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WO2000031134A1 - Regulation de la morphogenese des follicules pileux a partir de beta-catenine - Google Patents

Regulation de la morphogenese des follicules pileux a partir de beta-catenine Download PDF

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WO2000031134A1
WO2000031134A1 PCT/US1999/027490 US9927490W WO0031134A1 WO 2000031134 A1 WO2000031134 A1 WO 2000031134A1 US 9927490 W US9927490 W US 9927490W WO 0031134 A1 WO0031134 A1 WO 0031134A1
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catenin
hair
expression
skin cell
skin
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WO2000031134A9 (fr
WO2000031134A8 (fr
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Uri Gat
Ramanuj Dasgupta
Linda Degenstein
Elaine Fuchs
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Arch Development Corp
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Arch Development Corp
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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
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • 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
    • 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
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering

Definitions

  • the present invention relates generally to the fields of the hair growth and morphogenesis. More particularly, it concerns the ability of ⁇ -catenin to induce the post natal de novo formation of hair follicles replete with sebaceous glands and dermal papilla.
  • Hair morphogenesis is dependent upon a series of mesenchymal-epithelial interactions in embryonic skin (Hardy, 1992).
  • the first signal is mesenchymal, instructing ectoderm to form a hair germ.
  • An ectodermal signal then alters associated mesenchyme to condense and form a dermal papilla, developed by the ectoderm.
  • a second dermal signal initiates proliferation and differentiation within the developing epithelial component to form the follicle and its sebaceous gland appendages.
  • the dermal papilla and upper portion of the follicle are permanent in postnatal life, but the remainder of the follicle undergoes cycles of growth (anagen), regression (catagen), and rest (telogen) (FIG. 1 ; Hardy, 1992). Each time a follicle regresses, the dermal papilla contracts upward, coming into transient contact with the permanent epithelial base, called the bulge.
  • Lef-1 lymphoid enhancer factor
  • Lef-1 mRNAs are first expressed in El 2.5 ectodermal placodes, and as the developing follicle assembles a dermal papilla, epithelial Lef-1 is maintained at the growing tip of the hair germ (Zhou et al., 1995). During hair germ formation, it is also induced in the mesenchyme at the base of the follicle (Zhou et ⁇ /., 1995; Kratchowil et al., 1996).
  • Lef-1 plays some role in follicle development is demonstrated convincingly by Lef-1 null mice, which display a reduction and shortening of hair coat follicles and lack whiskers (van de Genderen et al., 1994).
  • Mesenchymal Lef-1 may be important in whisker formation, as the process is blocked in embryonic organ culture even if wild-type ectoderm is combined with Lef-1 null mesenchyme (Kratchowil et al., 1996).
  • Epithelial Lef-1 may be important in some aspect of the hair coat, since K14-Le -i transgenic mice display perturbations in the orientation of whisker and body hairs (Zhou et al., 1995).
  • Activation by Wnt/wingless leads to inhibition of the GSK3 kinase that phosphorylates ⁇ -catenin's N-terminus and typically targets its cytoplasmic pool for ubiquitin-mediated degradation (Munemitsu et al., 1996; Aberle et ⁇ /., 1997).
  • ⁇ -catenin Upon accumulation, ⁇ -catenin then interacts with members of the Lefl/Tcf family of DNA binding proteins to generate a functional transcription factor complex (Behrens et al., 1996; van de Wetering et al., 1997; for review, see Clevers and van de Wetering, 1997; Willert and Nusse, 1998).
  • This form can still interact with Lef-1/Tcf family members and function as a transcription co- factor (Molenaar et al., 1996), which is likely to account for its effects on proliferation in intestinal epithelia (Wong et al., 1998) and on axis duplication in Xenopus development (Funuyama et al, 1995; Wylie et ⁇ /., 1996; Fagotto et al., 1998). Whether the wnt/wingless pathway functions in hair follicle morphogenesis and/or hair cycling has not yet been explored.
  • Lef-1/Tcf members and ⁇ -catenin can be activated independently of a Wnt/wingless signal (Cadigan and Nusse et al., 1997; Willert and Nusse, 1998; Novak et al., 1998).
  • ⁇ -catenin can also act independently of Tcf/Lef, where it plays an essential role in cell-cell junction formation (for reviews, see Klymkowsky and Parr, 1995; Barth et al., 1997).
  • ⁇ -catenin binds to cell surface E- and P-cadherins and also to ⁇ -catenin, forming a bridge to the actin cytoskeleton (Pasdar and Nelson, 1988; Adams et ⁇ /., 1996). Microinjection of antibodies against P- and E-cadherins interfere with hair follicle morphogenesis in vitro (Hirai et ⁇ /., 1989), reflective of the extensive remodeling of epithelial cell junctions that occurs during this process.
  • Cicatrical alopecias result from the malformation or destruction of follicles so that they cannot produce hair caused by developmental defects, e.g., aplasia cutis congentia, infection, neoplasms and chemical and physical agents, e.g., burns and mechanical trauma.
  • Noncicatrical alopecias result from either the unscheduled entry of many follicles into the telogen stage of the hair cycle or the transformation of terminal to vellus follicles resulting in short, fine, unpigmented hairs.
  • noncicatrical alopecias include male pattern baldness (androgenetic alopecia) and alopecia areata.
  • minoxidil can help prevent or retard hair loss in male pattern baldness. It must be used constantly and tends to be effective only on individuals who have early balding of the vertex that is less than 10 cm in diameter.
  • Treatments for alopecia areata include high-potency corticosteriods and contact allergens, primary irritants, and the photosensitizing drug psoralen. All these therapies have limited efficacy and potential side effects.
  • ⁇ -catenin activity may be used to increase the density of hair growth in sheep and other livestock.
  • methodologies that improve wool production from livestock are examples of these therapies.
  • the present invention provides a method for inducing hair growth by providing ⁇ -catenin activity to a skin cell. This may be achieved by, but not limited to, providing a ⁇ -catenin polypeptide, providing a ⁇ -catenin agonist, providing a polynucleotide encoding a ⁇ -catenin polypeptide, enhancing the de novo synthesis of ⁇ -catenin, increasing the stability of ⁇ -catenin polypeptides or otherwise decrease the degradation of ⁇ -catenin polypeptides.
  • a polynucleotide encoding a ⁇ -catenin polypeptide may be provided by gene therapy and, in the case of sheep and other livestock, by production of a transgenic animal.
  • ⁇ -catenin and ⁇ -catenin polypeptide is intended to refer to a polypeptide based on the native sequence of ⁇ -catenin. This includes wild-type ⁇ -catenin and analogs and derivatives of wild-type ⁇ -catenin which retain the activity to induce hair growth.
  • the ⁇ -catenin polypeptide is ⁇ N87 ⁇ cat.
  • a ⁇ -catenin polypeptide including but not limited to, a ⁇ -catenin polypeptide, a ⁇ -catenin agonist, a polynucleotide encoding a ⁇ -catenin polypeptide, enhancing the de novo synthesis of ⁇ -catenin, increasing the stability of ⁇ -catenin polypeptides or otherwise decrease the degradation of ⁇ -catenin polypeptides, for use as a medicament is contemplated.
  • an inhibitor of the Wnt or Shh signaling pathway such as an inhibitor of mitogen-activated protein kinase, recombinant FrzB, constitutively active forms of GSK, dominant negative ⁇ -catenin polypeptides and dominant negative Lef-1 polypeptides, for use as a medicament is contemplated.
  • a ⁇ -catenin polypeptide a ⁇ -catenin agonist, a polynucleotide encoding a ⁇ -catenin polypeptide, enhancing the de novo synthesis of ⁇ -catenin, increasing the stability of ⁇ -catenin polypeptides or otherwise decrease the degradation of ⁇ -catenin polypeptides, for the manufacture of a medicament for the treatment of disease, including but not limited to a condition such as reduced hair growth or morphogenesis (i.e., alopecia).
  • a condition such as reduced hair growth or morphogenesis (i.e., alopecia).
  • an inhibitor of the Wnt or Shh signaling pathway such as an inhibitor of mitogen-activated protein kinase, recombinant FrzB, constitutively active forms of GSK, dominant negative ⁇ -catenin polypeptides and dominant negative Lef-1 polypeptides, for the manufacture of a medicament for the treatment of disease, including but not limited to a condition such as excess or undesired hair growth or morphogenesis and cancer, particularly hair tumors.
  • the ⁇ -catenin activity may be provided in a pharmaceutically acceptable therapeutic composition.
  • Administration of therapeutic compositions according to the present invention may be via any route so long as the target tissue is available via that route.
  • the therapeutic composition may be applied topically.
  • a topical composition may be formulated as, but not limited to, a cream, lotion, emulsion or be aqueous or non-aqueous based.
  • the topical formulation contains liposomes.
  • the liposomes may contain a ligand.
  • the ligand may be a galactose-terminal asialganglioisde.
  • the invention also provides that liposomes may contain proteins including, but not limited to, HJV and nuclear nonhistone chromosomal proteins.
  • the topical formulation contains cationic lipid-DNA lipoplexes.
  • the invention also provides for the use of methods to maximize the delivery of ⁇ -catenin activity by topical application. Both chemical and biophysical methods may be used to enhance the bioavailability of topically applied therapeutic agents.
  • the superficial layers of the skin are removed by tape stripping.
  • the invention provides for the use of intophoresis or related methods using electrical fields to enhance percutaneous absorption.
  • the ⁇ -catenin activity may be delivered by parenteral administration. Parenteral administration may be achieved by, but not limited to, intradermal, subcutaneous, or intravenous injection.
  • the invention also provides that the ⁇ -catenin activity may be provided by ballistic transfer.
  • the invention further provides for ex vivo delivery of ⁇ -catenin activity.
  • a patient's skin cells are cultured, transduced in vitro, then returned to the patient.
  • the skin cells can be returned to the patient by intradermal injection or by autlogous skin graft after culturing skin cells as epithelial sheet.
  • the ⁇ -catenin activity may be provided to the skin cell by means of a polynucleotide.
  • the polynucleotide may encode a ⁇ -catenin polypeptide or a ⁇ -catenin polypeptide analog or derivative.
  • the polynucleotide is operably linked to a promoter.
  • the invention provides that the promoter may be skin specific and may be selected from the group consisting of keratin promoters, involucrin promoters, filagrin promoters and loricrin promoters.
  • the invention further provides that keratin promoters may be selected from the group consisting of K3, K5, K10, K14 and K16.
  • the keratin promoter is K5 or K14.
  • the promoter may be selected from the group consisting of CMV IE, SV40 IE, RSV, ⁇ -actin, tetracyline regulatable and ecdysone regulatable. The promoter may also be inducible.
  • a polynucleotide may be contained in a vector.
  • the vector may be a viral vector.
  • the invention provides that a viral vector may be selected from the group consisting of adeonvirus, retrovirus, adeno-associated virus, vaccinia virus and polyomavirus.
  • the vector may be a non-viral vector.
  • Non-viral vectors include, but are not limited to, DNA loaded liposomes, cationic lipid-DNA lipoplexes and cationic polymer-DNA complexes.
  • the invention also provides for the delivery of plasmids or naked DNA.
  • non-viral in vivio and/or ex vivo transfer of expression constructs may be achieved by, but not limited to, calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection. cell sonication, ballistic transfer, receptor medicated transfection or use of liposomes. lipoplexes or polymer complexes.
  • the ⁇ -catenin activity may be provided by stabilising endogenous ⁇ -catenin.
  • Endogenous ⁇ -catenin may be stabilized by inhibition of glycogen synthase kinase 3 by such factors as, but not limited to, the products of Wnt genes, insulin, epidermal growth factor and integrin linked kninase.
  • the invention also provides a method where ⁇ -catenin activity is provided in conjunction with a factor that triggers the induction of hair growth by ⁇ -catenin.
  • This factor may be provided by a method that induces existing hair follicles to enter a synchronous hair cycle. Hair follicles may be induced to synchronously enter the anagen stage of the hair cycle by depilation or by administration of chemical agents such as cyclosporin or FK506.
  • the invention also provides that ⁇ -catenin activity may be provided with an agent that provides Lef-1 or TCF-3activity to the skin cell.
  • the invention provides a method for screening for hair growth disorders and/or the propensity for developing alopecia comprising evaluation of ⁇ -catenin expression in a skin cell.
  • the expression of ⁇ -catenin polypeptides can be evaluated by immunodetection methods such as enzyme linked immunosorbent assays and immunohistochemistry.
  • ⁇ -catenin mRNA may be evaluated by methods such as RT-PCRTM.
  • the invention also provides methods for screening for hair growth disorders and the propensity for developing alopecia comprising evaluation of the responsiveness of a skin cell to ⁇ -catenin activity.
  • This screening may consist of providing the skin cell with ⁇ -catenin activity and evaluating the localization of ⁇ -catenin or Lef-1 polypeptides in the nucleus of the skin cell by immunohistochemical techniques or by evaluating the activation of the c-MYC gene, a newly identifed target gene of the ⁇ -catenin pathway.
  • the invention provides for a screening method for compounds that may deliver ⁇ -catenin activity. Induction by a compound of the translocation of Le/-1 to the nucleus of a cell is indicative of the delivery of ⁇ -catenin activity. Translocation of Lef-1 to the nucleus of a cell can be established by immunohistochemical techniques and can be adapted for high through-put screening of candidate compounds.
  • This screening may be facilitated by the use of the reporter construct TOPFLASH.
  • This reporter contains the luciferase gene under the control of an enhancer containing Lef-lfTcf elements.
  • an enhancer containing Lef-lfTcf elements e.g., KCl, KCl, KCl, KCl, KCl, KCl, KCl, KCl, KCl, KCl, KCl-catenin, etc.
  • This assay is readily adaptable for large scale screening using, for example, microtiter plate assay methodologies.
  • the same enhancer linked to the GFP protein may also be used for screening.
  • the invention provides a method for inducing de novo hair morphogensis comprising providing ⁇ -catenin activity to a skin cell.
  • the invention also provides methods for the treatment of alopecia, stimulating hair growth, and preventing hair loss comprising providing ⁇ -catenin activity to a skin cell.
  • the invention provides for a method of inhibiting hair growth. Any method that leads to an inhibition of the Wnt signaling pathway in hair should lead to an inhibition of hair growth.
  • Such inhibitors may include, but not be limited to, inhibitors of mitogen-activated protein kinase, recombinant FrzB, constitutively active forms of GSK, dominant negative ⁇ -catenin polypeptides and dominant negative Lef-1 polypeptides.
  • the above described high through-put screening methods could be used for identification of inhibitors of hair growth, which would decrease reporter activation.
  • the transgenic ⁇ N87 ⁇ cat phenotype mouse exemplified in the present invention provides a model for screening compounds and therapies for human hair tumors, including trichofolliculoma and pilomatricoma.
  • the benefit of this model is that ⁇ N87 ⁇ cat mice exhibited numerous trichofolliculomas and pilomatricomas were numerous and occurred in a predictable fashion.
  • the invention additionally provides a method of sensitizing at least one cell to at least one nucleic acid damaging agent, comprising administering to the patient(s) at least one effective amount of at least one dominant/negative APE ⁇ ef-1 polypeptide or nucleic acid that encodes the dominant negative APE/Ref-1 polypeptide(s).
  • FIG. 1 Hair structure. The sebaceous gland, upper outer root sheath
  • Matrix cells are transit amplifying cells which differentiate upward in concentric rings of cells, giving rise to precortex, cortex and medulla (hair shaft), and surrounded by IRS and ORS.
  • FIG. 2 pTOPFLASH luciferase reporter assays.
  • Mouse keratinocytes were transfected with pTOPFLASH, containing four consensus Tcf/Lef binding motifs, a minimal Fos promoter and the luciferase reporter gene (van de Wetering et al., 1997; Korinek et ⁇ /., 1998a).
  • Transfections were done with or without K14- ⁇ N87 ⁇ cat and CMV-hLef-1. Forty-eight hour posttransfection, cells were lysed and protein extracts were assayed for luciferase. Studies were repeated 3 times, each time with a second reporter construct, CMV- lacZ, to correct for plasmid transfection efficiency.
  • Luciferase activity values represent an average of the three studies, with variations shown by error bars.
  • FIG. 3 Stick diagram of K14- ⁇ N87 ⁇ cat. Abbreviations: K 14, 2100 bp of K14 promoter sequence (Vassar et al., 1989); arm; armadillo repeats; B, Bam ⁇ ; X, Xhol; Bg, BglR; int, ⁇ -globin intron; pA, polyadenylation signal.
  • FIG. 4 Activation of the LEF TCF-dependent TOPGAL transgene in keratinocytes and in transgenic mice that also express a stabilized form of p-catenin.
  • A TOPGAL construct. The promoter contains three consensus LEF-1 /TCF-binding motifs (L) and a minimal c-fos promoter to drive transcription of the lacZ gene encoding p-galactosidase.
  • B pTOPGAL reporter assays.
  • the mouse keratinocyte line, UG1 was transfected with pTOPGAL, pCMV-luciferase (as an internal control gene) and equimolar amounts of plasmids pK14- # • • • • peat (K14-b # beat), pK14-Lefl or empty expression vector, as indicated (see Gat et al., 1998 for method). 48 hours later, cells were lysed and protein extracts were assayed for p-galactosidase activity (test) and luciferase (to correct for transfection efficiency). Normalized activities represent an average of three experiments, with variations shown by error bars. FOPGAL had no activity (not shown). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • the inventors have used a tissue-specific K14 promoter to drive the expression of an N-terminally truncated human ⁇ -catenin mutant, ⁇ N87 ⁇ cat, in skin.
  • ⁇ -catenin is a key factor in controlling hair follicle morphogensis.
  • the expression of ⁇ N87 ⁇ cat in the basal layer of the epidermis and follicle outer root sheath reprograms these cells to induce hair follicle morphogenesis.
  • the inventors have shown that the process of hair follicle morphogenesis occurs, including the development of dermal papilla and sebaceous glands normally established only in embryogenesis, and hair shaft production typical of both initial and cycling follicles.
  • the invention will be useful therapy for hair growth and alopecia disorders in humans and the induction of hair growth in sheep and other livestock.
  • the wingless cascade first identified in Drosophila, has been implicated in a multitude of developmental processes. There are at least 11 known Wnt genes in vertebrates which are homologs of the Drosophila Wingless genes (Parr & McMahon, 1994).
  • the Wnt proteins are secreted polypeptides, about 350-380 amino acids long and have a signal sequence and a recognition site for a signal peptidase. They are known to tightly associate with the extracellular matrix thus they may function mostly in short distance signaling.
  • Mutations in Drosophila which have similar phenotypes to those of wingless and biochemical studies have identified the components of an entire pathway transducing the wingless signals. It is thought that wg/wnt bind and transduce their message to a family of seven transmembrane domain (serpentine) receptors called Frizzled (fz).
  • the message is then transmitted in an unknown way to a cytoplasmic protein called Disheveled (dsh) of which there also several homologs in vertebrates.
  • dsh Disheveled
  • GSK3 Glycogen Synthase Kinase 3
  • ZW3 Glycogen Synthase Kinase 3
  • GSK3 is an ubiquitous serine/threonine kinase involved in multiple diverse functions such as basic metabolism, growth control and cell fate decisions (Woodgett et al, 1993).
  • GSK is known to have many substrates, including the transcription factors Jun and Fos as well as Myc and Myb and the microtubule associated protein Tau.
  • phosphorylation by GSK causes inactivation of its substrate.
  • GSK's major target in the Wnt pathway is thought to be ⁇ -catenin, the mammalian homolog of the Drosophila Armadillo protein.
  • Beta-catenin has two known functions: (1) it localizes to intercellular adherens junctions, where it binds to cadherins and participates in stabilizing them by linking them to the actin cytoskeleton (through ⁇ -catenin); and (2) when cytoplasmic pools of ⁇ -catenin accumulate, it can associate with the Lef-1/Tcf family of DNA binding proteins, and translocate to the nucleus, and transactivate the expression of genes that are regulated by Lef-1/Tcf sequence motifs.
  • GSK causes the phosphorylation of ⁇ -catenin, which in turn leads to its ubiquitination and proteolysis, thereby preventing accumulation of free cytoplasmic pools of the protein.
  • Wnt target genes Upon activation of the Wnt pathway, GSK is inhibited, leading to stabilization of cytoplasmic ⁇ -catenin and its ability to associate with Lef-1/Tcf proteins to become a transcription factor. This in turn causes Wnt target genes to be transcribed and a Wnt developmental program is executed.
  • ⁇ N87 ⁇ cat Using a ⁇ N87 ⁇ cat expression vector, the inventors demonstrated the functionality of ⁇ N87 ⁇ cat in keratinocytes.
  • ⁇ -Catenin accumulated in the cytoplasm of transfected keratinocytes.
  • Lef-1 Upon elevating the levels of Lef-1 in these keratinocytes, most of the ⁇ -catenin concentrated in the nuclei.
  • ⁇ N87 ⁇ cat can localize to sites of epidermal cell-cell adhesion, and if sufficient Tcf/Lef is present, it can translocate to the nucleus. This is a key component of the wingless signaling pathway (Molenaar et al., 1996; Larabell et al., 1997; Fagotto et al., 1998).
  • transient activation of ⁇ -catenin may be the long-sought "epidermal message” which has been predicted to receive a "dermal message” and respond by assembling a dermal papilla and forming an epithelial bulb around it (Sengel, 1990; Hardy, 1992).
  • Lef-1/Tcf expression Another factor clearly important in eliciting a Wnt signal in hair follicle induction is Lef-1/Tcf expression.
  • ⁇ -catenin can collaborate to transactivate a reporter gene containing Lef-1/Tcf sequence motifs 5' from a minimal enhancer inkeratinoctyes.
  • Overexpression of Lef-1 in transgenic mice can elicit epithelial invaginations (Zhou et al, 1995).
  • ⁇ N87 ⁇ cat function in embryogenesis can be attributed to either or the combination two factors: 1) the K14 promoter, while active at E9.5, is not dramatically elevated until E13.5-E14.5, after the major onset of follicle morphogenesis (Byrne et ⁇ /., 1994; Wang et ⁇ /., 1997), and 2) to elicit a response, ⁇ N87 ⁇ cat seemed to require a transiently induced factor, which in postnatal skin appeared to be released at the start of the hair cycle. That major morphological changes in transgenic mice were not observed until initiation of the hair cycle, indicates that ⁇ -catenin stabilization is not required at other times during follicle morphogenesis. Thus only the transient expression of ⁇ -catenin activity, when such expression occurs in conjunction with other appropriate factors, is required to induce post-natal hair morphogenesis.
  • a stable ⁇ -catenin is delivered to epidermal cells by using an epidermal keratin promoter/enhancer to drive expression of an N-terminally truncated form of ⁇ -catenin, lacking the phosphorylation/ubiquination sites.
  • endogenous ⁇ -catenin expressed by epidermal cells may be stabilized by inhibition of GSK3. Wnts can act on a cell with the appropriate receptor and lead to inactivation of GSK3 and stabilization of ⁇ -catenin.
  • GSK3 is also inhibited by insulin and epidermal growth factor signaling via its phosphorylation primarily by the MAPK (mitogen activated protein kinase) cascade (Seedorf et al., 1995). Inhibition of GSK3 is also implicated in the mechanism by which integrin linked kinase increases cytoplasmic levels of ⁇ -catenin (Novak et al., 1998). This kinase is activated by cell-extracellular matrix interactions and is a proto-oncogene whose overexpression causes anchorage independent growth of cells
  • Wnt 10b expression has been reported in skin (Wang et ⁇ /., 1996) and during development of the hair follicle (St-Jacques, 1998). Although this particular member seems to be the prime candidate to be involved in hair formation other Wnt genes such as Wnt7a, Wnt3a may also be involved in one the multiple stages and may activate ⁇ -catenin stabilization in the skin.
  • ⁇ -catenin degradation is enhanced by association with negative regulatory proteins, including the tumor suppressor gene product APC and recently described proteins axin and conductin. Inactivating mutations of APC cause the accumulation of ⁇ -catenin in a Wnt induced like fashion (Polakis, 1997). Thus, ⁇ -catenin stabilization can be enhanced in an epidermal cell by inhibition of these and other negative regulators.
  • ⁇ -catenin activity can be used to induce hair growth in sheep and other livestock in addition to humans.
  • transgenic sheep have been produced (e.g., Schnike et ⁇ /., 1997; Clements et ⁇ /., 1994. 1996; Wall et al., 1996). Genes have already been directed to the hair follicle in transgenic sheep (Damak et ⁇ /., 1996a, 1996b; Su et ⁇ /., 1998).
  • ⁇ -catenin activity is delivered to the epidermal cells of livestock by production of a transgenic animal with a transgene expressing a ⁇ -catenin polypeptide.
  • the reverse approach i.e., use of an inhibitor or suppressor of ⁇ -catenin activity, may be used to inhibit hair growth.
  • hair growth inhibition is desirable to suppress hair growth in certain body sites. Any method that leads to an inhibition of the Wnt pathway/growth factor pathway should lead to an inhibition of hair growth.
  • the pathway could be inhibited by using known inhibitors, such as are available for mitogen-activated protein kinase (Carter et al., 1998; Potchinsky et al., 1998; Nair et ⁇ /., 1997).
  • gene therapy of the Wnt cascade may be used, such as application of recombinant FrzB, a Frizzled receptor inhibitor (Lin et al, 1997); use of a constitutive active form of GSK, which could be elicited by phosphorylation; a dominant negative ⁇ -catenin, which does not contain the trans-activating C-terminal domain or a dominant negative Lef-1 which does not contain the N-terminal domain required for ⁇ -catenin binding. Inhibition of ⁇ -catenin activity will stop progression of hair through the next hair cycle and thereby reduce hair density.
  • FrzB Frizzled receptor inhibitor
  • the transgenic ⁇ N87 ⁇ cat phenotype mouse model can be used as a screen to identify the second factor.
  • Candidate factors can be delivered to the epidermis prior to the initiation of the first postnatal hair cycle. As ⁇ N87 ⁇ cat is already being expressed, delivery of the second agent or a compound with equivalent activity will initiate hair morphogenesis prior to that initiated by the first postnatal hair cycle.
  • the second factor may normally be present in adolescent and adult human and adult skin.
  • alopecia covers a diverse range of pathologies and it is possible that the second factor may be present in some cases and not others.
  • endogenous second agent activity may be induced by initiation of a synchronous hair cycle. Human hair follicles cycle asynchronously, but follicles can be induced to synchronously move into anagen by depilation or treatment with cyclosporin or FK506 (Vans et al., 1998; Jiang et al,
  • ⁇ -catenin activity is delivered to skin cells in conjunction with a physical or chemical treatment that induces existing hair follicles to synchronously enter the anagen stage of the hair cycle.
  • ⁇ -catenin is given in concert with an agent that upregulates or delivers Lef-1 activity to a skin cell.
  • Shh pathway stimulation would be most useful for enhancing hair growth, rather than for hair follicle formation per se. Nevertheless, in cases where hair growth stimulation is sufficient (e.g., as in minoxidil applications) and where hair follicle formation (e.g., as in hair transplants) is not required, Shh pathway stimulation would be expected to provide some benefit for hair growth, based upon the studies.
  • Shh pathway inhibitors can be expected to inhibit hair growth.
  • Shh pathway inhibitors such as cyclopin, may be useful for the treatment or prevention of hair tumors.
  • Wnt pathway activation leads to stimulation of cell growth, uncontrolled or too much stimulation via the Wnt pathway has been suspected of causing cancer.
  • the tumor suppressor adenomatous polyposis coli (“APC”) gene is lost or inactivated. Inactivation of the APC gene leads to ⁇ -catenin accumulation in the nucleus and presumably stimulation of tumor cell growth. Wnt signaling has now been linked to activation of the c-MYC oncogene (Pennisi, 1998).
  • mice As ⁇ N87 ⁇ cat mice aged, they developed visible, large tumors (>1 cm dia.), which by histology were much less differentiated in appearance than trichofolliculomas.
  • mice in contrast to basal cell carcinoma, centers of these less differentiated ⁇ N87 ⁇ cat tumors were Ptc negative. In addition, the centers were filled with enucleated cellular ghosts, which bore a striking resemblance to "shadow” cells, the hallmarks of pilomatricomas, common hair matrix tumors in humans (Fitzpatrick et al., 1993). As the ⁇ N87 ⁇ cat phenotype mice develop two types of hair tumor, they can be used a model for human trichofolliculoma and pilomatricoma formation and serve as a screen for compounds that prevent the formation of such tumors as well as a screen for compounds that inhibit or suppress the Wnt signaling pathway.
  • ⁇ -catenin activity will require that such activity is delivered and/or expressed transiently so as to obviate potential stimulation of tumor growth by long term delivery of ⁇ -catenin activity.
  • ⁇ -catenin activity can be delivered in a transient and/or tightly regulated fashion.
  • Use of many conventional gene delivery approaches have failed to uniformly sustain transgene expression in genetically engineered skin past 4 weeks (Lu et al., 1997; Fenjves et ⁇ /., 1996; Gerard et al, 1993; Setoguchi et al, 1994; Choate and Khavari, 1997).
  • the present invention envisages taking advantage of the tendency for transgenes to be expressed transiently in skin.
  • inducible promoters such as steroid-inducible promoters (White, 1997) can be used to drive expression of ⁇ -catenin transgenes.
  • Controlled delivery of the inducing agent in vivo will result in concomitant controlled expression of the ⁇ -catenin polypeptide.
  • the delivery over a defined period of time of agents that stabilize or otherwise enhance the activity or expression of endogenous ⁇ -catenin will result in the required regulated and transient delivery of ⁇ -catenin activity.
  • An advantage of using ⁇ -catenin gene therapy is that use of an endogenous protein should obviate immune reactions, a major problem in gene therapy.
  • transgenic ⁇ N87 ⁇ cat phenotype mice appeared healthy and normally active as far as they were grown, up to one year.
  • the bigger tumors are rare and appear only in a few animals at relatively "old age.”
  • transgenic expression of ⁇ N87 ⁇ cat in domestic animals could be used to enhance hair follicle formation.
  • the present invention involves the manipulation of genetic material to produce expression constructs that encode therapeutic ⁇ -catenin genes.
  • Such methods involve the generation of expression constructs containing, for example, a heterologous DNA encoding a gene of interest and a means for its expression, replicating the vector in an appropriate helper cell, obtaining viral particles produced therefrom, and infecting cells with the recombinant virus particles.
  • the invention also envisages the use of multigene constructs wherein a ⁇ -catenin gene is administered with another hair growth or morphogenesis regulatory therapeutic gene.
  • ⁇ -catenin is intended to refer to a polypeptide based on the native sequence of ⁇ -catenin. This includes analogs and derivatives of wild-type ⁇ -catenin which retain the wild-type ⁇ -catenin's activity to induce hair growth.
  • the ⁇ -catenin derivative is ⁇ N87 ⁇ cat, which is a N-terminally truncated ⁇ -catenin mutant.
  • a " ⁇ -catenin gene" is a polynucleotide that encode a " ⁇ -catenin” polypeptide.”
  • Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • a specialized kind of insertional variant is the fusion protein.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
  • Another aspect of the present invention includes novel compositions comprising isolated and purified ⁇ -catenin-derived peptides, synthetic modifications of these, epitopic peptides, peptides derived from site-specifically-mutagenized nucleic acid segments encoding such peptides.
  • Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular protein.
  • site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 15, or 16, or 17 or 18 nucleotides, or even up to and including about 25, or 35, or 45, or even about 75 nucleotides or more in length is preferred, with about 14 to about 20 or 30 or more residues being highly preferred.
  • about 8 or 9 or 10 or so unmodified nucleotides will flank the particular sequence being altered, which itself may be 10 or 15, or 20, or even 25-35 or so nucleotides in length.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • a genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones incorporating the mutagenic oligonucleotide.
  • thermostable enzymes such as Taq polymerase
  • PCRTM -mediated mutagenesis procedures of Tomic et al (1990) and Upender et al. (1995) provide two examples of such protocols.
  • a PCRTM employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector.
  • the mutagenesis procedure described by Michael (1994) provides an example of one such protocol.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
  • oligonucleotide directed mutagenesis procedure is intended to refer to a process that involves the template-dependent extension of a primer molecule.
  • template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing.
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent 4,237,224, specifically incorporated herein by reference in its entirety.
  • Codons are selected from a preferred codon table so as to avoid codons which are rarely found in ⁇ -catenin genomes. Regions with many consecutive A+T bases or G+C bases are predicted to have a higher likelihood to form hairpin structures due to self-complementary. Disruption of these regions by the insertion of heterogeneous base pairs is preferred and should reduce the likelihood of the formation of self-complementary secondary structures such as hairpins which are known in some organisms to inhibit transcription (transcriptional terminators) and translation (attenuators). Alternatively, a completely synthetic gene for a given amino acid sequence can be prepared. Restriction sites found in commonly used cloning vectors are preferably avoided. However, placement of several unique restriction sites throughout the gene is useful for analysis of gene expression or construction of gene variants.
  • the therapeutic expression constructs of the present invention contain nucleic acid constructs whose expression may be identified in vitro or in vivo by including a marker in the expression construct.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants.
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT. zeocin and histidinol are useful selectable markers.
  • Deng et al (1998), has shown that blasticidin is superior to the more commonly used neomycin in the selection of transduced keratinocytes.
  • enzymes such as herpes simplex virus thymidine kinase (tk) may be employed.
  • Immunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art and include reporters such as EGFP, ⁇ -gal or chloramphenicol acetyltransferase (CAT).
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for
  • RNA synthesis The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
  • Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • a transgene or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it may be desirable to prohibit or reduce expression of one or more of the transgenes.
  • transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes.
  • Several inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.
  • An indelible promoter may be used to control the expression of a ⁇ -catenin polypeptide. Topical or other application of the inducing compound in vivo can be used to regulate the expression of ⁇ -catenin activity Steroid indelible promoters (White, 1997) may be used for this purpose.
  • the ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained.
  • both monomers of the heterodimeric receptor are constituitively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid.
  • Engineering of this type of system into the gene transfer vector of interest would therefore be useful.
  • Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
  • expression of the transgene could be activated with ecdysone or muristeron A.
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-OnTM system gene expression is turned on in the presence of doxycycline
  • Tet-OffTM system gene expression is turned on in the absence of doxycycline.
  • the tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein is cloned into a plasmid behind a promoter that has tetracycline -responsive elements present in it.
  • a second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-OfF system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • transcription is constituitively on.
  • the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription.
  • the Tet-OfF M system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constituitively on.
  • a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired.
  • the CMV immediate early promoter if often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • Other viral promoters that may be used depending on the desired effect include SV40,
  • the invention provides embodiments wherein skin tissue specific promoters are used to effect ⁇ -catenin transcription in epidermal cells.
  • Skin specific promoters include the keratin promoters including, but not limited to, K3, K5, K10, K14, K16.
  • K5 and K14 promoters are used.
  • the keratins K5 and K14 are major proteins expressed by mitotically active cells of the epidermis and epidermal appendages.
  • the genes encoding K5 and K14 are abundantly transcribed in cultured keratinocytes (Stellmach et al, 1991).
  • the K14 promoter directs the expression of a luciferase reporter in keratinocytes much more potently than in breast cancer cells. (Staggers et al, 1995).
  • hair specific ultra-high-sulfur keratin promoters are used. (McNab et ⁇ /., 1990; Damak et ⁇ /., 1996a). This list of skin specific promoters should not be construed to be exhaustive or limiting, those of skill in the art will know of other promoters that may be used in conjunction with the promoters and methods disclosed herein.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human or bovine growth hormone and SV40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • IRES elements are used to create multigene polycistronic messages.
  • IRES elements are able to bypass the ribosome scanning model of 5'-methylated, Cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker. IRES sequences have been used to maximize the gene-transfer efficiency for skin gene therapy (Deng et al, 1998).
  • the expression construct comprise a virus or engineered construct derived form a viral genome. It is contemplated that a variety of viral particles may be employed according to the present invention. To determine whether other viral vectors could be manipulated in this manner, one of skill in the art can perform simple molecular biology techniques and assays. For example, replacement of promoter elements can be achieved through various cloning techniques well known in the art. Assays for the detection of gene expression such as Northern or Western blots can then be used to determine expression levels of the viral gene products in various cell types, either in vitro, or in vivo, through the use of biopsies.
  • ELISA enzyme-linked immunosorbent assays
  • CTL cytotoxic T Lymphocyte
  • In vitro infectivity assays also measure cytotoxic or cytopathic effects.
  • viral vectors with a marker gene such as ⁇ -galactosidase or luciferase as the transgene, can be employed. Measurement of the expression of the marker gene can then be done either qualitatively (e.g., microscopically) or quantitatively (e.g., flow cytometrically).
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
  • the roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis-ac ⁇ ng elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal repeats
  • the early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.
  • the El region encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990).
  • the products of the late genes (LI, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • the MLP (located at 16.8 map units) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence which makes them preferred mRNAs for translation.
  • TL tripartite leader
  • adenovirus In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products.
  • the two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease.
  • Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al, 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication.
  • packaging signal for viral encapsidation is localized between
  • adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants.
  • Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus.
  • helper viruses that are packaged with varying efficiencies.
  • the mutations are point mutations or deletions.
  • helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper.
  • helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions.
  • the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved.
  • First generation adenovirus vectors contain deletions in the El region, and the replication of these defective vectors is supported by packaging cell lines such as 293 cells that provide the El region gene products. Similarly, adenovirus vectors with the
  • El and E4 gene deleted but provided by 293 cells expressing both viral gene products, have been made. It is possible to make even larger deletions on the adenovirus genome and then provide the deleted genes in trans, either by a helper virus or helper cell, or both.
  • adenovirus desirable as a gene transfer vector.
  • the complete removal of these adenoviral genes would obviously eliminate the host immune response, however, it can be difficult to establish packaging cell lines because of viral gene-mediated toxicity.
  • the replacement of viral promoter elements with a synthetic promoter significantly reduces the level of viral gene expression.
  • Adenoviral vectors with synthetic promoters will have at least i) a deletion of the E1B and or E1A region and ii) an inducible or synthetic promoter substituted for the normal viral promoter that regulates expression of one or more of the early adenoviral genes E2, E3, E4 and E5.
  • a synthetic promoter could be substituted for the adenoviral major late promoter, which regulates expression of the late adenoviral gene LI, L2, L3, L4 and L5. Any vector with at least one promoter replacement is envisioned.
  • combinations of early region replacement include E1A and E1B, E1A and E2, E1B and E2, E1A and E4, E1B and E4, E1A and E5, E1B and E5, E1A and E1B and E2, E1A and E1B and E4, E1A and E1B and E5, E1A and E1B and E2 and E4, E1A and E1B and E2 and E5, E1A and E1B and E4 and E5, and E1A and E1B and E2 and E4 and E5.
  • one or more inducible or synthetic promoters may be used together to regulate transcription of viral genes.
  • the GAL4/TATA promoter may be substituted for the adenoviral E4 promoter, as well as the adenoviral E2 promoter, so that expression of the E2 and E4 genes is only possible in producer cells expressing the GAL4/VP16 transactivating polypeptide.
  • the GAIA ⁇ TATA promoter could be substituted for the E2 promoter, and the GAL4-estrogen receptor response element promoter could be linked to the E4 gene.
  • the producer cells would express the GAL4/VP16 fusion polypeptide to drive expression of the E2 gene, as well as the GAL4/ER (estrogen receptor) fusion polypeptide to drive expression of the E4 gene.
  • GAL4/VP16 fusion polypeptide to drive expression of the E2 gene
  • GAL4/ER estrogen receptor
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene, termed ⁇ functions as a signal for packaging of the genome into virions.
  • LTR long terminal repeat
  • a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol and env genes but without the LTR and ⁇ components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al, 1975).
  • Deng et ⁇ /. (1997) have described methods for to sustain retro viral-induced transgene expression in keratinocytes. Conventional viral vectors tends to fail to uniformly sustain transgene expression in skin past 1 to 4 weeks. This is the period spanning the time for epidermal turnover. Methylation of the retroviral long-term repeat (LTR) is implicated as the mechanism of loss of retroviral gene expression (Chakraborty et al, 1993). Deng et ⁇ /. (1997) describe the deletion and of promoter and enhancer sequences at the 3' LTR, to produce self-inactivating retroviral vectors. The 5' LTR ultimately replaces the deleted 3' LTR and the vector is transcriptionally inactivated. An internal promoter is used to drive gene expression. Transgene expression in genetically engineered skin resulting from use of such vectors persisted longer as compared to conventional retroviral vectors.
  • LTR long-term repeat
  • Adeno-associated Virus AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.
  • the three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
  • the splice site derived from map units 42-46, is the same for each transcript.
  • the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
  • AAV is not associated with any pathologic state in humans.
  • AAV requires "helping" functions from viruses such as herpes simplex virus I and ⁇ , cytomegalovirus, pseudorabies virus and, of course, adenovirus.
  • the best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication.
  • Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.
  • the terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al. 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV.
  • the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
  • AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, 1996 ; Chatterjee et al, 1995: Ferrari et al.. 1996; Fisher et al, 1996; Flotte et ⁇ /., 1993; Goodman et al, 1994; Kaplitt et a , 1994; 1996, Kessler et ⁇ /., 1996; Koeberl et al, 1997; Mizukami et a . 1996; Xiao et al, 1996).
  • AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1996; Flotte et al, 1993).
  • the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor LX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al, 1996; Flotte et al, 1993; Kaplitt et al, 1994; 1996; Koeberl et al, 1997; McCown et al, 1996; Ping et al, 1996; Xiao et al, 1996).
  • herpes simplex virus is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non-dividing neuronal cells without integrating in to the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency makes HSV an attractive vector. And though much attention has focused on the neurotropic applications of HSV, this vector also can be exploited for other tissues given its wide host range.
  • HSV Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance
  • HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings.
  • HSV as a gene therapy vector, see Glorioso et al. (1995).
  • HSV HSV
  • subtypes 1 and 2 are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide.
  • the large, complex, double-stranded DNA genome encodes for dozens of different gene products, some of which derive from spliced transcripts.
  • the virus encodes numerous other proteins including a protease, a ribonucleotides reductase, a DNA polymerase, a ssDNA binding protein, a helicase/primase, a DNA dependent ATPase, a dUTPase and others.
  • HSV genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion (Honess and Roizman, 1974; Honess and Roizman 1975; Roizman and Sears, 1995).
  • the expression of ⁇ genes is enhanced by the virion protein number 16, or ⁇ -transinducing factor (Post et al, 1981; Batterson and Roizman, 1983; Campbell, et al, 1984).
  • the expression of ⁇ genes requires functional ⁇ gene products, most notably ICP4, which is encoded by the ⁇ .4 gene (DeLuca et al, 1985).
  • ⁇ genes a heterogeneous group of genes encoding largely virion structural proteins, require the onset of viral DNA synthesis for optimal expression (Holland et al, 1980).
  • HSV In line with the complexity of the genome, the life cycle of HSV is quite involved. In addition to the lytic cycle, which results in synthesis of virus particles and, eventually, cell death, the virus has the capability to enter a latent state in which the genome is maintained in neural ganglia until some as of yet undefined signal triggers a recurrence of the lytic cycle. Avirulent variants of HSV have been developed and are readily available for use in gene therapy contexts (U.S. Patent No. 5,672,344).
  • Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA.
  • Vaccinia contains a linear, double-stranded DNA genome of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal repeats of about 10.5 kb flank the genome. The majority of essential genes appear to map within the central region, which is most highly conserved among poxviruses.
  • Estimated open reading frames in vaccinia virus number from 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common.
  • Prototypical vaccinia vectors contain transgenes inserted into the viral thymidine kinase gene via homologous recombination. Vectors are selected on the basis of a tk-phenotype. Inclusion of the untranslated leader sequence of encephalomyocarditis virus, the level of expression is higher than that of conventional vectors, with the transgenes accumulating at 10% or more of the infected cell's protein in 24 h (Elroy-Stein et al, 1989).
  • Simian virus 40 was discovered in 1960 as a contaminant in polio vaccines prepared from rhesus monkey kidney cell cultures. It was found to cause tumors when injected into newborn hamsters.
  • the genome is a double-stranded, circular DNA of about 5000 bases encoding large (708 AA) and small T antigens (174 AA), agnoprotein and the structural proteins VP1, VP2 and VP3.
  • the respective size of these molecules is 362, 352 and 234 amino acids.
  • T antigen one of the early proteins, plays a critical role in replication and late gene expression and is modified in a number of ways, including N-terminal acetylation, phosphorylation, poly-ADP ribosylation, glycosylation and acylation.
  • the other T antigen is produced by splicing of the large T transcript.
  • the corresponding small T protein is not strictly required for infection, but it plays a role in the accumulation of viral DNA.
  • DNA replication is controlled, to an extent, by a genetically defined core region that includes the viral origin of replication.
  • the SV40 element is about 66 bp in length and has subsequences of AT motifs, GC motifs and an inverted repeat of 14 bp on the early gene side.
  • Large T antigen is required for initiation of DNA replication, and this protein has been shown to bind in the vicinity of the origin. It also has ATPase, adenylating and helicase activities.
  • Late region expression initiates.
  • the transcripts are overlapping and, in some respect, reflect different reading frames (VP1 and VP2/3).
  • Late expression initiates is the same general region as early expression, but in the opposite direction.
  • the virion proteins are synthesized in the cytoplasm and transported to the nucleus where they enter as a complex. Virion assembly also takes place in the nucleus, followed by lysis and release of the infectious virus particles.
  • the present invention will encompass SV40 vectors lacking all coding sequences.
  • the region from about 5165-5243 and about 0-325 contains all of the control elements necessary for replication and packaging of the vector and expression of any included genes.
  • minimal SV40 vectors are derived from this region and contain at least a complete origin of replication.
  • the promoter driving the heterologous gene be a polyomavirus early promoter, or more preferably, a heterologous promoter.
  • the SV40 promoter and enhancer elements are dispensable.
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as lentivirus, poxvirus, alphavirus and coxsackie virus. These viruses offer several features for use in gene transfer into various mammalian cells.
  • DNA constructs of the present invention are generally delivered to a cell, in certain situations, the nucleic acid to be transferred is non-infectious, and can be transferred using non-viral methods.
  • the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites.
  • the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991a)
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA has been very successful in vitro. Lipofection is an efficient system of DNA delivery to primary keratinocytes (Jaing et al, 1991). Liposomes may also be used for the in vivo topical application of cutaneous gene therapies. Topical application of liposomes has been successfully used to target the delivery of a retroviral vector to cells in the hair follicle (Li and Hoffman, 1995; Hoffman 1998). Sawamura et al, (1997) have used a hemagglutinating virus of Japan (HVJ)-liposome combination for the topical delivery of DNA.
  • HVJ hemagglutinating virus of Japan
  • the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et /., 1991).
  • HMG-1 nuclear nonhistone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • Lipoplexes have been widely used as gene transfer vectors (Escriou et ⁇ / 1998a, 1998b; Zelphati et al, 1996, 1998; Dodds et ⁇ /., 1998).
  • a cationic lipid vector has been used in the Phase I study of direct gene transfer of HLA-B7 in patients with metastatic melanoma (Stopeck et al, 1997).
  • cationic polymer-DNA complexes can be used as delivery vehicles for DNA.
  • Simple cationic polymers will bind to DNA and assemble into discrete complexes (Wolfert et al, 1996).
  • Cationic-hydrophilic block copolymers can be used to increase aqueous solubility (Toncheva et /., 1998).
  • DNA-cationic polymer complexes have been widely used as synthetic vectors for delivery of genes in vitro and in vivo (Wagner et al, 1991; Trubetskoy et al, 1992; Pocet et ⁇ /., 1996; Coll et al, 1997; Goldman et al, 1997; Abdallah et al, 1996; Ferrari et al, 1997).
  • U.S. Patent 5,679,559 discloses the use of a lipoprotein containing cationic polymer system for gene delivery.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferring Wang and Wu, 1990
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • the human prostate-specific antigen (Watt et al, 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Naked DNA is internalized and expressed in skin after in vivo intradermal injection. (Hengge et al, 1996). Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of CaPO precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a ⁇ -catenin polypeptide may also be transferred in a similar manner in vivo and express ⁇ -catenin polypeptide.
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. The ballistic approach has been employed in vivo to achieve expression in skin (Cheng., et al, 1993)
  • compositions of the present invention will have an effective amount of agent to induce hair growth.
  • Administration of therapeutic compositions according to the present invention will be via any route so long as the target tissue is available via that route.
  • a preferred route of administration are topical and intradermal injection.
  • administration may be subcutaneous, ballistic, intramuscular, intraperitoneal or intravenous injection.
  • Such compositions generally will be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • the therapeutic formulations of the invention are prepared in forms suitable for topical administration, such as cremes and lotions.
  • the active compounds of the present invention may be formulated for parenteral administration, for example, formulated for injection via the intradermal, subcutaneous intravenous routes.
  • parenteral administration for example, formulated for injection via the intradermal, subcutaneous intravenous routes.
  • the preparation of an aqueous composition that contains an effective amount of therapeutic agents to induce hair growth will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can be emulsified.
  • other pharmaceutically acceptable forms include tablets or other solids for oral administration; time release capsules; and any other form currently used.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydoxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the complex For the application of a gene therapy, it will be necessary to prepare the complex as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.
  • the active compounds may be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts including the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethlyamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously-sterile-filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
  • aqueous solutions For parental administration in an aqueous solution, for example, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intradermal, subcutaneous or intravenous administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 100 ml of hypodermocylsis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variations in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the inventors contemplate the use of liposomes for the topical application or intradermal injection of one or more of the disclosed pharmaceutical composition into a host cell.
  • Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of ⁇ -catenin or ⁇ -catenin derivatives or analogs disclosed herein.
  • liposomes are generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). More recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Chonn, 1987).
  • the disclosed composition may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
  • the term "liposome” is intended to mean a composition arising spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991b).
  • gene gun-mediated skin transfection will be used as the route of administration (Rakhmilevich et al, 1996; Cheng et al, 1993).
  • This method involves coating DNA to gold particles directly into the skin at high velocity
  • the treatments may include various "unit doses."
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered, and the particular route and formulation are within the skill of those in the clinical arts.
  • Also of import is the subject to be treated, in particular, the state of the subject and the protection desired.
  • a unit dose need not be a single administration, as for example a single injection, but may comprise continuous administration over a set period of time, as for example a continuos perfusion.
  • unit doses of the present invention may conveniently may be described in terms of plaque forming units (pfu) of the viral construct. Unit doses range from 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10", 10 12 , 10 13 pfu and higher.
  • the invention provides for the delivery of ⁇ -catenin activity such that the resulting level of ⁇ -catenin polypeptide in an epidermal or ORS cell is 2-100 fold higher than level of endogenous ⁇ -catenin in an equivalent untreated epidermal or ORS cell.
  • Expression of a ⁇ -catenin polypeptide can be established by Westen blot and quantiifed by ELISA and raidoimmunoassays.
  • the delivery of an effective dose of ⁇ -catenin activity will be that sufficient to cause translocation of Lef-1 to the nucleus of an epidermal cell.
  • the translocation of Lef-1 can be monitored by immunohistochemical methodologies.
  • the invention provides for the ex vivo gene delivery.
  • Skin as a tissue is ideally suited to ex vivo gene therapy treatments (Wang et al, 1997).
  • the accessity of skin and the proliferative capacity of cultred epidermal cells makes keratinocytes ideal candiaites for transfection and gene therapy.
  • a patient's skin cells are cultured, transduced in vitro, then returned to the patient.
  • Large number of skin cells can be obtained from a small skin biopsy and the cell culturing conditions are well defined (Reinwald and Green, 1975).
  • Cultured keratinocytes form an epithelial sheet when approaching confluence and can be as an autologous skin graft (Greenhalgh et al, 1994).
  • transduced cells can be injected intradermally into the patient's skin.
  • keratinocytes Prior to return to the patient, keratinocytes can be screened for the effective delivery of ⁇ -catenin activity by monitoring for the translocation of Lef-1 to the nucleus. If an inducible promoter, such as a steroid inducible promoter (White, 1997), is used, the effective delivery of ⁇ -catenin activity in vitro can be established by monitoring for nuclear translocation of Lef-1 after exposure of transfected keratinocytes to the inducing agent. Once the keratinocytes are returned to the patient, in vivo delivery of ⁇ -catenin activity can be initiated by delivery of the inducing agent by topical or other routes of delivery.
  • J. DIAGNOSTIC AND SCREENING TESTS Methods for screening for ⁇ -catenin expression and responsiveness of skin cells to ⁇ -catenin are another aspect of the present invention. These parameters may also be predicative of the propensity to certain types of hair loss.
  • these methods can be used to screen compounds for the ability to deliver or induce ⁇ -catenin activty.
  • the translocation of Le/-1 to the nucleus is indicative of the effective delivery of ⁇ -catenin activty and can be used as the basis for a high-through-put screening methodologies for investigating candidate compounds for the ability to deliver or induce ⁇ -catenin activty. Such screening may be facilitated by the use of the reporter construct TOPFLASH.
  • This reporter contains the luciferase gene under the control of an enhancer containing Lef-1 /Tcf elements.
  • an enhancer containing Lef-1 /Tcf elements e.g., nuclear ⁇ -catenin together with Lef-1 will activate the reporter which is readily monitored by a luminometer.
  • This assay is readily adaptable for large scale screening using, for example, microtiter plate assay methodologies.
  • the same enhancer linked to the GFP protein may also be used for screening.
  • the present invention concerns immunodetection methods for binding, quantifying or otherwise generally detecting ⁇ -catenin and Lef-1/Tcf protein components.
  • the ⁇ -catenin Lef-1/Tcf antibodies prepared in accordance with the present invention may be employed to detect wild-type or mutant ⁇ -catenin proteins or peptides. As described throughout the present application, the use of wild-type or mutant specific antibodies is contemplated.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.
  • the immunobinding methods include obtaining a sample suspected of containing a ⁇ -catenin or Lef-1/Tcf protein or peptide, and contacting the sample with a first anti ⁇ -catenin or antiLefl/Tcf antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • these methods include methods for purifying wild-type or mutant proteins or peptides as may be employed in purifying wild-type or mutant proteins or peptides from patients' samples or for purifying recombinantly expressed wild-type or mutant ⁇ -catenin proteins or peptides.
  • the antibody removes the antigenic wild-type or mutant ⁇ -catenin protein or peptide component from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the wild-type or mutant protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which wild-type or mutant protein antigen is then collected by removing the wild-type or mutant protein or peptide from the column.
  • the immunobinding methods also include methods for detecting or quantifying the amount of a wild-type or mutant protein reactive component in a sample, which methods require the detection or quantification of any immune complexes formed during the binding process.
  • methods for detecting or quantifying the amount of a wild-type or mutant protein reactive component in a sample which methods require the detection or quantification of any immune complexes formed during the binding process.
  • the biological sample analyzed may be any sample that is suspected of containing a wild-type or mutant protein-specific antigen, such as a cancer tissue section or specimen, a homogenized cancer tissue extract, a cancer cell, separated or purified forms of any of the above wild-type or mutant protein-containing compositions, or even any biological fluid that comes into contact with cancer tissue, including blood and serum, although tissue samples and extracts are preferred.
  • a wild-type or mutant protein-specific antigen such as a cancer tissue section or specimen, a homogenized cancer tissue extract, a cancer cell, separated or purified forms of any of the above wild-type or mutant protein-containing compositions, or even any biological fluid that comes into contact with cancer tissue, including blood and serum, although tissue samples and extracts are preferred.
  • the chosen biological sample with the antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time lone enough for the antibodies to form immune complexes with, i.e., to bind to, any wild-type or mutant ⁇ -catenin protein antigens present.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the wild-type or mutant antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • the immunodetection methods of the present invention have evident utility in the diagnosis or prognosis of conditions such as various forms of cancer.
  • a biological or clinical sample suspected of containing a wild-type or mutant protein, peptide or mutant is used.
  • these embodiments also have applications to non-clinical samples, such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.
  • the detection of a ⁇ -catenin mutant, or an alteration in the levels of ⁇ -catenin in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with cancer.
  • a clinical diagnosis would not necessarily be made on the basis of this method in isolation.
  • Those of skill in the art are very familiar with differentiating between significant differences in types or amounts of biomarkers, which represent a positive identification, and low level or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off above which increased detection will be scored as significant or positive.
  • ELISAs As detailed above, immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • the anti-wild-type or mutant antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the wild-type or mutant protein antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound wild-type or mutant ⁇ -catenin protein antigen may be detected. Detection is generally achieved by the addition of another anti-wild-type or mutant antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti-wild-type or mutant antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the wild-type or mutant protein antigen are immobilized onto the well surface and then contacted with the anti-wild-type or mutant ⁇ -catenin antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-wild-type or mutant antibodies are detected. Where the initial anti-wild-type or mutant antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-wild-type or mutant antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the wild-type or mutant proteins or peptides are immobilized involves the use of antibody competition in the detection.
  • labeled antibodies against wild-type or mutant protein are added to the wells, allowed to bind, and detected by means of their label.
  • the amount of wild-type or mutant protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against wild-type or mutant polypeptide or protein before or during incubation with coated wells.
  • the presence of wild-type or mutant protein in the sample acts to reduce the amount of antibody against wild-type or mutant protein available for binding to the well and thus reduces the ultimate signal.
  • This is also appropriate for detecting antibodies against wild-type or mutant protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)tTween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS -containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • the antibodies of the present invention may also be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • each tissue block consists of 50 mg of residual "pulverized" diabetic tissue.
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • inverting the capsule and pelleting again by centrifugation snap-freezing in -70°C isopentane
  • cutting the plastic capsule and removing the frozen cylinder of tissue securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • RNA expression of ⁇ -catenin RNA can be measured by RT-PCRTM. Nucleic acids used as a template for amplification and detection are isolated from cells contained in biological samples according to standard methodologies (Sambrook et al, 1989). Where RNA is used, it may be desired to convert the RNA to a complementary DNA.
  • PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • modified primers are used in a PCR-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • an excess of labeled probes are added to a sample.
  • the probe In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Gingeras et al PCT Application WO 88/10315, incorporated herein by reference.
  • NASBA the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With propei choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCR” (Frohman, M.A., In: PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y., 1990 incorporated by reference).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification step of the present invention. Following any amplification, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989).
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
  • Amplification products must be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled, nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
  • kits This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair.
  • the pK14 ⁇ N87 ⁇ cat vector was constructed in two steps: (1) The sense primer included a BamHl site, an ideal Kozak sequence and nucleotides 462-482 of human ⁇ -catenin cDNA (obtained from Dr. Birchmeier, Univ. Berlin,
  • the anti-sense primer included nucleotides 2550-2532 of human ⁇ -catenin cDNA and a BamHl site. These were used with human ⁇ -catenin cDNA as template to synthesize a PCRTM product encoding an 87 amino acid N-terminally truncated ⁇ -catenin. This fragment was subcloned into the BamHl site of the K14 expression vector.
  • the Xhol-Bgl . fragment of the resulting plasmid, containing most of the ⁇ -catenin insert (1996 bp) was replaced with the original ⁇ -catenin cDNA, and all
  • PCRTM/primer-engineered segments were verified by sequencing.
  • the vector pCMV-hLefl encodes the full length hLef-1 product (Zhou et al, 1995).
  • the luciferase reporter construct pTOPFLASH was obtained from Dr. van de Wetering and Dr. Clevers (University Hospital, Utrecht, The Netherlands).
  • UDVIK epidermal primary keratinocyte culture of murine skin. This line, termed UDVIK, displays many of the characteristics of primary keratinocytes, including basal keratin expression and calcium-inducible differentiation (U. Gat, unpublished). Keratinocytes were cultivated as described (Hennings et al, 1980). Transfections were performed using the SuperFect polycationic reagent (Qiagen) or the lipid based FuGENE ⁇ reagent (BMB) according to manufacturer's protocol except that the SuperFect-DNA complex was left on the plates for 6 h instead of 2. Cells transfected with pTOPFLASH were processed and assayed for luciferase activity with the luciferase assay system kit (Promega, Madison, WI) and a luminometer.
  • Qiagen SuperFect polycationic reagent
  • BMB lipid based FuGENE ⁇ reagent
  • the K14- ⁇ N87 ⁇ cat transgene was isolated from the vector and injected into fertilized CD-I mouse embryos and transgenic mice were generated as described previously (Vassar et al, 1989). Mice positive for the transgene were identified by PCRTM analysis of toe genomic DNA and transgene expression was determined by RT-PCRTM.
  • Digoxygenin hybridization probes were synthesized according to the manufacturer's instructions (Boehringer Mannheim biochemicals, Indianapolis, IN).
  • a 517-bp mouse Lef-1 cDNA was made by excising a 700-bp BamHl fragment from the 1217-bp RT-PCRTM mouse Lef-1 cDNA clone (in pCRII) that was made as described (Zhou et al, 1995). This procedure eliminated the HMG sequences conserved among family members.
  • Antisense probe was made by linearizing the plasmid with EcoRV and transcribing with SP6 polymerase.
  • Sense cRNA was made by linearizing with BamHl and transcribing with T7 polymerase.
  • N-terminally truncated forms of ⁇ -catenin accumulate in the cytoplasm of a number of cell types, including mammalian fibroblasts and simple epithelial cells, but are not impaired in their ability to bind
  • Tcf family members function with ⁇ -catenin to repress other promoters in keratinocytes is an intriguing question beyond the scope of the present study.
  • the inventors used the K14 promoter, active in the mitotically active cells of the epidermis and the outer root sheath (ORS) of hair follicles (Vassar et al, 1989; Byrne et al, 1994; Wang et al, 1997).
  • This promoter has been tested extensively in conjunction with many different transgenes to give rise to highly restricted keratinocyte-specific expression in mice (Vassar et al,
  • the transgene construct engineered is illustrated in FIG. 3.
  • the inventors first used polymerase chain reaction (PCRTM) of tail DNAs to identify which mice carried the transgene, and RT-PCRTM to verify expression.
  • the inventors verified keratinocyte-specific protein expression of ⁇ N87 ⁇ cat by immunoblot analysis of proteins isolated from epidermis and dermis after their separation by 2M NaBr treatment of transgenic skin. An 80 kDa ⁇ N87 ⁇ cat was detected by anti- ⁇ -catenin antibody in transgenic epidermis. This band was not seen in control epidermis or in transgenic dermis.
  • the intensity of the ⁇ N87 ⁇ cat was comparable to that of the endogenous 91 kDa ⁇ -catenin band.
  • endogenous ⁇ -catenin was present ubiquitously throughout the skin epithelium, while the transgene was expressed only in the K14 promoter-active cells, constituting -10-20% of this population (Vassar et al, 1989; Wang et al. 1997). Therefore, the inventors estimate that the level of ⁇ N87 ⁇ cat is 5-10 fold higher than endogenous ⁇ -catenin in the basal epidermal and ORS follicle cells of the skin. This is consistent with the expected increased stability of the truncated version of ⁇ -catenin.
  • Lef-1 One obvious candidate for an internal change that could act in concert with ⁇ N87 ⁇ cat to elicit a response is Lef-1.
  • In situ hybridization revealed that despite the paucity of Lef-1 mRNAs in most areas of the epidermis and ORS, they were found in the aberrant invaginations of both interfollicular epidermis and ORS. Expression was maintained at the base of these hair germ-like structures, similar to that seen in mature wild-type follicles. Addressing whether this induction is direct or indirect is an issue beyond the scope of the present study. However, the inventors' data demonstrate that some factor(s) present in haired skin at the start of hair growth cycles causes a change in ⁇ N87 ⁇ cat-expressing cells that leads to their proliferation, invagination, and appearance of Lef-1 mRNAs.
  • the follicles Whether arising from dorsal paw skin or from backskin, the follicles arising de novo postnatally displayed characteristics that are normally only evident during embryonic follicle mo ⁇ hogenesis.
  • the bulbar portions of the follicles encased mesenchymal condensates, mo ⁇ hologically analogous to dermal papillae, and many areas of these cysts were plentiful with sebaceous glands and hair shafts. Although less frequent, hair shaft formation was evident in some interfollicular hair germs of the dorsal paw skin as early as d24 postnatally.
  • the epitheliod cysts with radial projection of hair follicle-like structures in ⁇ N87 ⁇ cat mice bore a striking resemblance to human trichofolliculomas, well-differentiated hair tumors each typified by an epithelioid cyst containing densely packed and misangled hair follicles, and small hairs that emanate from cysts at odd angles relative to the skin surface (Fitzpatrick et al, 1993). Scanning electron microscopy confirmed that of those hairs in the ⁇ N87 ⁇ cat mice that actually broke the skin surface, the angling was aberrant, and hairs were frequently irregularly positioned and of dramatically varied diameters.
  • Human trichofolliculomas generally arise as isolated tumors; in contrast, development of trichofolliculoma-like tumors was rampant in the inventors' transgenic mice, eventually giving rise to deep skin folds over the entire body.
  • the striking resemblance of ⁇ N87 ⁇ cat skin to these human tumors leads to the postulate that an alteration of a gene involved in ⁇ -catenin regulation may be causative for trichofolliculoma tumors in humans.
  • Mammalian hairs are polarized at a specific angle relative to the skin surface (Hardy, 1992).
  • a striking feature of the de novo hair follicles formed in ⁇ N87 ⁇ cat skin was their inappropriate angling and loss of polarity. While the mechanisms controlling this process have not yet been elucidated, it seems likely that follicles sense position by some factor(s) that is itself polarized.
  • Sonic hedgehog is a factor which is expressed in the hair bulb, but only in a patch at the side of the matrix which is closest to the skin surface (Bitgood and McMahon, 1995).
  • Shh mRNAs were restricted to the developing matrix cells in contact with the dermal papilla; however, in contrast to normal hair follicles, the patterns of Shh mRNA expression were most peculiar. In some hair germs, such as the one shown in frame B, expression appeared to be random, with some matrix cells expressing at significantly higher levels than others. In other cases, such as the one shown in the inset to frame B, expression occurred in discrete patches on both sides of the matrix.
  • Shh Activated Shh binds to patched, a membrane-bound receptor which indirectly represses its own gene transcription in the absence of Shh (Hooper and Scott, 1989; Ingham et ⁇ /., 1991).
  • upregulation of Ptc mRNA expression is a biochemical indicator of Shh activation (for review, see Ingham, 1993; Oro et al, 1997).
  • the matrix cells of developing de novo hair germs displayed marked hybridization to a didegoxygenin-labeled Ptc cRNA probe.
  • Shh and Ptc mRNAs were restricted to the matrix cells at the follicle base. However, as the follicles became progressively more irregular and trichofolliculomas began to develop, Shh and Ptc mRNA expression also became progressively more aberrant. Expression was still seen in matrix cells, but some irregular follicles displayed additional expression along the length of their outer root sheaths. This expanded pattern of Shh and Ptc expression arose in cells that were also positive for the basal keratins, and thus for K14 promoter activity and ⁇ N87 ⁇ cat expression. Like the formation of the hair germs themselves, expression required some additional, as yet unidentified factor, since not all cells positive for transgene expression displayed Shh and Ptc induction.
  • Shadow cells are normally a constituent of the hair medulla and are rarely seen in other skin tumors.
  • Pilomatricomas occurred in a number of the older ⁇ N87 ⁇ cat mice, but are extremely rare in normal mouse colonies. These data imply that pilomatricomas are derived from trichofolliculomas and are dependent upon ⁇ N87 ⁇ cat expression, but that an additional gene mutation is required for their formation.
  • Treatment of Alopecia This example describes a protocol to facilitate the treatment of patients suffering from alopecia using an agent providing ⁇ -catenin activity alone or in combination with other hair growth-promoting agents.
  • a composition of the present invention is typically administered topically or parenterally by intradermal or subcutaneous injection.
  • the agent providing ⁇ -catenin activity may be delivered to the patient before, after, or concurrently with other hair growth-promoting agents.
  • a typical treatment may comprise a single administration or a series of administrations of an agent providing ⁇ -catenin activity, either daily for several days, weekly for several weeks, or monthly for several months.
  • Patient groups may consist of men with good general health and suffering from androgenetic alopecia (male pattern baldness) or alopecia areata who have not used a hair growth-promoting agent such as minoxidil in the past 6 months.
  • Hair density can be measured by counting the number of terminal hair shafts per unit area or by evaluation of photographs (Olsen and DeLong, 1990; Gilhar et ⁇ /., 1990). Other methods of measuring hair growth such as use of optical microscopy and image analysis or by measuring the anagen ratio and hair diameters have been published (Tsuji et ⁇ /., 1994; Hayashi et al, 1991). Hair measurements will be taken prior to administration of the agent providing ⁇ -activity and at monthly intervals for a period of several months.
  • mice expressing a transgene whose expression is dependent upon activated Lef/TCF and ⁇ -catenin the inventors identifies which cells receive and respond to a Wnt or equivalent signal at specific times within the developing and postnatal hair follicle.
  • Transgene promoter activity in mice that are otherwise genetically wild type and in mice that express a constitutively active form of ⁇ -catenin was examined.
  • Plasmid pTOPGAL was engineered by replacing the luciferase reporter gene of pTOPFLASH (Korinek et al, 1998a) with the bacterial lacZ gene.
  • the TOP promoter consists of three multimerized Lef/TCF consensus binding sites and the c-fos minimal promoter Xbal fragment) with the promoter-less pNASS(m) (lacZ) plasmid (linearized with Spel).
  • Plasmid pK14- ⁇ N87 ⁇ cat was engineered as described herein. Transgenic mice harboring K14- ⁇ N87 ⁇ cat and TOPGAL or TOPGAL alone were made as previously described (Vassar et al, 1989), and transgene integration was verified using PCRTM analysis of tail DNAs. Transgene expression was verified by X-Gal staining of tail samples ⁇ -galactosidase; Byrne et al, 1994) and by phenotype ⁇ N87 ⁇ cat.
  • X-Gal assays on frozen tissue sections Frozen sections (10 ⁇ m) were fixed with 0.2% glutaraldehyde for 2 min. After washing 7-8 times in l ⁇ PBS, slides were then transferred into Tissue Stain Base solution (Specialty Media) with X-Gal substrate at a final concentration of 2 mg/ml. Staining was performed in the dark at 37°C for 6-8 hr and, after mounting in 80% glycerol, samples were visualized by Nomarski optics with a Zeiss axiophot microscope. After photographing slides, coverslips were removed and slides were treated sequentially with Hematoxylin for 2 min, water rinse, eosin for 5 min and a final water rinse. Slides were then remounted and tissue sections were photographed as before.
  • Frozen sections of tissues were subjected to indirect immunofluorescence as described herein.
  • antigen unmasking was performed by autoclaving 4% paraformaldehyde-fixed tissue sections in 10 mM sodium citrate, for 2 min.
  • Primary antibodies used were rabbit anti-Lefl (van Genderen et al. , 1994); mouse monoclonal anti-TCF3 (Barker et al, 1999); mouse monoclonal anti-trichohyalin (AE15; Manabe et al, 1996) and anti-hair keratin (AE13; Lynch et al, 1986); guinea pig anti-K5 (Byrne et al, 1994); rabbit anti-Ki67 (proliferation marker).
  • Fluorescence-conjugated secondary antibodies were obtained from Jackson Laboratories (West Grove, PA). Slides were mounted with antifade and visualized by Zeiss confocal microscopy.
  • Digoxigenin-labeled cRNAs were synthesized according to the manufacturers' instructions (Boeringer Mannheim Biochemicals, Indianapolis, IN). A 517 bp mouse Lefl cDNA, lacking the conserved DNA-binding domain, was subcloned as an
  • a transgenic mice expressing ⁇ N87 ⁇ cat, a constitutively activated form of ⁇ -catenin, under the control of the keratin 14 (K14) promoter which is upregulated at embryonic day 14.5 (E14.5) in the basal layer of epidermis, the ORS and the bulge has been generated.
  • the skin produces interfollicular epithelial invaginations, which sometimes resemble primordial hair germs and develop into bona fide hair follicles.
  • the transgene referred to here as TOPGAL, is a derivative of TOPFLASH, whose expression is dependent upon stabilized ⁇ -catenin and is comparably responsive to TCF3 and Lefl (Korinek et al, 1998a).
  • TOPGAL in cultured keratinocytes, TOPGAL, but not a version with mutated Lef/TCF binding sites (FOPGAL), behaved similarly and was superactivated when co-transfected with vectors expressing ⁇ N87 ⁇ cat and hLef 1 , but not with either alone (FIG. 4B).
  • FPGAL mutated Lef/TCF binding sites
  • Transgenic mouse skin was analyzed for ⁇ -galactosidase activity at 28 days of age, when many hair follicles were in anagen, and when there was a prevalence of interfollicular epithelial invaginations caused by ⁇ N87 ⁇ cat expression.
  • TOPGAL expression in skin from transgenic mice which were also positive for K14-pN8* peat.
  • the K14-pN8» peat transgene and its behavior in mice has been described herein. Sections (10 bm) of the toe skin from 28-day-old double transgenic mice were assayed for p-galactosidase activity using the X-gal assay. At all ages, control skins were negative for blue stain.
  • Epidermal basal cells were detected to have activated the TOPGAL transgene; as well as a de novo hair germ (hg), induced artifactually by pN8 » beat expression, along with an epidermal cell that has activated the TOPGAL transgene.
  • pN8» peat-induced epithelial invaginations expressing TOPGAL were detected.
  • ⁇ -galactosidase activity was detected in a number of epithelial cells in the skin of these transgenic mice.
  • K14- ⁇ N87 ⁇ cat/TOPGAL transgenic mouse skin scored positive for ⁇ -galactosidase activity.
  • postnatal de novo hair germs unique to ⁇ N87 ⁇ cat transgenic skin, stained blue, as did the flower-like epithelial invaginations that often followed the initial signs of ⁇ N87 ⁇ cat-induced hair germ formation.
  • TOPGAL activities in epidermis and in postnatal interfollicular epidermal invaginations were not seen when mice expressed only TOPGAL and not ⁇ N87 ⁇ cat.
  • TOPGAL is active in a subset of Lefl -expressing embryonic skin cells at the start of hair follicle morphogenesis
  • Lefl mRNAs are expressed early in embryonic mouse skin in both the ectodermal placodes and in the underlying dermal condensates (Zhou et al, 1995), and it was reported that of the Tcf family members, TCF3 is also expressed in hair follicles (Barker et al, 1999). Lefl and Tcf 3 behave very similarly in TOP promoter assays in at least some cells in vitro, and both are activated by the presence of stabilized ⁇ -catenin (Korinek et al, 1998a).
  • TOPGAL activation was used as an in vivo assay to understand how Lefl and TCF3, presumably in conjunction with endogenously activated ⁇ -catenin, might be involved in normal hair follicle mo ⁇ hogenesis during embryonic skin development. To this end, mice were engineered harboring only the TOPGAL and not the K14- ⁇ N87 ⁇ cat transgene.
  • TOPGAL transgenic mice were used for the remaining studies reported herein this example. Two lines were generated, and they behaved similarly in all assays.
  • Skin of a Lefl knockout newborn was used as a control for the antibody stainings. Sections were assayed for anti-K5 (K5) and/or anti-Lefl (Lefl) immunofluorescence or p-galactosidase activity. Sites of blue ectodermal and mesenchymal cells corresponded roughly in frequency and distribution to Lefl -intense sites, and the absence of blue staining in developing hair germ/plug were noted.
  • Lefl was readily detected in the nuclei of most if not all cells within the basal layer. Moreover, nuclear Lefl could be seen in these basal cells throughout embryonic development, i.e., encompassing the time when ectoderm was pluripotent and able to choose between epidermal and follicle cell fates. This finding indicated that either the threshold levels of Lef/TCF factors were not sufficient to activate TOPGAL in many of these cells, or that an additional signal, perhaps a Wnt, was necessary to stabilize ⁇ -catenin and enable downstream target genes to be expressed.
  • the strongest TOPGAL expression was detected in the differentiating cells above each hair bulb.
  • the two lines of cells expressing Lefl mRNAs also expressed TOPGAL, creating a narrow zone of overlap between the two patterns.
  • Biochemical evidence to establish the identity of the differentiating cells that express both TOPGAL and Lefl mRNAs was later obtained.
  • the differentiating cells at least included the precursor cells of the hair shaft, since clipped hairs of postnatal TOPGAL transgenic animals stained faintly blue when subjected to ⁇ -galactosidase activity assays.
  • Lefl mRNA expression persisted throughout anagen, but then declined dramatically at catagen and telogen, as hair growth ceased and follicles regressed, ⁇ -galactosidase activity persisted in the hair shaft cells into telogen but, by 18 days postnatally, only faint staining was detected. Similar patterns of blue staining were seen in postnatal follicles at different body sites, including body, tail and whisker follicles. This was su ⁇ rising, given that whiskers are completely lacking in Lefl knockout mice, whereas body hairs were only partially compromised (van Genderen et al, 1994). This difference might stem from variations in the importance of Lef/TCF activation in the waves of embryonic follicle development that give rise to different hair types (Headon and Overbeek, 1999).
  • TCF3 protein known to be expressed in skin (Barker et al, 1999), might be present in the precortex and account for the activation of TOPGAL in these cells.
  • Antibodies used were: TCF3 (green), Lefl (green), K5 (red), Ki67 (green), Dapi (blue).
  • K5 is a marker of the outer root sheath (ORS); Ki67 is a nuclear marker of proliferating skin cells. Brightly stained Lefl flat nuclei were visualized, more diffuse Lefl staining of matrix and also the immunofluorescence negative zone that separates the K5-positive ORS cells from the Lefl -stained cells was noted. This zone is likely the inner root sheath (IRS), which can be distinguished mo ⁇ hologically from the precortex.
  • IFS inner root sheath
  • TOPGAL expression occurs as matrix cells differentiate, concomitant with an increase in intensity of nuclear anti-Lefl immunofluorescence and in the apparent absence of TCF3.
  • TOPGAL which was expressed in the precortex of newborn tailskin follicles, TCF3 was confined to the outer root sheath.
  • TCF3 and Lefl behave similarly with stabilized ⁇ -catenin in TOP-promoter assays in lymphocytes in vitro (Korinek et al, 1998a)
  • TCF3 did not seem to coincide with TOPGAL expression in skin in vivo.
  • the inactivity of TCF3 may be a reflection of its interaction with the CtBP repressor protein, known to act selectively on TCF3 and not Lefl (Brannon et al, 1999).
  • TOPGAL/strongly nuclear Lefl -positive cells in the mature follicle were also the ones that expressed the hair specific keratins
  • well-characterized monoclonal antibodies that are highly specific for either the hair shaft keratins (AE13) or the IRS marker trichohyalin (AE15) were used (Manabe et al, 1996; Lynch et al., 1986).
  • Backskins from TOPGAL transgenic mice were taken at various ages (nb, day 6 and day 9), and tissues were processed for either p-galactosidase activity (X-gal, and/or counterstained with hematoxylin and eosin), or indirect immunofluorescence.
  • Antibodies used were: Lefl (green), AE13 against hair-specific keratins (pu ⁇ le), K5 specific for the ORS of the follicle (red), AE15 specific for the IRS (pu ⁇ le).
  • a filter was applied across the images of immunofluorescence. This distorts the actual coloring slightly, but allows the reader better visualization of the blue secondary antibody as pu ⁇ le and the overlap of blue AE13 and green Lefl labeling as turquoise (more Lef than AE13) and white (more AE13 than Lefl). Sections through the heart of the follicles and through the heart of the follicle bulbs, but at a slight angle grazing through the IRS rather than the hair shaft were examined.
  • Biochemical identification of the strongly Lef 1 -positive, TOPGAL-expressing cells as those that begin to express the hair-specific keratins and not inner root sheath markers Serial sections stained for ⁇ -galactosidase activity, intense nuclear anti-Lefl staining and anti-hair keratin (AE13) staining were clearly overlapping in the precortex region of newborn mouse skin. In contrast, another serial section of the same newborn follicle stained with anti-trichohyalin (AE15) and anti-Lefl revealed that these patterns were mutually exclusive.
  • the hair follicle is derived from a series of mesenchymal and epithelial cues transmitted in early embryonic development (Hardy, 1992). These signals have been predicted on the basis of tissue recombination experiments conducted by early developmental biologists (Sengel, 1976; Hardy, 1992). The postnatal hair cycles through periods of growth (anagen), rest (catagen) and regression (telogen). The transition from telogen to the new cycle is thought to depend upon a stimulus from the dermal papilla to the bulge, a permanent compartment of epithelial stem cells surrounding the dead hair club from the prior cycle. This results in epithelial proliferation to form the secondary hair germ (2° hg).
  • follicle stem cells Contiguous with the ORS and localized at the transition zone between permanent and cycling portions of the hair follicle is the bulge, the putative compartment of follicle stem cells (Lavker et al, 1991). This compartment of epithelial cells is not needed for establishing the embryonic hair follicle, but it is required for the initiation of each new hair cycle, as stem cells are stimulated by an as yet unidentified signal, most likely from the dermal papilla (Lavker et al, 1991). Wnt signaling and TCF/Lef transcription factors may be utilized at this stage of the hair cycle.
  • the bulge region was examined for the presence of TCF3 and Lefl. Anti-TCF3 staining was seen within the bulge, where it was localized in cell nuclei. Nuclear TCF3 persisted at this location throughout all stages of the hair cycle, even in telogen, when the ORS below the bulge was absent. In the upper portion of the follicle above the bulge, and in epidermis, anti-TCF3 labeling was dramatically diminished.
  • TOPGAL expression in this follicle was examined by staining another serial section of tissue, ⁇ -galactosidase activity was not detected in the growing hair germ. It was found at the base of the old hair club and, while this was never seen in non-transgenic skin, it was most likely due to residual enzymatic activity in the dead hair cells rather than new induction of TOPGAL. At a slightly later stage of anagen, as the secondary follicle began to form a bulb, ⁇ -galactosidase was detected in a triangular zone of cells, which appeared to be the developing precortex.
  • LEF1 Activation of LEF1 as a transcription factor in both ectoderm and mesenchyme at the initiation stage of hair follicle morphogenesis and Wnt signaling
  • this first mesenchymal cue could either be a Wnt directly, or alternatively, a signal such as BMP-4, known to induce Lefl gene expression and influence hair organogenesis in vitro (Kratchowil et al, 1996; Keranen et al. , 1998). While there may be additional Lef/TCF factors in this process, Lefl but not TCF3 was detected at this early stage of development.
  • TOPGAL expression in the developing dermal condensate was observed and, since TOPGAL expression seemed to be predominantly in either ectoderm or dermal condensate, it seems that the two signals leading to Lef/TCF target gene expression are sequential, rather than simultaneous.
  • One or more of the mesenchymal-ectodermal signals that lead to hair germ formation may be Wnts.
  • the results indicate that the next dermal message, instructing committed epithelium to proliferate and form the hair follicle (Hardy, 1992), might not involve a Wnt, if indeed the TOP promoter provides a faithful and broad representation of Lef/TCF activation in vivo, as it is known to do in lymphocytes in vitro (Korinek et al. , 1998a). Rather, based upon the block in the proliferation step of embryonic Sonic hedgehog null follicles (St. Jacques et al, 1998; Oro et ⁇ /., 1997), Shh is a possible candidate for regulating this process.
  • Hair matrix cells are able to select mo ⁇ hologically and biochemically distinct differentiation pathways leading to the IRS and hair shaft (Hardy, 1992).
  • the present studies provide among the first biochemical insights into how matrix cells select these programs of differentiation.
  • Lefl mRNAs and some Lefl protein only cells at the center of the bulb appear to accumulate Lefl and activate downstream target genes concomitant with commitment to differentiate.
  • the TOPGAL-expressing cells give rise to the hair shaft, while the surrounding cells choose an alternative pathway of differentiation and give rise to the IRS.
  • the studies indicate that induction of Lefl expression is a characteristic of undifferentiated matrix cells, and that utilizing Lefl to activate downstream target genes is a feature of matrix cells that become committed to a hair shaft differentiation program.
  • TOPGAL is expressed in precortical cells that give rise to the hair shaft provides compelling evidence that activated Lefl is important in regulating genes that specifically give rise to the hair shaft.
  • Wnt mRNAs have been found in mature hair follicles. Of these, Wnt-3 is of special interest in that its mRNAs are expressed in the precursor cells of the hair medulla (Millar et al, 1999).
  • TCF3 or LEF1 A role for TCF3 or LEF1 in activating follicle stem cells
  • a model to explain the hair cycle is that stem cells in the bulge become activated at the start of each new cycle, when they receive an as yet unidentified signal transmitted by dermal papilla cells that have retracted upwards at the end of the previous cycle (Hardy, 1992).
  • the studies here have shown that nuclear TCF3 resides continually in the putative stem cell compartment within the bulge.
  • co-expression of ⁇ N87 ⁇ cat was needed to detect strong TOPGAL activation in the bulge, a few cells within the bulge area of both TOPGAL and TOPGAL/ ⁇ N87 ⁇ cat follicles expressed ⁇ -galactosidase at or around the initiation of the first postnatal hair follicle.
  • Lefl also appeared to be activated at or soon thereafter, as judged by the fact that intense nuclear staining was concentrated in the secondary hair germ epithelial cells maintaining direct contact with the dermal papilla. This evidence indicates the possibility that TCF3/Lefl -mediated target genes may be expressed as an important step in stem cell activation. Additional studies using factors such as plucking to stimulate bulge cells (Wilson et al. , 1994), as well as measurements of TCF3's transactivation potential in ORS keratinocytes and/or bulge cells may be conducted.
  • activation of proliferation-associated target genes by activated Lef/TCF factors may occur only when the natural mechanisms governing Lef/TCF/ ⁇ -catenin regulation are subverted, as in the case of ⁇ -catenin activating mutations.
  • the interfollicular downgrowths induced by the constitutively active ⁇ -catenin transgene, K14- ⁇ N87 ⁇ cat displayed strong TOPGAL activation in the double transgenic mice. These invaginating epithelial pockets grow very rapidly and ultimately lead to pilomatricoma tumorigenesis.
  • Lef/TCF transcription factor complexes When taken together with the finding that in normal postnatal follicles, activation of Lef/TCF transcription factor complexes seems to arise to change an epithelial cell's fate or differentiation state, it is contemplated that different threshold levels of Lef 1 /TCF activation may explain how TCF/Lef-mediated activation can induce a terminal differentiation program on the one hand, and tumorigenesis on the other.
  • Feather mo ⁇ hogenisis and hair mo ⁇ hogenesis are regulated by the same signalling pathways.
  • the present example demonstrates that ⁇ -catenin and its signalling pathway are important in stimulating hair mo ⁇ hogenisis and the mo ⁇ hogenisis of other epidermal structures, such as feathers.
  • ⁇ -catenin signaling in the skin of the experimentally accessible chicken embryo was examined.
  • the change in the stability and subcellular localization of ⁇ -catenin can be monitored by immunohistochemical techniques to chronicle the activity of this pathway during development of a tissue (Schneider et al, 1996).
  • This signaling pathway was activated in a dynamic pattern throughout skin development from the onset of tract patterning through the generation of polarized buds. This pattern indicates roles for ⁇ -catenin signaling in placode formation, intra-bud patterning, and polarized outgrowth of the bud.
  • Endogenous ⁇ -catenin was detected with a monoclonal antibody (15b8, Sigma) diluted 1:500 in PBST followed by secondary detection with fluorescein-conjugated anti-mouse IgG. Sections were counterstained with 7-amino-actinomycin D or TOPRO 3 and images were captured on a Leica confocal microscope employing sequential scanning to eliminate bleedthrough between channels. In situ hybridization was as described by Morgan et al (1998). Infected cells were detected with a polyclonal antisera to the viral p27 protein (SPAFAS), or with a monoclonal raised against the HA epitope tag (Babco).
  • SPAFAS polyclonal antisera to the viral p27 protein
  • Bobco monoclonal raised against the HA epitope tag
  • the RCAS- ⁇ -catenin retrovirus was the generous gift of C. Tabin.
  • Stocks of viral innoculum were generated as described by Morgan and Fekete (1996).
  • a stock of 2xl0 8 infectious units/ml was injected and embryos harvested as described by Noramly and Morgan (1998).
  • the expression of viral transcripts alone was examined in a total of 31 embryos. Of these, 13 were harvested at day 6 of incubation, 9 at day 7, 6 at day 8 and 3 at day 9. Prior to day 8, no spatial restriction in infection was observed.
  • the TCF-1 probe was derived from nt 630-1400 of the chTCF-1 plasmid (Gastrop et al, 1992). Six embryos were sectioned after whole-mount in situ hybridization as described by Noramly and Morgan (1998) to examine the mo ⁇ hology of the induced placodes. Representative samples were also sectioned to confirm the localization of induced gene expression to the ectoderm or dermis. Finally, virus was detected immunohistochemically on sections from an additional 8 embryos to confirm conclusions based on whole-mount analysis.
  • the construct was sufficient to induce bud formation since it does so both within presumptive feather tracts and in normally featherless regions where tract-specific signals are absent. It was also insensitive to the lateral inhibition that mediates the normal spacing of buds and can induce ectopic buds in interfollicular skin. However, additional patterning signals cooperate with this pathway to regulate gene expression within domains of stabilized ⁇ -catenin expression.
  • compositions, methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, methods and apparatus of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and apparatus, and in the steps or in the sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Barker et al Am. J. Pathol, 154:29-35, 1999. Barth et al, Curr. Opin. Cell. Biol, 9:683-690, 1997.

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La présente invention concerne une méthode permettant d'induire la pousse des cheveux grâce à l'activité de la β-caténine dans une cellule de la peau. Cette invention consiste à utiliser un polypeptide β-caténinique, un agoniste de la β-caténine, un polynucléotide codant pour un polypeptide β-caténinique, à renforcer le nouveau cycle de synthèse de la β-caténine, à augmenter la stabilité ou à diminuer la dégradation des polypeptides β-caténinique.
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WO2003057912A3 (fr) * 2002-01-07 2004-04-29 Max Planck Gesellschaft Trichogramme moleculaire
JP2008537940A (ja) * 2005-03-29 2008-10-02 ザ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・ペンシルバニア 新しい毛包の形成、はげ頭症の治療、及び除毛をするための方法
US7476512B2 (en) 2004-02-27 2009-01-13 The General Hospital Corporation Methods of identifying dermal papilla cells
US8252749B2 (en) 2006-09-28 2012-08-28 Follica, Inc. Methods, kits, and compositions for generating new hair follicles and growing hair
CN110101592A (zh) * 2019-06-10 2019-08-09 上海龙禧投资有限公司 一种促进毛囊生长的组合物
WO2020081838A1 (fr) * 2018-10-17 2020-04-23 Frequency Therapeutics, Inc. Procédés pour la prolifération des cellules souches des follicules pileux
WO2020130878A1 (fr) * 2018-12-21 2020-06-25 CELL and GENE THERAPY Ltd Vecteur d'adn de thérapie génique basé sur un vecteur d'adn de thérapie génique vtvaf17
WO2021242826A1 (fr) * 2020-05-27 2021-12-02 The Regents Of The University Of California Compositions et procédés de transdifférenciation de cellules
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth

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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
EP1267616A4 (fr) * 2000-03-31 2003-07-16 Gen Hospital Corp Procede modulant la croissance du cheveu
WO2003057912A3 (fr) * 2002-01-07 2004-04-29 Max Planck Gesellschaft Trichogramme moleculaire
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
US9700502B2 (en) 2005-03-29 2017-07-11 The Trustees Of The University Of Pennsylvania Methods for generating new hair follicles, treating baldness, and hair removal
JP2008537940A (ja) * 2005-03-29 2008-10-02 ザ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・ペンシルバニア 新しい毛包の形成、はげ頭症の治療、及び除毛をするための方法
US9220926B2 (en) 2005-03-29 2015-12-29 The Trustees Of The University Of Pennsylvania Methods for generating new hair follicles, treating baldness, and hair removal
US9642789B2 (en) 2005-03-29 2017-05-09 The Trustees Of The University Of Pennsylvania Methods for generating new hair follicles, treating baldness, and hair removal
US8252749B2 (en) 2006-09-28 2012-08-28 Follica, Inc. Methods, kits, and compositions for generating new hair follicles and growing hair
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth
WO2020081838A1 (fr) * 2018-10-17 2020-04-23 Frequency Therapeutics, Inc. Procédés pour la prolifération des cellules souches des follicules pileux
WO2020130878A1 (fr) * 2018-12-21 2020-06-25 CELL and GENE THERAPY Ltd Vecteur d'adn de thérapie génique basé sur un vecteur d'adn de thérapie génique vtvaf17
RU2731514C2 (ru) * 2018-12-21 2020-09-03 Селл энд Джин Терапи Лтд Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген, выбранный из группы генов SHH, CTNNB1, NOG, WNT7A для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli SCS110-AF/VTvaf17-SHH, или Escherichia coli SCS110-AF/VTvaf17-CTNNB1, или Escherichia coli SCS110-AF/VTvaf17-NOG, или Escherichia coli SCS110-AF/VTvaf17-WNT7A, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора
CN110101592A (zh) * 2019-06-10 2019-08-09 上海龙禧投资有限公司 一种促进毛囊生长的组合物
WO2021242826A1 (fr) * 2020-05-27 2021-12-02 The Regents Of The University Of California Compositions et procédés de transdifférenciation de cellules

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