least 10 contiguous amino acids in length encoded by a polynucleotide region representing the junction resulting from the splicing of exon 12 to exon 14 of the HMGCR gene. When HMGCRsv2 is used as an immunogen, preferably it consists of a polypeptide derived from SEQ ID NO 4 or fragments thereof of having at least 10 contiguous amino acids in length encoded by a polynucleotide region representing the junction resulting from the splicing of exon 7 to exon 9 of the HMGCR gene. When HMGCRsv3 is used as an immunogen, preferably it consists of a polypeptide derived from SEQ ID NO 6 or fragments thereof of having at least 10 contiguous amino acids in length encoded by a polynucleotide region representing the junction resulting from the splicing of exon 17 to exon 19 of the HMGCR gene. When HMGCRsv4 is used as an immunogen, preferably it consists of a polypeptide derived from SEQ ID NO 8 or fragments thereof of having at least 10 contiguous amino acids in length encoded by a polynucleotide region representing the junction resulting from the splicing of exon 16 to exon 19 of the HMGCR gene.

In some embodiments where, for example, HMGCRsvl is used to develop antibodies that bind specifically to HMGCRsvl and not to HMGCR or to other HMGCR isoforms, the HMGCRsvl polypeptide comprises at least 10 contiguous amino acids of HMGCRsvl encoded by a junction polynucleotide region created by the splicing of exon 12 to exon 14 of a transcript of the HMGCR gene (see Figure 3). For example, the amino acid sequence: amino terminus-YNYSLLGGGA-carboxy terminus [SEQ ID NO 13], represents one embodiment of such an inventive HMGCRsvl polypeptide wherein the first 5 amino acid region is encoded by a nucleotide sequence at the 3' end of exon 12 of the HMGCR gene and a second 5 amino acid region is encoded by nucleotides at the 5' end of exon 14 (see Figure 3). Preferably, the at least 10 amino acids of the HMGCRsvl polypeptide comprises a first continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 3' end of exon 12 and a second continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 5' end of exon 14.

In other embodiments where, for example, HMGCRsv2 is used to develop antibodies that bind specifically to HMGCRsv2 and not to HMGCR or to other HMGCR isoforms, the HMGCRsv2 polypeptide comprises at least 10 contiguous amino acids of HMGCRsv2 encoded by a junction polynucleotide region created by the splicing of exon 7 to exon 9 of the primary transcript the HMGCR gene (see Figure 4). For example, the amino acid sequence: amino terminus- VSLVLESLGLV-carboxy terminus [SEQ ID NO 14], represents one embodiment of such an inventive HMGCRsv2 polypeptide wherein the first 5 amino acid region is encoded by a nucleotide sequence at the 3' end of exon 7 of the HMGCR gene and a second 5 amino acid region is encoded by nucleotides at the 5' end of exon 9 (see Figure 4). Preferably, at least 10 amino acids of the HMGCRsv2 polypeptide comprises a first continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 3' end of exon 7 and a second continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 5' end of exon 9.

In another embodiments where, for example, HMGCRsv3 is used to develop antibodies that bind specifically to HMGCRsv3 and not to HMGCR or other HMGCR isoforms, the HMGCRsv3 polypeptides comprise at least 10 contiguous amino acids of the HMGCRsv3 encoded by a junction polynucleotide region created by the alternative splicing of exon 17 to exon 19 of a transcript the HMGCR gene (see Figure 5). For example, the amino acid sequence: amino terminus -IACGQMLGVQ- carboxy terminus [SEQ ID NO 15], represents one embodiment of such an inventive HMGCRsv3 polypeptide wherein the first 5 amino acid region is encoded by a nucleotide sequence at the 3' end of exon 17 of the HMGCR gene and a second 5 amino acid region is encoded by nucleotides at the 5' end of exon 19 (see Figure 5). Preferably, at least 10 amino acids of the HMGCRsv3 polypeptide comprises a first continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 3' end of exon 17 and a second continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 5' end of exon 19. In yet another embodiments where, for example, HMGCRsv4 is used to develop antibodies that bind specifically to HMGCRsv4 and not to HMGCR or other HMGCR isoforms, the HMGCRsv4 polypeptides comprise at least 10 contiguous amino acids of the HMGCRsv4 encoded by a junction polynucleotide region created by the alternative splicing of exon 16 to exon 19 of a transcript of the HMGCR gene (see Figure 6). For example, the amino acid sequence: amino terminus-KVVREMLGVQ-carboxy terminus [SEQ ID NO 16], represents one embodiment of such an inventive HMGCRsv4 polypeptide wherein the first 5 amino acid region is encoded by a nucleotide sequence at the 3' end of exon 16 of the HMGCR gene and a second 5 amino acid region is encoded by nucleotides at the 5' end of exon 19 (see Figure 6). Preferably, at least 10 amino acids of the HMGCRsv4 polypeptide comprises a first continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 3' end of exon 16 and a second continuous region of 2 to 8 amino acids that are encoded by nucleotides at the 5' end of exon 19.

In other embodiments, HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4-specific antibodies are made using an HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptide, respectively, that comprises at least 20, 30, 40 or 50 amino acids of the respective HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 sequences, that correspond to a isoform specific exon junction polynucleotide region created by alternative splicing of the HMGCR transcript. For example, in the case of HMGCRsvl, an amino acid region is selected that is encoded by a polynucleotide that spans the exon junction created by splicing of exon 12 to exon 14 in a transcript of the HMGCR gene. Alternatively, in the case of HMGCRsv2, an amino acid region is selected that is encoded by a polynucleotide that spans the exon junction created by splicing of exon 7 to exon 9 in a transcript of the HMGCR gene. In the case of HMGCRsv3, an amino acid region is selected that is encoded by a polynucleotide that spans the exon junction created by splicing of exon 17 to exon 19 in a transcript of the HMGCR gene. In the case of HMGCRsv4, an amino acid region is selected that is encoded by a polynucleotide that spans the exon junction created by splicing of exon 16 to exon 19 in a transcript of the HMGCR gene.

In other embodiments, an HMGCRsvl polypeptide is selected to comprise a first continuous region of at least 5 to 15 amino acids that is encoded by nucleotides at the 3' end of exon 12 and a second continuous region of at least 5 to 15 amino acids that is encoded by nucleotides at the 5' end of exon 14 of the HMGCR gene. A person skilled in the art can use a similar strategy to design HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides that are selected to comprise a 3' end and 5' end of exon junction sequences that are unique to each respective HMGCR splice variant. Antibodies to HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 have different uses such as being used to identify the presence of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides, respectively, and to isolate HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides, respectively. Identifying the presence of HMGCRsvl can be used, for example, to identify cells producing HMGCRsvl. Such identification provides an additional source of HMGCRsvl and can be used to distinguish cells known to produce HMGCRsvl from cells that do not produce HMGCRsvl. For example, antibodies to HMGCRsvl can distinguish human cells expressing HMGCRsvl proteins or polypeptides from human cells not expressing HMGCRsvl or non-human cells (including bacteria) that do not express HMGCRsvl. Such HMGCRsvl antibodies can also be used to determine the effectiveness of HMGCRsvl ligands, using techniques well known in the art, to detect and quantify changes in the protein levels of HMGCRsvl in cellular extracts, and in situ immunostaining of cells and tissues. In addition, the same above- described utilities also exist for HMGCRsv2, HMGCRsv3, and HMGCRsv4-specific antibodies.

Techniques for producing and using antibodies are well known in the art. Examples of such techniques are described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998; Harlow, et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; and Kohler, et al., 1975 Nature 256:495-7.

HMGCR Splice Variant Isoform Binding Assays

A number of compounds known to act as inhibitors that block HMG-CoA reductase activity have been disclosed (see for example, U.S. Patent Numbers 3,883,140; 4,231,938; 4,346,227; 4,444,784; 4,450,171; 4,450,171; 4,739,073 and 5,273,995). Organic compounds, such as statins, that block HMG-CoA reductase activity have been used for treating hypercholesterolemia and dyslipidemia (For a review, Mahley, R.W. and Bersot, T.P., In, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw-Hill, New York, 1996, Ch. 36, pp. 984-995). In addition, a variety of high throughput assays are known in the art that can be used to screen for compounds that bind to, and functionally alter HMGCR proteins as a class or in an isoform-specific fashion.

HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4, or fragments thereof can be used in binding studies to identify compounds binding to each respective protein. In other embodiment, a first HMGCR splice variant isoform or fragments thereof is used in binding studies with a second HMGCR isoform, or a fragment thereof, that is different from the first HMGCR isoform to identify compounds that bind to both the first and second HMGCR isoform polypeptides; compounds that bind the first HMGCR isoform protein but do not bind to the second HMGCR isoform protein; or compounds that bind to the second HMGCR isoform protein but do not bind to the first HMGCR isoform protein. Such binding methods can be performed using different formats including competitive and non-competitive formats. Further competition binding assays can also be carried out using additional compounds to determine binding to HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4, HMGCR or fragments thereof.

The particular HMGCR splice variant isoform amino acid sequences involved in ligand-binding can be readily identified by using labeled compounds that bind to the protein and to different protein fragments. Different strategies can be employed to select fragments to be tested to narrow down the binding region(s). Examples of such strategies include testing consecutive fragments about 15 amino acids in length starting at the N- terminus, and testing longer length fragments. If longer length fragments are tested, a fragment binding to a compound can be subdivided to further locate the binding region. Fragments used for binding studies can be generated using recombinant nucleic acid techniques.

Preferably, binding studies are performed using HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides expressed from recombinant nucleic acids. More preferably, recombinantly expressed HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptide consists of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, respectively.

Binding assays can be performed using individual compounds or preparations containing different numbers of compounds. A preparation containing different numbers of compounds having the ability to bind to an HMGCR splice variant isoform of the present invention can be divided into smaller groups of compounds that can be tested to identify the compound(s) binding to the selected HMGCR splice variant isoform.

Binding assays can be performed using recombinantly produced HMGCR splice variant isoform proteins present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing HMGCRsvl, HMGCRsv2, HMGCRsv 3, ox HMGCRsv4 recombinant nucleic acids; and also include, for example, the use of purified HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 polypeptides produced by recombinant means which are introduced into different environments.

In one embodiment of the invention, a binding method is provided for screening for a compound able to bind selectively to an HMGCR isoform polypeptide. The method comprises the steps: providing a first HMGCR isoform polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6; and SEQ ID NO 8; providing a second HMGCR isoform polypeptide that is not the first HMGCR isoform polypeptide; contacting the first HMGCR isoform polypeptide and the second HMGCR polypeptide with a test preparation comprising one or more test compounds; and then determining the binding of the test preparation to the first HMGCR isoform polypeptide and to the second HMGCR isoform polypeptide, wherein a compound which binds to the first HMGCR isoform polypeptide but does not bind to the second HMGCR isoform polypeptide, is a compound that selectively binds the first HMGCR isoform polypeptide.

In another embodiment of the invention, the compound binding method comprises the steps: providing a first and a second HMGCR isoform polypeptide wherein the first HMGCR isoform polypeptide has an amino acid sequence that is different from the second HMGCR polypeptide and wherein the second HMGCR isoform comprises an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6; and SEQ ID NO 8; contacting the first HMGCR isoform polypeptide and the second HMGCR polypeptide with a test preparation comprising one or more test compounds; and then determining the binding of the test preparation to the first HMGCR isoform polypeptide and to the second HMGCR isoform polypeptide, wherein a compound which binds to the first HMGCR isoform polypeptide but does not bind to the second HMGCR isoform polypeptide, is a compound that selectively binds the first HMGCR isoform polypeptide.

The above-described selective binding assays can also be performed with polypeptide fragments of an HMGCR isoform polypeptide. In particular, when using a fragment of HMGCRsvl, the polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that bridges the splice junction created by the splicing of the 3' end of exon 12 to the 5' end of exon 14 in HMGCR mRNA. When using a fragment of HMGCRsv2, the polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that bridges the splice junction created by the splicing of the 3' end of exon 7 to the 5' end of exon 9 in HMGCR mRNA. When using a fragment of HMGCRsv3, the polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that bridges the splice junction created by the splicing of the 3' end of exon 17 to the 5' end of exon 19 in HMGCR mRNA. When using a fragment of HMGCRsv4, the polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that bridges the splice junction created by the splicing of the 3' end of exon 16 to the 5' end of exon 19 in HMGCR mRNA.

Similarly, the selective binding assays may also be performed using a polypeptide fragment of a HMGCR polypeptide that is not found in another of the HMGCR isoform polypeptides being screened in the same binding assay, e.g., HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4. For example, when HMGCRsvl or a fragment thereof is in the same binding assay with a fragment of the HMGCR polypeptide, the HMGCR polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that is contained within exon 13 of HMGCR mRNA. When HMGCRsv2 or a fragment thereof is in the same binding assay with a fragment of the HMGCR polypeptide, the HMGCR polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that is contained within exon 8 of HMGCR mRNA. When HMGCRsv3 or a fragment thereof is in the same binding assay with a fragment of the HMGCR polypeptide, the HMGCR polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that is contained within exon 18 of HMGCR mRNA. When HMGCRsv4 or a fragment thereof is in the same binding assay with a fragment of the HMGCR polypeptide, the HMGCR polypeptide fragment comprises at least 10 consecutive amino acids that are encoded by a nucleotide sequence that is contained within exon 17 and/or exon 18 of HMGCR mRNA.

HMGCR Functional Assays

The identification of splice variants of HMGCR provides a means for screening for compounds that bind to one or more of the HMGCR splice variant isoform proteins thereby altering the ability of the HMGCR splice variant isoform polypeptide to bind to NADPH and or HMG-CoA or any other reaction intermediate compound, or to bind to a statin compound, or to perform as a HMG-CoA reductase enzyme, including any HMGCR sub-reactions as described, for example by Frimpong and Rodwell (1994 J. Biol. Chem. 269:11478-83). Assays involving a functional HMGCR splice variant isoform polypeptide can be employed for different purposes such as selecting for compounds active on one or more HMGCR isoform polypeptide; evaluating the ability of a compound to alter HMG-CoA reductase activity; and mapping the activity of different HMGCR splice variant polypeptide regions. HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 activity can be measured using different techniques such as: detecting a change in the intracellular conformation of the HMGCR splice variant protein being tested; detecting the amount of binding of NADPH, HMG-Co-A, or a statin compound to HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4 compared to a different HMGCR isoform protein; or measuring the level of HMG-CoA reductase activity of the selected HMGCR isoform variant, e.g., one or more of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4.

Recombinantly expressed HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be used to facilitate determining whether a compound is active on a selected HMGCR splice variant isoform polypeptide. For example, HMGCRsvl can be expressed by an expression vector containing an HMGCRsvl polynucleotide in a cell line and used in a co- culture growth assay, such as described in WO 99/59037, to identify compounds that bind to HMGCRsvl as compared to binding to second HMGCR isoform protein, e.g., HMGCR or another HMGCR splice variant isoform protein. A similar assay can be used with recombinantly expressed HMGCRsv2, HMGCRsv3, or HMGCRsv4.

Techniques for measuring HMG-CoA reductase activity are well known in the art (Heller and Shrewsbury, 1976 J. Biol. Chem., 251:3815-22; Edwards et al., 1979 J. Lipid Res. 20:40-6; Kleinsek et al, 1981 in Methods in Enzymology (Lowenstein, Edl), 71:462-79, Academic Press, San Diego). In particular, Mayer, et al. (1988 Arch. Biochem. and Biophys., 267:110-18) report methods for expressing a recombinant fragment of the HMGCR gene in E. coli to produce truncated HMGCR polypeptides comprising the catalytic domain of HMGCR. Mayer, et al., also describes methods for purifying the expressed HMGCR polypeptide from E. coli extracts using an HMG-CoA reductase enzyme assay. Large varieties of other assays have been used to investigate the properties of HMGCR and therefore would also be applicable to the measurement of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 function.

Functional assays of HMGCR isoform protein can be performed using cells expressing HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4, respectively, at high levels, and then contacted the cells or protein purified from the cells with individual compounds or preparations containing different compounds. A preparation containing different compounds where one or more compounds affects a test HMGCR splice variant isoform polypeptide (e.g., HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4) in cells over-producing the test HMGCR isoform protein as compared to control cells containing an expression vector lacking the test HMGCR isoform protein; can be divided into smaller groups of compounds to identify the compound(s) affecting the activity of the test HMGCR isoform polypeptide.

HMGCR isoform protein functional assays can be performed using a recombinantly produced HMGCR isoform protein, such as HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4, that is present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the selected HMGCR isoform protein expressed from recombinant nucleic acid and an appropriate membrane for the polypeptide; and the use of a purified HMGCR isoform protein produced by recombinant means that is introduced into a different environment suitable for measuring HMG-CoA reductase. MODULATING HMGCR ISOFORM EXPRESSION HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 protein expression can be modulated as a means for increasing or decreasing HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 activities, respectively. Such modulation includes inhibiting the activity of nucleic acids encoding the target HMGCR isoform peptide to reduce HMGCR isoform protein expression, or supplying HMGCR nucleic acids to increase the level of expression of the target HMGCR isoform polypeptide thereby increasing the target HMGCR activity.

Inhibition of HMGCR Isoform Activities

HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 nucleic acid activities can be inhibited using nucleic acids recognizing HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 nucleic acids, respectively, and affecting the ability of such nucleic acids to be transcribed or translated. Inhibition of HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 nucleic acid activities can be used, for example, in target validation studies.

A preferred target for inhibiting HMGCRsvl, HMGCRsv2, HMGCRsv3, HMGCRsv4 translation is mRNA. The ability of mRNA encoding HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 to be translated into a protein can be effected by compounds such as anti-sense nucleic acid, RNA interference (RNAi) and enzymatic nucleic acid.

Anti-sense nucleic acid can hybridize to a region of a target mRNA. Depending on the structure of the anti-sense nucleic acid, anti-sense activity can be brought about by different mechanisms such as blocking the initiation of translation, preventing processing of mRNA, hybrid arrest, and degradation of mRNA by RNAse H activity.

RNAi also can be used to prevent protein expression of a target transcript. This method is based on the interfering properties of double-stranded RNA derived from the coding regions of gene that disrupts the synthesis of protein from transcribed RNA.

Enzymatic nucleic acid can recognize and cleave another nucleic acid molecule. Preferred enzymatic nucleic acids are ribozymes.

General structures for anti-sense nucleic acids, RNAi and ribozymes, and methods of delivering such molecules, are well known in the art. Modified and unmodified nucleic acids can be used as anti-sense molecules, RNAi and ribozymes. Different types of modifications can modify certain anti-sense activities such as the ability to be cleaved by RNAse H, and can alter nucleic acid stability. Examples of references describing different anti-sense molecules, and ribozymes, and the use of such molecules, are provided in U.S. Patent Nos. 5,849,902; 5,859,221; 5,852,188; and 5,616,459. Examples of organisms in which RNAi has been used to inhibit expression of a target gene include: C. elegans (Tabara, et al., 1999 Cell 99:123-32; Fire, et al., 1998 Nature 391:806-11), plants (Hamilton and Baulcombe, 1999 Science 286:950-52), Drosophila (Hammond, et al., 2001 Science 293:1146-50; Misquitta and Patterson, 1999 Proc. Nat. Acad. Sci. 96:1451-56; Kennerdell and Carthew, 1998 Cell 95:1017-26), and mammalian cells (Bernstein, et al, 2001 Nature 409:363-6; Elbashir, et al., 2001 Nature 411:494-8).

Increasing HMGCR Isoform Expression

Nucleic acid coding for HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 can be used, for example, to cause an increase in HMG-CoA reductase or to create a test system (e.g., a transgenic animal) for screening for compounds affecting HMGCRsvl, HMGCRsv2, HMGCRsv3, or HMGCRsv4 expression. Nucleic acids can be introduced and expressed in cells present in different environments.

Guidelines for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences, 18^th Edition, supra, and Modern Pharmaceutics, 2^nd Edition, supra. Nucleic acid can be introduced into cells present in different environments using in vitro, in vivo, or ex vivo techniques. Examples of techniques useful in gene therapy are illustrated in Gene Therapy & Molecular Biology: From Basic Mechanisms to Clinical Applications, Ed. Boulikas, Gene Therapy Press, 1998.

EXAMPLES Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.

Example 1: Identification of HMGCR Splice Variants Using Tiling Microarrays

To identify variants of the "normal" splicing of the exon regions encoding HMGCR, an exon junction microarray, comprising probes complementary to each splice junction resulting from splicing of the 20 exon coding sequences in HMGCR heteronuclear RNA, was hybridized to a mixture of cRNAs prepared from samples obtained from 79 different human tissues. Exon junction microarrays are described in PCT patent applications WO 02/18646 and WO 02/16650. Materials and methods for preparing hybridization samples from purified RNA, hybridizing the microarrays, detecting hybridization signals, and data analysis are described in van't Veer, et al. (2002 Nature 415:530-536) and Hughes, et al. (2001 Nature Biotechnol. 19:342-7). Inspection of the exon junction microarray hybridization data (not shown) suggested that the mRNA structure of HMGCR was altered at several different exon splice junctions. In particular, the splice junctions of exons 12 and 14, exons 7 and 9, exons 17 and 19, and exons 16 and 19 of HMGCR mRNA were altered in a number of tissues examined, suggesting the presence of at least four different HMGCR splice variant mRNA populations within the normal HMGCR mRNA population. An RT-PCR assay was then performed using oligonucleotide primers complementary to selected exons, as described in more detail below, to confirm the exon junction array results and to allow the sequence structure of the HMGCR splice variants to be determined.

Example 2: Confirmation of HMGCR Splice Variants Using RT-PCR

The structures of HMGCR mRNA in the regions implicated by the exon junction microarray data were determined for a panel of human tissues using a reverse transcription and polymerase chain reaction (RT-PCR) based assay (Figures 1 and 2). PolyA purified mRNA isolated from 79 different human tissues was obtained from BD Biosciences Clontech (Palo Alto, CA), Biochain Institute, Inc. (Hayward, CA), and Ambion Inc. (Austin, TX). In addition, one monkey mRNA sample (brain, from Biochain Institute, Inc.) was also obtained and assayed. RT-PCR primers of 28 to 30 nucleotides were selected that were complementary to sequences in a variety of HMGCR (NMJ300859) exons.

Based upon the nucleotide sequence of HMGCR mRNA, an HMGCR exon 12 and exon 15 primer set (hereafter HMGCRu-is primer set) was expected to amplify a 417 base pair amplicon representing the "normal" HMGCR mRNA exon 12 to exon 15 mRNA region. The HMGCR exon 12 primer has the sequence: 5' AAACTCTGATGGAAACTCAT GAGCGTGG 3' [SEQ ID NO 17]; and the HMGCR exon 15 primer has the sequence: 5' TCCTTTATCACTGCGAACCCTTCAGATG 3' [SEQ ID NO 18].

Similarly, RT-PCR primers of 28 nucleotides were selected that were complementary to sequences in exons 5 and 9 in HMGCR (NM_000859). Based upon the nucleotide sequence of HMGCR mRNA, the HMGCR exon 5 and exon 9 primer set (hereafter HMGCR₅.₉ primer set) was expected to amplify a 417 base pair amplicon representing the "normal" HMGCR mRNA exon 5 to exon 9 mRNA region. The HMGCR exon 5 primer has the sequence: 5' GACCTTTCCAGAGCAAGCACATTAGCAA 3' [SEQ ID NO 19]; and the HMGCR exon 9 primer has the sequence: 5' ACTGTGAGCATGAACA AGAACCAAGCCT 3' [SEQ ID NO 20].

RT-PCR primers of 28 nucleotides were selected that were complementary to sequences in exons 16 and 20 in HMGCR (NM_000859). Based upon the nucleotide sequence of HMGCR mRNA, the HMGCR exon 16 and exon 20 primer set (hereafter HMGCR₁₆-₂₀ primer set) was expected to amplify a 787 base pair amplicon representing the "normal" HMGCR mRNA exon 16 to exon 20 mRNA region. The HMGCR exon 16 primer has the sequence: 5' ATTTCCCTGAAATGCAGATTCTAGCCGT 3' [SEQ ID NO 21]; and the HMGCR exon 16 primer has the sequence: 5' CCAAAAACCAAGTGGCTGTCTC AGTGAT 3' [SEQ ID NO 22].

Twenty-five ng of polyA mRNA from each tissue was subjected to a one-step reverse transcription-PCR amplification protocol using the Qiagen, Inc. (Valencia, CA), One- Step RT-PCR kit, using the following conditions:

Cycling conditions were as follows: 50°C for 30 minutes; 95°C for 15 minutes; 35 cycles of:

95°C for 1 minutes;

60°C for 1 minutes;

72°C for 1 minutes; then 72°C for 15 minutes.

RT-PCR amplification products (amplicons) were size fractionated on a 2% agarose gel (see FIG. 1). Selected amplicon fragments were manually extracted from the gel and purified with a Qiagen Gel Extraction Kit. Purified amplicon fragments were sequenced from each end (using the same primers used for RT-PCR) by Qiagen Genomics, Inc. (Bothell, Washington).

Several different RT-PCR amplicons were obtained from human mRNA samples using the HMGCR_\2-_\5 primer set (Figure 1A). Every human tissue assayed, except peripheral leukocytes (Figure 1A, lane 40), exhibited the expected amplicon size of 417 base pairs for normally spliced HMGCR mRNA (reference form). In addition, the monkey brain mRNA sample (e.g., Figure 1 A, lane 80) also exhibited the expected 417 base pair amplicon. However, in addition to the expected HMGCR amplicon of 417 base pairs, all of the human tissues also exhibited a second amplicon of 255 base pairs (short form). Indeed, the peripheral leukocyte mRNA sample (Figure 1 A, lane 40) appears to have mRNA that predominantly results in amplification of the short, 255 base pair amplicon form. Interestingly, skeletal muscle mRNA (FIG. 1 A, lane 47), appears to exhibit three different HMGCR mRNA forms; the reference form, the 255 base pair short form, and an intermediate sized amplicon of about 325 base pairs. The intermediate sized amplicon was only observed in mRNA derived from human skeletal tissue and upon further sequence analysis was found to represent an amplification artifact.

Similar RT-PCR analysis of the samples using the HMGCRsv primer set reveals an additional splice variant of HMGCR mRNA (Figure 2A). All three samples shown in Figure 2A (lands 1-3) exhibited the expected amplicon size of 417 base pairs for normally spliced HMGCR mRNA. However, in addition to the expected HMGCR amplicon of 417 base pairs, a second amplicon of about 300 base pairs (labeled HMGCRsv2) was also observed in human peripheral leukocytes (Figure 2A, lane 3) and in human skeletal muscle (data not shown). Sequence analysis of the 300 base pair amplicon from peripheral leukocytes (Figure 2A, lane 3) of revealed that this amplicon form is due to splicing of exon 7 of the HMGCR hnRNA to exon 9. That is, the 300 base pair HMGCR amplicon is due to the complete absence of the exon 8 nucleotide sequence. The new splice variant of HMGCR was named HMGCRsv2.

RT-PCR analysis of the human and monkey samples using the HMGCRu-2₀ primer set revealed an additional two splice variants of HMGCR mRNA (Figure 2B). All four samples shown in Figure 2B (lanes 1-4) exhibited the expected amplicon size of 787 base pairs for normally spliced HMGCR mRNA. However, in addition to the expected HMGCR amplicon of 787 base pairs, a second amplicon of about 650 base pairs (labeled HMGCRsv3) was also observed in human ileocecum, transverse colon, descending colon and human peripheral leukocytes (Figure 2B, lanes 1-4). An additional third amplicon of about 500 base pairs (labeled HMGCRsv4) was also observed in human peripheral leukocytes (Figure 2B, lane 4). Sequence analysis of the 650 base pair amplicon of HMGCR from the descending colon sample revealed a nucleotide sequence that is consistent with splicing of exon 17 of the HMGCR hnRNA to exon 19. That is, the 650 base pair HMGCR amplicon is due to the complete absence of the exon 18 nucleotide sequence. The new splice variant of HMGCR was named HMGCRsv3.

Sequence analysis of the 500 base pair amplicon of HMGCR from the peripheral leukocyte RNA sample revealed that this amplicon form is consistent with splicing of exon 16 of the HMGCR hnRNA to exon 19. That is, the 500 base pair form HMGCR amplicon is due to the complete absence of the exons 17 and 18 nucleotide sequences. The new splice variant of HMGCR was named HMGCRsv4.

Example 3: Cloning of HMGCRsyl Polynucleotides

Microarray and RT-PCR data indicate that in addition to normal HMGCR mRNA sequence, NM_000859, encoding HMGCR protein, NP_000850, splice variant forms of HMGCR mRNA also exist in some tissues. Indeed, inspection of the amplicon band intensities in Figures 1 A suggests that the HMGCRsvl form of HMGCR mRNA is present in an amount that is about equal to or slightly less than the "reference" HMGCR mRNA containing exon 13.

A full-length clone having a nucleotide sequence comprising the HMGCRsvl was isolated using a combination of reverse transcription (RT) and polymerase chain reaction (PCR). More specifically, human liver-left lobe polyA mRNA was reverse transcribed using Superscript II (Gibco/Invitrogen,, Carlsbad, CA) according to the Superscript II manufacturer's instruction. PCR was performed using a 5' "forward" HMGCR primer, designed to have a nucleotide sequence of 5' ATGTTGTCAAGACTTTTTCGAATGCA TGGC 3' (SEQ ID NO 22), and a 3' "reverse" HMGCR primer, designed to have the nucleotide sequence of 5' TCAGGCTGTCTTCTTGGTGCAAGCTCCTT 3' (SEQ ID NO 23). After an initial 95°C denaturation of 1 minute, 35 cycles of amplification were performed using a 30 second denaturation at 95°C followed by a 3 minute annealing and synthesis at 68°C. The 35 cycles of PCR were followed by a 3 minute extension at 68°C. The RT-PCR amplification products (amplicons) were size fractionated on a 1% agarose gel. A product of about 2.7 kilobases (Kb) was manually extracted from the gel and purified using the QIAquik Gel extraction Kit (Qiagen, Valencia, CA).

Twenty transformants were screened by using the HMGCR_{U S} primer set to identify clones containing the HMGCRsvl structure. Three clones produced the 255 base pair amplicon representing HMGCRsvl, whereas the other seventeen clones produced the 417 base pair amplicon representing the reference HMGCR. DNA sequence analysis of the about 2.7 Kb inserts of the three HMGCRsvl clones was performed. The consensus sequence of the three clones produced the polynucleotide sequence set forth in SEQ ID NO 1. SEQ ID NO 1 presents an HMGCRsvl polynucleotide sequence having an open reading frame that encodes a HMGCRsvl protein (SEQ ID NO 2) identical to the HMGCR (NP_000850), but lacking a 53 amino acids region encoded by exon 13 of the full length, non-splice variant HMGCR mRNA (NM_000859).

Example 4: Cloning of HMGCRsv2. HMGCRsv3, and HMGCRsv4 Polynucleotides

Microarray and RT-PCR data also indicate that in addition to normal HMGCR mRNA sequence, NM_000859, encoding HMGCR protein, NP_000850, splice variant forms sv2, sv3, and sv4 of HMGCR mRNA also exist in some tissues. Indeed, inspection of the amplicon band intensities in Figures 2 A and B, similarly show that sv2, sv3 and sv4 splice variant forms of HMGCR mRNA are also present in samples as mixed populations in amounts much lower than the reference HMGCR containing all of the NM_000859 exons.

A full length HMGCR clone having a nucleotide sequence comprising the above-described HMGCR splice variants HMGCRsv2, HMGCRsv3, and HMGCRsv4 are isolated using the same method as was used to obtain the HMGCRsvl clone. HMGCRsv2, HMGCRsv3, and HMGCRsv4 are cloned from appropriate mRNA samples that were shown in Example 2 to have the largest amount of the target HMGCR splice variant mRNA.

RT-PCR The HMGCRsv2, HMGCRsv3, and HMGCRsv4 cDNA sequences are cloned using a combination of reverse transcription (RT) and polymerase chain reaction (PCR). More specifically, about 25 ng of human polyA mRNA from a human sample shown in Example 2 to have the largest amount of the target HMGCR splice variant mRNA is reverse transcribed using Superscript II (Gibco/Invitrogen,, Carlsbad, CA) and oligo d(T) primer (RESGEN/Invitrogen,, Huntsville, AL) according to the Superscript II manufacturer's instructions. For PCR, 1 μl of the completed RT reaction is added to 40 μl of water, 5 μl of 10X buffer, 1 μl of dNTPs and 1 μl of enzyme from the Clontech (PaloAlto, CA) Advantage 2 PCR kit. PCR is done in a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) using the HMGCR "forward" and 'reverse" primers. After an initial 94°C denaturation of 1 minute, 35 cycles of amplification are performed using a 30 second denaturation at 94°C followed by a 3 minute annealing at 65°C and synthesis at 68°C. The 35 cycles of PCR are followed by a 7 minute extension at 68°C. The 50 μl reaction is then chilled to 4°C. 40 μl of the RT-PCR amplification products (amplicons) is size fractionated on a 1% agarose gel (see Figures 1 and 2). A product of about 2.7 kilobases (Kb) is manually extracted from the gel and purified using the QIAquik Gel extraction Kit (Qiagen, Valencia, CA).

Cloning of RT-PCR Products

About 4 μl of the 40 μl of purified RT-PCR products from either peripheral leukocytes (for, HMGCRsv2 and HMGCRsv4) or descending colon (for HMGCRsv3) are used in a cloning reaction using the reagents and instructions provided with the TOPO XL cloning kit (Invitrogen, Carlsbad, CA). About 2 μl of the cloning reaction is used following the manufacturer's instructions to transform TOP 10 chemically competent E. coli provided with the cloning kit. After the 1 hour recovery of the cells in SOC medium (provided with the TOPO XL cloning kit), 200 μl of the mixture is plated on LB medium plates (Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989) containing 50 μg/ml Kanamycin (Sigma, St. Louis, MO). Plates are incubated overnight at 37°C. Fifty colonies resulting from each splice variant cloning reaction are picked from the plates into 2 ml of LB medium containing 50 μg of Kanamycin per ml. These liquid cultures are incubated overnight on a roller at 37°C. Plasmid DNA is extracted from these cultures using the Qiagen (Valencia, CA) Qiaquik Spin Miniprep kit.

Plasmid DNA is purified from each set of 50 putative HMGCR splice variant clones and subjected to PCR using the appropriate HMGCR primer set (e.g., HMGCR₇-₉ for HMGCRsv2; and HMGCR₁₆.₂o for HMGCRsv3 and HMGCRsv4). The HMGCRη- primer set is used to identify clones containing the HMGCRsv2 structure by amplification of a 300 base pair amplicon, whereas the reference HMGCR clone will give rise to a 417 base pair amplicon. The

primer set is used to identify clones containing the HMGCRsv3 structure by amplification of a 650 base pair amplicon, whereas the reference HMGCR clone will give rise to a 787 base pair amplicon. The HMGCRιβ-20 primer set is also used to identify clones containing the HMGCRsv4 structure by amplification of a 500 base pair amplicon. Twelve clones giving rise to the RT-PCR products diagnostic for each of the four HMGCR splice variants are selected for further analysis.

The predicted size for a cDNA insert for each of the HMGCR splice variants is about 2.4 to 2.5 Kb. Twelve clones having the proper RT-PCR reaction products upon use of the appropriate HMGCR exon primer pairs for HMGCRsv2, HMGCRsv3, and HMGCRsv4 are identified and prepared for a second RT-PCR reaction to confirm the presence of the expected splice variant structure within the full length HMGCR splice variant cDNA clone.

DNA sequence analysis of the about 2.5 Kb insert from at least one of the HMGCRsv2 clones produces a polynucleotide sequence set forth in SEQ ID NO 3. SEQ ID NO 3 presents an HMGCRsv2 polynucleotide sequence having an open reading frame that encodes a HMGCRsv2 protein (SEQ ID NO 4) identical to the HMGCR (NP_000850), but lacking a 39 amino acids region encoded by exon 8 of the full length, non-splice variant HMGCR mRNA (NM_000859).

DNA sequence analysis of the about 2.5 Kb insert from at least one of the HMGCRsv3 clones produces a polynucleotide sequence set forth in SEQ ID NO 5. SEQ ID NO 5 presents an HMGCRsv3 polynucleotide sequence having an open reading frame that encodes a HMGCRsv3 protein (SEQ ID NO 6) identical to the HMGCR (NP_000850), but lacking a 53 amino acids region encoded by exon 18 of the full length, non-splice variant HMGCR mRNA (NM_000859).

DNA sequence analysis of the about 2.4 Kb insert from at least one of the HMGCRsv4 clones produces a polynucleotide sequence set forth in SEQ ID NO 7. SEQ ID NO 7 presents an HMGCRsv4 polynucleotide sequence having an open reading frame that encodes a HMGCRsv4 protein (SEQ ID NO 8) identical to the HMGCR (NP_000850), but lacking a 100 amino acids region encoded by exons 17 and 18 of the full length, non-splice variant HMGCR mRNA (NM_000859).

All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are shown and described, persons skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. Various modifications may be made to the embodiments described herein without departing from the spirit and scope of the present invention. The present invention is limited only by the claims that follow.

Claims

WHAT IS CLAIMED:

1. A purified human nucleic acid comprising SEQ ID NO 1, or the complement thereof.

2. The purified nucleic acid of claim 1, wherein said nucleic acid comprises a sequence encoding SEQ ID NO 2.

3. The purified nucleic acid of claim 1, wherein said nucleic acid encodes a polypeptide consisting of SEQ ID NO 2.

4. A purified polypeptide comprising SEQ ID NO 2.

5. The polypeptide of claim 4, wherein said polypeptide consists of SEQ ID NO 2.

6. An expression vector comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO 2, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.

7. The expression vector of claim 6, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 2.

8. The expression vector of claim 6, wherein said nucleotide sequence comprises SEQ ID NO 1.

9. The expression vector of claim 6, wherein said nucleotide sequence consists of SEQ ID NO 1.

10. A recombinant cell comprising the expression vector of claim 6, wherein said cell comprises an RNA polymerase recognized by said promoter.

11. The recombinant cell of claim 10, wherein said cell is made by a process comprising the step of introducing the expression vector of claim 6 into said cell.

12. A purified antibody preparation comprising an antibody that binds specifically to HMGCRsvl.

13. A method of preparing an HMGCRsvl polypeptide comprising the step of growing the recombinant cell of claim 10 under conditions wherein said polypeptide is expressed from said expression vector.

14. A method for screening for a compound able to bind to HMGCRsvl comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 2 from recombinant nucleic acid;

(b) providing to said polypeptide a test preparation comprising one or more different test compounds; and

(c) measuring the ability of said test preparation to bind to said polypeptide.

15. The method of claim 14, wherein said steps (b) and (c) are performed in vitro.

16. The method of claim 14, wherein said steps (a), (b) and (c) are performed using a whole cell.

17. The method of claim 14, wherein said polypeptide is expressed from an expression vector.

18. The method of claim 14, wherein said polypeptide consists of the amino acid sequence of SEQ ID NO 2.

19. A method of screening for a compound able to bind selectively to HMGCRsvl comprising the steps of:

(a) providing HMGCRsvl polypeptide comprising SEQ ID NO 2;

(b) providing HMGCR isoform polypeptide that is not HMGCRsvl,

(c) contacting said HMGCRsvl polypeptide and said HMGCR isoform polypeptide that is not HMGCRsvl with a test preparation comprising one or more different test compounds; and (d) determining the binding of said test preparation to said HMGCRsvl polypeptide and to said HMGCR isoform polypeptide that is not HMGCRsvl, wherein a test preparation which binds to said HMGCRsvl polypeptide but does not bind to said HMGCR isoform polypeptide that is not HMGCRsvl, contains a compound that selectively binds to said HMGCRsvl polypeptide.

20. The method of claim 19, wherein said HMGCRsvl polypeptide is obtained by expression of said polypeptide from an expression vector comprising a polynucleotide encoding SEQ ID NO 2.

21. The method of claim 20, wherein said polypeptide consists of SEQ ID NO 2.

22. A method of screening for a compound able to bind to or interact with ^'• HMGCRsvl or a fragment thereof comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 2 or a fragment thereof from recombinant nucleic acid;

(b) providing to said polypeptide a labeled HMGCR ligand that binds to said polypeptide and a test preparation comprising one or more different test i compounds; and

(c) measuring the effect of said test preparation on binding of said labeled HMGCR ligand to said polypeptide, wherein a test preparation that alters the binding of said labeled HMGCR ligand to said polypeptide contains a compound that binds or interacts with said polypeptide.

23. The method of claim 22, wherein said steps (b) and (c) are performed in vitro.

24. The method of claim 22, wherein said steps (a), (b) and (c) are i preformed using a whole cell.

25. The method of claim 22, wherein said polypeptide is expressed from an expression vector.

^; 26. The method of claim 22, wherein said HMGCR ligand is selected from the group consisting of a statin, HMG-CoA and NADPH.

27. The method of claim 25, wherein said expression vector comprises SEQ ID NO 1.

28. The method of claim 22, wherein said polypeptide consists of SEQ ID NO 2 or a fragment of SEQ ID NO 2.

29. The method of claim 22, wherein said test preparation contains one compound.

30. A method of screening for HMGCRsvl activity comprising the steps of:

(a) contacting a cell expressing a recombinant nucleic acid encoding HMGCRsvl, said HMGCRsvl comprising SEQ ID NO 2 with a test preparation comprising one or more different test compounds; and

(b) measuring the effect of said test preparation on HMG-CoA reductase activity of said HMGCRsvl.

31. A purified human nucleic acid comprising SEQ ID NO 3 , or the complement thereof.

32. The purified nucleic acid of claim 31, wherein said nucleic acid comprises a sequence encoding SEQ ID NO 4.

33. The purified nucleic acid of claim 31, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 4.

34. A purified polypeptide comprising SEQ ID NO 4.

35. The polypeptide of claim 34, wherein said polypeptide consists of SEQ ID NO 4.

36. An expression vector comprising a nucleotide sequence encoding SEQ ID NO 4, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.

37. The expression vector of claim 36, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 4.

38. The expression vector of claim 36, wherein said nucleotide sequence comprises SEQ ID NO 3.

39. The expression vector of claim 36, wherein said nucleotide sequence consists of SEQ ID NO 3.

40. A recombinant cell comprising the expression vector of claim 36, wherein said cell comprises an RNA polymerase recognized by said promoter.

41. The recombinant cell of claim 40, wherein said cell is made by a process comprising the step of introducing the expression vector of claim 36 into said cell.

42. A purified antibody preparation comprising an antibody that binds specifically to HMGCRsv2.

43. A method of preparing an HMGCRsv2 polypeptide comprising the step of growing the recombinant cell of claim 40 under conditions wherein said polypeptide is expressed from said expression vector.

44. A method for screening for a compound able to bind to HMGCRsv2 comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 4 from recombinant nucleic acid;

(b) providing to said polypeptide a test preparation comprising one or more different test compounds; and

(c) measuring the ability of said test preparation to bind to said polypeptide.

45. The method of claim 44, wherein said steps (b) and (c) are performed in vitro.

46. The method of claim 44, wherein said steps (a), (b) and (c) are performed using a whole cell.

47. The method of claim 44, wherein said polypeptide is expressed from an expression vector.

48. The method of claim 44, wherein said polypeptide consists of SEQ ID NO 4.

49. A method of screening for a compound able to bind selectively to HMGCRsv2 comprising the steps of: i (a) providing HMGCRsv2 polypeptide comprising SEQ ID NO 4;

(b) providing HMGCR isoform polypeptide that is not HMGCRsv2,

(c) contacting said HMGCRsv2 polypeptide and said HMGCR isoform polypeptide that is not HMGCRsv2 with a test preparation comprising one or more different test compounds; and

(d) determining the binding of said test preparation to said HMGCRsv2 polypeptide and to said HMGCR isoform polypeptide that is not HMGCRsv2, wherein a compound which binds said HMGCRsv2 polypeptide but does not bind to said HMGCR isoform polypeptide that is not HMGCRsv2 is a compound that selectively binds to

> said HMGCRsv2 polypeptide.

50. The method of claim 49, wherein said HMGCRsv2 polypeptide is obtained by expression of said polypeptide from an expression vector comprising a polynucleotide encoding SEQ ID NO 4.

51. The method of claim 50, wherein said polypeptide consists of SEQ ID NO 4.

52. A method of screening for a compound able to bind to HMGCRsv2 or ) a fragment thereof comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 4 or a fragment thereof from recombinant nucleic acid;

(b) providing to said polypeptide a labeled HMGCR ligand that binds to said polypeptide and a test preparation comprising one or more different test

: compounds; and (c) measuring the effect of said test preparation on binding of said labeled HMGCR ligand to said polypeptide.

53. The method of claim 52, wherein said steps (b) and (c) are performed in vitro.

54. The method of claim 52, wherein said steps (a), (b) and (c) are preformed using a whole cell.

55. The method of claim 52, wherein said polypeptide is expressed from an expression vector.

56. The method of claim 52, wherein said HMGCR ligand is selected from the group consisting of a statin, HMG-CoA and NADPH.

57. The method of claim 55, wherein said expression vector comprises SEQ ID NO 3.

58. The method of claim 52, wherein said polypeptide consists of SEQ ID i NO 4 or a fragment of SEQ ID NO 4.

59. The method of claim 52, wherein said test preparation contains one compound.

60. A method of screening for HMGCRsv2 activity comprising the steps of:

(a) contacting a cell expressing a recombinant nucleic acid encoding HMGCRsv2 comprising SEQ ID NO 4 with a test preparation comprising one or more different test compounds; and ) (b) measuring the effect of said test preparation on HMG-CoA reductase activity of said HMGCRsv2.

61. A purified human nucleic acid comprising SEQ ID NO 5, or the complement thereof.

62. The purified nucleic acid of claim 61 , wherein said nucleic acid comprises a sequence encoding SEQ ID NO 6.

63. The purified nucleic acid of claim 61 , wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 6.

64. A purified polypeptide comprising SEQ ID NO 6.

65. The polypeptide of claim 64, wherein said polypeptide consists of SEQ ID NO 6.

66. An expression vector comprising a nucleotide sequence encoding SEQ ID NO 6, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.

67. The expression vector of claim 66, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 6.

68. The expression vector of claim 66, wherein said nucleotide sequence comprises SEQ ID NO 5.

69. The expression vector of claim 66, wherein said nucleotide sequence consists of SEQ ID NO 5.

70. A recombinant cell comprising the expression vector of claim 66, wherein said cell comprises an RNA polymerase recognized by said promoter.

71. The recombinant cell of claim 70, wherein said cell is made by a process comprising the step of introducing the expression vector of claim 66 into said cell.

72. A purified antibody preparation comprising an antibody that binds specifically to HMGCRsv3.

73. A method of preparing a HMGCRsv3 polypeptide comprising the step of growing the recombinant cell of claim 70 under conditions wherein said polypeptide is expressed from said expression vector.

74. A method of screening for a compound able to bind to HMGCRsv3 comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 6 from recombinant nucleic acid;

(b) providing to said polypeptide a test preparation comprising one or more different test compounds; and

(d) measuring the ability of said test preparation to bind to said polypeptide.

75. The method of claim 74, wherein said steps (b) and (c) are performed in vitro.

76. The method of claim 74, wherein said steps (a), (b) and (c) are performed using a whole cell.

77. The method of claim 74, wherein said polypeptide is expressed from an expression vector.

78. The method of claim 74, wherein said polypeptide consists of SEQ ID NO 6.

79. A method of screening for a compound able to bind selectively to HMGCRsv3 comprising the steps of:

(a) providing HMGCRsv3 polypeptide comprising SEQ ID NO 6;

(b) providing HMGCR isoform polypeptide that is not HMGCRsv3,

(c) contacting said HMGCRsv3 polypeptide and said HMGCR isoform polypeptide that is not HMGCRsv3 with a test preparation comprising one or more different test compounds; and

(d) determining the binding of said test preparation to said HMGCRsv3 polypeptide and said HMGCR isoform polypeptide that is not HMGCRsv3, wherein a test preparation which binds to said HMGCRsv3 polypeptide but does not bind to said HMGCR isoform polypeptide that is not HMGCRsv3 contains a compound that selectively binds to said HMGCRsv3 polypeptide.

80. The method of claim 79, wherein said HMGCRsv3 polypeptide is obtained by expression of said polypeptide from an expression vector comprising a polynucleotide encoding SEQ ID NO 6.

81. The method of claim 80, wherein said polypeptide consists of SEQ ID NO 6.

82. A method of screening for a compound able to bind to HMGCRsv3 or a fragment thereof comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 6 or a fragment thereof from recombinant nucleic acid;

(b) providing to said polypeptide a labeled HMGCR ligand that binds to said polypeptide and a test preparation comprising one or more different test compounds; and

(c) measuring the effect of said test preparation on binding of said labeled HMGCR ligand to said polypeptide.

83. The method of claim 82, wherein said steps (b) and (c) are performed in vitro.

84. The method of claim 82, wherein said steps (a), (b) and (c) are preformed using a whole cell.

85. The method of claim 82, wherein said polypeptide is expressed from an expression vector.

86. The method of claim 82, wherein said HMGCR ligand is selected from the group consisting of a statin, HMG-CoA and NADPH.

87. The method of claim 85, wherein said expression vector comprises SEQ ID NO 5.

88. The method of claim 82, wherein said polypeptide consists of SEQ ID NO 6 or a fragment of SEQ ID NO 6.

89. The method of claim 82, wherein said test preparation contains one compound.

90. A method of screening for HMGCRsv3 activity comprising the steps of:

(a) contacting a cell expressing a recombinant nucleic acid encoding HMGCRsv3 comprising SEQ ID NO 6 with a test preparation comprising one or more different test compounds; and

(b) measuring the effect of said test preparation on HMG-CoA reductase activity of said HMGCRsv3.

91. A purified human nucleic acid comprising SEQ ID NO 7, or the complement thereof.

92. The purified nucleic acid of claim 91, wherein said nucleic acid comprises a sequence encoding SEQ ID NO 8.

93. The purified nucleic acid of claim 91 , wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 8.

94. A purified polypeptide comprising SEQ ID NO 8.

95. The polypeptide of claim 94, wherein said polypeptide consists of SEQ ID NO 8.

96. An expression vector comprising a nucleotide sequence encoding SEQ ID NO 8, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.

97. The expression vector of claim 96, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 8.

98. The expression vector of claim 96, wherein said nucleotide sequence comprises SEQ ID NO 7.

99. The expression vector of claim 96, wherein said nucleotide sequence consists of the sequence of SEQ ID NO 7.

100. A recombinant cell comprising the expression vector of claim 96, wherein said cell comprises an RNA polymerase recognized by said promoter.

101. The recombinant cell of claim 100, wherein said cell is made by a process comprising the step of introducing the expression vector of claim 96 into said cell.

102. A purified antibody preparation comprising an antibody that binds specifically to HMGCRsv4.

103. A method of preparing a HMGCRsv4 polypeptide comprising the step of growing the recombinant cell of claim 100 under conditions wherein said polypeptide is expressed from said expression vector.

104. A method for screening for a compound able to bind to HMGCRsv4 comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 8 from recombinant nucleic acid;

(b) providing to said polypeptide a test preparation comprising one or more different test compounds; and

(c) measuring the ability of said test preparation to bind to said polypeptide.

105. The method of claim 104, wherein said steps (b) and (c) are performed in vitro.

106. The method of claim 104, wherein said steps (a), (b) and (c) are performed using a whole cell.

.

107. The method of claim 104, wherein said polypeptide is expressed from an expression vector.

108. The method of claim 104, wherein said polypeptide consists of SEQ ID NO 8.

109. A method of screening for a compound able to bind selectively to HMGCRsv4 polypeptide comprising the steps of:

(a) providing HMGCRsv4 polypeptide comprising SEQ ID NO 8;

(b) providing HMGCR isoform polypeptide that is not HMGCRsv4,

(c) contacting said HMGCRsv4 polypeptide and said HMGCR isoform polypeptide that is not HMGCRsv4 with a test preparation comprising one or more different test compounds; and

(d) determining the binding of said test preparation to said HMGCRsv4 polypeptide and to said HMGCR isoform polypeptide that is not HMGCRsv4, wherein a compound which binds to said HMGCRsv4 polypeptide but does not bind to said HMGCR isoform polypeptide that is not HMGCRsv4 is a compound that selectively binds said HMGCRsv4 polypeptide.

110. The method of claim 109, wherein said HMGCRsv4 polypeptide is obtained by expression of said polypeptide from an expression vector comprising a polynucleotide encoding SEQ ID NO 8.

i 111. The method of claim 110, wherein said polypeptide consists of SEQ ID

NO 8.

112. A method of screening for a compound able to bind to HMGCRsv4 or a fragment thereof comprising the steps of:

(a) expressing a polypeptide comprising SEQ ID NO 8 or a fragment thereof from recombinant nucleic acid;

(b) providing to said polypeptide a labeled HMGCR ligand that binds to said polypeptide and a test preparation comprising one or more different test compounds; and i (c) measuring the effect of said test preparation on binding of said labeled HMGCR ligand to said polypeptide.

113. The method of claim 112, wherein said steps (b) and (c) are performed in vitro.

114. The method of claim 112, wherein said steps (a), (b) and (c) are preformed using a whole cell.

115. The method of claim 112, wherein said polypeptide is expressed from an expression vector.

116. The method of claim 112, wherein said HMGCR ligand is selected from the group consisting of a statin, HMG-CoA and NADPH.

117. The method of claim 115, wherein said expression vector comprises SEQ ID NO 7.

118. The method of claim 112, wherein said polypeptide consists of SEQ ID NO 8 or a fragment of SEQ ID NO 8.

119. The method of claim 112, wherein said test preparation contains one compound.

120. A method of screening for HMGCRsv4 activity comprising the steps of:

(a) contacting a cell expressing a recombinant nucleic acid encoding HMGCRsv4 comprising SEQ ID NO 8 with a test preparation comprising one or more different test compounds; and

(b) measuring the effect of said test preparation on HMG-CoA reductase activity of said HMGCRsv4.