WO2002088348A2 - Lipid producing cells and uses thereof - Google Patents

Abstract

This invention relates to the production of lipids which are required for the barrier function of the skin and eye, more particularly, to the identification of a particular type of cell in sebaceous-related glands and hair follicles which is responsible for the production of esterified very long chain fatty acids (VLCFAs) which play an integral role in this barrier function. Cells which expresses Elov13 are isolated from a populaiton of cells of a hair follicle and/or a sebaceous/related gland, and can be used in the manufacture of a medicament for the use in the treatment of a disease associated with aberrant barrier lipid function. The isolated cells can also be used for compounds that modulates formation and/or properties of a barrier lipid mixture.

Description

Lipid Producing Cells and Uses Thereof

The present invention relates to the production of lipids which are required for the barrier function of the skin and eye, more particularly, the cells which are responsible for the production of esterified very long chain fatty acids (VLCFAs) which play an integral role in this barrier function.

VLCFA have been recognized as structural components in a variety of fat molecules such as glycerophospholipids, sphingolipids, and wax- and sterol-esters . Depending on their chain length and degree of unsaturation, they contribute to fluidity, permeability and other physical and chemical properties of the membrane. VLCFA are found in virtually all cells and are major constituents of the brain, skin and testis .

Esterified or as free acyl chains, VLCFA comprise more than 50% of the fatty acids found in epidermis, the outermost layer of the skin, which is the highest amount present in any mammalian tissue, and are indispensable for the formation and function of the permeability layer of the skin. VLCFA are also found in gross amounts in glands such as sebaceous and meibomian glands . The sebaceous glands are appendages of the epidermis which are connected to hair follicles and secrete a mixture of lipids (sebum) onto the skin surface. This mixture, especially in mouse, is rich in esters of cholesterol and other sterols which are esterified with VLCFA.

Three genes, Elovll , Elovl2 and Elovl , have recently been identified in mouse [Tvrdik, (2000) J. Cell. Biol . 149 707- 717] . All three mouse genes show a distinct tissue specific expression pattern and recent results have suggested that the corresponding proteins are involved in the synthesis of very long chain fatty acids (VLCFA) , as well as ceramide and sphingolipid formation [Tvrdik, 2000] .

Elovl3 encodes a 30 kDa glycoprotein and was originally termed Cig30 (Cold-inducible g_lycoprotein ^of jD kDa) since its mRNA level is increased in brown adipose tissue upon cold stimulation [Tvrdik, (1997) J.Biol. Chem. 272 31738-31746]. Elovl3 is also expressed in liver and skin where it is unaffected by cold. Elovl3 gene encodes a product which belongs to a highly conserved family of microsomal enzymes involved in the formation of VLCFA and sphingolipids from yeast to man. ELOVL3 is believed to elongate fatty acids up to 24 carbon atoms [Tvrdik, 2000 supra] .

The present invention relates to the finding that the Elovl3 protein plays a vital role in the formation of esterified and free VLCFAs in the hair follicles and the sebaceous glands and that the lipids thus formed are required for the barrier function of the skin and other surfaces. Furthermore, expression analysis of the Elovl3 gene has revealed that expression is specific to sebocytes in the sebaceous glands and modified-sebaceous glands and certain epithelial cells of the hair follicles.

Previously reported cell cultures derived from sebaceous glands (reviewed in Fujie, T. et al . (1996) Arch . Dermatol . Res . 288, 703-708) have had the major disadvantage that they comprise a heterogeneous mixture of different types of cells at different states of differentiation. Such cell cultures are difficult to use for the isolation of physiological important lipids or in assay methods for substances regulating lipid production and release. Methods for the isolation of Elovl3 expressing cells and uses and applications of such cells are described herein.

An aspect of- the present invention provides an isolated cell which secretes esterified or free VLCFAs obtainable by a method comprising; dissociating tissue of a hair follicle and/or a sebaceous-related gland into a population of constituent cells, identifying one or more cells within said population which express Elovl3, and; separating said one or more cells from said population.

Another aspect of the present invention provides an isolated cell which secretes esterified or free VLCAs obtainable by a method comprising; providing a population of cells of a hair follicle and/or a sebaceous-related gland, identifying one or more cells within said population which expresses Elovl3, and; separating said one or more cells from said population.

Hair follicle and sebaceous-related glands may originate or be obtained from a mammal, such as a rodent or a human.

A sebaceous-related gland may be a sebaceous, meibomian, preputial, perianal, clitoral or ceruminous gland. Preferably, a cell which expresses Elovl3 is a sebocyte from a sebaceous- related gland or an epithelial cell of the hair follicle, more preferably a cell of the inner layer of the outer root of the hair follicle.

A cell which expresses Elovl3 may be identified using standard techniques in the art, for example FACS, Northern blotting, and Western blotting. Full details of these methods are provided in, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell., 2001, Cold Spring Harbor Laboratory Press NY and Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

Cells identified as lipid producing cells through the expression of Elovl3 (at the nucleic acid or the protein level) may then be screened for the ability to form specific lipids (using techniques such as TLC, GC and MS) i.e. the ' lipid production of the cells may be determined. In particular, the ratio of 20:1 to 18:1 monounsaturated fatty acids, which is shown herein to be indicative of Elovl3 activity, may be determined. In particular, an increase in the 20:1 to 18:1 ratio in the presence of a test compound relative to the absence is indicative of an inhibitory effect on Elovl3 activity.

The esterified or free VLCFAs produced by the Elovl3 expressing cells described herein are essential, as components of the barrier lipid mixture, in conferring the water repulsion and thermoregulation properties of the skin.

Methods for isolating cells are described in more detail below.

Cells identified and/or obtained as described herein may be cultured and/or subjected to clonal expansion using techniques well-known in the art to increase the homogeneity of said cells.

An isolated cell may be provided free or substantially free from contaminants with which it is naturally associated, for example, other cell types which do not express Elovl3. The isolated and/or purified cell may be used in the formulation of a composition, which may include at least one additional component, for example, a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.

A composition including a cell according to the invention may be used in prophylactic and/or therapeutic treatment of diseases associated with aberrant barrier lipid production or formation in the skin or eyes, as discussed below.

A barrier lipid mixture is a mixture of lipids which is secreted onto a body surface such as the skin or eye and has a protective effect. Examples include sebum secreted by the sebaceous glands and hair follicles and ocular lubricant secreted by the meibomian glands.

Pharmaceutical research leading to the identification of a new drug may involve the screening of very large numbers of candidate substances, both before and even after a lead compound has been found. This is one factor which makes pharmaceutical research very expensive and time-consuming. Means for assisting in the screening process can have considerable commercial importance and utility.

Means for screening for substances potentially useful in treating or preventing diseases associated with aberrant barrier lipid production in the skin or eyes are provided by isolated cells according to the present invention.

Substances found to be modulators of Elovl3 protein expression and/or activity of esterified or free VLCFA production represent an advance in the fight against such diseases since they provide basis for design and investigation of therapeutics for in vivo use.

In various further aspects the present invention relates to screening and assay methods and means and substances and compounds obtained by such methods .

A further aspect of the present invention therefore provides an assay method which comprises : (a) treating one or more isolated cells as described herein with a test substance or compound;

(b) determining the presence or absence of an effect on the one or more isolated cells as a result of the treatment with the test substance.

The nature of an effect when detected may be investigated.

Potential end-points for detection include visual effects, effects determined immunologically or biochemically, and effects determined by means of determination of gene expression, for instance by means of Southern or Northern blotting of nucleic acid extracts or derivatives from appropriate cells. The test substance is preferably membrane permeable .

The effect may be a modulation of the expression or activity of Elovl3 protein or a modulation of the formation or secretion of components of barrier lipid mixture, particularly esterified or free VLCFAs. In some embodiments, the ratio of 20:1 to 18:1 monounsaturated fatty acids may be determined.

The skilled person is well aware of the need for control experiments and well able to design appropriate controls, both positive and negative. An end-point indicative of a positive result may be chosen in view of the therapeutic application in mind.

Thus, further aspects of the present invention provide the use of an isolated cell as described herein in screening or searching for and/or obtaining/identifying a substance, e.g. peptide or chemical compound, which modulates the formation, composition, secretion or properties of a barrier lipid mixture comprising esterified or free VLCFAs.

For instance, a method according to one aspect of the invention includes providing an isolated cell of the invention and bringing it into contact with a substance or compound, which contact may result in modulation of the expression and/or activity of Elovl3 protein by the substance or compound. Expression and/or activity may be determined by any of a number of techniques available in the art, both qualitative and quantitative.

In various aspects the present invention is concerned with provision of assays for substances which modulate the activity of Elovl3 and thereby the production or properties of barrier lipid mixtures which comprise esterified or free VLCFAs, in particular, substances which modulate the 20:1/18:1 monounsaturated lipid ratio.

Further assays are for substances which modulate one or more cellular activities related to the formation or secretion of esterified or free VLCFA components of the barrier lipid mixture (e.g. sebum).

Expression of the Elovl3 gene is essential for the production of essential skin lipids, as described herein. Substances which modulate the production of skin lipids such as esterified or free VLCFA components may therefore be identified or obtained by determining Elovl3 gene expression, for example by Northern blotting, Western blotting techniques or immunoassays . Lipid profiling may also be used to determine the production of such lipids.

A method of screening for a substance which modulates the formation and/or properties of a barrier lipid mixture comprising esterified or free VLCFAs produced by a cell as described herein, for example, by modulating the expression and/or activity of Elovl3, may include; contacting one or more test substances with a cell or an population of cells as described herein in a suitable reaction medium; and, determining the expression and/or activity of Elovl3.

A method of screening for a substance which modulates the formation and/or properties of a barrier lipid mixture comprising esterified or free VLCFAs produced, may include; contacting the skin cells of a mammal with one or more test substances; and, determining the expression and/or activity of Elovl3 in said cells .

Activity may be compared with the expression and/or activity of Elovl3 in comparable reaction medium untreated with the test substance or substances. A difference in Elovl3 expression or activity between the treated and untreated cells is indicative of a modulating effect of the relevant test substance or substances.

Such methods may further comprise determining the esterified or free VLCFAs produced in the cell or cells, in particular the levels of 20:1 and 18:1 monounsaturated fatty acids. A method of screening for a compound or substance which modulates the formation and/or properties of a barrier lipid mixture comprising esterified or free VLCFAs may include; contacting one or more test compounds with a cell as described herein or an population of said cells in a suitable reaction medium; and, determining the esterified or free VLCFAs formed in the cell or cells.

The esterified or free VLCFAs may be compared with the esterified or free VLCFAs in comparable reaction medium untreated with the test compound or compounds. A difference in the esterified or free VLCFAs formed in treated and untreated cells is indicative of a modulating effect of the relevant test compound or compounds. In particular a difference in the ratio of 20:1 to 18:1 monounsaturates may be determined, for example in the total lipid extract or a fraction thereof, such as the triglyceride or sterol-wax ester fraction.

Esterified or free VLCFAs may be determined by lipid profiling using conventional techniques.

For example, an increase in the proportion of 20:1 fatty acids and/or a decrease in the proportion of 16:0, 16:1, 18:0, or 18:1 fatty acids in the secreted lipid may be indicative of an inhibition of the Elovl3 protein by the test substance. In particular, the ratio of monounsaturated fatty acids 20:1 and 18:1 produced by said cell may be determined.

The present inventors have recognised that the ratio of monounsaturated fatty acids 20:1 and 18:1 (i.e. 20:1/18:1) in both the total lipid, triglyceride and the cholesterol- and wax esters fraction produced by cells described herein increases in cells with reduced or absent Elovl3 activity. For example, the ratio might be less than 0.5 in normal cells and greater than 0.5 in Elovl3 ablated cells.

An increase in the ratio of 18:1 to 20:1 fatty acids in a treated cell relative to an untreated cell is indicative that the test substance or compound is an inhibitor of the expression or activity of Elovl3.

The ratio of 18:1 to 20:1 fatty acids may be determined, for example, in the total lipid fraction, the cholesterol- and wax esters fraction or the triglyceride fraction.

Lipid production and composition may be monitored, for example, using Thin Layer Chromatography as described herein.

A substance which modulates cellular activity as described may be isolated and/or purified, manufactured and/or used to modulate its activity as discussed.

The precise format of the assay of the invention may be varied by those of skill in the art using routine skill and knowledge .

Suitable screening methods are conventional in the art. They include techniques such as radioimmunosassay, scintillation proximetry assay, thin layer chromatography, and ELISA methods .

Elovl3 expression may be determined by determining the level of Elovl3 mRNA or the level of Elovl3 protein. Suitable techniques are well known in the art and include Northern blotting (for mRNA) , Western blotting (for protein) and immunoassays (for example, radioimmunoassays and ELISA) .

Esterified or free VLCFA barrier lipid components may be identified or determined using techniques well known in the art such as Thin-layer-chromatography (TLC) , Gas Chromatography-Mass Spectroscopy (GC-MS) and High Pressure Liquid Chromatography (HPLC) .

Of course, the person skilled in the art will design any appropriate control experiments with which to compare results obtained in test assays.

Lipid producing cells may be isolated as described herein from an Elovl3 ablated animal (e.g. Elovl3 (-/-) mice). Such cells are useful for determining lipid production in the absence of Elovl3 expression, for example as a negative control in the experiments described herein. The effect of a test compound on the expression and/or activity of Elovl3 may, for example, be determined by comparing the production of lipids in the presence and absence of test compound in cells expressing Elovl3 and cells not expressing Elovl3.

A method as described herein may therefore further comprise the step of identifying a test compound, substance or molecule as a modulator of the formation and/or properties of a barrier lipid mixture.

Performance of a method as described above may be followed by purification and/or isolation of a test compound, substance or molecule which tests positive for ability to modulate the formation and/or secretion of esterified or free VLCFAs from said cells and/or the manufacture or synthesis of such a compound Combinatorial library technology (Schultz, JS (1996) Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.

The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM. Greater concentrations may be used when a peptide is the test substance.

Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used. A further class of putative inhibitor compounds can be derived from the Elovl3 polypeptide product. Peptide fragments of from 5 to 40 a ino acids, for example from 6 to 10 amino acids from the region of the Elovl3 polypeptide responsible for interaction, may be tested for their ability to disrupt cellular activity.

Other candidate inhibitor compounds may be based on modelling the 3 -dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.

Assay methods and methods of screening as described herein may be useful in identifying and/or obtaining agents for treatment of eye problems, and skin disorders, which may be selected from disorders associated with aberrant barrier lipid function such as atopic dermatitis, hair loss, psoriasis, cachexia, atopic cataract, blepharitis, atopic eczema, corneal inflammation and wound healing and others disclosed herein and apparent to the skilled person based on the present disclosure.

Following identification of a substance which modulates or affects polypeptide activity, the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug i.e. it may be formulated with a pharmaceutically acceptable excipient. These may be administered to individuals.

Thus, the present invention extends in various aspects not only to a substance, compound or molecule identified as a modulator of cellular activity as disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a condition associated with aberrant barrier lipid function, use of such a substance in manufacture of a composition or medicament for administration, e.g. for treatment of a condition associated with aberrant barrier lipid function, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients .

A substance identified using as a modulator of polypeptide or promoter function may be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimick of the substance (particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound.

This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it.

Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of substances identified as having ability to modulate cellular activity using a screening method as disclosed herein are included within the scope of the present invention. A polypeptide, peptide or substance able to modulate cellular activity according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment . Such a kit may include instructions for use.

Another aspect of the present invention provides a method of isolating a cell which secretes esterified or free VLCFA components of barrier lipid mixtures comprising; dissociating tissue of a hair follicle and/or a sebaceous-related gland of a mammal into a population of constituent cells, identifying one or more cells within said population which express Elovl3, and; separating said one or more cells from said tissue.

Methods for isolating such cells are described herein.

Sebaceous-related glands may include sebaceous, meibomian, preputial, perianal, clitoral or ceruminous glands of a mammal. An Elovl3 expressing cell may include a sebocyte or epithelial cell of the hair follicle, more preferably a cell of the inner layer of the outer root of the hair follicle.

The identification and recovery of an Elovl3 expressing cell may be achieved by screening for expression of the Elovl3 gene, for example by detecting mRNA encoding the Elovl3 protein product or by detecting the Elovl3 protein product itself, according to methods standard in the art.

Cells isolated as described herein may be immortalised and cultured according to standard techniques known in the art. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook et al . , 2001, Cold Spring Harbor Laboratory Press NY and Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

Cells as described herein may complement dysfunctional cells in vivo and may therefore be used to treat diseases associated with aberrant lipid barrier function, such as atopic dermatitis, hair loss, psoriasis, cachexia, atopic cataract, blepharitis, atopic eczema, corneal inflammation and wound healing and others disclosed herein. Thus, the present invention therefore extends in various aspects not only to an isolated cell which secretes barrier lipids, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a cell, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a condition associated with aberrant barrier lipid function, use of such a cell in manufacture of a composition or medicament for administration, e.g. for treatment of a disease associated with aberrant barrier lipid function, and a method of making a pharmaceutical composition comprising admixing such a cell with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

Whether it is a cell, polypeptide, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound or composition according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Aspects of the present invention will now be illustrated with reference to the accompanying figures described below and experimental exemplification, by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art. All documents mentioned in this specification are hereby incorporated herein by reference.

Figure 1 shows the water content of the body hair of Elovl3 - /- (o) and Elovl3 +/+ (•) mice after immersion.

Figure 2 shows the core body temperature of Elovl3 -/- (o) and Elovl3 +/+ (•) mice after immersion

Figure 3 shows the Trans-Epidermal Water Loss (TEWL) of Elovl3 -/- (o) and Elovl3 +/+ (•) mice.

Figure 4 shows the w/w proportion of lipid to body hair of Elovl3 -/- (o) and Elovl3 +/+ (•) mice.

Figure 5 shows the fatty acid composition of the sebum of Elovl3 -/- (o) and Elovl3 +/+ (•) mice.

Figure 6 shows the relative elongation activity of fatty acyl CoAs in the liver microsomes of Elovl3 -/- (o) and Elovl3 +/+

(•) mice.

Table 1 shows fatty acid composition of triglycerides and sterol- and wax esterss from murine sebum and hair lipids: A+B

(wild type mice) and C+D (EL0VL3 -/- mice) .

EXPERIMENTAL

Transgenic mice in which the Elovl3 gene was disrupted were used to investigate the role of the Elovl3 protein in the formation of sebum. Further analysis of the particular cells which express Elovl3 was undertaken in both transgenic and wild type mice.

The production of Elovl3 -deficient transgenic mice was described by the present inventors in WO 00/70945.

MATERIALS AND METHODS Gene Targeting Vector A genomic liver DNA library from mouse strain 129/Sv cloned into the Lambda FIXII vector was used to isolate a DNA fragment containing the entire Elovl3 gene [Tvrdik, 1999] . A 2.75 kb fragment between Sad and Sail upstream of the first exon was sub-cloned into the polylinker of the pBluescript SK (+/-) plasmid in order to become the left arm (LA) in the Elovl3-KO construct. The Sad site was blunt-ended by T4 DNA polymerase (New England Biolab) and turned into a Notl site by ligation of Notl primers (Bio Source International) . A 1.2 kb fragment containing a neomycin^r (neo^r) gene was cut out with Xhol from the KT1 LoxA plasmid and inserted into the compatible Sail site of the plasmid containing the 2.75 kb left arm fragment. The ligation between Sail and Xhol ends consequently destroyed the recognition site for respective digestion enzyme. A 5.30 kb Sail/Sail fragment downstream of the second exon of the Elovl3 gene was ligated into the pBluescript SK(+/-) plasmid and further digested with Sail and Xhol to receive a 2.77 kb fragment corresponding to the right arm in the Elovl3-KO construct. This fragment was subcloned into the Xhol site of the vector containing the 2.75 kb left arm fragment and the neo gene. The Notl/Xhol fragment from the resulting plasmid was ligated into the corresponding sites in the TK1-TK2 vector. Gene-targeting in ES cells and Blastocyst Injection The knockout construct was linearized by Notl and electroporated into the RI embryonic stem (ES) cells [Nagy, A. et al (1993) Proc . Natl. Acd. Sci . USA 90 8424-8428] derived from male 129/Sv agouti mice. ES cells were cultured as earlier described and subjected to positive/negative selection using G418 and FIAU as previously described [Thomas, K. R., and Capecchi, M. R. (1987) Cell 51, 503-512; Mansour, S. L. et al (1988) Nature 336, 348-352] . ES cell DNA was prepared according to standard procedures [Nagy A. et al (1993) supra] . Probes 1 and 2 were used to confirm ES cell clones heterozygous for the Elovl3-KO construct.

Elovl3 targeted ES cells were injected into C57BL/6J blastocysts and implanted into white foster mothers (FI,

CBAxC57BL6) according to standard procedures [Gossler, A. et al. (1986) Proc. Natl . Acad. Sci . USA 83, 9065-9069] The male offspring being the most chimeric, approximately 80% agouti and 20% black in the coat colour, were bred with C57B1/6J female in order to generate offspring heterozygous for the mutation. Genomic DΝA was prepared from mouse tails by the simplified mammalian DΝA isolation as described earlier [Laird, P. W. et al (1991) Nucleic Acids Res . 19, 4293-4293] . Tail biopsies were collected from 3 -week-old mice and used directly for DΝA isolation.

The Elovl3 ablated mice were back-crossed with C57B1/6 (B & K Universal, Stockholm Sweden). As control mice, we used heterozygote littermates or age matched C57B1/6 which were bred under the same conditions as the Elovl3 ablated mice.

Animals were fed ad libitum (Rat and Mouse Standard Diet Νo.l, BeeKay Feeds, B & K Universal; Stockholm, Sweden) , had free access to water and were kept on a 12:12 h light: dark cycle in single cages: Wild-type and Elovl3 -ablated mice were bred in room temperature. Before the cold-exposure experiments, the mice were stored at 30°C (thermoneutrality) for ten days if nothing else is indicated. The mice were then placed in 4°C for three days (cold stressed) or 3-4 weeks (cold-acclimated) as indicated in results.

Southern blot Analysis of DNA from ES cells and Mouse Tails The DNA for the probes were purified according to the Jetsorb Gel extraction kit (Genomed Inc.) and were labelled with [α-

32P] dCTP using a random primed labelling kit (Boehringer Mannheim) . Ten μg of digested DNA from mouse tails and ES cells was separated on 0.8% agarose gel and transferred to Hybond-N membrane (Amersham) in 20 x SSC. The hybridisation procedure were identical to those for Northern blot analysis, except that hybridisation was carried out over night at 55°C. Hybond-N membrane was then washed twice in 2 x SSC, 0.2% SDS at 30°C for 20 min each and then once in 0.1 x SSC, 0.2% SDS at 55°C for 30 min. The filters were analysed on a Molecular Dynamics Phosphorlmager with the ImageQuant program.

RNA Analysis

Total RNA was isolated using Ultraspec (Biotech lab.) from 50- 100 mg (w/w) of each tissue as described earlier [Tvrdik, 1997] . For Northern blot analysis, 20 mg of total RNA was separated on a 1.2% (w/v) formaldehyde agarose gel and blotted onto Hybond-N membrane (Amersham) in 20 x SSC. The membrane was prehybridised with a solution containing 5 x SSC, 5 x Denhardt's, 0.5% SDS, 50 mM sodium phosphate, 50% formamide and 100 mg/ml of degraded DNA from herring sperm (Sigma) at 45°C. After pre-hybridisation, the membrane was transferred to a similar solution containing the denatured probe. The hybridisation was carried out over night at 45°C. The membrane was then washed twice in 2 x SSC, 0.2% SDS at 30°C for 20-30 min each and then twice in 0.1 x SSC, 0.2% SDS at 50°C for 45 min. The filters were analysed on a Phosphorlmager (Molecular Dynamics) and quantified with the ImageQuant program (Molecular Dynamics) .

Histology

Skin biopsies of age- and sex-matched animals were taken from similar body sites. Skin samples were fixed overnight at 4°C in a phosphate buffered, pH 7.4, 4% formaldehyde solution. Semi- thin sections were stained with hematoxylin and eosin and examined by light microscopy.

In situ Hybridization

RNA probes were prepared from Elovl3 ORF mouse cDNA. In si tu hybridization was performed according to methods well-known in the art. Briefly, a 871-bp fragment corresponding to nt 162- 1056 in the Elovl3 cDNA was cloned into a pCI-neo vector, appropriately linearized and in vitro transcribed to obtain antisense and sense probes. Sections were treated with proteinase K (Sigma Chemical Co.) and washed in 0.1M triethanolamine buffer containing 0.25% acetic anhydride.

Subsequently, sections were hybridized overnight with 2.5xl0⁶ cpm of labeled antisense or sense probe at 55°C. Autography was for 14 days . After development of the photographic emulsion, slides were stained with hematoxylin and eosin.

Water Retention Assay and Temperature Measurement Weight or colonic temperature was measured (Model BAT-12 thermometer, Physitemp Instruments Inc.) once for each adult mouse prior to swim. Mice were let to swim in 30°C for 2 min. Excessive water was eliminated by allowing the mice to walk on paper towels for a few seconds. Weight or colonic temperature was recorded every 5 min in 22°C. Between each measurement the mice were kept individually in empty plastic cages. The hair water content was calculated by subtracting the preswim weight .

Trans epidermal water loss (TEWL) analysis Mice were anaestesized with 2.5% avertin by intraperitoneal injection at a concentration of 0.014 ml/gr body weight. The evaporimeter (EP 1C, Servomed, Gόteborg, Sweden) was run for at least 15 min. prior to use. The evaporimeter was placed on a 1 cm² of shaved skin on the back of the animals in an open chamber. During the measurement the evaporimeter was allowed to stabilize for 30 seconds before the value was recorded. All measurements were performed according to the guidelines from the standardization group of contact dermatitis [Pinnagoda, J. et al (1990) Contact Dermati tis 22, 164-178] .

Hair lipid Analysis

Hair from adult mice was extracted and filtered twice with 20 ml acetone for 15 min. The two extracts were combined and allowed to evaporate to dryness in glass vials. The amount of dry lipids was calculated by subtracting the predetermined weight of empty vials. Equal amounts of lipids (120 μg) were dissolved in acetone and applied to each lane on a Whatman HPTLC Silica G plate and separated according to Downing (1968) (Downing, D. T. (1968) J^". Chromatogr . 38, 91-99)

Briefly, the plate was previously run with chloroform, placed in the oven 105°C for 30 min and immediately placed into a dessicator to cool down to room temperature. The samples were spotted on the plate and resolved in hexane . The plates were removed and air dried for 15 min. Secondly, the plates were run in toluene until the solvent front reached the end of the plate. After the plates were air dried for 15 min a third phase containing hexane/ether/acetic acid (70:30:1), was run. The TLC plates were stained with sulfuric acid/ethanol (1:1) and charred at 150°C. Scanning densitometry on lipids were analyzed on digital pictures taken from the plate with a Molecular Dynamics PhosphorImager with the ImageQuant program.

GC-MS Analysis

For the fatty acid determinations 2 ml of chloroform-methanol- water (60:30:4.5) extract was evaporated and dissolved in 2 ml of 2.5 % sulfuric acid in methanol. Acidic methanolysis was performed at 80°C for 16 hours. Formed fatty acid methyl esters were purified by preparative TLC in dichloromethane . Gas chromatographic-mass spectrometrical analysis was performed on a Fison MD 800 equipment with on-column injection. A DB 1 (J&W) capillary column 30mx.32 mm was used for the separation.

Preparations of Microsomes and Fatty Acid Elongation Assay About 100 mg liver tissue pieces was dissected and homogenized in 4 ml of ice-cold 0.25 M sucrose. Following a 30 min stepwise centrifugation (10 min at each 700 g, 8000 g and 17000 g) at 4 to 10°C, the supernatant was carefully transferred to fresh tubes and microsomes were sedimented at 105000 g for 45 min. The pellet was resuspended in 20 mM Tris- HCl, pH 7.4, containing 0.4 M KCl , and centrifuged at 105000 g for 45 min. The final microsomal pellet was resuspended in 200 μl of 0.1 M Tris-HCl, pH 7.4, and the protein concentration was determined according to the Bradford method.

Total fatty acid elongation activity was measured essentially according to [Suneja, S. K. , et al (1991) J. Neurochem . 57, 140-146] . The assay mixtures (0.5 ml total, including a 25 μg protein addition) contained 0.1 M Tris-HCl, pH 7.4; either 50 μM palmitoyl-CoA, 15 μM arachidoyl-CoA or 15 μM lignoceroyl- CoA; substrate/BSA ratio of 2:1; 1 mM NADPH; and 50 μM malonyl-CoA (containing 0.050 μCi of [2-¹C] malonyl-CoA) . The reaction was carried out for 17 min at 37°C and terminated by addition of 0.5 ml of 15% KOH in methanol and saponified at 65°C for 45 min. Then the samples were cooled and acidified with 0.5 ml of cold 5 M HC1. Free fatty acid were extracted from the mixture three times with 3 ml of n-hexane and dried under vacuum. The extract was dissolved in 1 ml of chloroform and measured after addition of 10 ml of scintillation mixture in a Beckman liquid scintillation system 3801.

Isolation of Lipid Producing Cells

Lipid producing cells are isolated as follows;

Skin containing the sebaceous, meibomian, preputial, perianal, clitoral or ceruminous gland is surgically removed from a mammal. The tissue is cut into small pieces and treated with dispase in dissociation buffer (such as PBS or Hanks' or Cell culture medium) or other enzymes widely used for dissociation of tissues such as collagenase or trypsin. The tissue is kept in the dissociation solution (usually 5 min - 4 hours in 37°C or in 4°C overnight) until sebaceous glands can be removed from the epidermis. The sebaceous glands are further treated with the dissociation solution with gentle agitation until cells are detached from each other.

The cells are centrifuged at 700-1000 rpm for 2 -10 min. After removing the dissociation solution, the cells are re-suspended in standard cell culture medium (preferentially MCDB-104, 131, 153 or keratinocyte-SFM or DMEM, supplemented with 10% newborn calf serum for inactivation of dissociation enzymes) .

The cells are kept in the medium supplemented with 10% newborn calf serum for a period of time (for example, 10 minutes) and then centrifuged again. After removing the medium, the cell pellet is re-suspended in medium (see above) supplemented with 0.1 ng/ml Epidermal Growth Factor, 5 μg/ml insulin, 5 μg/ml hydrocortisone or dexamethasone, 0.4 % Bovine Pituitary Extract, Leukemia inhibitory factor (LIF) , antibiotics and antimycotics .

The primary cultured cells are grown for a number of days until confluence.

The medium is then changed to standard serum free medium supplemented with 5 μg/ml insulin, antibiotics and antimycotics .

Cells may be kept in this medium for some time and used in assay methods as described herein. Such methods may be performed, for example, by adding test-substances to the medium and measuring expression of Elovl3 (gene expression or the protein) and the ability of these cells to form lipids (TLC, GC and MS) .

Cultures may be enriched for Elovl3 expressing cells by clonal expansion i.e. passage and dilute the cultured cells into several culture flasks and screened for Elovl3 expression upon administration of test-substances .

Cells are sub-cultured before full confluence (50-80%) . The medium is then changed to standard serum free medium supplemented with 5 μg/ml insulin, antibiotics and antimycotics. Over a period of time, one or more adrenergic and/or androgenic agonists are added to produce a culture of a more homogenous population of Elovl3 expressing cells. Suitable agonists include vitamins, especially vitamin D and vitamin A, Fetal calf serum, Newborne calf serum, transferrin, IGF,TGF, Cytosine b-D-arabinofuranoside, EGF, insulin, glucocorticoids such as dexamethasone, hydrocortisone, LIF, PPARα ( 'phytanic acid-like') ligands and bovine pituitary extract.

This is accomplished by adding an agonist as described to the medium (which affect proliferation and differentiation both positive and negative) and measuring expression of Elovl3 (gene expression or the protein) and the ability of these cells to form specific lipids (for example, using one or more of TLC, GC, HPLC and MS) .

Alternatively, a homogeneous population may be obtained by identifying a single Elovl3 expressing cell and sub-cultured through several passages to expand (proliferate) the homogeneous population.

Cell Lines and Primary Cultures of Brown Adipocytes Brown fat precursor cells were isolated from 3 - -week-old mice. The cells were seeded into six-well dishes and cultured in medium consisting of DMEM with physiological levels of glucose (5 mM, ICN) , supplemented with 4 M glutamine, 10% newborn calf serum, 4 nM insulin, 10 mM HEPES, and with 50 IU/ml penicillin, 50 μg/ml streptomycin and 25 μg/ml sodium ascorbate. The cells were grown at 37°C in a humidified atmosphere of 8% C02 in air for 7-9 days, with medium exchange on day 1, 3 and 6. Upon confluence (day 6), the cells were chronically treated for 1-3 days with 0.1 μM norepinephrine, 1 μM dexamethasone or a combination of both. Control cells were left untreated. Dexamethasone was added in a single dose, freshly prepared norepinephrine was added every 12 hours . The day before harvesting, the cells were exposed to 200 mM phytanic acid for 16 hours. RESULTS

Generation of Eloyl3 Deficient Mice

A Sail fragment including the transcription start site, exon 1 and 2 of the Elovl3 gene was deleted by homologous recombination in RI ES cells. Targeted ES cells, confirmed by Southern analysis, were microinjected into blastocysts of C57B1/6J (B6) mice. Two independent chimeric lines were transmitting the Elovl3 disruption through the germline which was confirmed by Southern blot analysis. The 5.6 kb Seal genomic fragment representing the wild-type Elovl3 allele is absent in Elovl3 -ablated mice and is replaced by a 11.6 kb fragment that hybridizes to probe 1.

In order to determine the Elovl3 expression Northern blot analysis were performed with total RNA isolated from brown adipose tissue, skin and liver, i.e. organs which have been shown to express Elovl3 [Tvrdik, 1997 supra] , from wild-type and mutant mice. The gene disruption was especially clear upon cold stimulation, where no Elovl3 mRNA was detected in BAT from mutant mice, a site with the highest reported Elovl3 expression [Tvrdik, 1997 supra] .

Phenotype characteristics

The striking feature that distinguished Elovl3 -deficient mice from wild-type or heterozygous littermates was a tousled and a marked reduced fur content over the whole body which is noticeable at about two weeks of age when the hair is formed.

This is a phenotypic distinction which is sustained throughout the life span of these animals. In addition, mice older than approximately six months showed irritated skin and distinct scratch marks on the neck which resembles disorders such as atopic dermatitis or atopic eczema. Another distinct phenotype of several Elovl3 -deficient mice is an impaired ability in opening and closing their eyelids which is most pronounced during their first months of life. The phenomena resembles disorders such as atopic cataract or blepharitis, which is often linked to atopic eczema (Driver, P. J., and Lemp, M. A. (1996) Surv. Ophthalmol . 40, 343-367) and might be associated with a dysfunction in the meibomian gland to lubricate their eyes. However, no sign of char or soared eye lens was visible.

Offspring genotypes obtained from heterozygous FI intercrosses showed normal mendelian distribution. Within litters from heterozygous pairs, Elovl3 -/- offspring showed a 28% decrease in weight upon weaning (day 23 - 34) compared to wild-type or heterozygous littermates (not shown) . However, this discrepancy is not found when Elovl3 -/- litters are born and raised separately from normal mice which may suggest that within a heterogeneous litter a competitive food intake is in favor for the wild-type mice.

Microscopic analysis of the skin in Eloyl3 deficient mice There were striking differences in the skin between the Elovl3 -ablated mice and the wild type mouse. Grossly, the epidermis of the Elovl3 deficient mice seems normal, but parts of the epidermis is thicker than normal with observed hypergranulation. Three to four cell-layers is occasionally detected in the mutant mice. However, most evident finding is a general hyperplasia of the pilosebaceous system seen in the mutant mice. In the Elovl3 knock out mice both the sebaceous glands and the hair follicles are localized deep down in the subcutaneous fat. The sebaceous glands and the meibomian glands are enlarged and increased in numbers . The hair follicles are also increased in numbers and enlarged and, as in the epidermis, scattered hypergranulation is seen. The nuclei of the hair follicles cells seem irregularly shaped especially cells in the inner layer of the outer root sheath. In the majority of hair follicles normal hair is missing in the upper part, though, the hair seems normal in the more lower parts. The dermis seems thinner than normal consisting of loose collagen fibers.

Cell-specific expression of Eloyl3 in the skin The Elovl3 expression was found to be increased in wild-type mouse skin during the first weeks of life and reached a steady state level around three weeks of age.

In-situ hybridisation analysis of the skin of the wild type mouse showed a strong Elovl3 mRNA signal in the sebocytes in the sebaceous glands and in the epithelial cells of the hair follicles.

In the hair follicle, there is a distinct signal in the matrix cells but the strongest Elovl3 mRNA signal is specifically seen in the cells in the inner layer of the outer root sheath. In the normal epidermis the Elovl3 mRNA signal is very low and in the fibroblasts no detectable signal is seen. In the Elovl3 -ablated mouse there is no detectable mRNA signal in any cell type.

Defective water repulsion and thermoregulation in Eloyl3 deficient mice

In order to investigate the relevance of Elovl3 for normal function of the skin, we performed a water repulsion experiment in which the animals were allowed to swim at 30°C for 2 minutes .

Elovl3-ablated mice needed more time for their fur to dry than their wild-type counterparts: more than 60 min versus 15 min respectively (Figure 1) . This was due to an impaired ability in water repulsion in the mutant mice since they absorbed three times as much water as wild-type mice (Figure 1) , about 14% of their body mass, compared to 4% for the wild-type. However, the rate of water evaporating from the skin during the following 40 minutes of the test proved to be equal between the Elovl3 -ablated and wild-type mice (Figure 1) . Removal of lipids by a 5% SDS wash increased water absorption up to 10% of body weight in sacrificed wild-type mice. Further, washing Elovl3 -ablated mice with SDS did not increase their ability to absorb water.

Surprisingly, although Elovl3 mRNA level has been shown to be increased about hundred fold in brown adipose tissue in cold exposed wild-type mice [Tvrdik et al, (1997) supra] , the

Elovl3 -ablated mice did not show impaired brown fat activity, (i.e. loss of body temperature), even when exposed to cold for several months .

In order to see if the impaired water repulsing in the Elovl3- ablated mice also lead to impaired thermoregulation, body temperature was measured in wild-type and knockout mice after a two minutes swim in 30°C. The Elovl3 -ablated and wild-type animals had the same core body temperature at an ambient temperature (22°C) before the swim (Figure 2) . After the swim, wild-type mice experienced a drop in core temperature to a lowest value of 35°C after 5 minutes. The core body temperature returned to baseline value after approximately 20 minutes which also coincided with the return of a completely dry fur (Figure 2) . On the contrary, the core temperature in Elovl3-ablated mice decreased to 27°C and remained at that temperature for at least one hour which also correlated with a wet fur. So, even if the Elovl3-ablated mice can sustain a 4°C cold-exposure, they were unable to keep up the body temperature with a wet fur, even at room temperature.

Increased Trans-Epidermal Water Loss in Eloyl3 -ablated mice The diffusion of water through the skin was quantified by a TEWL test. As shown in Figure 3 the Elovl3 -ablated mice had a 70% higher rate of evaporation compared to wild-type mice, indicating a higher water loss due to skin barrier impairment .

Disturbed lipid content in the hair of Eloyl3 -ablated mice

Hair lipids were analysed by thin layer chromatography (TLC) . The total amount of acetone-extractable lipids did not significantly differ between the wild-type and the Elovl3- ablated mice (Figure 4) . However, the triglycerides and the two most mobile fractions, which were a mixture of wax esters, diol esters and sterol- and wax esterss, formed a different pattern in the two strains of mice (Figure 5) .

The most obvious difference was a shift in the amount of specific lipid components in the ablated mice in the area were the triglycerides localized. Densitometric analysis displayed a tenfold decrease in a relatively more hydrophilic component and a corresponding tenfold increase in a more hydrophobic component. In addition, a nine-fold increase in lipid compounds which resided in the vicinity of wax and diol esters was measured and a fourfold increase of lipid compounds which resided within the area of sterol- and wax esterss.

An increase in a specific lipid fraction may have a corresponding band found in normal mice in very low amounts, or whether may be new type of lipid compound specific for the knockout mice. In general, Elovl3 -ablated animals seem to accumulate more hydrophobic esters than normal mice due to a shift in polarity, i.e. a lower Rf value. Mass-Spectrometrical Analysis of Hair Lipids

The ELOVL3 protein may be involved in the formation of fatty acyl chains containing up to 24 carbon atoms. When we compared the acyl chain content in acetone extracted lipids from hair from wild type and Elovl3 -ablated mice by mass-spectrometrical analysis, there was a remarkable increase of 20:1 lipids in the Elovl3-ablated mice. The number increased from about 15% to about 50% of total lipids. On the contrary, there was about a 40% decrease in saturated and monounsaturated lipids with 16 and 18 carbon chain lengths. This may explain the dramatic shift in mobility of specific lipid compounds observed in the TLC experiment. However, no significant reduction of specific acyl chains longer than 20 carbon atoms was observed in the Elovl3 -ablated mice compared with wild-type mice, but there is a possibility -that ELOVL3 controls the further elongation of 20:1 into other acyl chains which were not detected in the present system (Fig 5) .

Decreased long-chain fatty acyl CoA elongation activity in liver from Eloyl3 -ablated mice

Synthesis of VLCFA occurs in the ER (Dickson, R. C, and Lester, R. L. (1999) Biochim. Biophys . Acta 1426, 347-357), so the ability of microsomes from wild-type and Elovl3-ablated mice to elongate fatty acyl -CoAs of different length was investigated. No significant elongation activity could be detected in skin microsomes isolated from either mutant or wildtype mice.

Liver has been shown to have significant Elovl3 expression [Tvrdik, 1997 supra] and was used as a microsomal source.

As seen in Figure 6, diminished chain elongation with palmitoyl CoA (C16:0 CoA) and arachidoyl CoA (C20:0 CoA) as substrates was observed in Elovl3-ablated mice relative to wild-type mice. The relative activity of palmitoyl CoA chain elongation in liver microsomes from ablated mice was decreased by 39% compared with wild-type microsomes. A 73% reduction in fatty acid acid elongation was seen in the Elovl3 -ablated microsomes with arachidoyl CoA as substrate compared to wild- type microsomes. No difference was seen with lignoceroyl CoA as substrate. The microsomal activity for elongating lignoceroyl CoA was 2% of the activity seen with palmitoyl CoA as substrate in normal mice, compared with 17% for arachidoyl CoA compared with palmitoyl CoA. This makes the measurements of elongation activities of fatty acids longer than C24 difficult to interpret.

These results are in line with the suggested function of the Elovl3 protein, which is to participate in the formation of fatty acids with 24 carbon atoms [Tvrdik, 2000 supra] .

Isolation of Eloyl3 expressing Cells The in si tu hybridisation analysis described above indicated that Elovl3 is expressed by a particular cell type within the serbaceous gland and hair follicle.

These cells were isolated as described above for use in assay methods of the present invention.

Lipid Profiling

Lipid profiles were obtained for extracts of sebum and hair lipid from Elovl3 (-/-) and wild type mice.

The triglyceride and sterol-ester profiles are shown in table 1. Levels of 20:1 sterol- and wax- esters and triglycerides were dramatically increased in Elovl3 deficient mutants relative to the wild type, while levels of 18:1 were decreased in the Elovl3 deficient mutants.

The ratio of 20:1 to 18:1 in the wild type mice was observed to be about 3.1 for the triglyceride fraction and about 0.1 for the sterol- and wax-esters fraction and in the Elovl3 (-/- ) mice to be about 9.1 for the triglyceride fraction and about 1.5 for the sterol- and wax-esters fraction.

DISCUSSION

A novel mechanism involving the Elovl3 gene product in the formation of hair follicles and the function of skin barrier is described herein.

The hair phenotype of Elovl3 deficient mice was explained by the absence of Elovl3 expression in the inner cell layer of the outer root sheath. These cells lose their normal function in the mutant mice. Abnormal cells like those found in the hair follicles of the Elovl3 deficient mice are seen in human skin tumors or during the aging process in adults.

Cells in the outer root sheath normally express an array of keratins, adhesion molecules, cytokines, and growth factor receptors that are distinct from those expressed by epidermal cells (Paus, R., and Cotsarelis, G. (1999) N. Engl . J. Med. 341, 491-497) These factors migrate out of the follicle and regenerate the epidermis e.g. after injury or loss.

However, in hyperproliterative states, such as psoriasis, and during wound healing, epidermal cells produce specific keratins which are normally found in the outer root sheath of hair follicles. The marked increase in TEWL of the Elovl3- ablated mice indicates that the stratum corneum does not provide a normal barrier in these animals . The increased thickness of the epidermis and the hypergranulation indicate a more general deficiency of the epidermal layer including hyperkeratosis and scaling rather than a thin stratum corneum.

Apart from in the hair follicles, a significant amount of Elovl3 mRNA was found in the sebaceous glands . From the TLC experiment it was clear that the Elovl3 -ablated mice had reduced levels of specific ester components in their sebum. However, there was also a dramatic increase of specific ester components in these mice. The fact that the sebaceous glands were hyperplastic in these mice, may suggest a compensatory mechanism which fits with the high amount of 20:1 found in the hair lipids of these mice. The lower amounts of 16:0, 16:1, 18:0 and 18:1 provide indication that these acyl chains are consumed on behalf of increased 20:1 production as they are a substrate for this synthetic pathway. In particular, the ratio of 20:1 to 18:1, for example in the total lipid extract or sterol- and wax-ester fraction thereof, is indicative of Elovl3 activity. Inhibition or ablation of Elovl3 leads to a large increase in this ratio. For example, a ratio below 0.5 may be indicative of normal functioning and above 0.5 of aberrant functioning.

Surprisingly, although the Elovl3 -ablated mice had slightly more total lipids in their fur compared to normal mice it did not help the skin to repel water. Whether this is due to a deficiency in the coat texture or exclusively in the lipid composition of the sebum is still not known. Though, even if the Elovl3 -ablated mice have been shown to have the capability to induce heat production from the brown adipose tissue at 4°C in order to maintain their body temperature, the cold stress, experienced by being wet, was to detrimental for the mice. Since the meibomian glands in the Elovl3-ablated mice also were hyperplastic and the normal ocular barrier function of the eye was affected, it is apparent that all modified sebaceous glands - meibomian, preputial, perianal, clitoral and ceruminous glands - in these mice secrete a lipid composition which is different from normal mice.

VLCFA are fundamental membrane lipids as components of e.g. sphingolipids and ceramides which normally reduce the permeability of the skin. The deficiency in the barrier integrity of the stratum corneum of the Elovl3 ablated mice may be due to a direct effect of Elovl3 deficiency in epidermal cells or the disruption of the skin barrier may be indirect, due to e.g. deficient hair follicles or/and an injured epidermal layer because of imbalanced sebum.

No difference could be found in the fatty acyl chain profile (acyl chains with more than 16 carbons) between the sphingolipid fraction and the glycerolipid fraction in both the Elovl3 -ablated and the wild-type mice, which provides indication of a relatively similar distribution of VLCFA within both of these acyl chain depots. Other fractions such as the triglyceride fraction and sterol- and wax- ester fraction show differences in the fatty acid profile, in particular the 20:1/18:1 ratio.

However, the Elovl3 expression study showed that there was almost zero amount of detectable Elovl3 mRNA in the epidermal layer in normal mice, supporting an indirect effect of the epidermis in the knockout mice.

Previous studies on the expression of the Elovl3 gene have indicated that chronic exposure of glucocorticoids is needed for proper expression [Tvrdik, 1997 supra] . In view of glucocorticosteroids as important regulators of hair growth and skin formation in general (Stenn, K. S.et al . (1993) Skin . Pharmacol . 6, 125-134), Elovl3 may be under the control of several factors which regulate skin development under the influence of glucocorticoids.

The present application describes, for the first time, the role of Elovl3 in producing specific skin lipids and eye lubrication lipids essential for the barrier function of the skin and eye (e.g. sebum and ocular lubricant) . Furthermore, specific cells have been identified in the hair follicle, sebaceous gland and meibomian gland which express the protein Elovl3 and which are involved in the synthesis of these lipids. These cells have never been previously identified or isolated and are distinct from other cells in these tissues.

The isolation of these cells provides both approaches to therapy and assay methods for substances which modulate the formation of barrier lipids and are therefore also useful in therapy.

Table 1

Claims

Claims :

1. An isolated cell which secretes esterified or free very long chain fatty acids (VLCFAs) obtainable by a method comprising; providing a population of cells of a hair follicle and/or a sebaceous-related gland, identifying one or more cells within said population which expresses Elovl3, and; separating said one or more cells from said population.

2. An isolated cell according to claim 1 wherein said population of cells is provided by dissociating tissue of a hair follicle and/or a sebaceous-related gland into a population of constituent cells.

3. An isolated cell according to claim 1 or claim 2 wherein the sebaceous-related gland is selected from the group consisting of a sebaceous, meibomian, preputial, perianal, clitoral or ceruminous gland.

4. An isolated cell according to any one of the preceding claims wherein said one or more cells which express Elovl3 are sebocytes from a sebaceous-related gland or epithelial cells of the hair follicle.

5. An isolated cell according to any one of the preceding claims wherein the one or more cells which express Elovl3 are cells of the inner layer of the outer root of the hair follicle.

6. An isolated cell according to any one of the preceding claims wherein said method further comprises determining the lipid production of said one or more cells.

7. An isolated cell according to any one of the preceding claims wherein said method further comprises culturing and//or clonally expanding said one or more cells.

8. A composition comprising an isolated cell according to any one of the preceding claims and a pharmaceutically acceptable excipient.

9. A composition according to claim 8 for use in a method of treatment of the human or animal body.

10. A composition according to claim 9 for use in the treatment of a disease associated with aberrant barrier lipid function.

11. Use of an isolated cell according to any one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of a disease associated with aberrant barrier lipid function.

12. A method of making a pharmaceutical composition comprising admixing a cell according to any one of claims 1 to 7 with a pharmaceutically acceptable excipient.

13. A method for obtaining an isolated cell which secretes esterified or free VLCFAs comprising; providing a population of cells of a hair follicle and/or a sebaceous-related gland, identifying one or more cells within said population which expresses Elovl3, and; separating said one or more cells from said population.

14. A method according to claim 13 wherein said population of cells is provided by dissociating tissue of a hair follicle and/or a sebaceous-related gland into a population of constituent cells.

15. A method according to claim 13 or claim 14 wherein the sebaceous-related gland is selected from the group consisting of a sebaceous, meibomian, preputial, perianal, clitoral or ceruminous gland.

16. A method according to any one of claims 13 to 15 wherein said one or more cells which express Elovl3 are sebocytes from a sebaceous-related gland or epithelial cells of the hair follicle.

17. A method according to any one of claims 13 to 16 wherein the one or more cells which express Elovl3 are cells of the inner layer of the outer root of the hair follicle.

18. A method according to any one of claims 13 to 17 wherein further comprising determining the lipid production of said one or more cells.

19. A method according to any one of claims 13 to 18 wherein further comprising culturing and/or clonally expanding said one or more cells.

20. A method for obtaining a compound which modulates formation and/or properties of a barrier lipid mixture which comprises:

(a) contacted one or more isolated cells according to any one of claims 1-7 with a test compound; (b) determining the presence or absence of an effect on the one or more isolated cells.

21. A method according to claim 20 comprising determining the presence or absence of an effect on the expression and/or activity of Elovl3.

22. A method according to claim 20 comprising determining the esterified or free VLCFAs formed in the one or more cells.

23. A method according to claim 22 wherein the ratio of 20:1 to 18:1 fatty acids is determined.

24. A method according to any one of claims 20 to 23 comprising identifying said test compound as a modulator of formation and/or properties of a barrier lipid mixture.

25. A method according to claim 24 comprising isolating and/or purifying said test compound.

26. A method according to claim 25 comprising formulating said test compound with a pharmaceutically acceptable excipient .

27. A modulator of formation and/or properties of a barrier lipid mixture obtained by a method according to any one of claims 20-25.

28. A pharmaceutical composition comprising a modulator according to claim 27 and a pharmaceutically acceptable excipient .

29. Use of a modulator according to claim 27 in the manufacture of a medicament for treatment of a condition associated with aberrant barrier lipid function

30. A method of treatment of a condition associated with aberrant barrier lipid function comprising administering a composition according to claim 9 or claim 28 to a patient in need thereof .