Methods and compositions employing a novel stearoyl-coa desaturase-hscd5

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of human stearoyl-CoA desaturase enzymes and the effects of modulation of such enzymes with various disease processes, including the use of such enzymes, and the genes encoding them, in the development of therapeutic screening processes.

BACKGROUND OF THE INVENTION

[0003] Acyl desaturase enzymes catalyze the formation of double bonds in fatty acids derived from either dietary sources or de novo synthesis in the liver. Mammals synthesize four desaturases of differing chain length specificity that catalyze the addition of double bonds at the Δ9, Δ6, Δ5 and Δ4 positions. Stearoyl-CoA desaturases (SCDs) introduce a double bond in the Δ9-position of saturated fatty acids. The preferred substrates are palmitoyl-CoA (16:0) and stearoyl-CoA (18:0), which are converted to palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1), respectively. The resulting mono-unsaturated fatty acids are substrates for incorporation into phospholipids, triglycerides, and cholesterol esters, and also function in gene transcription, and as precursors for other biologically active compounds such as thromboxanes and prostaglandins.

[0004] A number of mammalian SCD genes have been cloned. For example, two genes have been cloned from rat (SCD1, 2) 1,2 and four SCD genes have been isolated from mouse (SCD1, 2, 3, 4.)3,4 Until now, a single SCD gene, SCD1, has been characterized in humans.5

[0005] Alterations in SCD gene expression and/or enzyme activity have been correlated with disease states. These disease states may fall into two classes: those that are direct effects of decreased. SCD enzyme activity and those that have been correlated with changes in SCD activity where the causal relationship between enzyme activity and the disease state is not known.

[0006] Mutations in mouse SCD1 cause disturbances in skin lipids, abnormal differentiation of sebaceous glands and go on to develop an alopecia similar to clinical scarring alopecias seen in humans6. In addition, mutants homozygous for a disruption of SCD1 show corneal defects, suggesting that SCD1 is required for ocular barrier function in the eye7. These animals also show striking lipid abnormalities, including decreased levels of liver cholesterol esters and triglycerides, and reportedly increased plasma HDL levels8.

[0007] Approximately half of all human patients with coronary-artery disease have a low concentration of high density lipoprotein-cholesterol (HDL-C)24. Clustering studies have shown that high triglycerides, low HDL-C, diabetes, hypertension and hyperuricemia were related to insulin resistance/high insulin levels and central and/or obesity25. Thus, the inventor hereto has recognized that modulation of SCD activity in humans may have an effect on lipid metabolism, and play a role in propensity to develop atherosclerosis and cardiovascular disease.

[0008] In accordance with the present invention, there is disclosed herein sequence and gene expression data confirming the existence of an additional SCD gene in humans (hSCD5).

BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention relates to isolated polynucleotides comprising polynucleotide sequences having at least 60% identical, preferably at least 80% identical, most preferably at least 90% identical, especially at least 95% identical, and most especially sequences identical, to the sequence of SEQ ID NO: 1 or the complement thereof. Such polynucleotide sequences may be genomic or non-genomic, with the latter including DNA, RNA, cDNA or any type of wholly synthetic sequence prepared by cloning or direct chemical synthesis thereof and include complements of any of these.

[0010] In another aspect, the present invention relates to isolated polynucleotides comprising a polynucleotide sequence encoding the same polypeptide as the polynucleotide with the sequence of SEQ ID NO: 1. Such polynucleotide sequences may be genomic or non-genomic, with the latter including DNA, RNA, cDNA or any type of wholly synthetic sequence prepared by cloning or direct chemical synthesis thereof. The complements of all polynucleotides disclosed herein are also specifically contemplated. Such polypeptide will commonly have desaturase activity.

[0011] An additional aspect of the present invention relates to an isolated stearoyl-CoA desaturase (hSCD5—SEQ ID NO: 2) encoded by the isolated polynucleotides disclosed according to the invention, to vectors comprising such polynucleotides and to recombinant eukaryotic cells, and cell lines, preferably mammalian cells, and cell lines, and most preferably human cells, and cell lines, transfected so as to comprise such vectors and/or said polynucleotides and wherein said cells express hSCD5.

[0012] Using the polynucleotides, enzymes, and cells disclosed herein, it is an object of the present invention to provide a process for determining the ability of an agent to modulate the activity of said human stearoyl-CoA desaturase (hSCD5).

[0013] It is also an object of the present invention to provide agents capable of modulating the activity and/or expression of human stearoyl-CoA desaturase (hSCD5) as disclosed herein, especially where said modulating ability was first determined using an assay comprising hSCD5 or a gene encoding hSCD5. Compositions comprising such agents are specifically contemplated.

[0014] It is another object of the present invention to provide screening processes, including high throughput screening processes, for determining the efficacy of potential therapeutic and diagnostic drugs for treating the diseases described herein, preferably diseases in which increased or decreased activity or expression of stearoyl-CoA desaturase (hSCD5 of the invention) plays a key role in mediating such disease.

[0015] It is a still further object of the present invention to provide agents wherein said agent is useful in treating, preventing and/or diagnosing a disease or condition.

[0016] It is a yet further object of the present invention to provide a process for preventing or treating a disease or condition in a patient afflicted therewith comprising administering to said patient a therapeutically or prophylactically effective amount of a composition as disclosed herein.

[0017] In other aspects, the present invention also provides a process for diagnosing a disease or condition in a patient, commonly a human being, suspected of being afflicted therewith, or at risk of becoming afflicted therewith, comprising obtaining a tissue sample from said patient and determining the level of activity of hSCD5 in the cells of said tissue sample and comparing said activity to that of an equal amount of the corresponding tissue from a patient not suspected of being afflicted with, or at risk of becoming afflicted with, said disease or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]
FIG. 1 shows the nucleotide sequence of a cDNA encoding the novel human stearoyl-CoA desaturase enzyme (hSCD5) of the present invention (the sequence of SEQ ID NO: 1). The sequence is 3042 nucleotides in length and shows start and stop codons in bold and with northern blot underlined.

[0019]
FIG. 2 shows a putative amino acid sequence (SEQ ID NO: 2) derived from the cDNA sequence of FIG. 1.

[0020]
FIG. 3 shows a cross species comparison of amino acid sequences for the species shown in Table 1. FIGS. 3A and 3B follow in sequence. The compared sequences are as follows: mouse mHCD1 (SEQ ID NO: 3), mHCD2 (SEQ ID NO: 4), human hSCD1 (SEQ ID NO: 5), carp SCD1 (SEQ ID NO: 6), carp SCD2 (SEQ ID NO: 7), chicken (SEQ ID NO: 8), Drosophila melanogaster (SEQ ID. NO: 9) and Caenorhabditis elegans (SEQ ID NO: 10).

[0021]
FIG. 4 shows a comparison of amino acid sequences of human and mouse SCD family members with conserved catalytic domains in bold. FIGS. 4A and 4B follow in sequence. Additional sequences are mouse mSCD3 (SEQ ID NO: 11) and mSCD4 (SEQ ID NO: 12).

[0022]
FIG. 5 shows a northern blot showing the presence or absence of hSCD5 mRNA transcripts in a variety of human tissues.

[0023]
FIG. 6 demonstrates that delta-9 desaturase activity is substantially increased in cells transfected with hSCD5 cDNA, thus confirming the functional role of hSCD5 as a delta-9 desaturase.

DETAILED SUMMARY OF THE INVENTION

[0024] The present invention relates to human stearoyl-CoA desaturase enzymes and uses thereof relying on the effects of modulation of such enzymes with various disease processes, including the use of such enzymes, and the genes encoding them, in the development of therapeutic screening processes.

[0025] In one aspect the present invention relates to an isolated polynucleotide comprising a nongenomic polynucleotide having at least 90% identity, preferably 95% identity, most preferably at least a 98% identity to the sequence of SEQ ID NO: 1, especially where said sequences are the same and including any of the complements of any of the foregoing.

[0026] In accordance with the present invention, the term “percent identity” or “percent identical,” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”). The Percent Identity is then determined according to the following formula:

Percent Identity=100[1−(C/R)]

[0027] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

[0028] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.

[0029] The present invention further relates to an isolated polynucleotide comprising a non-genomic polynucleotide encoding the same polypeptide as the polynucleotide with the sequence of SEQ ID NO: 1 and represented by the reading frame starting with the initiating (ATG) codon represented in boldface in FIG. 1. In specific embodiments thereof this non-genomic polynucleotide is a DNA, preferably a cDNA and complements thereof.

[0030] In accordance with the present invention, there is disclosed herein the nucleotide sequence of a non-genomic DNA, here a cDNA, encoding a novel hSCD5. Bacterial artificial chromosomes (BAC's) containing the full length human genomic sequence of hSCD5 have also been isolated. The cDNA sequence bears a 56% sequence homology with the known human sequence the known human gene coding for Stearoyl-CoA Desaturase (hSCD1). In addition, the sequence disclosed according to the invention herein maps to chromosome location 4q21 (hSCD1 maps to chromosome 10) and exhibits a high degree of divergence at the 5′-end. Heretofore, the full length sequence has not been available in the public domain.

[0031] Further to the disclosure herein at least three overlapping BACs containing the human genomic sequence have been identified that are commercially available (Research Genetics Inc., Huntsville, Ala.) as (comment BAC RP11 141F19 might be chimeric and is no longer being studied), RP11-791 G16 (GenBank Accession No. AC067942), and RP11-57B24 (GenBank Accession No. AC073413). And additional BAC, not found in GenBank, named RP11-683C18 has also been identified using a BAC fingerprint map. 791G16 contains only 3′UTR and exon 6 of SCD5. C18683 contains 3′UTR, as well as exons 3, 4, 5, 6. 57B24 contains at least 3′UTR, as well as exons 2, 3, 4, 5. 57B24 extends further 5′, and therefore may contain exon 1 and 5′UTR as well. This BAC is being further analyzed to see if it encodes the entire SCD5 gene. Further, review of the publicly available morbidity map fails to indicate any disease linked to the chromosomal position of hSCD5 as disclosed herein. Sequence homology with related polypeptides was found to be as follows for the full length sequences:

1TABLE 1Cross-Species Sequence HomologiesGene SequencehSCD1hSCD5hSCD1—57%hSCD557%—Chicken SCD66%61%Carp SCD260%61%CarpSCD162%60%mSCD185%56%mSCD282%56%Drosophila SCD48%45%C. elegans45%41%

[0032] By analogy, mutations in hSCD1 have been identified as the basis for the Asebia mouse phenotype which, inter alia, have no hair (due to a defect in their sebaceous glands) and have decreased levels of TG (triglycerides) and VLDL (very low density lipoproteins). No consistent elevation of HDL was observed in these animals; however, this may be due to the fact that mice lack the enzyme CETP (cholesterol ester transfer protein). In mammals with CETP activity, decreased VLDL causes a resultant increase in HDL, since CETP functions to transfer cholesterol from HDL to VLDL. In the absence of substrate, that is VLDL, it would be expected that CETP positive mammals would show increased levels of HDL. Elevated HDL levels provide considerable cardiovascular protection.

[0033] Thus, modulators of hSCD5 are expected to effectively treat cholesterol disorders, lipidemias, cardiovascular disease, atherosclerosis, diabetes, obesity, kidney dysfunction, and related disorders. In particular such modulators are expected to be useful in increasing HDL levels in a patient and/or for reducing triglyceride levels in a patient. Human subjects having increased HDL levels and/or decreased triglyceride levels have a highly significant correlation with reduced cardiovascular disease and reduced coronary artery disease24,25. Such modulators may also be effective in treating baldness (see, for example, WO 00/09754) and various skin diseases, as well as such diverse maladies as cancer and multiple sclerosis.

[0034] A search of the patent databases reveals that numerous ESTs (expressed sequence tags) are available for this gene but none has identified it specifically as an hSCD gene for a desaturase. The EST database profile indicates organs that might be affected by modulators of hSCD5 as including muscle, colon, adrenal, aorta, prostate, stomach, kidney, ovary, germ cell, lung, foreskin, brain, eye, parathyroid gland, ear, breast, uterus, placenta, and tests, all of which may be either adult or embryonic tissues. There is a Unigene entry NM—024906 (Mar. 18, 2001) that describes this gene with only a partial amino acid sequence and makes no mention of any desaturase activity.

[0035] As used herein and except as noted otherwise, all terms are defined as given below.

[0036] In accordance with the present invention, the term “DNA segment” refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non-translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

[0037] The nucleic acids and polypeptide expression products disclosed according to the present invention, as well as expression vectors containing such nucleic acids and/or such polypeptides, may be in “enriched form.” As used herein, the term “enriched” means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form.

[0038] “Isolated” in the context of the present invention with respect to polypeptides or polynucleotides means that the material is removed from its original environment (e.g., the natural environment if it Is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

[0039] The polynucleotides, and recombinant or immunogenic polypeptides, disclosed in accordance with the present invention may also be in “purified” form. The term “purified” does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Furthermore, claimed polypeptide which has a purity of preferably 0.001%, or at least 0.01% or 0.1%; and even desirably 1% by weight or greater is expressly contemplated.

[0040] The term “coding region” refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene. The coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

[0041] In accordance with the present invention, the term “nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides. Generally, DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

[0042] The term “expression product” means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).

[0043] The term “fragment,” when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.

[0044] The term “primer” means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3′OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.

[0045] The term “promoter” means a region of DNA involved in binding of RNA polymerase to initiate transcription.

[0046] The term “open reading frame (ORF)” means a series of triplets coding for amino acids without any termination codons and is a sequence (potentially) translatable into protein (and wherein any one of these is a “reading frame”).

[0047] As used herein, reference to a DNA sequence includes both single stranded and double stranded DNA. Thus, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.

[0048] The present invention further relates to a polypeptide which has the deduced amino acid sequence of SEQ ID NO: 2, as well as fragments, analogs and derivatives of such polypeptide that exhibit hSCD activity.

[0049] The terms “fragment” “derivative” and “analog” when referring to the polypeptide, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Such fragments, derivatives and analogs must have sufficient similarity to the polypeptide of SEQ ID NO:2 so that activity of the native polypeptide is retained.

[0050] The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

[0051] The fragment, derivative or analog of the polypeptide (SEQ ID NO:2) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0052] As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

[0053] In accordance with the foregoing, the present invention also relates to an isolated stearoyl-CoA desaturase encoded by the isolated polynucleotide of the invention.

[0054] Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

[0055] As used herein, the terms “portion,” “segment,” and “fragment,” when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to polynucleotides, such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.

[0056] The present invention also relates to vectors which contain polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention, especially where such cells result in a cell line that can be used for assay of hSCD, especially hSCD5, activity and production of polypeptides of the invention by recombinant techniques.

[0057] Host cells, preferably insect cells of Spodoptera species, most especially SF9 cells, are genetically engineered (transduced or transformed or transfected) with the vectors, especially baculovirus) of this invention which may be, for example, a cloning vector or an expression vector. Such vectors can include plasmids, viruses and the like. The engineered host cells are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0058] The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

[0059] The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0060] The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[0061] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

[0062] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. Such transformation will be permanent and thus give rise to a cell line that can be used for further testing. Such cell lines used for testing will commonly be mammalian cells, especially human cells.

[0063] As representative examples of appropriate hosts, there may be mentioned Spodoptera Sf9 (and other insect expression systems) and animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, and even bacterial cells, etc, all of which are capable of expressing the polynucleotides disclosed herein. The selection of an appropriate host is deemed to be within the knowledge of those skilled in the art based on the teachings herein. For use in the assay methods disclosed herein, mammalian, especially human, cells are preferred.

[0064] More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, especially where the Baculovirus/SF9 vector/expression system is used, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

[0065] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0066] In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L, Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). A preferred embodiment utilizes expression from insect cells, especially SF9 cells from Spodoptera frugiperda.

[0067] The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

[0068] Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wu et al, Methods in Gene Biotechnology (CRC Press, New York, N.Y., 1997), Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997), and Current Protocols in Molecular Biology, (Ausabel et al, Eds.), John Wiley & Sons, NY (1994-1999), the disclosures of which are hereby incorporated by reference in their entirety.

[0069] Transcription of the DNA encoding the polypeptides of the present invention by eukaryotic cells, especially mammalian cells, most especially human cells, is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0070] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae Trp1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0071] Use of a Baculovirus-based expression system is a preferred and convenient method of forming the recombinants disclosed herein. Baculoviruses represent a large family of DNA viruses that infect mostly insects. The prototype is the nuclear polyhedrosis virus (AcMNPV) from Autographa californica, which infects a number of lepidopteran species. One advantage of the baculovirus system is that recombinant baculoviruses can be produced in vivo. Following co-transfection with transfer plasmid, most progeny tend to be wild type and a good deal of the subsequent processing involves screening. To help identify plaques, special systems are available that utilize deletion mutants. By way of non-limiting example, a recombinant AcMNPV derivative (called BacPAK6) has been reported in the literature that includes target sites for the restriction nuclease Bsu36I upstream of the polyhedrin gene (and within ORF 1629) that encodes a capsid gene (essential for virus viability). Bsf36I does not cut elsewhere in the genome and digestion of the BacPAK6 deletes a portion of the ORF1629, thereby rendering the virus non-viable. Thus, with a protocol involving a system like Bsu36I-cut BacPAK6 DNA most of the progeny are non-viable so that the only progeny obtained after co-transfection of transfer plasmid and digested BacPAK6 is the recombinant because the transfer plasmid, containing the exogenous DNA, is inserted at the Bsu36I site thereby rendering the recombinants resistant to the enzyme. [see Kitts and Possee, A method for producing baculovirus expression vectors at high frequency, BioTechniques, 14, 810-817 (1993). For general procedures, see King and Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall, New York (1992) and Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997), at Chapter 19, pp. 235-246.

[0072] In accordance with the foregoing, the present invention further relates to vectors comprising a polynucleotide of the invention and to recombinant eukaryotic cells expressing the stearoyl-CoA desaturase of the present invention, preferably wherein said cell is a mammalian cell, most preferably a human cell.

[0073] The present invention further relates to processes for using the polynucleotides, enzymes, and cells disclosed herein in a process for determining the ability of an agent to modulate the expression of said human stearoyl-CoA desaturase in cells expressing said human stearoyl-CoA desaturase of the invention, comprising the steps of:

[0074] (a) contacting the agent under suitable conditions with a eukaryotic cell expressing the human stearoyl-CoA desaturase of the invention at a predetermined level of said agent;

[0075] (b) determining if the expression level of said stearoyl-CoA desaturase changes after said contact,

[0076] thereby determining if said agent has modulated said expression level.

[0077] In one such embodiment, the present invention relates to a process for identifying a compound that modulates the biological activity of a human stearoyl-CoA desaturase 5 (hSCD5), comprising:

[0078] (a) contacting a compound with a cell expressing the human stearoyl-CoA desaturase of the invention under conditions promoting said expression, and

[0079] (b) detecting a difference in expression of said desaturase as compared to when said compound is not present,

[0080] thereby identifying a compound that modulates human desaturase biological activity.

[0081] In specific embodiments, said modulation is an increase in said expression level or a decrease in said expression level and the cells employed may be mammalian cells, especially human cells, including the recombinant cells disclosed herein. Said cells may also be recombinant cells genetically engineered to produce the desaturase of the invention, such as where a polynucleotide of the invention is inserted into such cell, for example, using one of the vectors disclosed herein. In one such embodiment, the cell is engineered to contain more than one such gene expressing hSCD5. In a separate embodiment, the recombinant cell may not otherwise express such a gene, such as where the cell does not normally contain such a gene as part of its genetic complement but is specifically engineered to contain, and to express, such a gene, thereby expressing hSCD5. In accordance with the present invention, said expression level may be determined by determining the level of messenger RNA produced after contact of said cell with said agent The difference in the expression levels so measured may involve a difference in the overall amount of polypeptide or mRNA produced from a given gene or the rate of said production over time.

[0082] Factors that may modulate gene expression include transcription factors such as, but not limited to, retinoid X receptors (RXRs), peroxisomal proliferation-activated receptor (PPAR) transcription factors, the steroid response element binding proteins (SREBP-1 and SREBP-2), REV-ERBα, ADD-1, EBPα, CREB binding protein, P300, HNF 4, RAR, LXR, and RORα.

[0083] Physiological benefits of an increase or decrease in the activity or expression of hSCD5 include, but are not limited to, increased plasma HDL and/or decreased triglycerides leading to cardioprotective benefit, therapeutic benefit in Type II diabetes, weight loss, and decreased chance of malignancy. Thus, the determination of the ability of agents to modulate such activity or expression affords an opportunity to discover useful therapeutic agents producing such effects.

[0084] In addition, variations in hSCD5 gene expression, function, stability, catalytic activity and other characteristics may be due to allelic variations in the polynucleotide sequences encoding such enzymes. The processes disclosed according to the present invention may likewise be used to determine such genomic effects on expression of hSCD5. Using the processes of the present invention, such variations may be determined at the level of DNA polymorphism within the hSCD5 gene and/or promoter sequences. Such effects lead to the elucidation of associations between such polymorphisms and predisposition to cancer, neurological disease, skin disease, obesity, diabetes, immune function and lipid metabolism through both population and family-based genetic analysis.

[0085] In specific embodiments, the present invention contemplates a process wherein said modulation is an increase or decrease in said expression level and where said cell may be a mammalian cell, especially a human cell, including any of the recombinant cells disclosed herein. In one embodiment, the expression level is determined by determining the level of messenger RNA produced after contact of said cell with said agent.

[0086] In another aspect, the present invention relates to a process for determining the ability of an agent to modulate the activity of a human stearoyl-CoA desaturase, comprising the steps of:

[0087] (a) contacting the agent under suitable conditions with the human stearoyl-CoA desaturase of the invention at a predetermined level of said agent;

[0088] (b) determining if the activity of said stearoyl-CoA desaturase changes after said contact,

[0089] thereby determining if said agent has modulated said activity.

[0090] In one such embodiment, the present invention relates to a process for identifying a compound that modulates hSCD5 biological activity, comprising:

[0091] (a) contacting a compound with the human stearoyl-CoA desaturase of the invention in the presence of a lipid that can act as a substrate of said desaturase under conditions promoting desaturation of the lipid by said desaturase, and

[0092] (b) detecting a difference in the desaturation of said lipid by said desaturase as compared to when said compound is not present,

[0093] thereby identifying a compound that modulates human stearoyl-CoA desaturase activity.

[0094] Such an assay may be carried out as a cell free assay employing a cellular fractional, such as a microsomal fraction, obtained by conventional methods of differential cellular fractionation, most commonly by ultracentrifugation methods. In specific embodiments, such modulation may be an increase or decrease in the activity of the desaturase.

[0095] In a further aspect, the present invention relates to a process for determining the ability of an agent to modulate the activity of a human stearoyl-CoA desaturase in cells expressing the human stearoyl-CoA desaturase of the invention, comprising the steps of:

[0096] (a) contacting the agent under suitable conditions with a eukaryotic cell expressing the human stearoyl-CoA desaturase of the invention at a predetermined level of said agent and under conditions where said agent may or may not modulate the expression level of said desaturase;

[0097] (b) determining if the activity of said stearoyl-CoA desaturase changes after said contact,

[0098] thereby determining if said agent has modulated said expression level.

[0099] In specific embodiments of said processes, the modulation may be an increase or decrease in activity of the desaturase and cells useful in these processes are preferably mammalian cells, most preferably human cells, and include any of the recombinant cells disclosed herein.

[0100] This invention teaches screening assays employing hSCD5 for use in identifying prophylactic and/or therapeutic agents for use in treating diseases or conditions, and in particular for use in treating a disease or condition which includes, but is not limited to, cholesterol disorders, lipidemias, cardiovascular disease (including a low HDL disorder or a high triglyceride disorder), diabetes, obesity, weight disorders, kidney dysfunction, baldness, skin diseases, cancer and multiple sclerosis, especially where the disease is a cardiovascular disease or a skin disease or where the condition is baldness. On the basis of the disclosure herein, those skilled in the art are able to develop the claimed screening assays based on known types of assays available. Known types of assays include cell based, cell extract (i.e. microsomal assays) or cell free (i.e. transcription) assays. Such assays are typically radioactivity or fluorescence based (i.e. fluorescence resonance transfer or FRET), or they may measure cell behavior (growth, activity, shape, etc). Alternatively, screening may employ multicellular organisms, including genetically modified organisms such as knock-out or knock-in mice, or naturally occurring genetic variants. Screening assays may be manual or low throughput assays, or they may be high throughput screens which are mechanically/robotically enhanced.

[0101] In one such embodiment, the present invention provides a process for identifying a compound that modulates triglyceride levels, comprising administering to an animal an effective amount of a compound identified as a modulator of hSCD5 biological activity using a process of the invention and detecting a difference in the triglyceride level in said animal as compared to when said compound is not administered, thereby identifying a compound that modulates triglyceride levels.

[0102] In another such embodiment, the invention provides a process for identifying a compound that modulates cholesterol levels, comprising administering to an animal an effective amount of a compound identified as a modulator of hSCD5 biological activity using a process of the invention and detecting a difference in the cholesterol level in said animal as compared to when said compound is not administered, thereby identifying a compound that modulates cholesterol levels.

[0103] The tissues monitored for such activity conveniently include plasma and the animal may be a mammal, especially a human being.

[0104] The aforementioned processes afford the basis for screening processes, including high throughput screening processes, for determining the efficacy of potential therapeutic and diagnostic drugs for treating the diseases described herein, preferably diseases in which increased or decreased activity or expression of stearoyl-CoA desaturase (hSCD5 of the invention) plays a key role in mediating such disease.

[0105] In accordance with the foregoing, the present invention also relates to therapeutic and/or diagnostic agents, regardless of molecular size or weight, effective in treating and/or diagnosing and/or preventing any of the diseases disclosed herein, preferably where such agents have the ability to modulate activity and/or expression of the hSCD5 disclosed herein, and most preferably where said agents have been determined to have such activity through at least one of the screening assays disclosed according to the present invention. In one specific embodiment said agent is an antibody with specificity for hSCD5 that, when administered to a patient, has the effect of neutralizing said hSCD5 and thereby decreasing the activity of such enzyme.

[0106] Thus, in one aspect the present invention relates to agents capable of modulating the activity and/or expression of human stearoyl-CoA desaturase (hSCD5) as disclosed herein, especially where said modulating ability was first determined using an assay of comprising hSCD5 or a gene encoding hSCD5, or an assay which measures hSCD5 activity. As used herein the term “capable of modulating” refers to the characteristic of such an agent whereby said agent has the effect of changing the activity of hSCD5, either by increasing or decreasing said activity, under suitable conditions of temperature, pressure, pH and the like so as to facilitate such modulation to a point where it can be detected either qualitatively or quantitatively and wherein such modulation may occur in either an in vitro or in vivo environment. In addition, while the term “modulation” is used herein to mean a change in activity, more specifically either an increase or decrease in such activity, the term “activity” is not to be limited to specific enzymatic activity alone (for example, as measured in units per milligram or some other suitable unit of specific activity) but includes increases in enzyme activity due not to changes in specific enzyme activity but due to changes (i.e., modulation) of expression of polynucleotides encoding and expressing said hSCD5 enzyme. Human SCD5 activity may also be influenced by agents which bind specifically to substrates of hSCD5. Thus, the term umodulations as used herein means a change in hSCD5 activity regardless of the molecular genetic level of said modulation, be it an effect on the enzyme per se or an effect on the genes encoding the enzyme or on the RNA, especially mRNA, involved in expression of the genes encoding said enzyme. Thus, modulation by such agents can occur at the level of DNA, RNA or enzyme protein and can be determined either in vivo or ex vivo.

[0107] In specific embodiments thereof, said assay is any of the assays disclosed herein according to the invention. In addition, the agent(s) contemplated by the present disclosure includes agents of any size or chemical character, either large or small molecules, including proteins, such as antibodies, nucleic acids, either RNA or DNA, and small chemical structures, such as small organic molecules.

[0108] In other aspects, the present invention contemplates agents wherein said agent is useful in treating, preventing and/or diagnosing a disease or condition. Specific embodiments are directed to situations wherein the disease or condition includes, but is not limited to, cholesterol disorders, lipidemias, cardiovascular disease, diabetes, obesity, baldness, skin diseases, cancer, kidney dysfunction, and multiple sclerosis, especially where the disease is a cardiovascular disease or a skin disease or where the condition is baldness. In a preferred embodiment, such agents will increase HDL levels in a patient and/or decrease triglyceride levels in a patient Either or both effects are directly associated with reduced risk of cardiovascular disease and coronary artery disease.

[0109] In another aspect the present invention is directed to compositions comprising and of the polynucleotides, polypeptides or other chemical agents, including therapeutic, prophylactic or diagnostic agents, such as small organic molecules, disclosed herein according to the present invention wherein said polynucleotides, polypeptides or other agents are suspended in a pharmacologically acceptable carrier, which carrier includes any pharmacologically acceptable diluent or excipient.

[0110] Thus, the pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).

[0111] In another aspect the present invention further relates to a process for preventing or treating a disease or condition in a patient afflicted therewith comprising administering to said patient a therapeutically or prophylactically effective amount of a composition as disclosed herein.

[0112] In specific embodiments thereof, disease or condition includes, but is not limited to, cholesterol disorders, lipidemias, cardiovascular disease, diabetes, obesity, baldness, skin diseases, cancer, kidney dysfunction, and multiple sclerosis, especially where said disease is a cardiovascular disease or a skin disease or where said condition is baldness. In a preferred embodiment, such disease or condition is a low HDL level or an elevated triglyceride level in a subject at risk of cardiovascular disease.

[0113] In an additional aspect, the present invention also relates to a process for diagnosing a disease or condition in a patient, commonly a human being, suspected of being afflicted therewith, or at risk of becoming afflicted therewith, comprising obtaining a tissue sample from said patient and determining the level of activity of hSCD5 in the cells of said tissue sample and comparing said activity to that of an equal amount of the corresponding tissue from a patient not suspected of being afflicted with, or at risk of becoming afflicted with, said disease or condition. In specific embodiments thereof, said disease or condition includes, but is not limited to, cholesterol disorders, lipidemias, cardiovascular disease, diabetes, obesity, baldness, skin diseases, cancer and multiple sclerosis, especially wherein said disease is a cardiovascular disease or a skin disease or said condition is baldness.

[0114] In an additional aspect, this invention teaches that hSCD5 has pharmacogenomic significance. Variants of hSCD5 including SNPs (single nucleotide polymorphisms), cSNPs (SNPs in a cDNA coding region), polymorphisms and the like may have dramatic consequences on a subject's response to administration of a prophylactic or therapeutic agent Certain variants may be more or less responsive to certain agents. In another aspect, any or all therapeutic agents may have greater or lesser deleterious side-effects depending on the hSCD5 variant present in the subject

[0115] In general, the invention discloses a process of selecting a prophylactic and/or therapeutic agent for administration to a subject in need thereof comprising,

[0116] (a) determining at least a part of the hSCD5 nucleic acid sequence of said subject; and

[0117] (b) comparing said hSCD5 nucleic sequence to known variants of hSCD5 nucleic acids;

[0118] wherein said known variants are correlated with responsiveness to said agent and said agent is selected for said subject on the basis of a desired correlation. In this method the correlation may be a prophylactic and/or therapeutic effect or it may be avoidance of a deleterious side effect, or any other desired correlation.

[0119] Those skilled in the art are able to confirm the relevance of hSCD5 to human health by analogy to animal models. Models that may be used to ascertain the role of hSCD5 in growth, development, diseases or disease processes include genetically modified multicellular animals, such as knock-out or knock-in mice; and yeast recovery assays, where SCD deficient yeast are recovered by administration of an expression construct bearing the hSCD5′ gene.

[0120] The present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent For example, the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention. For example, the user of the assay (user 1) may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results). This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.

[0121] In carrying out the procedures of the present invention it is of course to be understood that reference to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein.

EXAMPLE 1

Expression Analysis of Human SCD5 Transcripts in Multiple Human Tissues

[0122] Methods: A 627 bp EcoRI/Hind III fragment of the human SCD5 cDNA was restricted from EST clone AW131469. This corresponds to a 5′ region of the cDNA, including the initiating ATG. This region of the cDNA was chosen to maximize sequence divergence between human SCD1 and SCD5 so as to avoid cross hybridization. The probe was labeled by RediPrimer II according to the manufactures instructions with alpha-[32P]dCTP supplied by NEN. The labeled probes were purified with Centri•Sep spin columns from Princeton Separation Inc. For the Northern Blot, the Human 12-lane MTN Blot cat#7780-1 from Clontech was used with the ExpressHyb protocol. Labeled blots were exposed to minus 70 degrees for an average of 17 hours.

[0123] Results of the hSCD5 northern blot are shown in FIG. 5. Human tissues analyzed were brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and leukocyte. The expected transcript size for human SCD5 is 3.0 kb, based on characterization of the cDNA. A 3.0 kb transcript was detected and expressed fairly ubiquitously, with the highest levels of expression in the brain and kidney. A larger 4.5 kb transcript was also observed. This may reflect a differentially spliced SCD5 transcript or cross hybridization with other desaturases. Notably, hSCD5 was not highly expressed in the liver.

[0124] Transcripts identified with the SCD5 probe are unlikely to correspond to SCD1. Published transcript sizes for SCD1 are 3.9 kb and 5.2 kb. A comparison blot was also performed with an SCD1 cDNA, confirming published reports of widespread expression with the highest levels in the liver and the brain.

EXAMPLE 2

Transfection of Cells with hSCD5 Construct Leads to Significant Gain in Delta-9-Desaturase Activity

[0125] Methods: A human SCD5 construct was created using a vector for mammalian expression, pCDNA3.1/V5-His-TOPO. The cDNA coding for hSCD5 was amplified by PCR from hSCD5 EST A1815730 using primers hSCD5 pcDNA3.1F (caccatgccaggcccggccaccga; SEQ ID NO:13) and hSCD5 pcDNA3.1R (agcactgctgtccagtcc; SEQ ID NO:14). PCR was carried out using Stratagene's Herculase™ Hotstart DNA Polymerase. Amplified PCR product was run out on a TAE gel and purified by GENECLEAN SPIN Kit cat#1101-200. The amplified PCR product of the hSCD5 fragment was ligated into the mammalian expression vector pcDNA3.1/V5-His-TOPO® supplied by Invitrogen and transformed into NovaBlue Singles™ from Novagen, according to the manufactures instructions. Plasmids were isolated by Qiagen's MiniPrep protocol and their inserts were sequenced using standard 17 primers internal primers. The correct orientation and sequence of the cloned hSCD5 constructs were verified by restriction digestion and sequence analysis.

[0126] This construct was transfected into human 293K cells (or HEK 293 cells) which are known to demonstrate low intrinsic delta-9 desaturase activity. Confluent cells were split in ¼ in DMEM with 10% FBS into small plates with 20 ml media. Prior to transfection, media was replaced with fresh media, with 10 ul 50 mM chloroquine. After chloroquine was added 2 mll transfection mixture, including 20 μg hSCD5/Topo construct DNA was added to the media (H2O; 450 μl-μl DNA added DNA; 10 μg pCMV-Lac; 500 μl 2×HBS buffer 500 μl; 50 μl 2.5 M CaCl2). Cells were incubated at 37° C. for 6 hours, prior to aspiration of media and addition of 20 ml fresh media. Cells were incubated for an additional 48 hours at 37° C. Transfection efficiency was judged based on LacZ staining. Controls employed were maximally induced mouse liver microsomes (having increased SCD1 activity), and HEK 293 cells with or without mock transfection. Cells were washed 2× with PBS and then scraped off plates in 10 ml PBS. Cells were centrifuged at 1200 prm for 10 min at 4 C. PBS was aspirated and cells were resuspended in 1 ml of fresh PBS and transferred to an eppendorf. A small aliquot of suspended cells was removed to test for expression of SCD5 by Western blot. The remainder of the cells were centrifuged at 1200 rpm for 10 min, 4° C. PBS was aspirated and the cell pellet was frozen and stored at −70° C. prior to the microsomal preparation.

[0127] In all cases, the microsomal assay used to measure delta-9-desaturase biological activity was the tritium desaturation assay set out in WO 01/62954. Basically, the assay measures the release of tritium from stearoyl-CoA having tritium incorporated at C9 and C10. Cell pellets were homogenized using a motorized Potter-Elvehjem homogenizer on an intermediate power setting so as not to overheat preparation. Cells were homogenized for 1 min in 0.1 M PK buffer on ice. Homogenate was spun for 15 min, 10,000×g, 4° C. Supernatant was transferred to a clean tube and the pellet material was discarded. Microsome solution was centrifuged for 60 min, 100,000×g, 4° C. Supernatant was discarded and pellet, containing microsomes, was rinsed with 1 ml of cold 0.1 M PK buffer. PK buffer was removed and pellet was resuspended in cold 0.1 M PK buffer (2 μl/mg cell pellet). Microsome solution was transferred to tube and rehomogenized for 1 min at an intermediate setting. Protein protein concentration was determined and microsomes diluted to yield a concentration of 20 mg/ml. The Activity assay pre-mix contains 2 μl 1.5 mM stearoyl-CoA (0.03 mM final), 1 μl 1 mCi/ml radioactive 3H stearoyl-CoA (1 μCi), 10 μl 20 mM NADH (2 mM final), 67 μl 0.1 M PK buffer per assay point. Assay premix (80 μl/assay point) was added 20 μl of microsomes to initiate reaction. Reaction proceeded at room temperature for 30 minutes. Reactions were then quenched with 10 μl of 6% PCA. Following vortexing, 100 μl of charcoal suspension was added to sediment the unused substrate. Following vortexing, samples were centrifuged at 13,000 rpm, 10 min, 4° C. 50 μl supernatant was removed and transferred to a counting vial for liquid scintillation counting

[0128] Results are shown in FIG. 6. The results show that transient expression of human SCD5 in HEK 293 cells can increase delta-9 desaturase activity significantly above mock transfected HEK 293 cells. The increase in activity is substantial, amounting to about 10% of that measured in maximally induced mouse liver microsomes.

[0129] In applying the disclosure, it should be kept clearly in mind that other and different embodiments of the methods disclosed according to the present invention will no doubt suggest themselves to those of skill in the relevant art.

REFERENCE LIST

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Methods and compositions employing a novel stearoyl-coa desaturase-hscd5

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