WO2000009754A2 - COMPOSITIONS ASSOCIEES A LA STEAROYL-CoA DESATURASE HUMAINE ET PROCEDES DE TRAITEMENT D'AFFECTIONS CUTANEES - Google Patents

COMPOSITIONS ASSOCIEES A LA STEAROYL-CoA DESATURASE HUMAINE ET PROCEDES DE TRAITEMENT D'AFFECTIONS CUTANEES Download PDF

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WO2000009754A2
WO2000009754A2 PCT/US1999/018387 US9918387W WO0009754A2 WO 2000009754 A2 WO2000009754 A2 WO 2000009754A2 US 9918387 W US9918387 W US 9918387W WO 0009754 A2 WO0009754 A2 WO 0009754A2
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stearoyl
coa desaturase
human
skm
scd
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PCT/US1999/018387
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WO2000009754A3 (fr
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Kurt Stenn
Stephen M. Prouty
Satish Parimoo
Lin Zhang
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Johnson & Johnson Consumer Companies, Inc.
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Priority to JP2000565188A priority Critical patent/JP2003533965A/ja
Priority to CA002339748A priority patent/CA2339748A1/fr
Priority to AU57746/99A priority patent/AU5774699A/en
Priority to EP99945051A priority patent/EP1105538A2/fr
Publication of WO2000009754A2 publication Critical patent/WO2000009754A2/fr
Publication of WO2000009754A3 publication Critical patent/WO2000009754A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the diagnosis and treatment of skin disorders characterized by abnormal stearoyl-CoA desaturase expression and activity.
  • the invention also relates to various means of identifying agents useful for treating such disorders.
  • Fatty acid desaturases are enzymes that catalyze the insertion of a double bond in fatty acids.
  • This enzyme introduces a cis-double bond at the delta- 9 position of fatty acyl-CoA's to produce palmitoleoyl and oleoyl-CoA.
  • Mammalian fatty acid desaturases are structurally similar to each other. They are integral membrane, iron-containing enzymes that catalyze the NADH- and 0 2 - dependent formation of double bonds into methylene- interrupted fatty acyl chains .
  • Various forms of mammalian stearoyl-CoA desaturase (“SCD”) have been isolated from rat, mouse, human, bovine (St. John, et al . 1991), ovine, porcine, and hamster.
  • SCD mammalian stearoyl-CoA desaturase
  • the coding regions of mouse, human and rat SCD sequences show over 80% nucleotide sequence identity. They apparently share a similar exonic structure, but differ markedly in upstream regulatory regions (Mihara 1990) .
  • Stearoyl-CoA desaturase is responsible for the production of unsaturated fatty acids, which are required for energy and lipid metabolism, membrane structure, and signal transduction.
  • the expression of stearoyl-CoA desaturase in the skin suggests an important role for unsaturated fatty acids in skin homeostasis, and specifically in the function of the pilosebaceous unit and the eccrine sweat gland.
  • SCD is the enzyme responsible for the committed step in unsaturated fatty acid ("UFA") synthesis, and as such is the key point of metabolic control in this pathway.
  • UFA'S are important for three major reasons. UFA'S are key components of cellular membranes. Triglycerides contain UFA'S and are a major component of energy metabolism. Finally, UFA'S play an important role in stimulating lipid- activated kinases during signal transduction.
  • SCD1 SCD1
  • M-SCD2 SCD2
  • a critical step in the biosynthesis of unsaturated fatty acids is the introduction of the first cis-double bond in the ⁇ 9 position (between carbons 9 and 10) (Ntambi 1995) .
  • the iron-containing stearoyl-CoA desaturase enzyme catalyzes this oxidative step. In this reaction, electrons flow from NADPH through NADH- cytochrome b5 reductase to cytochrome b5. Cytochrome b5 is the direct electron donor to the desaturase.
  • Stearoyl-CoA desaturase is believed to utilize iron in an oxidative-reduction reaction transferring electrons to molecular oxygen with the production of H 2 0 (Strittmatter, et al . 1974).
  • the rate-limiting step in this reaction is at the desaturase. It is this factor which is cyanide-sensitive and limits the overall desaturation rate (Oshino 1972; Oshino, et al . 1972).
  • Acyl CoA derivatives of fatty acids containing 12 to 19 carbon residues are required for desaturase activity. (Enoch, et al . 1976). Shorter chain acyl CoA derivatives, free CoA and free fatty acids do not appear to bind to the enzyme (Enoch, et al . 1976) . Additionally, SCD activity is affected by metal ions (Wahle, 1975) .
  • Palmitate and stearate are converted into palmitoleoyl -CoA and oleoyl-CoA, respectively, by the enzyme.
  • Palmitoleic and oleic acids are the major unsaturated fatty acid constituents of phospholipids and triacylglycerols . The former is central to membrane structure and the latter to the lipid store found in adipocytes. The ratio of stearic to oleic acid is one of the factors influencing cell membrane fluidity.
  • M-SCDl Two mouse SCD genes have been identified, and designated M-SCDl and M-SCD2.
  • M-SCDl cDNA was isolated from 3T3-L1 preadipocytes which had been shown to express M-SCDl upon differentiation (preadipocytes into adipocytes) (Ntambi, et al . 1988).
  • the M-SCDl gene encodes a 4.9kb mRNA.
  • the predicted amino acid sequence of the mouse 3T3 adipocyte SCD exhibits 92% similarity to rat liver SCDl.
  • the SCDl gene spans 15 kb and contains 6 exons and 5 introns .
  • the 5' end of the SCD-1 gene shows a canonical TATA box (Ntambi, et al . 1988).
  • a region similar to the binding site for the nuclear transcription factor, Spl, is present.
  • Upstream of the transcription initiation site are regions homologous to the fat- specific transcription element FSE2.
  • Core consensus sequences for cAMP and glucocorticoid regulatory elements are present (Ntambi, et al . 1988) .
  • the PPAR receptor localizes to an
  • C/EBP can bind to the M- SCD1 promoter and activate transcription during the late stage of adipocyte differentiation (Christy, et al. 1989) .
  • the M-SCD2 gene spans approximately 15 kb and, like M-SCDl and rat SCD, consists of 6 exons and 5 introns (Kaestner, et al . 1989; Mihara 1990).
  • the promoter regions for M-SCD2 have also been characterized (Kaestner, et al . 1989).
  • the 5' end of M-SCD2 lacks a typical 5' TATA box, but has two CCAAT boxes.
  • the M-SCD2 promoter contains a region (located between nucleotides -54 to -201) which shows a 77% sequence identity to a region in the M-SCDl promoter. It contains a site similar to the nuclear transcription factor, Spl, and an element with homology to the core consensus sequence of the glucocorticoid regulatory element (Kaestner, et al . 1989).
  • the SCD genes have tissue-specific expression patterns. Under normal dietary states, M-SCDl mRNA' s are expressed constitutively in adipose, but not hepatic, tissue. Their expression is induced in liver in response to a fat-free, high-carbohydrate diet. M- SCD2 mRNA's are constitutively expressed in brain and induced in kidney, lung, spleen and adipose tissue in response to a high carbohydrate diet, but not expressed in liver under either condition (Kaestner, et al .
  • the asejia mutation was first described in mice by Gates, et al . (1965) .
  • the mutation arose as an autosomal recessive trait in the BA B/c mouse strain.
  • Prominent elements of the phenotype include early loss of hair, scaly skin, and sebaceous glands that fail to fully develop. Since the meibomian glands are also hypoplastic, these mice also have eye problems.
  • the ocular findings have been described as eye inflammation, photophobia and, finally, scarring of the eyelid with resultant blindness. Histologically (Josefowicz, et al .
  • a human stearoyl-CoA desaturase cDNA of 712 bp was identified by PCR from adipose tissue (Li, et al . 1994) .
  • This form is referred to herein as human adipose SCD ("HA-SCD").
  • H-SCD human adipose SCD
  • From the determined sequence it was seen that at the nucleotide level, the homology to the various mouse and rat SCD's is between 80-84%. The similarity in deduced peptide sequence between human and mouse SCD's is approximately 93%.
  • RNAse protection demonstrates either no, little, or variable expression in normal esophagus, colon, and liver, respectively. Increased expression is seen in tumors derived from these three tissues.
  • a human cDNA from liver is present in the database (Wisconsin Package Version 9.1, Genetics Computer Group, GCG, Madison, Wise. : accession number: Y13647) that contains part of the 5' and 3' UTR's and the complete ORF of SCD.
  • This form is referred to herein as human liver SCD ("HL-SCD") .
  • HL-SCD human liver SCD
  • the identity of HL-SCD to HA-SCD is 98.6% (over the known sequence of HA-SCD); to M-SCDl is 76.2%; and to mouse SCD2 is 75.1%.
  • identity of HL-SCD to HA-SCD is 98.7%, and is 83.9% to the M-SCDl.
  • UFA'S are important for biological systems in the following ways: (1) as intermediates in lipid and energy metabolism (Neely, et al . 1974); (2) as components of triglycerides , which are a major form of cellular energy storage and the major component of circulating lipoprotein particles (Rosseneu, et al .
  • Membrane fluidity has also been related to cell- cell signalling and cancer formation.
  • the degree of membrane fluidity can affect the function of receptors (Clandimn, et al . 1991), and increased concentrations of membrane UFA'S have been detected in tumor cells, suggesting a role in neoplasia (Li, et al . 1994) .
  • Many tumors show altered fatty acid profiles, especially an increase m oleate (Hrelia, et al . 1994).
  • This change in membrane fluidity may confer changes in response to cell-cell and/or intra-cellular signalling. Indeed, the expression of high levels of yeast SCD in mammalian tumor cells increases membrane fluidity and greatly increases tumor necrosis factor signaling (Gyorfy, et al. 1997) .
  • UFA'S are implicated in the regulation of cellular growth and differentiation.
  • UFA's can co-activate various isoforms of protein kinase C (PKC) (Shinomura, et al . 1991). They can also alter the subcellular localization of PKC (Diaz-Guerra, et al . 1991), a process known to activate this enzyme.
  • PKC plays an important role in the normal growth and differentiation of epidermal cells (Dlugosz, et al .
  • UFA's are now recognized as activators of gene expression via transcription factors that bind to UFA's, such as peroxisome proliferator-activated receptor (Bocos, et al . 1995) and fatty acid-activated receptor (Ailhaud, et al . 1995).
  • Phosphatidylinositol (Ptdlns) -3 , 4 , 5-triphosphate (P3) is a lipid second messenger that is formed by the phosphorylation of the 3 position of the inositol ring of PtdIns-4,5- bisphosphate (located in plasma membranes) by the receptor-activated phosphatidylinositol-3 -OH kinase [PI(3)K].
  • PtdIns-3 , 4 , 5-P3 activates downstream kinases PDK1 , PDK2 , and PKB by recruiting these enzymes to the plasma membrane (Alessi, et al . 1998) . It has been shown that the most effective form of PtdIns-3 , 4 , 5-P3 for activating PKB is that with oleate at the 2 position of the phospholipid.
  • the biological processes regulated by PI3 kinase and PKB pathways are pleiotropic, and include membrane trafficking, adhesion, cell growth, and survival (Toker, et al . 1997). Recently, Cadena, et al .
  • MLD membrane fatty acid lipid desaturase
  • the skin is recognized as a lipid-rich organ, the proper function of which depends on the integrity of lipid metabolism. It has long been known that essential fatty acid deficiency has profound effects on the skin. Prominent effects include scaling of skin and increased trans-epidermal water loss (Holman 1993). It is notable that the asebia mouse also manifests these changes .
  • Atopic dermatitis a chronic inflammatory skin disease, is ameliorated by the administration of ⁇ - linolenic acid, indicating involvement of fatty acid metabolism in its pathogenesis (Youn, et al . 1998) .
  • the integrity of the stratum corneum is heavily dependent on the lipid composition of the corneocytes, which act to promote hydrophobicity as well as maintain adhesion (Chen, et al . 1996).
  • the maintenance of adhesion between the cuticle and the cortex of the hair shaft is dependent on specialized fatty acids, whose defects are manifest in hair from patients with Maple Syrup Urine Disease (Jones, et al . 1996) .
  • the pilosebaceous unit is sensitive to alterations in fatty acid composition. This is futher demonstrated by data indicating that local deficiency of linolenic acid in the sebaceous gland leads to proliferation of the keratinocytes lining its duct. This in turn leads to the formation of comedones, the precursor to acne vulgaris (Downing, et al. 1986) .
  • Hair matrix keratinocytes are the highly proliferative cells that give rise to the shaft and sheath of the hair. They are termed transient amplifying stem cells to indicate that their proliferation correlates with the growth phase of the hair, and that their quiescence correlates with the resting phase (i.e., no growth) of the hair follicle (Cotsarelis, et al . 1990).
  • Hypertrichosis (Olsen, 1994) and hirsutism (Hughes, Jr. 1994) are diseases of the hair follicle that result from excessive growth. Alopecia encompasses several distinct diseases that result in a lack of hair growth (Rietscel 1996) .
  • This invention provides a nucleic acid molecule encoding the human stearoyl-CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof .
  • This invention also provides a nucleic acid molecule which, under suitable conditions, specifically hybridizes to a nucleic acid molecule encoding the human stearoyl-CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof.
  • This invention further provides a method of diagnosing a human subject for a skin disorder characterized by an abnormal level of stearoyl-CoA desaturase expression, which comprises (i) obtaining a sample of skin mRNA from the subject; (ii) contacting the sample so obtained with an excess of the instant labeled nucleic acid molecule under conditions permitting hybridization of the labeled nucleic acid molecule with stearoyl-CoA desaturase mRNA present in the sample; (iii) removing un-hybridized labeled nucleic acid molecule from the sample; (iv) quantitatively determining the amount of hybridized labeled nucleic acid molecule present in the sample; and (v) comparing the amount determined in step (iv) with the amount determined using a skin mRNA sample from a normal human subject, a difference in these amounts being correlative of an abnormal level of stearoyl-CoA desaturase expression in the skin of the subject being diagnosed.
  • This invention further provides an
  • This invention still further provides a eukaryotic cell line which expresses human stearoyl-CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, wherein the cell is transfected with an expression vector encoding the desaturase.
  • This invention provides a method for determining whether an agent increases the expression level of human stearoyl-CoA desaturase in skin cells already expressing same, which comprises the steps of (i) contacting the agent under suitable conditions with a eukaryotic cell line expressing human stearoyl-CoA desaturase at a known level; and (ii) determining whether the stearoyl-CoA desaturase expression level increases after cellular contact with the agent, thereby determining whether the agent increases the expression level of human stearoyl-CoA desaturase in skin cells already expressing same.
  • This invention also provides a method for determining whether an agent decreases the expression level of human stearoyl-CoA desaturase in skin cells already expressing same, which comprises the steps of (i) contacting the agent under suitable conditions with a eukaryotic cell line expressing human stearoyl-CoA desaturase at a known level; and (ii) determining whether the stearoyl-CoA desaturase expression level decreases after cellular contact with the agent, thereby determining whether the agent decreases the expression level of human stearoyl-CoA desaturase in skin cells already expressing same.
  • This invention further provides a method for determining whether an agent decreases the activity of human stearoyl-CoA desaturase in skin cells, which comprises the steps of (i) contacting the agent under suitable conditions with human stearoyl-CoA desaturase having a known level of activity; and (ii) determining whether the desaturase activity decreases after contact with the agent , thereby determining whether the agent decreases human stearoyl-CoA desaturase activity in skin cells.
  • This invention further provides a method for determining whether an agent increases the activity of human stearoyl-CoA desaturase in skin cells, which comprises the steps of (i) contacting the agent under suitable conditions with human stearoyl-CoA desaturase having a known level of activity; and (ii) determining whether the desaturase activity increases after contact with the agent, thereby determining whether the agent increases human stearoyl-CoA desaturase activity in skin cells.
  • This invention provides an antibody which specifically binds to human stearoyl-CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, and thereby inhibits the activity thereof .
  • This invention also provides a pharmaceutical composition for treating a human skin disorder characterized by an excess of stearoyl-CoA desaturase activity, which comprises a therapeutically effective amount of the instant antibody and a pharmaceutically acceptable carrier for use m topical administration.
  • This invention further provides an expression vector suitable for use in gene therapy, which vector encodes a nucleic acid molecule capable of specifically inhibiting the expression of human skin stearoyl-CoA desaturase .
  • This invention further provides a pharmaceutical composition for treating a human skin disorder characterized by an excess of skin stearoyl-CoA desaturase activity, which comprises the instant expression vector, and a pharmaceutically acceptable carrier for use m topical administration.
  • This invention still further provides a method for treating a human subject afflicted with a skin disorder characterized by an excess of stearoyl-CoA desaturase activity, which comprises topically administering to the subject a therapeutically effective dose of the instant SCD activity-reducing pharmaceutical composition.
  • This invention provides an SCD-encodmg DNA expression vector suitable for use in gene therapy.
  • This invention also provides a pharmaceutical composition for treating a human skin disorder characterized by insufficient skin stearoyl-CoA desaturase activity, which comprises the instant SCD- encodmg expression vector, and a pharmaceutically acceptable carrier for use m topical administration.
  • This invention further provides a method for treating a human subject afflicted with a skin disorder characterized by insufficient stearoyl-CoA desaturase activity, which comprises topically administering to the subject a therapeutically effective dose of the instant SCD activity-increasing pharmaceutical composition.
  • This invention provides the instant antibody labeled with a detectable marker.
  • This invention also provides an antigen suitable for use in generating the instant antibody, which comprises at least a portion of human stearoyl-CoA desaturase.
  • This invention also provides a method of producing the instant antibody, which comprises the steps of administering to a suitable animal an antigenic portion of human stearoyl-CoA desaturase, and after a suitable length of time, isolating the antibody generated by the animal against the antigenic portion so administered.
  • This invention further provides a method of diagnosing a human subject for a skin disorder characterized by an abnormal level of stearoyl-CoA desaturase expression, which comprises (i) obtaining a stearoyl-CoA desaturase-containing sample from the subject's skin; (ii) contacting the sample so obtained with an excess of the instant antibody under conditions permitting binding of the antibody with stearoyl-CoA desaturase present in the sample; (iii) removing unbound antibody from the sample; (iv) quantitatively determining the amount of bound antibody present in the sample; and (v) comparing the amount determined in step (iv) with the amount determined using a skin stearoyl- CoA desaturase sample from a normal human subject, a difference in these amounts being correlative of an abnormal level of stearoyl -CoA desaturase expression in the skin of the subject being diagnosed.
  • this invention provides a transgenic mouse whose skin cells do not express any gene encoding mouse skin stearoyl -CoA desaturase having the amino acid sequence shown in Figure 1 or 2 , or any polymorphism thereof.
  • Figure 1 shows the sense strand sequence (mRNA sequence) of M-SCD3 cDNA obtained after sequencing 5' RACE cDNA clone from mouse skin mRNA.
  • the sequence corresponding to the coding sequence i.e., the protein sequence
  • the M-SCD3 -specific in situ hybridization (ISH) probe is boxed.
  • Figure 2 shows the sense strand sequence (mRNA sequence) of M-SCD4vl cDNA from two overlapping novel cDNA clones obtained by screening a mouse skin cDNA library with the M-SCD3 probe.
  • the sequence corresponding to the coding sequence (protein sequence) is underlined.
  • the boxed sequence corresponds to the 3' half of the ISH probe for M-SCD4v2 that has 100% identity with M-SCDvl.
  • Figure 3 shows the homology between M-SCD3 cDNA sequence and the M-SCD4vl cDNA sequence. The regions of homology are shown with vertical lines. The protein- coding sequence is underlined. The boxed sequence on the M-SCD4vl sequence corresponds to the 3' half of the ISH probe for M-SCD4v2 that has 100% identity with M- SCDvl. The bases that are boxed on the M-SCD3 sequence correspond to the M-SCD3 -specific ISH probe.
  • FIG 4 shows a comparison of the four mouse SCD cDNA sequences, i.e., M-SCD's 1, 2, 3 and 4.
  • the M-SCD4 cDNA sequence is that of M-SCD4vl.
  • the protein-coding region is underlined.
  • the first nucleotide beginning significant homology between a sequence and one or more of the other sequences is boxed and ends with the coding region.
  • Figure 5 shows the deduced protein sequence from the M- SCD3 sense strand cDNA.
  • the sequence is from amino acids 1-289.
  • the single code designation of amino acids is the standard biochemical single code designation for amino acids from the GCG computer program of Wisconsin Package (Genetics Computer Group, Madison, Wisconsin) .
  • Figure 6 shows the complete protein-coding sequence of mouse skin SCD4vl (359 amino acids) deduced from its cDNA sequence .
  • Figure 7 shows a comparison of mouse protein sequences derived from four SCD genes. The amino acid residues which are not common in all the four protein sequences are underlined. The boxed histidine residues are conserved in evolution from yeast to mammals.
  • Figure 8 shows the cDNA sequence (sense strand) and protein sequence of human SCD obtained from skin.
  • the ORF extends from bp 229 to bp 1308 and encodes a predicted protein sequence of 359 amino acids.
  • the boxed sequence corresponds to the human ISH probe.
  • Figure 9 shows a comparison between human skin cDNA and database-deposited human liver cDNA encoding SCD. Identical bases are indicated with vertical lines. All bases differing between skin and liver are indicated with boxes. The protein-coding sequence is underlined.
  • Figure 10 shows a comparison between human skin cDNA and database-deposited human adipose cDNA. Identical bases are indicated with vertical lines. All bases differing between skin and adipose are indicated with boxes. The protein-coding sequence is underlined.
  • Figure 11 shows a comparison of predicted amino acid sequences derived from human skin SCD, human liver SCD, and human adipose SCD. Amino acid differences are boxed. The conserved histidine residues are underlined. The adipose sequence does not contain the complete ORF.
  • Figure 12 shows the cDNA sequence homology (5' end) of sense strands of the two mouse skin SCD4 variant species. The regions of homology are connected by vertical bold lines. The protein-coding region is underlined. The boxed sequence corresponds to an ISH probe that recognizes both variant forms (vl and v2) of M-SCD4.
  • Figure 13 shows the cDNA sequence homology (3' end) of sense strands of the two mouse skin SCD4 variant species. The regions of homology are connected by vertical bold lines. The region of a 6-nucleotide difference at the 3' end is boxed. The protein-coding region is underlined.
  • This invention relates to the diagnosis and treatment of skin disorders characterized by abnormal stearoyl-CoA desaturase expression and activity.
  • the invention also relates to various means of identifying agents useful for treating such disorders.
  • Underlying this invention is the surprising discovery that stearoyl -CoA desaturase in mice and, more importantly, in humans, is expressed in skin.
  • Skin is a lipid-rich organ, and many skin disorders such as atopic dermatitis and acne involve lipid imbalances.
  • this invention provides a nucleic acid molecule encoding the human stearoyl-CoA desaturase having the amino acid sequence shown in
  • Figure 8 or a polymorphism thereof.
  • a polymorphism of the SCD whose sequence is shown in Figure 8 means any naturally occurring human SCD whose amino acid sequence varies therefrom due to one or more intra-species mutations.
  • the instant SCD-encoding nucleic acid molecule can be any type of nucleic acid molecule, such as mRNA and DNA.
  • the instant nucleic acid molecule is a DNA molecule.
  • DNA molecules envisioned in this invention include, by way of example, cDNA molecules, which can optionally be isolated molecules.
  • the nucleic acid molecule is a cDNA molecule comprising the sequence shown in Figure 8.
  • the instant SCD- encoding DNA molecule can be any form of DNA permitting the expression thereof, or any form of DNA, such as an insert, which serves as a precursor to a form permitting expression.
  • the DNA molecule is in an expression vector.
  • SCD Stearoyl-CoA desaturase
  • H-SCD human SCD
  • H-SCD human SCD
  • M-SCDl mouse SCDl
  • M-SCD2 SCD3
  • M- SCD4 SCD4
  • M-SCD3 and M-SCD4 are novel genes discovered as disclosed hereinbelow.
  • H-SCD sequence alternatively identified herein either as “skin H-SCD” or “HS-SCD” is novel and is expressed in skin, as well as other tissues.
  • the terms “skin SCD”, “HS- SCD”, “liver SCD”, “HL-SCD”, “adipose SCD” and “HA-SCD” are intended solely to indicate the organ from which their respective SCD-encoding mRNA's were obtained, and not to indicate that these organs are the only ones in which those SCD's are respectively expressed.
  • H-SCD' s identified herein as HA-SCD and HL- SCD not only differ in sequence from the instant HS- SCD, but, based on experimental discrepancies, may be polymorphisms of HS-SCD, erroneously sequenced non-HS- SCD's, or non-human SCD's entirely.
  • the instant HS-SCD is the first human SCD gene ever identified.
  • the terms "human stearoyl-CoA desaturase", "H-SCD” and "HS-SCD”, as they relate to the instant invention shall mean only the protein whose sequence is provided in Figure 8, and polymorphisms thereof.
  • HL-SCD and HA-SCD are included herein solely for the sake of comparison to the instant HS-SCD.
  • This invention also provides a nucleic acid molecule which, under suitable conditions, specifically hybridizes to a nucleic acid molecule encoding the human stearoyl -CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof.
  • this nucleic acid molecule which is preferably a DNA molecule, functions as a probe to detect and/or quantitate human stearoyl-CoA desaturase- encoding nucleic acid molecules in a sample. Accordingly, in the preferred embodiment, the molecule is labeled with a detectable marker.
  • the instant nucleic acid molecule "specifically hybridizes" with the H-SCD sequence if it hybridizes to that sequence, but not to any other SCD sequence to a significant degree. Ideally, the instant nucleic acid molecule hybridizes to the H-SCD-encoding molecule at least 10- fold more strongly than to any other human mRNA or cDNA. Detectable markers such as radiolabels and fluorescent labels, and methods of using same to label nucleic acid molecules, are well known in the art
  • the condition suitable for hybridizing is a stringent hybridizing condition described in Sambrook, J., et al . (1989).
  • This invention further provides a method of diagnosing a human subject for a skin disorder characterized by an abnormal level of stearoyl-CoA desaturase expression, which comprises (i) obtaining a sample of skin mRNA from the subject; (ii) contacting the sample so obtained with an excess of the instant labeled nucleic acid molecule under conditions permitting hybridization of the labeled nucleic acid molecule with stearoyl-CoA desaturase mRNA present in the sample; (iii) removing un-hybridized labeled nucleic acid molecule from the sample; (iv) quantitatively determining the amount of hybridized labeled nucleic acid molecule present in the sample; and (v) comparing the amount determined in step (iv) with the amount determined using a skin mRNA sample from a normal human subject, a
  • Skin disorders that can be diagnosed by the instant method include, for example, skin cancer, acne, atopic dermatitis, alopecia, hirsutism, and hypertrichosis.
  • Methods for obtaining tissue-specific mRNA samples e.g. skin mRNA
  • conditions permitting hybridization therewith by the instant labeled nucleic acid molecule and methods of quantitatively determining the amount of hybridization by the labeled molecule, are all well known in the art (Farrell Jr. , 1993).
  • This invention further provides an isolated human stearoyl -CoA desaturase encoded by the instant nucleic acid molecule.
  • the isolated desaturase has the amino acid sequence shown in Figure 8.
  • the "isolated" H-SCD protein is free of any other SCD protein.
  • the isolated H-SCD protein is free of any other protein.
  • Methods that can be used for making the H-SCD protein such as recombinant protein production and transfected cell culturing, are well known (Sambrook, et al . 1989) .
  • Methods that can be used for isolating H-SCD protein such as column chromatography and gel electrophoresis, are also well known (Sambrook, et al. 1989) .
  • This invention further provides a eukaryotic cell line which expresses human stearoyl-CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, wherein the cell is transfected with an expression vector encoding the desaturase.
  • Suitable eukaryotic cell lines include, but are not limited to, yeast cells, insect cells and animal cells.
  • Suitable animal cells include, but are not limited to HeLa cells, Cos cells, CV1 cells and various primary mammalian cells. Numerous mammalian cells may be used as hosts, including, but not limited to, the mouse fibroblast cell NIH-3T3 cells, CHO cells, HeLa cells, Ltk " cells and COS cells.
  • the eukaryotic cell line is a mammalian cell line, ideally one comprising, or derived from, skin tissue cells.
  • the eukaryotic cell lines may be transfected by methods well known in the art such as calcium phosphate precipitation, electroporation, lipofection, and microinj ection.
  • This invention provides several methods of screening agents for therapeutic and prophylactic use in connection with SCD-related disorders.
  • this invention provides a method for determining whether an agent increases the expression level of human stearoyl- CoA desaturase in skin cells already expressing same, which comprises the steps of (i) contacting the agent under suitable conditions with a eukaryotic cell line expressing human stearoyl -CoA desaturase at a known level; and (ii) determining whether the stearoyl -CoA desaturase expression level increases after cellular contact with the agent, thereby determining whether the agent increases the expression level of human stearoyl- CoA desaturase in skin cells already expressing same.
  • this invention provides a method for determining whether an agent decreases the expression level of human stearoyl-CoA desaturase in skin cells already expressing same, which comprises the steps of
  • the amount by which the H-SCD expression level is increased or decreased can be any quantifiable amount. In the preferred embodiment, this amount is at least a 50% increase or decrease in expression level .
  • Methods which can be used for quantitatively determining such increase or decrease include, for example, labeled probe hybridization with skin mRNA Northern blots, and are well known in the art (Farrell, Jr. 1993) .
  • the eukaryotic cell line can be either (a) a cell line transfected with an expression vector encoding a human stearoyl -CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, or (b) a non-transfected cell line.
  • this invention provides a method for determining whether an agent decreases the activity of human stearoyl-CoA desaturase in skin cells, which comprises the steps of (i) contacting the agent under suitable conditions with human stearoyl -CoA desaturase having a known level of activity; and (ii) determining whether the desaturase activity decreases after contact with the agent, thereby determining whether the agent decreases human stearoyl -CoA desaturase activity in skin cells.
  • this invention provides a method for determining whether an agent increases the activity of human stearoyl-CoA desaturase in skin cells, which comprises the steps of (i) contacting the agent under suitable conditions with human stearoyl -CoA desaturase having a known level of activity; and (ii) determining whether the desaturase activity increases after contact with the agent, thereby determining whether the agent increases human stearoyl -CoA desaturase activity in skin cells.
  • "activity of H-SCD” means the rate at which the SCD introduces a cis-double bond in its substrate palmitate to produce palmitoleoyl -CoA.
  • Methods that can be used to quantitatively measure SCD activity include, for example, measuring thin layer chromatographs of SCD reaction products over time. This method and others methods suitable for measuring SCD activity are well known (Henderson, et al . 1992).
  • This invention also provides an antibody which specifically binds to human stearoyl -CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, and thereby inhibits the activity thereof.
  • the instant antibody can be a polyclonal antibody, a monoclonal antibody, or an SCD-binding fragment thereof.
  • the antibody is an isolated antibody, i.e., an antibody free of any other antibodies.
  • the term "antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies.
  • the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof. Methods of making and isolating antibodies are well known in the art (Harlow, et al . 1988).
  • This invention further provides a pharmaceutical composition for treating a human skin disorder characterized by an excess of stearoyl -CoA desaturase activity, which comprises a therapeutically effective amount of the instant antibody and a pharmaceutically acceptable carrier for use in topical administration.
  • topically administering the instant pharmaceutical compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • the topical administration can be performed, for example, transdermally and via topical injection.
  • compositions for topical administration are well known in the art, as are methods for combining same with active agents to be delivered.
  • topical carriers and their uses are well known in the art (Ramchandani ; Barry; Wenniger; Martindale's Parmacopoeia; U.S. Pharmacopeoia) .
  • the following dermal delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the instant composition.
  • Transdermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) .
  • the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
  • liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,N I ,N II ,N III -tetramethyl-N,N I ,N II ,N III -tetrapalmityl- spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL) ; (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N- [1- (2 , 3 -dioleoyloxy) -N,N,N- trimethyl-ammoniummethylsulfate) (Boehringer Manheim) ; and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL) .
  • DOPE diole
  • This invention still further provides an expression vector suitable for use in gene therapy, which vector encodes a nucleic acid molecule capable of specifically inhibiting the expression of human skin stearoyl-CoA desaturase.
  • the nucleic acid molecule is an anti-sense molecule which is complementary to, and specifically hybridizes with, at least a portion of human stearoyl-CoA desaturase mRNA.
  • anti-sense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable.
  • the anti-sense approach employing a nucleic acid molecule that hybridizes with an mRNA molecule encoding an undesirable gene, leads to the inhibition of gene expression.
  • Methods of making and using anti -sense molecules against known target genes are known in the art (Agrawal , 1996) .
  • This invention still further provides a pharmaceutical composition for treating a human skin disorder characterized by an excess of skin stearoyl - CoA desaturase activity, which comprises the instant expression vector, and a pharmaceutically acceptable carrier for use in topical administration.
  • This invention also provides a method for treating a human subject afflicted with a skin disorder characterized by an excess of stearoyl -CoA desaturase activity, which comprises topically administering to the subject a therapeutically effective dose of the instant SCD activity-reducing pharmaceutical composition.
  • the disorder is selected from skin cancer, hypertrichosis, and hirsutism.
  • Determining a therapeutically effective dose of the instant pharmaceutical composition can be done based on animal data using routine computational methods.
  • the therapeutically effective dose contains between about 1 ⁇ g and about 1 g of the instant activity-reducing vector.
  • the therapeutically effective dose contains between about 10 ⁇ g and about 100 mg of the vector.
  • the therapeutically effective dose contains between about 100 ⁇ g and about 10 mg of the vector.
  • This invention provides an SCD-encoding DNA expression vector suitable for use in gene therapy.
  • This invention also provides a pharmaceutical composition for treating a human skin disorder characterized by insufficient skin stearoyl -CoA desaturase activity, which comprises the instant SCD- encoding expression vector, and a pharmaceutically acceptable carrier for use in topical administration.
  • This invention further provides a method for treating a human subject afflicted with a skin disorder characterized by insufficient stearoyl -CoA desaturase activity, which comprises topically administering to the subject a therapeutically effective dose of the instant SCD activity-increasing pharmaceutical composition.
  • the disorder is selected from acne, atopic dermatitis and alopecia.
  • the therapeutically effective dose contains between about 1 ⁇ g and about 1 g of the instant SCD-encoding vector. In another embodiment, the therapeutically effective dose contains between about 10 ⁇ g and about 100 mg of the vector. In a further embodiment, the therapeutically effective dose contains between about 100 ⁇ g and about 10 mg of the vector.
  • This invention provides the instant antibody labeled with a detectable marker.
  • This invention also provides an antigen suitable for use in generating the instant antibody, which comprises at least a portion of human stearoyl -CoA desaturase.
  • This invention also provides a method of producing the instant antibody, which comprises the steps of administering to a suitable animal an antigenic portion of human stearoyl -CoA desaturase, and after a suitable length of time, isolating the antibody generated by the animal against the antigenic portion so administered.
  • Suitable animals include, by way of example, mammals such as mice, rabbits, goats, and monkeys.
  • Suitable lengths of time for generating antibodies are well known in the art, and often include one or more "booster" administrations subsequent to the initial antigen administration.
  • the antigen is administered along with an adjuvant according to well known methods .
  • This invention further provides a method of diagnosing a human subject for a skin disorder characterized by an abnormal level of stearoyl-CoA desaturase expression, which comprises (i) obtaining a stearoyl -CoA desaturase-containing sample from the subject's skin; (ii) contacting the sample so obtained with an excess of the instant antibody under conditions permitting binding of the antibody with stearoyl-CoA desaturase present in the sample; (iii) removing un- bound antibody from the sample; (iv) quantitatively determining the amount of bound antibody present in the sample; and (v) comparing the amount determined in step (iv) with the amount determined using a skin stearoyl- CoA desaturase sample from a normal human subject, a difference in these amounts being correlative of an abnormal level of stearoyl -CoA desaturase expression in the skin of the subject being diagnosed.
  • Conditions required for antibody binding are well known.
  • the antibody can be labeled or unlabeled
  • transgenic mouse whose skin cells do not express any gene encoding mouse skin stearoyl-CoA desaturase having the amino acid sequence shown in Figure 1 or 2 , or any polymorphism thereof.
  • This type of transgenic mouse is also known in the art as a "knock-out mouse” , in that its transgenic status inhibits the expression of an undesired gene.
  • the transgenic mouse has operably integrated into its chromosomes a DNA sequence encoding human stearoyl -CoA desaturase having the amino acid sequence shown in Figure 8 or a polymorphism thereof, which desaturase is expressed in the mouse's skin cells.
  • this invention also provides, mutatis mutandis, the corresponding embodiment of each of mouse SCD genes 3 and 4.
  • mutant mouse known as aseJbia, discussed in more detail hereinabove, was chosen as an experimental focal point.
  • the most obvious phenotype is early loss of hair and hypoplasia of the sebaceous gland.
  • M-SCD3 and M-SCD4 are located in the keratinocyte matrix cells of the hair follicle and in the sebaceous gland.
  • HSDCD human SCD
  • SCD4 SCD4 sequence was derived from overlapping sequences of two clones - 15g and 7d. A variant of the SCD4 sequence was identified after sequencing another clone - clone 5a. The former is identified herein as "SCD4vl" (variant 1) and the latter (derived from 5a clone) as “SCD4v2" (variant 2) .
  • M-SCD3 clone The entire sequence of M-SCD3 clone (up to exon 5) is shown in Figure 1.
  • An open reading frame for protein starts from nucleotide 285, and that part of the sequence is underlined. In addition to the coding sequence, it has 284 nucleotides of 5' non-coding sequence.
  • Sequence comparison with the known M-SCDl indicates that -145 nucleotides at the 5' end of SCD3 are unique, whereas the rest of the sequence is highly homologous to the mouse SCDl sequence (-99% identity) .
  • sequence comparisons with the known mouse SCD2 showed that the region of dissimilarity (unique 5' end of SCD3) stretched down to about 280 nucleotides.
  • the region downstream of 280 nucleotides of SCD3 which encompasses the protein-coding region, shows only -90% identity with the SCD2 sequence.
  • the region of homology between SCD3 and SCDl includes both the protein-coding region and a part of 5' non-coding region, the identity between SCD3 and SCD2 is limited to the protein-coding region of the genes.
  • the nucleotide sequence of one of the isoforms of SCD4, SCD4vl is shown in Figure 2.
  • the sequence encompasses the entire protein-coding sequence (underlined) with 317 and 179 nucleotides of 5' and 3' UTR, respectively.
  • Sequence comparison with the M-SCDl sequence reveals that that the region of homology is limited to only the protein-coding sequence (underlined) ( ⁇ 91%identity) , with no significant homology in either 5' or 3 ' non-coding regions.
  • the sequence comparison with the M-SCD2 cDNA sequence again indicated a homologous region limited to the protein- coding segment (underlined) , with sequence identity of about 88%.
  • SCD4 cDNA Two distinct clones of SCD4 cDNA were identified, as indicated by unique sequences in both the 5' untranslated region ( Figure 12) and 3' untranslated region ( Figure 4) . These two species may be alternative splice forms of the SCD4 gene, both of which are expressed in mouse skin. Alternatively, they may represent sequences from two SCD4 genes. These clones are unlikely to be cloning artifacts, since the difference was also noted at the 3' end just prior to the poly A stretch in the two sequences as shown in Figure 4 (boxed region of 6 nucleotides) . The protein- coding regions are underlined, and are identical as between these two variants.
  • M-SCD3 cDNA The longest ORF of M-SCD3 cDNA that also has a high degree of homology to SCDl protein-coding sequence is shown in Figure 5. Although this sequence lacks the exon 6 sequence, it represents the sequence up to the end of exon 5, as indicated by homology to the M-SCDl sequence. This identifies it as a bonafide member of the SCD family. M-SCD3 cannot be a splice variant of the SCDl gene, since the differences m nucleotides that lead to a single ammo acid change of alan e to cysteme at position 97 of the SCD3 protein sequence occur withm an exon. In addition, SCD3 cDNA has a unique 3' non-coding sequence.
  • Figure 7 snows a comparison of mouse SCDl, SCD2 , SCD3 and SCD4 protein sequences.
  • SCDl has an identical protein sequence to that of SCD3 , except for one ammo acid position wnere the alanme in SCDl is replaced by cysteme m SCD3 (position 101 in Figure 7) .
  • SCD2 and SCD4 are more divergent and have more ammo acid differences, wnich are not shared by SCDl or SCD3. Those ammo acids which differ between all four SCD's are underlined.
  • the conserved histidme ammo acid residues m mammals, yeast and other lower species are boxed at positions 120, 125, 157, 160, 161, 298, 301, and 302 in Figure 7.
  • the positions and the neighboring ammo acid residues of these histidme regions are conserved m all four mouse protein sequences (SCD1-4) as shown m Figure 7.
  • a 129 bp fragment (Notl digest of 5 ⁇ 5 plasmid) containing the sequence from the 5' UTR that is unique o M-SCD3 was cloned into the PBluescript KS vector (Stratagene) , and was used for generating ⁇ boprobes .
  • the M-SCD3 -specific sequence is shown as boxed in Figure 1.
  • the relationship of the M-SCD3 -specific sequence (boxed) to that of M-SCD4vl is shown in Figure 3.
  • the dorsal skin of an adult C57B1/6 mouse was excised and frozen in OCT embedding compound. Frozen sections were collected on positively-charged glass slides.
  • M-SCD3 -unique plasmid was first linearized with Sacl and labeled with digoxigenm using T7 RNA polymerase according to manufacturer's instructions (Boehr ger-Manheim) .
  • a sense, digoxigenm- labeled ⁇ boprobe was generated jsmg T3 RNA polymerase with a BamHl - linearized plasmid.
  • In situ hybridization was carried out according to standard procedures (Hebert, et al . 1991). The results, not shown here, indicate that M-SCD3 is primarily expressed m matrix keratinocytes of the hair follicle and not m the sebaceous gland.
  • M- SCD3 in matrix keratinocytes suggests an important role for M-SCD3 either proliferation and/or differentiation of these cells.
  • Cotsarelis, et al . (1990) have reported that, using t ⁇ tiated thymidme, these cells are highly proliferative and comprise the transien -amplifying ste cells of the hair follicle.
  • M-SCD3 may not function at this site.
  • M-SCD4 may have a role m sebaceous function.
  • a 223 bp fragment (EcoRl-Sphl digest of clone 5a) containing the entire 5' UTR of M-SCD4v2 was cloned into the PBluescript SK vector (Stratagene) . Position 130 to 225 of the M-SCD4v2 sequence is 100% identical to that of M-SCD4vl. Thus, this probe recognizes both variant forms of M-SCD4.
  • Mouse skin for ISH was prepared as described above.
  • An anti-sense, digoxigenm- labeled riboprobe was generated using T7 RNA polymerase with a EcoRl- linearized plasmid.
  • a sense, digoxigenm- labeled riboprobe was generated using T3 RNA polymerase with a Kpnl -digested plasmid.
  • ISH was carried out as described above.
  • M-SCD4 is strongly expressed the matrix keratinocytes of the hair follicle.
  • Sebaceous glands express M-SCD4.
  • the follicular papilla ("FP") of hair follicles and epidermis do not express M-SCD4.
  • Adjacent follicular papilla fibroblascs are negative for M-SCD4 mRNA.
  • Sebaceous glands specifically express M-SCD4 in the lower aspect of the gland, although some sebaceous glands do not express significant amounts of M-SCD4. The reasons for this are not clear at present, but may be related to heterogeneity of M-SCD4- express g cells within the gland, cyclical expression in glands, or plane of section of the tissue sample.
  • M-SCD4 Based on the expression pattern and putative function of SCD, M-SCD4 apparently plays a role m both hair follicle growcn and sebaceous gland function. That M-SCD4, and not M-SCD3, is expressed sebaceous glands is consistent with the expression of FAR17c, which is the hamster homolog of M-SCD4. FAR17c is more related to M-SCD4 than to M-SCD3 m cDNA and protein sequence. FAR17c was isolated from hamster flank organ, a tissue comprised mostly of sebaceous cells. No localization data are presented in the work on FAR17c, thus this localization of M-SCD4 to sebaceous glands represents the first anatomical localization of SCD to vertebrate sebaceous glands.
  • M-SCD4 and M-SCD3 in the matrix keratinocytes suggests that redundancy may be related to a critical role for this enzyme in matrix cell physiology. It is notable that no expression is seen in the epidermis, a site of putative epidermal stem cells, suggesting that M-SCD3 and 4 may have a function specific to hair follicle (and possible sebaceous gland) stem cells.
  • PCR was used to amplify the complete open reading frame (ORF) , as well as to generate probes, from cDNA of human scalp PSU's. These probes were used to screen a human foreskin keratmocyte cDNA library, from which the 5' untranslated region (UTR) , the complete ORF, and part of the 3' UTR were cloned.
  • ORF open reading frame
  • UTR 5' untranslated region
  • HS-SCD Expression of HS-SCD is present in the matrix keratinocytes of the hair follicle and m the sebaceous gland.
  • mice do not have eccrme sweat glands in their hair-bearing skin.
  • a pair of primers forward: 5 ' GATATCTCAAGCTCCTATACC3 ' , reverse : 5 ' CTCCTCTGGAACATCACCAGTT3 '
  • HA-SCD Figure 10
  • the PCR fragment was cloned into the PBluescript vector and then used to generate a cDNA probe to screen a human foreskin keratinocyte (HFKC) cDNA lambda library constructed in ⁇ gtll (Clontech #HL1110B) .
  • HFKC human foreskin keratinocyte
  • cDNA sequences from the HFKC library indicate a C at position 898 (see Figure 8) , which results the ammo acid leuc e.
  • cDNA sequences of "uncloned" PCR products from the hair plugs indicate both a C and A at position 898, indicating that individual SCD transcripts from hair plug have either a C (producing leucme at ammo acid 224) or an A (producing methion e at ammo acid 224) at position 898 of the cDNA sequence.
  • Figures 8-11 all HS-SCD sequences are reported as C at position 898 of cDNA.
  • the ammo acid sequence in Figure 11 is reported with leucme at position 224. Since the hair plug samples are pooled from several individuals, the changes seen at bp 898 may be due to polymorphism.
  • the ORF s 1080 bp and generates a predicted protein of 359 ammo acids. Included m the human skin cDNA is 228 bp of 5' UTR and 689 bp of 3' UTR.
  • the cDNA sequences of human skm and liver SCD are 97.9% identical at the nucleotide level and 98.3% identical at the protein level.
  • the most significant base transitions are bp 301 and bp 304.
  • the substitution at these sites in liver vs skm result in ammo acid substitutions of prolme to serme (ammo acid 25) and m glycine to arginine (ammo acid 26) , respectively ( Figure 11) .
  • the substitution of serme for prolme in the skm cDNA may increase the polarity and may alter the secondary structure conferred by prolme.
  • the dramatic substitution of arginine for glycine in the skm cDNA replaces a nonpolar ammo acid with a postively charged ammo acid, which may result m an altered surface profile.
  • the 5' UTR contains 20 bp of unique sequence not present the liver cDNA.
  • the 3' UTR contains an additional 508 bp not contained in the liver cDNA.
  • the significance of the base pair differences m the UTR's is unknown. However, these differences may result m altered stability of the mRNA.
  • the 17 differences in the 3' UTR are mostly associated with the stretch of A's present in the liver cDNA.
  • the human adipose SCD represents a partial cDNA that is completely contained within the ORF of HS-SCD and HL-SCD.
  • the skin cDNA sequence that overlaps the adipose cDNA sequence is 97.8% identical.
  • the overlapping protem-codmg sequence is 97.4% identical.
  • the cDNA sequence of skm contains 15 base changes from the adipose cDNA sequence as seen in Figure 10. Of the 15 differences in the ORF, 6 lead to ammo acid changes. Of these 6 changes, 5 of 6 base substitutions occur either at the first or second position of the codon.
  • the base substitutions m the ORF of adipose compared to skm are as follows (using skm as the reference) : bp 241: A to T, bp 246: C to G, bp 249: A to G, bp 252: G to C, bp 261: A to T, bp 300: T to C, bp 301: C to T, bp 303: A to C, bp 304: G to A, bp 393: C to T, bp 888: C to T, bp 898: A to C, bp 936: C to T, bp 938: G to T, and bp 945: C to T ( Figure 10) .
  • substitutions are bp 301 and 304.
  • the substitution at 301 results in prolme to serme, and the substitution at 304 results m glycine to arginine (Figure 10) .
  • These are the same changes m ammo acids 25 and 26 that are seen m HL-SCD cDNA as compared to HS-SCD cDNA.
  • the suost tution of T for G at bp 938 of HS-SCD results in replacement of cysteme with phenylalanme at ammo acid 237, which may change secondary structure via altered disulfide bridges.
  • the substitions (in skm cDNA from adipose cDNA) at bp 241, 252, and 898 result m the replacement of ammo acid residues of similar biochemical profile. These replacements are: methionme to leucme (nonpolar), glutamate to aspartate (acidic) , and methionme to leucme (nonpolar) , respectively.
  • the base changes at 246, 249, 261, 393, 888, 936, and 945 do not result in ammo acid changes.
  • HS-SCD is a highly related, but unique sequence from HL-SCD and HA-SCD.
  • HS-SCD contains a serme and arginine at ammo acids 24 and 25, wnere both HL-SCD and HA-SCD contain prolme and glycine at these positions.
  • These particular substitutions, resulting m substitutions of adjacent ammo acids, are corroborated by the same serme and arginine found in the porcine SCD. No substitution between skm, liver, and adipose SCD disrupts or occurs withm the conserved histidme motifs as indicated by underlining m Figure 11.
  • the ISH probe used for localization of HS-SCD m skm is the same as that used to screen the HFKC cDNA library, as described above, and is shown boxed in Figure 8.
  • This region is highly homologous to the corresponding regions of liver SCD and adipose SCD, and thus would be expected to cross react in any procedure utlizing hybridization.
  • liver or adipose SCD sequences be detected in hair plug cDNA or in the HFKC library when sequencing both cloned and un-cloned PCR products. Nevertheless, a possible polymorphism was detected at bp 898. No other base positions were called ambiguously (an N in the base sequence, indicating "any" base) .
  • skm only expresses the skm SCD sequence as given m Figure 8.
  • the ISH probe although based on a common region of cDNA, snould only detect the skm SCD on tissue sections of hair plug samples .
  • the matrix keratinocytes of the hair bulb strongly and specifically expresses HS-SCD as indicated by data not shown here.
  • Adjacent FP fibroolasts ⁇ o not express HS-SCD. This expression pattern is strikingly similar to that of mouse. Although expressed less prominently than in the matrix cells, HS-SCD is specifically expressed in the human sebaceous gland.
  • Eccrine sweat glands specifically express HS-SCD.
  • the presence of HS-SCD in eccrine sweat gland suggests that HS-SCD may function in the growth regulation of the eccrine sweat gland cells and/or modification of the lipid contained m sweat .
  • SCD mRNA is highly expressed in the hair matrix keratinocytes of both mouse and human, suggesting a phylogenetically conserved function.
  • Cell division is highly conserved throughout evolution. Increased SCD has been found in several human tumors (Li et al . , 1994), and is up- regulated in cells tnat are placed m culture and undergoing rapid growth as determined here by experiment. Similarly, it is down-regulated in cells that have stopped dividing, as determined here by experiment .

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Abstract

La présente invention concerne une molécule d'acide nucléique codant la stéaroyl-CoA désaturase humaine, et une protéine codée par ladite molécule. L'invention concerne aussi un procédé permettant de diagnostiquer chez un sujet humain des affections cutanées caractérisées par un niveau d'expression anormal de la stéaroyl-CoA désaturase. L'invention concerne également un procédé permettant de déterminer si un agent augmente le niveau d'expression de lastéaroyl-CoA désaturase dans des cellules cutanées qui l'expriment déjà, ainsi qu'un procédé permettant de déterminer si un agent augmente au abaisse le niveau d'expression ou l'activité de la stéaroyl-CoA désaturase dans des cellules cutanées qui l'expriment déjà. On décrit des compositions pharmaceutiques destinées à traiter des affections cutanées caractérisées par un niveau d'expression et/ou d'activité anormal de la stéaroyl-CoA désaturase, ainsi que des procédés de traitement associés. L'invention concerne en outre des anticorps associés, des vecteurs de thérapie génique et des souris transgéniques.
PCT/US1999/018387 1998-08-14 1999-08-12 COMPOSITIONS ASSOCIEES A LA STEAROYL-CoA DESATURASE HUMAINE ET PROCEDES DE TRAITEMENT D'AFFECTIONS CUTANEES WO2000009754A2 (fr)

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JP2000565188A JP2003533965A (ja) 1998-08-14 1999-08-12 皮膚の異常症状を治療するためのヒト・ステアロイル−ＣｏＡデサチュラーゼ−関連組成物および方法
CA002339748A CA2339748A1 (fr) 1998-08-14 1999-08-12 Compositions associees a la stearoyl-coa desaturase humaine et procedes de traitement d'affections cutanees
AU57746/99A AU5774699A (en) 1998-08-14 1999-08-12 Human stearoyl-coa desaturase-related compositions and methods for treating skin disorders
EP99945051A EP1105538A2 (fr) 1998-08-14 1999-08-12 COMPOSITIONS ASSOCIEES A LA STEAROYL-CoA DESATURASE HUMAINE ET PROCEDES DE TRAITEMENT D'AFFECTIONS CUTANEES

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US9652098P 1998-08-14 1998-08-14
US60/096,520 1998-08-14

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19955349A1 (de) * 1999-11-17 2001-08-02 Switch Biotech Ag Verwendung von Polypeptiden oder diese kodierende Nukleinsäuren zur Diagnose oder Behandlung von Hauterkrankungen sowie ihre Verwendung zur Identifizierung von pharmakologisch aktiven Substanzen
WO2001066758A2 (fr) * 2000-03-07 2001-09-13 Millennium Pharmaceuticals, Inc. Nouvelle desaturase d'acides gras appelee 25934 et ses utilisations
WO2002026944A2 (fr) * 2000-09-26 2002-04-04 Xenon Genetics, Inc. Techniques et compositions utilisant une nouvelle stearyle-coa desaturase hscd5
WO2003012031A2 (fr) * 2001-07-30 2003-02-13 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la stearoyl-coa desaturase
WO2003074662A2 (fr) * 2002-03-01 2003-09-12 Exelixis, Inc. Enzymes scd utilisees comme modificateurs de la voie p53 et procedes d'utilisation correspondants
EP1346042A2 (fr) * 2000-11-17 2003-09-24 Xenon Genetics, Inc. Genes a regulation lipidique, utilisations de ces genes et composes permettant leur modulation
WO2004047746A2 (fr) * 2002-11-21 2004-06-10 Bayer Pharmaceuticals Corporation Regulation de stearoyl-coa desaturase dans le traitement de l'obesite
EP1429784A2 (fr) * 2001-08-29 2004-06-23 Xenon Genetics, Inc. Essais de criblage a haut rendement mettant en oeuvre des enzymes synthetiques d'acide gras
FR2853910A1 (fr) * 2003-04-16 2004-10-22 Galderma Res & Dev Genes du psoriasis
US6987001B2 (en) 2000-02-24 2006-01-17 Xenon Pharmaceuticals Inc. Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents
WO2009106991A3 (fr) * 2008-02-25 2009-11-05 Xenon Pharmaceuticals Inc. Dérivés de pyridazine et leur utilisation comme agents thérapeutiques
US7960358B2 (en) 2001-07-30 2011-06-14 Isis Pharmaceuticals, Inc. Antisense modulation of stearoyl-CoA desaturase expression
US8802167B2 (en) 2010-06-11 2014-08-12 Avon Products, Inc. Use of Eclipta prostrata and other PPAR-gamma inhibitors in cosmetics and compositions thereof
KR20180081624A (ko) * 2016-07-08 2018-07-16 카오카부시키가이샤 핵산 시료의 조제 방법
US10537623B2 (en) 2011-05-19 2020-01-21 Toray Industries, Inc. Immunity induction agent
EP4119664A4 (fr) * 2020-03-11 2024-04-24 Kao Corp Procédé de préparation d'arn dérivé de lipides de surface cutanée

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] Accession number (AC: Y13647), 1997 AL JERYAN L ET AL.: "Characterization and expression of a stearoyl CoA desaturase from human liver" XP002139045 *
KAESTNER K H ET AL.: "Differentiation-induced gene expression in 3T3-L1 preadipocytes" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 25, 1989, pages 14755-14761, XP002139043 *
LI J ET AL.: "Partial characterization of a cDNA for human stearoyl-CoA desaturase and changes in its mRNA expression in some normal and malignant tissues" INTERNATIONAL JOURNAL OF CANCER, vol. 57, 1994, pages 348-352, XP000910652 *
MILLER C W AND NTAMBI J M: "Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase I gene expression" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 93, 1996, pages 9443-9448, XP002139044 cited in the application *
NTAMBI J M ET AL.: "A model cell line to study regulation of stearoyl-CoA desaturase gene 1 expression by insulin and polyunsaturated fatty acids" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 220, 1996, pages 990-995, XP002139042 *
PARIMOO S ET AL.: "Identification of a novel SCD gene and expression of the SCD family in mouse skin" JOURNAL OF INVESTIGATIVE DERMATOLOGY SYMPOSIUM PROCEEDINGS, vol. 4, 1999, pages 320-322, XP000911022 cited in the application *
ZHANG L ET AL.: "Human stearoyl-CoA desaturase: alternative transcripts generated from a single gene by usage of tandem polyadenylation sites" BIOCHEMICAL JOURNAL, vol. 340, 1999, pages 255-264, XP000913556 *

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DE19955349A1 (de) * 1999-11-17 2001-08-02 Switch Biotech Ag Verwendung von Polypeptiden oder diese kodierende Nukleinsäuren zur Diagnose oder Behandlung von Hauterkrankungen sowie ihre Verwendung zur Identifizierung von pharmakologisch aktiven Substanzen
US7816075B2 (en) 2000-02-24 2010-10-19 Xenon Pharmaceuticals Inc. Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents
US7696151B2 (en) 2000-02-24 2010-04-13 Xenon Pharmaceuticals Inc. Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents
EP2172560A2 (fr) 2000-02-24 2010-04-07 Xenon Pharmaceuticals Inc. Méthodes et compositions utilisant la stéaoryl-CoA desaturase pour identifier des agents thérapeutiques reduisant les trigycerides
US6987001B2 (en) 2000-02-24 2006-01-17 Xenon Pharmaceuticals Inc. Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents
US6686185B1 (en) 2000-03-07 2004-02-03 Millennium Pharmaceuticals, Inc. 25934, a novel fatty acid desaturase and uses therefor
WO2001066758A3 (fr) * 2000-03-07 2002-04-11 Millennium Pharm Inc Nouvelle desaturase d'acides gras appelee 25934 et ses utilisations
WO2001066758A2 (fr) * 2000-03-07 2001-09-13 Millennium Pharmaceuticals, Inc. Nouvelle desaturase d'acides gras appelee 25934 et ses utilisations
US7232662B2 (en) 2000-09-26 2007-06-19 Xenon Pharmaceuticals Inc. Methods and compositions employing a novel stearoyl-CoA desaturase-hSCD5
WO2002026944A3 (fr) * 2000-09-26 2002-10-03 Xenon Genetics Inc Techniques et compositions utilisant une nouvelle stearyle-coa desaturase hscd5
WO2002026944A2 (fr) * 2000-09-26 2002-04-04 Xenon Genetics, Inc. Techniques et compositions utilisant une nouvelle stearyle-coa desaturase hscd5
EP1346042A2 (fr) * 2000-11-17 2003-09-24 Xenon Genetics, Inc. Genes a regulation lipidique, utilisations de ces genes et composes permettant leur modulation
WO2003012031A3 (fr) * 2001-07-30 2003-11-06 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la stearoyl-coa desaturase
WO2003012031A2 (fr) * 2001-07-30 2003-02-13 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la stearoyl-coa desaturase
US7132529B2 (en) 2001-07-30 2006-11-07 Isis Pharmaceuticals, Inc. Antisense modulation of stearoyl-CoA desaturase expression
US7960358B2 (en) 2001-07-30 2011-06-14 Isis Pharmaceuticals, Inc. Antisense modulation of stearoyl-CoA desaturase expression
US6759208B2 (en) 2001-08-29 2004-07-06 Xenon Genetics Inc. High throughput screening assays using fatty acid synthetic enzymes
EP1429784A4 (fr) * 2001-08-29 2005-09-14 Xenon Genetics Inc Essais de criblage a haut rendement mettant en oeuvre des enzymes synthetiques d'acide gras
US7294480B2 (en) 2001-08-29 2007-11-13 Xenon Pharmaceuticals Inc. High throughput screening assays using fatty acid synthetic enzymes
EP1429784A2 (fr) * 2001-08-29 2004-06-23 Xenon Genetics, Inc. Essais de criblage a haut rendement mettant en oeuvre des enzymes synthetiques d'acide gras
WO2003074662A2 (fr) * 2002-03-01 2003-09-12 Exelixis, Inc. Enzymes scd utilisees comme modificateurs de la voie p53 et procedes d'utilisation correspondants
WO2003074662A3 (fr) * 2002-03-01 2004-11-25 Exelixis Inc Enzymes scd utilisees comme modificateurs de la voie p53 et procedes d'utilisation correspondants
US8771942B2 (en) 2002-03-01 2014-07-08 Exelixis, Inc. SCDs as modifiers of the p53 pathway and methods of use
WO2004047746A3 (fr) * 2002-11-21 2005-03-24 Bayer Pharmaceuticals Corp Regulation de stearoyl-coa desaturase dans le traitement de l'obesite
WO2004047746A2 (fr) * 2002-11-21 2004-06-10 Bayer Pharmaceuticals Corporation Regulation de stearoyl-coa desaturase dans le traitement de l'obesite
WO2004094661A2 (fr) * 2003-04-16 2004-11-04 Galderma Research & Development, S.N.C. Genes fancc, dad1, grim19 et hadhii marquers du psoriasis
WO2004094661A3 (fr) * 2003-04-16 2005-10-13 Galderma Res & Dev Genes fancc, dad1, grim19 et hadhii marquers du psoriasis
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US7759348B2 (en) 2003-07-30 2010-07-20 Xenon Pharmaceuticals Inc. Pyridazine derivatives and their use as therapeutic agents
WO2009106991A3 (fr) * 2008-02-25 2009-11-05 Xenon Pharmaceuticals Inc. Dérivés de pyridazine et leur utilisation comme agents thérapeutiques
US8802167B2 (en) 2010-06-11 2014-08-12 Avon Products, Inc. Use of Eclipta prostrata and other PPAR-gamma inhibitors in cosmetics and compositions thereof
US10537623B2 (en) 2011-05-19 2020-01-21 Toray Industries, Inc. Immunity induction agent
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