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WO1997017982A1 - Procedes et compositions accelerant la pousse des poils - Google Patents

Procedes et compositions accelerant la pousse des poils Download PDF

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Publication number
WO1997017982A1
WO1997017982A1 PCT/US1996/017928 US9617928W WO9717982A1 WO 1997017982 A1 WO1997017982 A1 WO 1997017982A1 US 9617928 W US9617928 W US 9617928W WO 9717982 A1 WO9717982 A1 WO 9717982A1
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Prior art keywords
lef
gene
composition
hair growth
hair
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PCT/US1996/017928
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English (en)
Inventor
Rudolf Grosschedl
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The Regents Of The University Of California
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Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU76729/96A priority Critical patent/AU7672996A/en
Publication of WO1997017982A1 publication Critical patent/WO1997017982A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present invention relates generally to methods and compositions for promoting hair growth and, more particularly, to compositions and methods which restore or enhance the activity of LEF-1 and proteins regulated by LEF-1 in hair follicles.
  • Alopecia can result from a variety of causes, including genetic factors, aging, local and systemic diseases, and trauma.
  • the most common form of alopecia is referred to as "male-pattern baldness," an apparently genetic condition which is characterized by increasing hair loss with advancing age .
  • male-pattern baldness an apparently genetic condition which is characterized by increasing hair loss with advancing age .
  • the most effective therapy is transplantation of hair follicles from hairy to bald areas of the skin.
  • minoxidil anti- hypertensive drug, has been approved for the treatment of male-pattern baldness, but significant hair growth as the result of topical application of minoxidil is very rare.
  • compositions incorporating active agent (s) which are effective when topically applied to the skin in a region where hair growth is desired. It would be even more desirable to provide such methods and compositions which are effective in treating male-pattern baldness as well as other common forms of alopecia.
  • compositions comprising nucleic acids.
  • WO 94/22468 describes liposomal vehicles for delivery genes and other substances to hair follicles.
  • Lymphoid enhancer factor 1 (LEF-1) and its biological activities are described in a number of publications, including Giese et al . (1995) GENES & DEV.
  • LEF-1 expression has been associated with the growth of hair. Van Genderen et al . (1994) GENES & DEV.
  • Van Genderen et al . (1994) Data presented in van Genderen et al . (1994) form part of the present application. Van Genderen et al . (1994) was published less than one year prior to the filing date of the present application.
  • the present invention is based at least in part on the discovery that expression of lymphoid enhancer factor-1 (LEF-1) is essential for the development of hair follicles and the growth of hair from hair follicles.
  • the method of the present invention thus comprises restoring or enhancing the activity of LEF-1 in hair follicles in the epidermis (skin) of patients suffering from alopecia.
  • a composition comprising LEF-1 and/or a biologically equivalent substance is provided.
  • the composition is then applied to a region of skin, typically the scalp, where hair growth promotion is desired.
  • LEF-1 may be in the form of the full length LEF-1 protein, or an active portion thereof, typically retaining at least the HMG box of the protein, more typically being in recombinant form.
  • the LEF-1 substance may be an analog, mimetic, or other molecule which displays a similar or identical activity to LEF-1 in promoting hair follicle generation and hair growth therefrom.
  • LEF-1 activity is restored or enhanced by providing a composition comprising an LEF-1 gene in a topical carrier.
  • the composition is then applied to the skin where hair growth promotion is desired.
  • the LEF-1 gene will typically encode the full length LEF-1 gene product or an active portion thereof, and will usually be delivered as part of a liposomal or retroviral vector.
  • the gene will usually be provided in reading frame with homologous or heterologous regulatory control sequences.
  • compositions according to the present invention comprise an LEF-1 polypeptide or LEF-1 gene in a topical carrier.
  • EF-1 polypeptides either the full length LEF-1 protein or a fragment, analog, or mimetic thereof may be employed.
  • the gene will usually be present in a liposomal or retroviral vector, and the gene will generally be in reading frame with homologous or heterologous regulatory control sequences.
  • the LEF-1 polypeptide and/or gene may be derived from or based on the reported sequences of murine, human or other mammalian sources. Specifically, it has been found that the murine form of LEF-1 is active in human cells. BRIEF DESCRIPTION OF THE DRAWINGS
  • Fig. 1 shows the pattern of expression of LEF-1 during mouse development.
  • Sites of abundant LEF-1 expression include mesencephalon (me) , diencephalon (d) , inner ear (ie) , tongue (t) , lower and upper lips (11, ul) , nasal process (np) .
  • Sites of LEF-1 expression include pituitary gland (p) , tooth buds (tb) , and the kidney (k) ,
  • FIG. 1 depicts the scheme of a targeted disruption of the murine LEF-1 locus.
  • A The LEF-1 cDNA with the HMG domain shown as a solid box is indicated at the top. The line below represents the wild-type LEF-1 locus in the region of the HMG domain.
  • the targeting vector includes a PGK-neo r gene, inserted into the Smal site residing in the 3' exon of the HMG domain, and a tk gene.
  • Transcriptional polarity of the neo r and tk genes are indicated by arrows.
  • pBR vector sequences are represented by a shaded box.
  • Sites for restriction enzymes are indicated above the lines, and the length of the fragments generated by a BamHI-EcoRI digest that hybridize with a genomic flanking probe are indicated below.
  • B DNA blot analysis of genomic DNA from a representative litter generated by the mating of heterozygous mutant LEF-1-deficient mice. DNA was digested with BamHl-EcoRI and probed with a fragment shown in A. The 12.9- and 4.0-kb DNA fragments are generated from the wild-type and mutant LEF-1 allele, respectively.
  • Fig. 3 shows a phenotype of LEF-1-deficient mouse.
  • the homozygous mutant mouse (left) at day 16 after birth lacks whiskers and body hair. Moreover, the snout has a pointed appearance and the animal is significantly smaller compared to a wild-type sibling mouse (right) .
  • Fig. 4 shows skin from wild-type (+/+) and mutant (-/-) postnatal day 3 mice.
  • A, B Paraffin sections through the skin in the head of a wild-type (A) and mutant (B) animal stained with hematoxylin-eosin. The skin from the mutant is considerably thinner, lacks dermal fat, and has fewer hair follicles. Bar, 200 ⁇ m.
  • hair and phrase “hair growth” refer to the emergence of keratinized cells from hair follicles.
  • Hair follicles are tubular epidermal structures that penetrate into the dermis and have a widened portion (the bulb) at their lower ends. Cell production within the matrix of the hair follicle results in the growth of a hair shaft which emerges from the skin surface.
  • alopecia is a clinical condition characterized by the partial or complete loss of hair from the patient. It may result from heredity, aging, local or systemic disease, or trauma.
  • the most common form of alopecia to be treated by the present invention is male-pattern baldness which is generally a hereditary condition.
  • Other forms of alopecia which may be treated include toxic and traumatic forms of alopecia.
  • the present invention will be particularly useful for treating alopecia occurring on the scalp, but in some cases may also find use with other regions of the dermis.
  • the phrase "lymphoid enhancer-binding factor 1" and designation “LEF-1” refer to a mammalian cell type- specific transcription factor that is expressed in lymphocytes and sites of organogenesis and responsible for a variety of activities.
  • LEF-1 is expressed in the epidermis and in hair follicles and is responsible for the embryogenic emergence of hair follicles.
  • LEF-1 is a member of the family of high mobility group (HMG) proteins, which have the capacity to induce a sharp bend in the DNA helix and to activate transcription only in collaboration with other DNA- binding proteins.
  • HMG high mobility group
  • the cloning of the murine LEF-1 gene and full gene sequence are provided in Travis et al . (1991) , supra .
  • the murine gene sequence together with corresponding amino acid sequence are also set forth in SEQ ID. No. 1 and SEQ ID. No. 2, respectively.
  • LEF-1 protein and various amino-terminal truncations thereof are described in Giese and Grosschedl (1993) , supra..
  • the methods and compositions of the present invention can utilize either the murine, human, or other mammalian LEF-1 gene and proteins.
  • the sequence, cloning, and expression of human LEF-1 are described in Waterman et al . (1991) GENES & DEV. 5:656-669.
  • compositions according to the present invention which incorporate the LEF-l gene or a portion thereof may be prepared as follows.
  • a DNA segment is prepared which encodes an amino acid sequence corresponding entirely or partially to at least an active fragment of the LEF-l gene product as set forth in SEQ ID No. 2.
  • the amino acid sequence will comprise at least the HMG box, and more usually comprise the entire wild-type protein. It will be appreciated, however, that variations of one or several amino acids, and in some cases deletions of relatively large inactive portions of the protein, may readily be made without significantly altering the biological activity of the protein expressed by the DNA sequence.
  • the DNA segment will have a sequence which corresponds to the nucleotide sequence set forth in SEQ ID No. 1, but this is not necessary because of the redundant nature of the genetic code. That is, a variety of different codons which code for the same amino acids may be substituted for those found in the wild-type LEF-l gene.
  • the DNA segment which encodes the LEF-l protein or fragment thereof will usually be combined with homologous or heterologous regulatory control sequences which enable expression of the gene after transduction into the host cells of the patient.
  • the recombinant DNA segments incorporating both the LEF-l coding sequence and the homologous or heterologous regulatory control sequences will then be incorporated into a suitable delivery vehicle for transduction of the host cells by topical application.
  • Useful transduction vehicles include both liposomal vehicles and retroviral vectors, both of which are well described in the patent and scientific literature. Suitable liposomal vehicles are described in, for example, U.S. Patent Nos.
  • compositions incorporating LEF-l polypeptides according to the present invention may be prepared as follows.
  • the LEF-l polypeptides may comprise full length LEF-l proteins or active fragments, analogs, or mimetics thereof.
  • the LEF-l polypeptides will be recombinantly produced based on the amino acid sequence set forth in SEQ ID No. 2, using recombinant protein production techniques that are now widely described in the technical and scientific literature. See, for example, MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al . , Eds . , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), vols. 1-3.
  • Such recombinantly produced LEF-l polypeptides will be purified to a high degree of purity using conventional protein separation techniques. Typically, a purity of at least about 90% by weight will be obtained, preferably at least about 95% by weight, and more preferably at least about 99% by weight.
  • Both the LEF-l-gene-containing and LEF-1- protein-containing compositions will usually be incorporated in a carrier suitable for topical application to the skin, more typically the scalp.
  • a carrier suitable for topical application to the skin may be prepared by well known techniques, such as those described in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Philadelphia.
  • the topical carriers may be in the form of creams, oils, ointments, gels, pastes, liquids, powders, sprays, transdermal patches, and other vehicle forms suitable for application to the skin.
  • the particular vehicles should be selected so that they do not substantially interfere with the hair growth promotion activity of the polypeptide or gene, and so that they do not cause undue side effects when administered to the patient .
  • the dosage or concentration of the polypeptide or gene delivery vehicle in the topical composition may vary widely.
  • concentration of polypeptide in the topical carrier will typically be in the range from about 0.01 weight percent to 10 weight percent, typically being about 0.1 weight percent to 1 weight percent.
  • the liposomal and/or retroviral vehicles will be present in the topical carrier usually from about 0.1 weight percent to 1 weight percent.
  • Topical application of the compositions of the present invention to the patient's skin will result in stimulation of the emergence of hair follicles in the skin and/or stimulation of the emergence of hair from the hair follicles.
  • LEF-l gene will be incorporated into either or both of the epithelium and subadjacent mesenchyme, where gene expression will result in stimulation of hair follicle formation.
  • direct application of the LEF-l polypeptide is expected to enhance hair follicle production and/or hair growth.
  • the following experimental data demonstrate the role of LEF-l in hair growth.
  • TNN trigeminal nerve
  • LEF-l between E12.5 and E16.5 by in si tu hybridization of embryo sections with an 35 S- labeled LEF-l anti-sense RNA probe.
  • LEF-l in a sagittal section of an E12.5 embryo, abundant expression of LEF-l was detected in the mesencephalon, in the mesenchyme of the snout, and at multiple sites of organo-genesis including the ear and dental placodes (Fig. IA) .
  • Fig. IB major sites of LEF-l gene expression were identified in the mesencephalon, tooth germs, whisker follicles, pituitary gland, and kidney.
  • LEF-l gene expression is reduced markedly at E16.5, but persists at high levels in the whisker follicles (Fig. IF) and thymus (data not shown) .
  • Previous in si tu hybridization studies at E10.5 identified abundant LEF-l transcripts in the presumptive dental epithelium (Oosterwegel et al . 1993) .
  • LEF-l in the cap stage of tooth development at E14.5, expression of LEF-l could be detected in both dental epithelium and dental papilla mesenchyme (Fig. ID) .
  • the pattern of LEF-l expression in the developing tooth was also confirmed at the cellular level by immunohistochemistry with purified antibodies directed against LEF-l protein (Fig. IE) .
  • This spatial and temporal expression pattern of LEF-l coincides with inductive interactions between epithelial and mesenchymal cells during tooth development (Slavkin 1974; Thesleff and Hurmerinta 1981; Kollar 1983) .
  • heterologous and heterochronic tissue recombination studies have shown that the presumptive dental epithelium has potential to induce ectopic partner tissue to form a tooth until day 12 of embryogenesis, after which time this developmental potential shifts to the condensing mesenchyme (Kollar and Baird 1970; Mina and Kollar 1987) .
  • the LEF-neo-tk targeting construct was linearized and electroporated into D3 embryonic stem (ES) cells, and clones were selected doubly for the presence of the neo gene and the loss of the tk gene, Ramirez-Solis et al .
  • Clones were analyzed by digesting genomic DNA with BamHl and hybridizing DNA blots with a radiolabeled probe derived from genomic sequences 5' of the LEF-l gene in the targeting vector.
  • Accuracy of the recombination events was also confirmed by DNA blot analysis of Ba Hl and Ndel-digested D ⁇ A that confirmed the integrity of the 3' homologous arms and presence of a single neo integrant (data not shown) .
  • Targeted ES clones were injected into C57BL/6 blastocysts, and resulting chimeric mice with ES cell contribution exceeding 70% were crossed with C57BL/6 wild-type mice. Multiple chimeras from one clone transmitted the targeted allele through the germ line Crossing the LEF-l heterozygous mutant mice, which showed no obvious defects, generated 74 litters with 23% of the offspring homozygous for the mutation.
  • the genotypes of the offspring were determined by D ⁇ A blot analysis of genomic D ⁇ A digested with BamHi and EcoRI and hybridized with a flanking probe (Fig. 2B) .
  • mice had a drastically impaired viability with only 63% surviving the first week after birth and only 5% surviving the second week. No homozygous mutant survived to weaning, indicating that they carry a recessive mutation in an essential gene.
  • pre-B cell lines were derived from the bone marrow was derived of 10-day old wild-type (+/+) , heterozygous (+/-) , and homozygous mutant (-/-) mice by transformation with Abelson murine leukemia virus (MuLV) .
  • Nuclear extracts from the pre-B cell lines were prepared and assayed for the presence of LEF-l protein by immunoblot analysis (Fig. 2C) .
  • a mouse genomic library from 129/Sv mice was screened with an LEF-l cDNA probe encompassing sequences that encode the DNA-binding domain of LEF-l.
  • One phage was isolated that included the second exon of the HMG domain (nucleotides 1899-2119 of the LEF-l cDNA) .
  • An 8- kb genomic LEF-l fragment was excised with Sail and Xhol and subcloned into a Bluescript vector (Stratagene) that lacked the Smal ⁇ Xmal ) site.
  • the targeting vector was linearized with Clal. Electroporation of ES cells and selection of G418- and FIAU- (from Bristol-Myers Squibb) resistant colonies were performed as described by Ramirez-Solis et al . (1993) ED. P.M. WASSARMAN AND M.L. DEPAMPHILIS, 225:855-877, with the exception that G418-resistant primary embryonic fibroblasts were used as feeders.
  • genomic DNA was prepared as described by Ramirez-Solis et al. (1993) , supra . DNA blot analysis
  • Genomic DNA was isolated from the livers of E16 embryos and 20 ⁇ g of each sample was digested with EcoRI and Ba Hl. The fragments were separated on a 0.9% agarose gel and transferred to GeneScreen Plus (DuPont) .
  • the probe a 180-bp Sfcyl-Xhol fragment from the LEF-l locus, was labeled with ( ⁇ - 32 P)dCTP by random priming
  • Blots were washed to a final stringency of 0.1 x SSC, 0.1% SDS at 65°C, and exposed to film overnight.
  • Pre-B cell lines were generated from bone marrow by transformation with Abelson MuLV. Nuclear extract from 5xl0 7 cells of each pre-B cell line were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. The blot was incubated with a rabbit anti-murine LEF-l antibody, Travis et al . (1991) GENES & DEV. 5:880-894 and developed with an alkaline phosphatase- conjugated secondary antibody (Boehringer Mannheim) .
  • Embryos were fixed in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours at 4°C, immersed in 20% (wt/vol) sucrose/PBS for 24 hours at 4°C, frozen in powdered dry ice, and then stored at -80°C until cryostat sectioning. Serial sections of 14- to 16 ⁇ m thickness were thaw-mounted onto microscope slides (Superfrost Plus, Fisher) and stored at -80°C until use.
  • PBS phosphate-buffered saline
  • RNA expression vectors containing 0.36-kb protein-coding region of the LEF-l cDNA were used to generate 35 S- labeled sense and antisense RNA probes as described by Tecott et al . (1993) PROC. NATL. ACAD. SCI. 90:1430-1434. Embryo sections were hybridized, washed, and developed essentially as described by Tecott et al . (1993) .
  • Hybridizations were carried out at 52°C in 50% (vol/vol) formamide, 0.3 M NaCl, 20mM Tris-HCl (pH 8.0) , 5 mM EDTA, 1 X Denhardt's solution, 10 mM NaH 2 P0 4 , 10 mM dithiothreitol, 0.5 mg/ml of yeast tRNA, 10% (wt/vol) dextran sulfate.
  • mice For immunocytochemistry using antibodies against p75 LNGFR , brains of P9-P10 mice were dissected out from perfused animals and left in the same fixative overnight. Sagittal sections at 60 ⁇ m were obtained with a vibratome and collected in TBS [10 mM Tris (pH 7.5) , 150 mM NaCl] .
  • Sections were incubated in 3% hydrogen peroxide and 10% methanol, to quench endogenous peroxidase activity, blocked 2 hours in a TBS solution containing 0.4% Triton X-100, 1% glycine, 3% bovine serum albumin, and 10% normal goat serum, and left overnight at 4°C in the same blocking solution containing polyclonal antibodies against p75 LNGFR at l ⁇ g/ml .
  • the ABC method (ABC Elite kit, Vector Labs) was used for immunodetection.
  • MOLECULE TYPE DNA (genomic)
  • AAAGAAGCTC TAACGCGGAC GTCTGCAGCC CGGTGGCTCT TTATTGTTTA CTCTGAAGGA 420

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Abstract

L'invention se rapporte à des procédés et des compositions accélérant la pousse des poils et consistant à rétablir ou améliorer l'activité du facteur 1 lymphoïde-activateur dans les follicules des poils. Il est possible de rétablir cette activité en appliquant une composition comprenant LEF-1 présent dans un excipient en une dose suffisante pour rétablir ou accélérer la pousse des poils, cette composition étant appliquée sur une région de la peau d'un patient. Selon une autre variante, ces procédés peuvent consister à appliquer une composition comprenant la totalité ou une partie du gène du LEF-1 présent dans un vecteur approprié tel qu'un vecteur liposomique ou rétroviral.
PCT/US1996/017928 1995-11-13 1996-11-12 Procedes et compositions accelerant la pousse des poils WO1997017982A1 (fr)

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AU76729/96A AU7672996A (en) 1995-11-13 1996-11-12 Methods and compositions for hair growth promotion

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US55812995A 1995-11-13 1995-11-13
US08/558,129 1995-11-13

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WO1997017982A1 true WO1997017982A1 (fr) 1997-05-22

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WO2000031134A1 (fr) * 1998-11-20 2000-06-02 Arch Development Corporation Regulation de la morphogenese des follicules pileux a partir de beta-catenine
US6924141B2 (en) 2000-03-31 2005-08-02 The General Hospital Corporation Methods of modulating hair growth
US7476512B2 (en) 2004-02-27 2009-01-13 The General Hospital Corporation Methods of identifying dermal papilla cells

Non-Patent Citations (3)

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Title
EMBO J., 1993, Vol. 12, No. 12, GIESE et al., "LEF-1 Contains an Activation Domain That Stimulates Transcription Only in a Specific Context of Factor-Binding Sites", pages 4667-4676. *
GENES & DEVEL., 1994, Vol. 8, VAN GENDEREN et al., "Development of Several Organs That Require Inductive Epithelial-Mesenchymal Interactions is Impaired in LEF-1-Deficient Mice", pages 2691-2703. *
GENES & DEVEL., 1995, Vol. 9, ZHOU et al., "Lymphoid Enhancer Factor 1 Directs Hair Follicle Patterning and Epithelial Cell Fate", pages 700-713. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031134A1 (fr) * 1998-11-20 2000-06-02 Arch Development Corporation Regulation de la morphogenese des follicules pileux a partir de beta-catenine
US6924141B2 (en) 2000-03-31 2005-08-02 The General Hospital Corporation Methods of modulating hair growth
US7175842B2 (en) 2000-03-31 2007-02-13 The General Hospital Corporation Methods of modulating hair growth
US7985587B2 (en) 2000-03-31 2011-07-26 The General Hospital Corporation Methods of culturing dermal papilla cells
US7476512B2 (en) 2004-02-27 2009-01-13 The General Hospital Corporation Methods of identifying dermal papilla cells
US7981629B2 (en) 2004-02-27 2011-07-19 The General Hospital Corporation Methods of isolating dermal papilla cells

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