Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity.

Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68: 185-199).

The origin of the nervous system in all vertebrates can be traced to the end of gastrulation. At this time, the ectoderm in the dorsal side of the embryo changes its fate from epidermal to neural. The newly formed neuroectoderm thickens to form a flattened structure called the neural plate which is characterized, in some vertebrates, by a central groove (neural groove) and thickened lateral edges (neural folds). At its early stages of differentiation, the neural plate already exhibits signs of regional differentiation along its anterior posterior (A-P) and mediolateral axis (M-L). The neural folds eventually fuse at the dorsal midline to form the neural tube which will differentiate into brain at its anterior end and spinal cord at its posterior end. Closure of the neural tube creates dorsal/ventral differences by virtue of previous mediolateral differentiation. Thus, at the end of neurulation, the neural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see, for example, Principes in Neural Science (3rd), eds.

Kandel, Schwartz and Jessell, Elsevier Science Publishing Company: NY, 1991; and Developmental Biology (3rd), ed. S. F. Gilbert, Sinauer Associates: Sunderland MA, 1991).

Inductive interactions that define the fate of cells within the neural tube establish the initial pattern of the embryonic vertebrate nervous system. In the spinal cord, the identify of cell types is controlled, in part, by signals from two midline cell groups, the notochord and floor

plate, that induce neural plate cells to differentiate into floor plate, motor neurons, and other ventral neuronal types (van Straaten et al. (1988) Anat. Embryol. 177: 317-324; Placzek et al. (1993) Development 117: 205-218; Yamada et al. (1991) Cell 64: 035-647; and Hatta et al. (1991) Nature 350: 339-341). In addition, signals from the floor plate are responsible for the orientation and direction of commissural neuron outgrowth (Placzek, M. et al., (1990) Development 110: 19-30). Besides patterning the neural tube, the notochord and floorplate are also responsible for producing signals which control the patterning of the somites by inhibiting differentiation of dorsal somite derivatives in the ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol. 188: 239-245; Porquie, O. et al., (1993) Proc. Natl. Acad.

Sci. USA 90: 5242-5246).

Another important signaling center exists in the posterior mesenchyme of developing limb buds, called the Zone of Polarizing Activity, or"ZPA". When tissue from the posterior region of the limb bud is grafted to the anterior border of a second limb bud, the resultant limb will develop with additional digits in a mirror-image sequence along the anteroposterior axis (Saunders and Gasseling, (1968) Epithelial-Mesenchymal Interaction, pp. 78-97). This finding has led to the model that the ZPA is responsible for normal anteroposterior patterning in the limb. The ZPA has been hypothesized to function by releasing a signal, termed a"morphogen", which forms a gradient across the early embryonic bud. According to this model, the fate of cells at different distances from the ZPA is determined by the local concentration of the morphogen, with specific thresholds of the morphogen inducing successive structures (Wolpert, (1969) Theor. Biol. 25: 1-47). This is supported by the finding that the extent of digit duplication is proportional to the number of implanted ZPA cells (Tickle, (1981) Nature 254: 199-202).

Although the existence of inductive signals in the ZPA has been known for years, the molecular identities of these signals are only now beginning to be elucidated. An important step forward has been the discovery that the secreted protein Sonic hedgehog (Shh) is produced in several tissues with organizing properties, including notochord, floor plate and ZPA (Echelard et al. (1993), Cell 75: 1417-1430; Bitgood, M. J. and A. P.

McMahon (1995) Dev. Biol. 172: 126-38). Misexpressing Shh mimics the inductive effects on ectopic notochord in the neural tube and somites (Echelard et al. (1993) supra) and also mimics ZPA function in the limb bud (Riddle et al. (1993) Cell 75: 1401-16; Chang et al.

(1994) Development 120: 3339-53).

The vertebrate hedgehog family of inducing molecules comprises three homologs named Sonic, Indian and Desert hedgehog (Riddle et al. (1993) supra). Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is

primarily involved in morphogenic and neuroinductive activities. Given the critical inductive roles of hedgehog polypeptides in the development and maintenance of vertebrate organs, the identification of hedgehog interacting proteins is of paramount significance in both clinical and research contexts.

Summary of the Invention The present invention relates to the discovery of a new class of serpentine receptors, referred to herein as smoothened proteins. The smoothened proteins of the present invention include polypeptides which affect the transmission of signals by the products of the hedgehog gene family. Hedgehog family members are known for their broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

In general, the invention features isolated smoothened polypeptides, preferably substantially pure preparations of the subject smoothened polypeptides, such as liposomal preparations. The invention also provides recombinantly produced smoothened polypeptides.

In one embodiment, the polypeptide is identical with or similar to a smoothened polypeptide represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8.

Related members of the smoothened family are also contemplated, for instance, a smoothened polypeptide preferably has an amino acid sequence at least 65%, 70%, 75% or 80% identical or similar to a polypeptide represented by SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 though polypeptides with higher sequence homologies of, for example, 85,90% and 95% or are also contemplated. In a preferred embodiment, the smoothened polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in any one or more of SEQ ID Nos: 1-4 and 9. Homologs of the subject smoothened proteins also include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with a smoothened ligand, e. g. a hedgehog polypeptide.

The smoothened polypeptide can comprise a full length protein, such as represented in SEQ ID No: 5, SEQ ID No: 6 or SEQ ID No: 7, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e. g., at least 5, 10,25,50,100,150 or 200 amino acids in length. In a preferred embodiment, the smoothened polypeptide includes at least a portion of a smoothened protein corresponding

to Met 13-Ser 1035 of SEQ ID No. 5. In other preferred embodiments, the smoothened polypeptide includes a sufficient portion of the protein to be able to specifically bind to patched. Truncated forms of the protein include, but are not limited to, soluble extracellular and/or intracellular fragments, e. g., which bind to ligand or signal transduction proteins, respectively.

The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the smoothened protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e. g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the smoothened polypeptide.

In yet another embodiment, the invention features nucleic acids encoding smoothened polypeptides, which have the ability to modulate, e. g., either mimic or antagonize, at least a portion of the activity of a wild-type smoothened polypeptide.

Exemplary smoothened-encoding nucleic acid sequences are represented by SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4.

In another embodiment, the nucleic acids of the present invention include coding sequences which hybridize under medium or high stringency conditions with all or a portion of the coding sequences designated in one or more of SEQ ID Nos: 1-4. The coding sequences of the nucleic acids can comprise sequences which are identical to coding sequences represented in SEQ ID Nos: 1-4, or it can merely be homologous to those sequences. In preferred embodiments, the nucleic acids encode polypeptides which specifically modulate, by acting as either agonists or antagonists, one or more of the bioactivities of wild-type smoothened polypeptides.

Furthermore, in certain preferred embodiments, the subject smoothened nucleic acids will include a transcriptional regulatory sequence, e. g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the smoothened gene sequences. Such regulatory sequences can be used in to render the smoothened gene sequences suitable for use as an expression vector. This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing smoothened proteins by employing said expression vectors.

In yet another embodiment, the nucleic acid hybridizes under stringent conditions to nucleic acid probes corresponding to at least 12 consecutive nucleotides of either sense or antisense sequences of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4; though preferably to at least 25 consecutive nucleotides ; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4.

Yet another aspect of the present invention concerns an immunogen comprising a smoothened polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a smoothened polypeptide; e. g. a humoral response, e. g. an antibody response; e. g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e. g. a unique determinant, from a protein represented by one of SEQ ID No: 5, SEQ ID No: 6. SEQ ID No: 7 and/or SEQ ID No: 8.

A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the smoothened immunogen.

The invention also features transgenic non-human animals, e. g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e. g., animals which include (and preferably express) a heterologous form of a smoothened gene described herein, or which misexpress an endogenous smoothened gene, e. g., an animal in which expression of one or more of the subject smoothened proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed smoothened alleles or for use in drug screening.

The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequences of any one or more of SEQ ID Nos: 1-4 and 9-14, or naturally occurring mutants thereof. In preferred embodiments. the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e. g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co- factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a smoothened protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a smoothened protein; e. g. measuring a smoothened mRNA level in a cell, or determining whether a genomic smoothened gene has been mutated or deleted. These so-called"probes/primers" of the invention can also be used as a part of"antisense"therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e. g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject smoothened proteins so as to inhibit expression of that protein, e. g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25,40,50, or 75 nucleotides in length are also contemplated.

In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a patched protein and a smoothened polypeptide receptor. In preferred embodiments, the step of detecting

interaction of a target molecule, such as patched, and smoothened polypeptides is a competitive binding assay. In other preferred embodiments, the step of detecting interaction of the target molecule and smoothened polypeptides involves detecting, in a cell-based assay, change (s) in the level of an intracellular second messenger responsive to signaling mediated by the smoothened polypeptide. In still another preferred embodiment, the ability to modulate the bioactivity of smoothened comprises detecting, in a cell-based assay, change (s) in the level of expression of a gene controlled by a transcriptional regulatory sequence responsive to signaling by the smoothened polypeptide.

In preferred embodiments, the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 103,104 or 105 different test compounds. The test compound can be, e. g., a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract (or fraction thereof).

Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by modulating smoothened bioactivity, e. g., by potentiating or disrupting certain protein-protein interactions. In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a smoothened therapeutic so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with smoothened therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a smoothened protein.

Other smoothened therapeutics include antisense constructs for inhibiting expression of smoothened proteins, and dominant negative mutants of smoothened proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type smoothened protein.

In one embodiment, the subject method of modulating smoothened bioactivity can be used in the treatment of testicular cells, so as to modulate spermatogenesis. In another embodiment, the subject method is used to modulate osteogenesis, comprising the treatment of osteogenic cells with an agent that modulates smoothened bioactivity. Likewise, where the treated cell is a chondrogenic cell, the present method is used to modulate chondrogenesis. In still, another embodiment, the subject method can be used to modulate the differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e. g., to prevent apoptosis or other forms of cell death. For instance the present method can be used to affect the differentiation of neuronal cells such as motor neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, and peptidergic neurons.

Another aspect of the present invention provides a method of determining if a subject, e. g. an animal patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation or apoptosis. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a smoothened protein; or (ii) the mis- expression of a smoothened gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of : a deletion of one or more nucleotides from a smoothened gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble smoothened protein.

For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a smoothened gene or naturally occurring mutants thereof, or 5'or 3'flanking sequences naturally associated with the smoothened gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e. g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the smoothened gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a smoothened protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the smoothened protein.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U. S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. 1. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),

Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of tlte Drawings Figure 1 (panels A-H) are phase-contrast images of the ventral cuticle of pharate first instar larvae; anterior is to the top. a, Wild type. &,/Ko, raised at 25° C. Naked cuticle is deleted sporadically between adjacent denticle belts. c, STMO, raised at 18° C. Naked cuticle is replaced by denticle belts with reversed polarity. d, smo-embryo derived from smoD16 germline clone females. All naked cuticle and polarity is lost; this phenotype is typical of all smo alleles (three) analyzed in germline clone analysis. e, hhl3C (severe phenotype [Mohler, J. (1988) Genetics 131: 643-653]); the phenotype is virtually identical to that of a smo-embryo in d. f, wgCX4 (null allele [van den Huevel, M. et al. (1993) supra]); note the complete loss of naked cuticle but vestiges of polarity, in contrast, to in smo-and hh null mutants remain visible. g, smo-, hGAL4-UASwg; naked cuticle is partially restored in alternate segments. h, smo-GAL4-AUShh ; phenotype is indistinguishable from that of smo-.

METHODS: All smo alleles have been described (Nusslein-Volhard, C. et al. (1984) supra) except smog16. This allele and two others (smoF5 and smoF] l) were isolated in an F, lethal screen following-ray mutagenesis. Isogenized cn bw sp males were irradiated with 4,000 Rad emitted by a 60Co source and mated to wild-type females. Individual F, males (12,000) were test-crossed to smolt43 cn bw sp/CyO females and the F, progeny screened for the absence of white-eyed flies. Germline clone females were generated by the dominant female sterile technique either by irradiating larvae with-rays or using flippase- induced mitotic recombination (Chou, T. B. & Perrimon, N. (1992) Genetics 131: 643-653).

Ectopic expression of wg and hh was induced by the GAL4-UAS system using the hairy GAL4 enhancer trap, which expresses GAL4 in every other segment (Capdevilla, J. & Guerrero, I. (1994) EMBO J. 13: 4459-4468). hGAL4 UASwgts, UAShh, and hGAL4 chromosomes have been described.

Figure 2 (panels A-H) show the pattern of expression of wg, hh, and En in wild-type and mutant embryos. a, Expression of wg in wild-type stage 10. b, Expression of wg

(blue/black) and hh (red) in hGAL4-UAShh embryo. The ectopic expression of hh induces the broadening of wg in all segments because of the overlap of endogenous wg and ectopic hh. c, wg and hh in smo- ; hGAL4 UAShh ; expression of eg in the segmented germband is completely lost as smo-embryos (see d). d, wg in smo-stage 10; all wg expression disappears from the segmented germband. e, hGAL4 UASwg stage 10 embryo; ectopic and endogenous (arrowheads) wg stripes are seen to overlap, with the h driven wg expression spanning every other segment. f, smo- ; hGAL4 UASwg stage 10 embryo; ectopic but no endogenous wg expression is detected. g, Stage 10 smo- ; En is lost from the ectoderm in all thoracic and abdominal segments. h, Stage 11 hGAL4 UASwg embryo, showing slightly broadened stripes of En (compared to wild-type embryos). i, smo-, hGAL4 UASwg stage 10. En expression is maintained in alternate segments (arrowheads), corresponding to the segments where wg expression is driven by hGAL4. In the intervening stripes, some En is rescued, presumably due to the paracrine action of the misexpressed wg.

METHODS: Embryos were collected for one hour and aged for the appropriate times.

Fixations, in situ hybridizations and antibody stainings (for En, using monoclonal 4D9) have been described (Ingham, P. W. et al. (1991) supra; van den Huevel, M. et al. (1993) supra). Double-labeling with an hh flourescein-labeled and a wg digoxigenin probe was performed with two sequential alkaline phosphate substrates, BCIP/NBT and Vector red; the enzyme was inactivated between the steps by treatment with 0.2M glycine, pH 2.5.

Genotypes are described in Fig. 1 legend.

Figure 3: Stainings of third instar imaginal discs. a, Dpp expression (green) anterior to the compartment boundary is shown, as marked by the expression of En (red) throughout the posterior compartment (as hh). b, Wing-blade area stained for Dpp (green) and Myc epitope (red). Arrow indicates a smo-clone in the dpp expression region. c-e, Details of disc in b.

Similar clones within the dpp domain were found in six cases among 350 dissected progeny, of which 1/8 were of the correct genotype. Loss of Myc staining represents cells that have lost smo-activity, whereas nearby cells expressing high levels of Myc represent the sister clone carrying two copies of the myc construct. In c, arrow points to a smo-clone within the posterior compartment. The absence of green staining (Dpp) corresponds cell for cell (e) with the absence of red staining smo-. f, dpp expression; g, as in f, except that clones lacking smo-and the catalytic subunit of PKA were induced; these express dpp ectopically in the compartment owing to the overexpression of dpp (Capdevilla J. & Guerrero I. (1994) EMBOJ. 13: 4459-4468).

METHODS: Imaginal discs were dissected in PBS on ice and fixed in PBS 4% paraformaldehyde. For a, discs were collected from dpp/LacZ animals and for c-e, from a cross of smoD16 16 ck FRT/CyO females with Hs-FLP; FRT dppLacZ/CyO males, the progeny of which were heat-shocked at several stages during larval development. Discs were stained with anti-En, anti-Dpp (Panganiban, G. et al. (1990) Mol. Cell. Biol. 10: 2669- 2677), anti--galactosidase (Promega) and anti-Myc epitope antibodies or stained for- galactosidase activity using X-gal, following standard procedures.

Figure 4 (panels A-D) show various steps in the cloning of smoothened. A: Southern blot of genomic DNA (digested with BamHl) from different smo alleles, hybridized with a subclone from a PI clone (see c). Six PI clones, covering the area of 21 B7-8, the region to which smo maps (based on the exclusion from DfZL (al) and DfZL (PMF), were screened for the detection of aberrations in smo alleles. B: Developmental northern blot hybridized with subclone as in C. C: Restriction map of the smo locus. Complementary DNAs for all three transcripts were isolate. The genomic DNA used to make a transgenic fly is indicated.

Bold bars indicate the smo gene exons. D: The one-letter amino-acid sequence of the open reading frame in the smo gene is shown. Three more methionine codons are found in-frame before the indicated start of translation, two of which are not surrounded by residues creating a good translation start site ( (Brown, N. H. & Kafatos, F. C. (1988) J. Mol. Biol.

203: 425-437); a third methionine, just one codon before, is interrupted by an intron.

Hydrophobic stretches are underlined, the first one probably representing a signal sequence.

Arrow denotes a putative signal peptidase cleavage site. The homology with the Drosophila Fz protein is confined to the putative transmembrane region.

METHODS: PI phage DNA was prepared by the alkaline lysis method and digested; DNA fragments were isolated from agarose gels by spinning through glasswool and subcloned in BlueScript) Stratagene). Genomic fly DNA was prepared, digested with restriction enzymes, separated on agarose gels and transferred to Hybond N (Amersham). PI fragments were labeled with [32P] dCTP (Amersham protocol) and hybridized to the filters under standard conditions. Total RNA from different stages was prepared by guanidine-HCl extraction and acetic acid precipitation. RNA samples were run under identical conditions and the amount of RNA loaded was controlled by staining the blot with methylene blue after hybridization. The restriction fragment indicated in c was isolated and cloned into pCaSpeR for transformation. Transgenic flies were generated and identified among the Fl progeny on the basis of their eye pigmentation, and balanced lines were established. A partial complementary DMA for smo was isolated from a 0-2 h cDNA library (Brown, N. H. and Kafatos, F. C. (1988) supra) by hybridization with a probe contained within the rescue

fragment. The smo cDNA and genomic subclones completely covering the region of the rescue fragment were sequenced using the dideoxy method with Sequenase (USB); both strands of the DNA were sequenced at least twice. The start of transcription and intron- exon boundaries were confirmed by sequencing fragments generated by RT-PCR, using primers designed from the genomic sequence.

Detailed Description of the Invention Of particular importance in the development and maintenance of tissue in vertebrate animals is a type of extracellular communication called induction, which occurs between neighboring cell layers and tissues. In inductive interactions, chemical signals secreted by one cell population influence the developmental fate of a second cell population. Typically, cells responding to the inductive signals are diverted from one cell fate to another, neither of which is the same as the fate of the signaling cells.

Inductive signals are key regulatory proteins that function in vertebrate pattern formation, and are present in important signaling centers known to operate embryonically, for example, to define the organization of the vertebrate embryo. For example, these signaling structures include the notochord, a transient structure which initiates the formation of the nervous system and helps to define the different types of neurons within it. The notochord also regulates mesodermal patterning along the body axis. Another distinct group of cells having apparent signaling activity is the floorplate of the neural tube (the precursor of the spinal cord and brain) which also signals the differentiation of different nerve cell types. It is also generally believed that the region of mesoderm at the bottom of the buds which form the limbs (called the Zone of Polarizing Activity or ZPA) operates as a signaling center by secreting a morphogen which ultimately produces the correct patterning of the developing limbs.

The regulation of hedgehog protein signaling is an important mechanism for developmental control. Members of the hedgehog family of secreted proteins control a number of important inductive interactions in the development of both vertebrates and Drosophila (Ingham, P. W. (1995) Opin. Gen. Dev. 5: 492-498). In Drosophila, hedgehog is one of the segment-polarity genes, mutations of which disrupt the pattern and polarity of individual embryonic segments (Nusslein-Volhard, C. & Wieschaus, E. (1980) Nature 287: 795-801) and their adult derivatives (Williams, J. A. & Carroll, S. B. (1993) Bioessays 15: 567-577).

The present invention concerns the discovery of a new family of cell surface proteins, referred to herein as"smoothened"proteins. Here we show that the smoothened gene product is required for the response of cells to hedgehog signaling during the

development. Sequence analysis of the smoothened transcription unit reveals a single open reading frame encoding a protein with seven putative transmembrane domains. This structure is typical of G-protein-coupled receptors. As described herein, the vertebrate smoothened proteins exhibit spatially and temporally restricted expression domains indicative of important roles in hedgehog-mediated induction.

The sequence of exemplary smoothened genes cloned from various metazoan organisms (cf, Table 1 below) indicates it encodes a receptor-like serpentine protein that may be anchored at the cell membrane. Comparison of smoothened sequences from drosophila, chicken, rat and human clones suggests that smoothened is an integral membrane proteins with seven membrane spanning a helices, a long cytoplasmic tail, and a conserved signal peptide sequence. Moreover, analysis of the protein sequences suggests potential sites for modification by N-linked glycosylation, as well as potential phosphorylation sites for G-protein coupled receptors and cAMP-dependent kinases (e. g., PKA). The vertebrate smoothened proteins also include potential protein-protein interaction modules such as leucine zippers and RGD sequences. The smoothened coding sequences has weak homology with the frizzled genes, particularly across the transmembrane domains.

However, within the serpentine receptor family, the evidence provided herein suggests that these genes comprise a novel sub-family of receptors.

The smoothened proteins, through their ability to associate with patched and/or hedgehog proteins, are apparently capable of modulating hedgehog signaling. The smoothened proteins may function as a constitutively active signaling protein whose signal transduction capabilities are inhibited by interaction with patched, a hedgehog receptor (or subunit thereof). Binding of hedgehog to patched alters the interaction of smoothened and patched, and relieves the patched-mediated inhibition of smoothened signal transduction.

Thus, the smoothened polypeptides of the present invention may affect a number of hedgehog-mediated biological activities including: an ability to modulate proliferation, survival and/or differentiation of mesodermally-derived tissue, such as tissue derived from dorsal mesoderm, cartilage and tissue involved in spermatogenesis; the ability to modulate proliferation, survival and/or differentiation of ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; the ability to modulate proliferation, survival and/or differentiation of endodermally-derived tissue, such as tissue derived from the primitive gut.

As described in the appended examples, a drosophila smoothened orf (open reading frame) was identified in a screen for the ability of expressed transgene genomic fragments to rescue a smo phenotype. A chicken smoothened cDNA was isolated by hybridization under low to medium stringency conditions with the drosophila smoothened coding sequence. In addition to the chicken smoothened clone, the art has recently reported cDNA

clones from other vertebrates, including human and rodent smoothened genes. According to the appended sequence listing, (see also Table 1) a drosophila smoothened polypeptide is encoded by SEQ ID No: 1; a human smoothened polypeptide is encoded by SEQ ID No: 2; a rat smoothened polypeptide is encoded by SEQ ID No: 3; and a chicken smoothened polypeptide is encoded by SEQ ID No: 4.

Table 1 Guide to smoothened sequences in Sequence Listing Nucleotide Amino Acid Drosophila smoothened SEQ ID No. 1 SEQ ID No. 5 Human smoothened SEQ ID No. 2 SEQ ID No. 6 Rat smoothened SEQ ID No. 3 SEQ ID No. 7 Chicken smoothened SEQ ID No. 4 SEQ ID No. 8 The overall sequence identity between the smoothened proteins is shown in Table 2.

Table 2 Amino acid sequence identity between smoothened proteins. Chicken Chicken-Human Human64%-Rat Rat63%93%-Drosophila Drosophila25%31%30%

It is contemplated by the present invention that the cloned smoothened genes set out in the appended sequence listing, in addition to representing an inter-species family of related genes, are also each part of an intra-species family. That is, it is anticipated that other paralogs of the human and mouse smoothened proteins exist in those animals in much the same manner as multiple frizzled proteins have been identified, and orthologs of each smoothened gene are conserved amongst other animals.

In addition to the sequence variation between the various smoothened homologs, the vertebrate smoothened proteins are apparently present naturally in a number of different forms, including a pro-form. The pro-form includes an N-terminal signal peptide for directed secretion of at least the first extracellular domain of the protein, while the full- length mature form may lack this signal sequence. Further processing of the mature form may also occur in some instances to yield biologically active extracellular or intracellular

fragments of the protein. The smoothened proteins may also be modified post- translationally, such as by O-, S-and/or N-linked glycosylation. Potential Asn- glycosylation sites are shown in figure 4.

Smo mutants display phenotypes similar to the hh mutants, both in embryos and in clones of mutant cells in drosophila imaginal disks. Moreover, the effects of ectopic hedgehog become negligible when cells lack smoothened. When embryos lack both smoothened and patched, they have a similar phenotype to smo single mutants, indicating that patched is located genetically upstream of smoothened. It is postulated that secreted hedgehog protein binds to patched. This binding relieves the patched-dependent inhibition of smoothened, and may involve direct contact between smoothened and patched. Once it is relived from inhibitory signals, smoothened activates the downstream genes wg (Wnts in vertebrates), dpp (TGFß proteins) and patched, through the signaling components fused, costal-2 and cubitus interruptus (Gli's). This pathway may also involve the inhibition of protein kinase A (PKA), though the latter may merely act in parallel. In this case, smoothened would have a constitutive (hedgehog-independent) activity in the absence of any inhibition by patched.

Accordingly, certain aspects of the present invention relate to nucleic acids encoding smoothened polypeptides, the smoothened polypeptides themselves (including various fragments), antibodies immunoreactive with smoothened proteins, and preparations of such compositions. Moreover, the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression (or loss thereof) of smoothened, smoothened ligands, or signal transducers thereof.

In addition, drug discovery assays are provided for identifying agents which can modulate the biological function of smoothened proteins, such as by altering the interaction of smoothened and patched proteins, or other extracellular/matrix factors, or the ability of smoothened proteins to transduce intracellular signals. Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue, particularly a mesodermally-derived tissue, such cartilage, tissue involved in spermatogenesis and tissue derived from dorsal mesoderm; ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; endodermally-derived tissue, such as tissue derived from the primitive gut. Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.

For convenience, certain terms employed in the specification and appended claims are collected here.

The term"smoothened"polypeptide refers to a family of polypeptides characterized at least in part by being identical or sharing a degree of sequence homology with all or a

portion of the a smoothened polypeptide represented in any of SEQ ID Nos: 5-8. The smoothened polypeptides can be cloned or purified from any of a number of eukaryotic organisms, especially vertebrates, and particularly mammals. Moreover, other smoothened polypeptides can be generated according to the present invention, which polypeptides do not ordinarily exist in nature, but rather are generated by non-natural mutagenic techniques.

A"transmembrane"region refers to sequence of amino acids that is located in the cellular membrane, e. g., retained in the membrane at the cell surface.

A"glycosylated"smoothened polypeptide is an smoothened polypeptide having a covalent linkage with a glycosyl group (e. g. a derivatized with a carbohydrate). For instance, the smoothened protein can be glycosylated on an existing residue, or can be mutated to preclude carbohydrate attachment, or can be mutated to provide new glycosylation sites, such as for N-linked or O-linked glycosylation.

As used herein, the term"vertebrate hedgehog protein"refers to vertebrate inter- cellular signaling molecules related to the Drosophilia hedgehog protein. Three of the vertebrate hedgehog proteins, Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including amphibians, fish, birds, and mammals. Other members of this family, such as Banded hedgehog, Cephalic hedgehog, tiggy-winkle hedgehog, and echidna hedgehog have been so far identified in fish and/or amphibians. Exemplary hedgehog polypeptides are described in PCT applications W096/17924, W096/16668, W095/18856.

As used herein, the term"nucleic acid"refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

As used herein, the term"gene"or"recombinant gene"refers to a nucleic acid comprising an open reading frame encoding a smoothened polypeptide, including both exon and (optionally) intron sequences. A"recombinant gene"refers to nucleic acid encoding a smoothened polypeptide and comprising smoothened-encoding exon sequences, though it may optionally include intron sequences which are derived from, for example, a chromosomal smoothened gene (see SEQ ID NO. 9) or from an unrelated chromosomal gene. Exemplary recombinant genes encoding the subject smoothened polypeptide are represented in the appended Sequence Listing. The term"intron"refers to a DNA sequence present in a given smoothened gene which is not translated into protein and is generally found between exons.

As used herein, the term"transfection"means the introduction of a nucleic acid, e. g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

"Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a smoothened polypeptide or, where anti- sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the smoothened protein is disrupted.

As used herein, the term"specifically hybridizes"refers to the ability of a nucleic acid probe/primer of the invention to hybridize to at least 15 consecutive nucleotides of a smoothened gene, such as a smoothened sequence designated in any one or more of SEQ ID Nos: 1-4, or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e. g., mRNA or genomic DNA) encoding a protein other than a smoothened protein, as defined herein.

An"effective amount"of a hedgehog polypeptide, or a bioactive fragment thereof, with respect to the subject method of treatment, refers to an amount of agonist or antagonist in a preparation which, when applied as part of a desired dosage regimen, provides modulation of growth, differentiation or survival of cells, e. g., modulation of spermatogenesis, neuronal differentiation, or skeletogenesis, e. g., osteogenesis, chondrogenesis, or limb patterning.

As used herein,"phenotype"refers to the entire physical, biochemical, and physiological makeup of a cell, e. g., having any one trait or any group of traits.

The terms"induction"or"induce", as relating to the biological activity of a hedgehog protein, refers generally to the process or act of causing to occur a specific effect on the phenotype of cell. Such effect can be in the form of causing a change in the phenotype, e. g., differentiation to another cell phenotype, or can be in the form of maintaining the cell in a particular cell, e. g., preventing dedifferentation or promoting survival of a cell.

A"patient"or"subject"to be treated can mean either a human or non-human animal.

As used herein, the term"vector"refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i. e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as"expression vectors". In general, expression

vectors of utility in recombinant DNA techniques are often in the form of"plasmids"which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification,"plasmid"and"vector"are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

"Transcriptional regulatory sequence"is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant smoothened gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring forms of smoothened genes.

As used herein, the term"tissue-specific promoter"means a DNA sequence that serves as a promoter, i. e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin. The term also covers so- called"leaky"promoters, which regulate expression of a selected DNA primarily in one tissue, but can cause at least low level expression in other tissues as well.

As used herein, the term"target tissue"refers to connective tissue, cartilage, bone tissue or limb tissue, which is either present in an animal, e. g., a mammal, e. g., a human or is present in in vitro culture, e. g., a cell culture.

As used herein, a"transgenic animal"is any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In an exemplary transgenic animal, the transgene causes cells to express a recombinant form of a smoothened protein, e. g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant smoothened gene is silent are also contemplated, as for

example, the FLP or CRE recombinase dependent constructs described below. Moreover, "transgenic animal"also includes those recombinant animals in which gene disruption of one or more smoothened genes is caused by human intervention, including both recombination and antisense techniques.

The"non-human animals"of the invention include vertebrates such as rodents, non- human primates, livestock, avian species, amphibians, reptiles, etc. The term"chimeric animal"is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term "tissue-specific chimeric animal"indicates that a recombinant smoothened gene is present and/or expressed or disrupted in some tissues but not others.

As used herein, the term"transgene"means a nucleic acid sequence (encoding, e. g., a smoothened polypeptide, or pending an antisense transcript thereto), which is partly or entirely heterologous, i. e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e. g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity. The term"DNA sequence encoding a smoothened polypeptide"may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individuals of the same species, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.

"Homology"refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An"unrelated"or "non-homologous"sequence shares less than 40 percent identity, though preferably less than 25 percent identity, with a smoothened sequence of the present invention.

The term"ortholog"refers to genes or proteins which are homologs via speciation, e. g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species. The term"paralog"refers to genes or proteins which are homologs via gene duplication, e. g., duplicated variants of a gene within a genome. See also, Fritch, WM (1970) Syst Zool 19: 99-113.

"Cells,""host cells"or"recombinant host cells"are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A"chimeric protein"or"fusion protein"is a fusion of a first amino acid sequence encoding a smoothened polypeptide with a second amino acid sequence defining a domain (e. g. polypeptide portion) foreign to and not substantially homologous with any domain of a smoothened protein. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an"interspecies","intergenic", etc. fusion of protein structures expressed by different kinds of organisms. In general, a fusion protein can be represented by the general formula X-smoothened-Y, wherein smoothened represents a portion of the fusion protein which is derived from a smoothened protein, and X and Y are, independently, absent or represent amino acid sequences which are not related to a smoothened sequences in an organism.

As used herein, a"reporter gene construct"is a nucleic acid that includes a"reporter gene"operatively linked to a transcriptional regulatory sequences. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these control sequences is directly or indirectly regulated by a signal transduction pathway involving a phospholipase, e. g., is directly or indirectly regulated by a second messenger produced by the phospholipase activity. The transcriptional regulatory sequences can include a promoter and other regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or regulatory sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or regulatory sequences that are recognized by effector molecules, including those that are specifically induced upon activation of a phospholipase. For example, modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.

Such sequences are herein collectively referred to as transcriptional regulatory elements or sequences. In addition, the construct may include sequences of nucleotides that alter the

stability or rate of translation of the resulting mRNA in response to second messages, thereby altering the amount of reporter gene product.

The term"isolated"as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding a smoothened polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the smoothened gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid"is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

As described below, one aspect of the invention pertains to isolated nucleic acids comprising nucleotide sequences encoding smoothened polypeptides, and/or equivalents of such nucleic acids. The term nucleic acid as used herein is intended to include fragments as equivalents. The term equivalent is understood to include nucleotide sequences encoding functionally equivalent smoothened polypeptides or functionally equivalent peptides having an activity of a smoothened protein such as described herein. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the smoothened coding sequences shown in any one or more of SEQ ID Nos: 1-4 due to the degeneracy of the genetic code. Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i. e., equivalent to about 20-27°C below the melting temperature (Tm) of the DNA duplex formed in about 1M salt) to the nucleotide sequences represented in SEQ ID No: 1-4 or 9. In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and/or SEQ ID No: 4.

Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of a smoothened polypeptide which function in a limited capacity as one of either an agonist (e. g.. mimics or potentiates a bioactivity of the wild-type smoothened protein) or an antagonist (e. g., inhibits a bioactivity of the wild-type smoothened protein), in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein. Thus, specific biological effects can be elicited by treatment with a homolog of limited function. For example. truncated forms of a

smoothened protein, e. g., soluble fragments of the extracellular domain, may competitively inhibit interaction of the wild-type smoothened protein with other proteins (such as patched or a ligand for smoothened).

Homologs of the subject smoothened protein can be generated by mutagenesis, such as by discrete point mutation (s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the smoothened protein from which it was derived. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to patched proteins and competing with wild-type smoothened, or binding to other smoothened interacting proteins. Thus, the smoothened protein and homologs thereof provided by the subject invention may be either positive or negative regulators of cell growth, death and/or differentiation.

In general, polypeptides referred to herein as having an activity of a smoothened protein (e. g., are"bioactive") are defined as polypeptides which include an amino acid sequence corresponding (e. g., identical or similar) to all or a portion of the amino acid sequences of the smoothened protein shown in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8, and which agonize or antagonize all or a portion of the biological/biochemical activities of a naturally occurring smoothened protein. Examples of such biological activity includes the ability to interact with patched. (optionally) interact with hedgehog, regulate fused, costal-2 or Gli activities. The bioactivity of certain embodiments of the subject smoothened polypeptides can be characterized in terms of an ability to promote differentiation and/or maintenance of cells and tissue from mesodermally-derived tissue, such as tissue derived from dorsal mesoderm; ectodermally- origin, such as tissue derived from the neural tube, neural crest, or head mesenchyme; or endodermally-derived tissue, such as tissue derived from the primitive gut.

Other biological activities of the subject smoothened proteins are described herein or will be reasonably apparent to those skilled in the art. According to the present invention, a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally- occurring form of a smoothened protein.

Preferred nucleic acids encode a smoothened polypeptide comprising an amino acid sequence at least 60%, 63%, 70% or 80% homologous, more preferably at least 85% homologous and most preferably at least 93% or 95% homologous with an amino acid sequence of a naturally occurring smoothened protein, e. g., such as represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8. Nucleic acids which encode polypeptides at least about 98-99% homology with an amino acid sequence represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8 are of course also within the scope of the invention, as are nucleic acids identical in sequence with the enumerated

smoothened sequence of the Sequence listing. In one embodiment, the nucleic acid is a cDNA encoding a polypeptide having at least one activity of the subject smoothened polypeptide.

In certain preferred embodiments, the invention features a purified or recombinant smoothened polypeptide. It will be understood that the smoothened protein can include certain post-translational modifications, e. g., glycosylation, phosphorylation and the like, and cleavage of certain sequences, such as pro-sequences.

Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to one or more of the nucleic acids represented by SEQ ID Nos: 1-4 and 9. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.

Nucleic acids, having a sequence that differs from the nucleotide sequences shown in any of SEQ ID Nos: 1-4 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent peptides (i. e., a peptide having a biological activity of a smoothened polypeptide) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in"silent"mutations which do not affect the amino acid sequence of a smoothened polypeptide. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject smoothened polypeptides will exist among, for example, humans. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of a smoothened polypeptide may exist among individuals of a given species due to natural allelic variation.

As used herein, a smoothened gene fragment refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire mature form of a smoothened protein yet which (preferably) encodes a polypeptide which retains some biological activity of the full length protein. Fragment sizes contemplated by the present invention include, for example, 5,10,25,50,75,100, or 200 amino acids in length. In a preferred embodiment of a truncated receptor, the polypeptide will include all or a sufficient portion of a patched-

interacting domain or, as appropriate, a ligand domain or intracellular domain involved in signal transduction.

As indicated by the examples set out below, smoothened protein-encoding nucleic acids can be obtained from mRNA present in cells of metazoan organisms. As further illustrated in the examples, it is also possible to obtain nucleic acids encoding smoothened polypeptides of the present invention from genomic DNA from both adults and embryos.

For example, a gene encoding a smoothened protein can be cloned from either a cDNA or a genomic library in accordance with protocols described herein, as well as those generally known to persons skilled in the art. A cDNA encoding a smoothened protein can be obtained by isolating total mRNA from a cell, such as a mammalian cell, e. g. a human cell, as desired. Double stranded cDNAs can be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. The gene encoding a smoothened protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acid of the invention can be DNA or RNA. A preferred nucleic acid is a cDNA including a nucleotide sequence represented by any one of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, or SEQ ID No: 4.

Another aspect of the invention relates to the use of the isolated nucleic acid in "antisense"therapy. As used herein,"antisense"therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e. g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a subject smoothened protein so as to inhibit expression of that protein, e. g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general,"antisense"therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a smoothened protein. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a smoothened gene. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e. g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U. S.

Patents 5,176,996; 5,264,564; and 5,256,775), or peptide nucleic acids (PNAs).

Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668.

Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, by antagonizing the normal biological activity of a smoothened protein, e. g., by reducing the level of its expression, can be used in the manipulation of tissue, e. g. tissue maintenance, differentiation or growth, both in vivo and ex vivo.

Furthermore, the anti-sense techniques (e. g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a smoothened mRNA or gene sequence) can be used to investigate the role of smoothened in developmental events, as well as the normal cellular function of smoothened in adult tissue.

Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals (described infra).

This invention also provides expression vectors containing a nucleic acid encoding a smoothened polypeptide, operably linked to at least one transcriptional regulatory sequence.

Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the subject smoothened proteins. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding smoothened polypeptides of this invention. Such useful expression control sequences, include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage X, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e. g., Pho5, the promoters of the yeast a-matin factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.

Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In one embodiment, the expression vector includes a recombinant gene encoding a polypeptide having an agonistic activity of a subject smoothened polypeptide, or alternatively, encoding a polypeptide which is an antagonistic form of the smoothened protein. Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein.

Moreover, the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids, e. g., encoding either an agonistic or antagonistic form of a subject smoothened proteins or an antisense molecule described above. Thus, another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of a smoothened polypeptide or antisense molecule in

particular cell types so as to reconstitute the function of, or alternatively, abrogate all or a portion of the biological function of smoothened-induced transcription in a tissue in which the naturally-occurring form of the protein is misexpressed (or has been disrupted); or to deliver a form of the protein which alters proliferation, maintenance or differentiation of tissue, or which inhibits neoplastic or hyperplastic proliferation.

Expression constructs of the subject smoothened polypeptides, as well as antisense constructs, may be administered in any biologically effective carrier, e. g. any formulation or composition capable of effectively delivering the recombinant gene to cells in vivo.

Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e. g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaP04 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e. g. locally or systemically.

Furthermore, it will be recognized that the particular gene construct provided for in vivo transduction of smoothened expression are also useful for in vitro transduction of cells, such as for use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e. g. a cDNA encoding the particular smoothened polypeptide desired. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e. g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid. Retrovirus vectors, adenovirus vectors, adeno-associated and herpes-based virus vectors are exemplary recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a subject smoothened polypeptide in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject smoothened genes by the targeted cell.

Exemplary gene delivery systems of this type include liposomal derived systems, poly- lysine conjugates, and artificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutic smoothened gene can be introduced into a patient-animal by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e. g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.

In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U. S. Patent 5,328,470) or by stereotactic injection (e. g.

Chen et al. (1994) PNAS 91: 3054-3057). A smoothened gene can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al.

( (1994) Cancer Treat Rev 20: 105-115).

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively. where the complete gene delivery system can be produced intact from recombinant cells, e. g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

In yet another embodiment, the subject invention provides a"gene activation" construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous smoothened gene. For instance, the gene activation construct can replace the endogenous promoter of a smoothened gene with a heterologous promoter, e. g., one which causes constitutive expression of the smoothened gene or which causes inducible expression of the gene under conditions different from the normal expression pattern of smoothened. A variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications W093/09222, W095/31560, W096/29411, W095/31560 and W094/12650.

In preferred embodiments, the nucleotide sequence used as the gene activation construct can be comprised of (1) DNA from some portion of the endogenous smoothened gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) heterologous transcriptional regulatory sequence (s) which is to be operably linked to the coding sequence for the genomic smoothened gene upon recombination of the gene activation construct. For use in generating cultures of

smoothened producing cells, the construct may further include a reporter gene to detect the presence of the knockout construct in the cell.

The gene activation construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to provide the heterologous regulatory sequences in operative association with the native smoothened gene. Such insertion occurs by homologous recombination, i. e., recombination regions of the activation construct that are homologous to the endogenous smoothened gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.

The terms"recombination region"or"targeting sequence"refer to a segment (i. e., a portion) of a gene activation construct having a sequence that is substantially identical to or substantially complementary to a genomic gene sequence, e. g., including 5'flanking sequences of the genomic gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.

As used herein, the term"replacement region"refers to a portion of a activation construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.

The heterologous regulatory sequences, e. g., which are provided in the replacement region, can include one or more of a variety elements, including: promoters (such as constitutive or inducible promoters), enhancers, negative regulatory elements, locus control regions, transcription factor binding sites, or combinations thereof. Promoters/enhancers which may be used to control the expression of the targeted gene in vivo include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.

Exp. Med., 169: 13), the human P-actin promoter (Gunning et al. (1987) PNAS 84: 4831- 4835), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al. (1984) Mol. Cell Biol. 4: 1354-1362), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), the SV40 early or late region promoter (Bernoist et al. (1981) Nature 290: 304-310; Templeton et al. (1984) Mol. Cell Biol., 4: 817; and Sprague et al. (1983) J. Virol., 45: 773), the promoter contained in the 3'long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto et al., 1980, Cell, 22: 787-797), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS 82: 3567-71), and the herpes simplex virus LAT promoter (Wolfe et al. (1992) Nature Genetics, 1: 379-384).

In still other embodiments, the replacement region merely deletes a negative transcriptional control element of the native gene, e. g., to activate expression, or ablates a positive control element, e. g., to inhibit expression of the targeted gene.

Another aspect of the present invention concerns recombinant forms of the smoothened proteins. Recombinant polypeptides preferred by the present invention, in addition to native smoothened proteins, are at least 60%, 63%, 64% or 70% homologous, more preferably at least 80% homologous and most preferably at least 85% homologous with an amino acid sequence represented by one or more of SEQ ID Nos: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8. Polypeptides which possess an activity of a smoothened protein (i. e. either agonistic or antagonistic), and which are at least 90%, more preferably at least 93% or 95%, and most preferably at least about 98-99% homologous with SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and/or SEQ ID No: 8 are also within the scope of the invention. Such polypeptides, as described above, include various truncated forms of the protein.

The term"recombinant smoothened polypeptide"refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding a smoothened polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase"derived from", with respect to a recombinant smoothened gene, is meant to include within the meaning of"recombinant protein"those proteins having an amino acid sequence of a native smoothened protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the protein.

The present invention further pertains to recombinant forms of the subject smoothened polypeptides which are encoded by genes derived from a mammal (e. g. a human), reptile or amphibian and which have amino acid sequences evolutionarily related to the smoothened protein represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8. Such recombinant smoothened polypeptides preferably are capable of functioning in one of either role of an agonist or antagonist of at least one biological activity of a wild-type ("authentic") smoothened protein of the appended sequence listing.

The present invention also provides methods of producing the subject smoothened polypeptides. For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the polypeptide to occur. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The recombinant smoothened polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.

In a preferred embodiment, the recombinant smoothened polypeptide is isolated in a

membrane fraction of a liposome. For soluble fragments of the protein. the fusion protein can include a domain which facilitates its purification, such as GST fusion protein or poly (His) fusion protein.

This invention also pertains to a host cell transfected to express recombinant forms of the subject smoothened polypeptides. The host cell may be any eukaryotic or prokaryotic cell, though eukaryotic cells are preferred, especially mammalian cells. Thus, a nucleotide sequence derived from the cloning of smoothened genes, encoding all or a selected portion of a full-length protein, can be used to produce a recombinant form of a smoothened polypeptide via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known proteins, e. g. patched, G protein coupled receptors, as well as a wide range of other transmembrane proteins. Similar procedures, or modifications thereof, can be employed to prepare recombinant smoothened polypeptides by microbial means or tissue-culture technology in accord with the subject invention.

The recombinant smoothened genes can be produced by ligating nucleic acid encoding a smoothened polypeptide into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vectors for production of recombinant forms of the subject smoothened polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of a smoothened polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX- derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used. In an illustrative embodiment, a smoothened polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of a smoothened gene represented in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, or SEQ ID No: 9.

The preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription

units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant smoothened polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).

When it is desirable to express only a portion of a smoothened protein, such as a form lacking a portion of the N-terminus, i. e. a truncation mutant which lacks the signal peptide, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben- Bassat et al. (1987) J. Bacteriol. 169: 751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al. (1987) PNAS 84: 2718-1722). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing smoothened-derived polypeptides in a host which produces MAP (e. g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e. g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of a smoothened protein. For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the smoothened polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of a subject. smoothened protein to which

antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising smoothened epitopes as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of a smoothened protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al. (1989) Nature 339: 385; Huang et al. (1988) J. Virol. 62: 3855; and Schlienger et al. (1992) J. Virol. 66: 2).

The Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of a smoothened polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263: 1719 and Nardelli et al. (1992) J. Immunol. 148: 914). Antigenic determinants of smoothened proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the. smoothened polypeptides of the present invention, particularly truncated forms of the smoothened protein. For example, soluble forms of smoothened polypeptides can be generated as glutathione-S-transferase (GST- fusion) proteins. Such GST-fusion proteins can enable easy purification of the smoothened polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N. Y.: John Wiley & Sons, 1991)).

Techniques for making fusion genes are known to those skilled in the art.

Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.

Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

The smoothened polypeptides may also be chemically modified to create smoothened derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of smoothened proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C- terminus of the polypeptide.

The present invention also makes available isolated smoothened polypeptides which are isolated from, or otherwise substantially free of other cellular proteins, especially receptors and/or other inductive polypeptides which may normally be associated with the smoothened polypeptide. The term"substantially free of other cellular proteins" (also referred to herein as"contaminating proteins") or"substantially pure or purified preparations"are defined as encompassing preparations of smoothened polypeptides having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein. Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein. By "purified", it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins. The term"purified"as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term"pure"as used herein preferably has the same numerical limits as"purified"immediately above."Isolated"and"purified"do not encompass either natural materials in their native state or natural materials that have been separated into components (e. g., in an acrylamide gel) but not obtained either as pure (e. g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e. g. acrylamide or agarose) substances or solutions. In preferred embodiments, purified smoothened preparations will lack any contaminating proteins from the same animal from that smoothened is normally produced, as can be accomplished by recombinant expression of, for example, a mammalian smoothened protein in a yeast or bacterial cell.

As described above for recombinant polypeptides, isolated smoothened polypeptides can include all or a portion of an amino acid sequences corresponding to a smoothened polypeptide represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 or homologous sequences thereto.

Isolated peptidyl portions of smoothened proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using

techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, a smoothened polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e. g., "authentic") smoothened protein, such as by binding to patched. For example, Roman et al.

(1994) EurJBiochem 222: 65-73 describe the use of competitive-binding assays using short, overlapping synthetic peptides from larger proteins to identify binding domains.

Modification of the structure of the subject smoothened polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e. g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications. Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the smoothened polypeptides (though they may be agonistic or antagonistic of the bioactivities of the authentic protein). Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i. e. isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur- containing = cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed. by L.

Stryer, WH Freeman and Co.: 1981). Whether a change in the amino acid sequence of a peptide results in a functional smoothened homolog (e. g. functional in the sense that the resulting polypeptide mimics or antagonizes the authentic form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion

similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.

This invention further contemplates a method for generating sets of combinatorial point mutants of the subject smoothened proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e. g. homologs) that are functional in modulating signal transduction and/or ligand binding. The purpose of screening such combinatorial libraries is to generate, for example, novel smoothened homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together. To illustrate, smoothened homologs can be engineered by the present method to provide selective, constitutive activation of hedgehog activity, or alternatively, to be dominant negative inhibitors of smoothened-dependent signal transduction. For instance, mutagenesis can provide smoothened homologs which are able to bind or signal through intracellular regulatory proteins.

In one aspect of this method, the amino acid sequences for a population of smoothened homologs from different species or other related proteins are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, smoothened homologs from one or more species. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In a preferred embodiment, the variegated library of smoothened variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential smoothened sequences are expressible as individual polypeptides, or as a library.

There are many ways by which such libraries of potential smoothened homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential smoothened sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos.

Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249: 386-390; Roberts et al.

(1992) PNAS 89: 2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al.

(1990) PNAS 87: 6378-6382; as well as U. S. Patents Nos. 5,223,409,5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for a smoothened clone in order to generate a variegated population of smoothened fragments for screening and subsequent selection of bioactive fragments. A variety of techniques are known in the art for generating such libraries, including chemical synthesis. In one embodiment, a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of a smoothened coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with S1 nuclease; and (v) ligating the resulting fragment library into an expression vector. By this exemplary method, an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of smoothened homologs. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.

The invention also provides for reduction of the smoothened protein to generate mimetics, e. g. peptide or non-peptide agents, which are able to disrupt a biological activity of a wild-type smoothened protein, e. g. as inhibitors of protein-protein interactions, such as with patched. Thus, such mutagenic techniques as described above are also useful to map the determinants of the smoothened proteins which participate in protein-protein interactions. Alternatively, a similar system can be used to derive fragments of a patched protein which bind to a smoothened protein and competitively inhibit binding of the full length patched protein.

To further illustrate, the critical residues of either a smoothened protein or a patched protein which are involved in molecular recognition of the other can be determined and used to generate smoothened-derived or patched-derived peptidomimetics which competitively inhibit patchedlsmoothened protein interactions. By employing, for example, scanning mutagenesis to map the amino acid residues of a protein which is involved in binding other proteins, peptidomimetic compounds can be generated which mimic those

residues which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of a smoothened protein. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e. g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e. g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.

(1986) J Med Chem 29: 295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26: 647; and Sato et al.

(1986) J Chem Soc Perkin Trans 1: 1231), and p-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et al. (1986) Biochem Biophys Res Commun 134: 71).

Another aspect of the invention pertains to an antibody specifically reactive with a smoothened protein. For example, by using immunogens derived from a smoothened protein, e. g. based on the cDNA sequences, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e. g., a smoothened polypeptide or an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a smoothened protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of a smoothened protein of a organism, such as a mammal, e. g. antigenic determinants of a protein represented by SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 or closely related homologs (e. g. at least 70% identical, preferably at least 80% identical, and more preferably at least 90% identical). In yet a further preferred embodiment of the present invention, in order to provide, for example, antibodies which are immuno-selective for discrete smoothened homologs, the anti-smoothened polypeptide antibodies do not substantially cross react (i. e. does not react specifically) with a protein which is, for example, less than 85%, 90% or 95% homologous with the selected smoothened. By"not substantially cross react". it is meant that the antibody has a binding affinity for a non-homologous protein which is at least one order of magnitude, more preferably at least 2 orders of magnitude, and even more preferably at least

3 orders of magnitude less than the binding affinity of the antibody for the intended target smoothened.

Following immunization of an animal with an antigenic preparation of a smoothened polypeptide, anti-smoothened antisera can be obtained and, if desired, polyclonal anti- smoothened antibodies isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a smoothened polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with a smoothened polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F (ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F (ab) fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules having affinity for a smoothened protein conferred by at least one CDR region of the antibody.

Both monoclonal and polyclonal antibodies (Ab) directed against authentic smoothened polypeptides, or smoothened variants, and antibody fragments such as Fab, F (ab) 2, Fv and scFv can be used to block the action of a smoothened protein and allow the study of the role of these proteins in, for example, differentiation of tissue. Experiments of this nature can aid in deciphering the role of smoothened proteins that may be involved in control of proliferation versus differentiation, e. g., in patterning and tissue formation.

Antibodies which specifically bind smoothened epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject smoothened polypeptides. Anti-smoothened antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate smoothened protein levels in tissue as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of proliferative or differentiative disorders. Likewise, the ability to

monitor smoothened protein levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of smoothened polypeptides may be measured from cells in bodily fluid, such as in samples of cerebral spinal fluid or amniotic fluid, or can be measured in tissue, such as produced by biopsy. Diagnostic assays using anti-smoothened antibodies can include, for example, immunoassays designed to aid in early diagnosis of a disorder, particularly ones which are manifest at birth. Diagnostic assays using anti-smoothened polypeptide antibodies can also include immunoassays designed to aid in early diagnosis and phenotyping neoplastic or hyperplastic disorders.

Another application of anti-smoothened antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as X gtll, kgtl8-23, kZAP, and RORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.

For instance, A. gtl 1 will produce fusion proteins whose amino termini consist of B- galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a smoothened protein, e. g. orthologs of the smoothened protein from other species, can then be detected with antibodies, as. for example, reacting nitrocellulose filters lifted from infected plates with anti-smoothened antibodies. Positive phage detected by this assay can then be isolated from the infected plate. Thus, the presence of smoothened homologs can be detected and cloned from other animals, as can alternate isoforms (including splicing variants) from humans.

Moreover, the nucleotide sequences determined from the cloning of smoothened genes from organisms will further allow for the generation of probes and primers designed for use in identifying and/or cloning smoothened homologs in other cell types, e. g. from other tissues, as well as smoothened homologs from other organisms. For instance, the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least 15,20,25 or 30 consecutive nucleotides of sense or anti-sense sequence selected from the group consisting of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4 or naturally occurring mutants thereof. For instance, primers based on the nucleic acid represented in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4, can be used in PCR reactions to clone smoothened homologs.

Likewise, probes based on the subject smoothened sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto and able to be detected, e. g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

Such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a smoothened protein, such as by measuring a level of a smoothened-encoding nucleic acid in a sample of cells from a patient-animal; e. g. detecting smoothened mRNA levels or determining whether a genomic smoothened gene has been mutated or deleted.

To illustrate, nucleotide probes can be generated from the subject smoothened genes which facilitate histological screening of intact tissue and tissue samples for the presence (or absence) of smoothened-encoding transcripts. Similar to the diagnostic uses of anti- smoothened antibodies, the use of probes directed to smoothened messages, or to genomic smoothened sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, degenerative disorders marked by loss of particular cell-types, apoptosis, neoplastic and/or hyperplastic disorders (e. g. unwanted cell growth) or abnormal differentiation of tissue. Used in conjunction with immunoassays as described above, the oligonucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a smoothened protein. For instance, variation in polypeptide synthesis can be differentiated from a mutation in a coding sequence.

Accordingly, the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant apoptosis, cell proliferation and/or differentiation. In preferred embodiments, method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding a smoothened-protein, or (ii) the mis-expression of the smoothened gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a smoothened gene, (ii) an addition of one or more nucleotides to a smoothened gene, (iii) a substitution of one or more nucleotides of a smoothened gene, (iv) a gross chromosomal rearrangement of a smoothened gene, (v) a gross alteration in the level of a messenger RNA transcript of a smoothened gene, (vii) aberrant modification of a smoothened gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a smoothened gene, (viii) a non-wild type level of a smoothened-protein, and (ix) inappropriate post-translational modification of a smoothened-protein. As set out below, the present invention provides a large number of assay techniques for detecting lesions in a smoothened gene, and importantly, provides the ability to discern between different molecular causes underlying smoothened-dependent aberrant cell growth, proliferation and/or differentiation.

In an exemplary embodiment, there is provided a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a smoothened gene, such as represented by any one of SEQ ID Nos: 1-4 and 9-14, or naturally occurring mutants thereof, or 5'or 3'flanking sequences or intronic sequences naturally associated with the subject smoothened genes or naturally occurring mutants thereof. The nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected. Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.

In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e. g. U. S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e. g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1944) PNAS 91: 360-364), the later of which can be particularly useful for detecting point mutations in the smoothened gene. In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e. g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to a smoothened gene under conditions such that hybridization and amplification of the smoothened gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.

In still another embodiment, the level of a smoothened-protein can be detected by immunoassay. For instance, the cells of a biopsy sample can be dispersed, and the level of a smoothened-protein present on the surface of each cell can be quantitated by standard immunoassay techniques. In yet another exemplary embodiment, aberrant methylation patterns of a smoothened gene can be detected by digesting genomic DNA from a patient sample with one or more restriction endonucleases that are sensitive to methylation and for which recognition sites exist in the smoothened gene (including in the flanking and intronic sequences). See, for example, Buiting et al. (1994) Human Mol Genet 3: 893-895. Digested DNA is separated by gel electrophoresis, and hybridized with probes derived from, for example, genomic or cDNA sequences. The methylation status of the smoothened gene can be determined by comparison of the restriction pattern generated from the sample DNA with that for a standard of known methylation.

A number of techniques exist in the art for identifying ligands to the smoothened receptor. For instance, expression cloning can be carried out on a cDNA or genomic library by isolating cells which are decorated with a labeled form of the receptor, such as present in

labeled liposomal preparations. In a preferred embodiment, the technique uses the smoothened receptor in an in situ assay for detecting smoothened ligands in cDNA cloned from tissue samples and whole organisms. For instance, the present invention makes use of the RAP-in situ assay (for Receptor Affinity Probe) of Flanagan and Leder (see PCT publications WO 92/06220; and also Cheng et al. (1994) Cell 79: 157-168). This system involves the use of an expression cloning system whereby a smoothened ligand can be cloned on the basis of a cDNA/alkaline phosphatase fusion protein binding to smoothened.

In general, the method comprises (i) providing a hybrid molecule (the affinity probe) including the cDNA-encoded protein covalently bonded to an enzymatically active tag, preferably for which chromogenic substrates exist, (ii) contacting a cell expressing smoothened to form complexes between the probe and smoothened, removing unbound probe, and (iii) detecting the affinity complex using a chromogenic substrate for the enzymatic activity associated with the affinity probe.

Furthermore, by making available purified and recombinant smoothened polypeptides, the present invention facilitates the development of assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subject smoothened proteins, or of their role in the pathogenesis of cellular maintenance, differentiation and/or proliferation and disorders related thereto. In a general sense, the assay evaluates the ability of a compound to modulate binding between a smoothened protein and a molecule, e. g., patched or a smoothened ligand, that interacts with the smoothened protein. Exemplary compounds which can be screened against such smoothened-mediated interactions include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries, such as isolated from animals, plants, fungus and/or microbes.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as"primary"screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with a ligand. Accordingly, in an exemplary screening assay of the present invention, a reaction mixture is generated to include a smoothened polypeptide, compound (s) of interest, and a"target molecule", e. g., a protein, which interacts with the smoothened polypeptide. As set out above, exemplary target molecules include patched, extracellular ligands, as well as other protein and non-protein interacting

molecules which interact with smoothened in the membrane or cytoplasm. Detection and quantification of interaction of the smoothened protein with the target molecule provides a means for determining a compound's efficacy at inhibiting (or potentiating) interaction between the smoothened and the target molecule. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, interaction of the smoothened polypeptide and target molecule is quantitated in the absence of the test compound.

Interaction between the smoothened polypeptide and the target molecule may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled smoothened polypeptides, by immunoassay, by chromatographic detection, or by detecting the intrinsic activity of the acetylase.

Accordingly, in an exemplary screening assay for smoothened therapeutics, the compound of interest is contacted with a mixture including a smoothened protein (e. g., a cell expressing smoothened) and a target molecule under conditions in which the two molecules are ordinarily capable of binding one another. To the mixture is then added a composition containing a test compound. Detection and quantification of smoothened complexes provides a means for determining the test compound's efficacy at inhibiting (or potentiating) complex formation between the molecules. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, formation of the complexes is quantitated in the absence of the test compound.

In an illustrative embodiment, the screening assay includes all or a suitable portion of the smoothened protein, which can be obtained from vertebrate or invertebrate sources.

The smoothened protein can be provided in the screening assay as a whole protein (preferably expressed on the surface of a cell), or alternatively as a fragment of the full length protein which binds to a target molecule, e. g., an extracellular or intracellular domain (s) as appropriate. In other embodiments, the protein can be provided as part of a liposomal preparation or expressed on the surface of a cell. The smoothened protein can derived from a recombinant gene, e. g., being ectopically expressed in a heterologous cell.

For instance, the protein can be expressed on oocytes, mammalian cells (e. g., COS, CHO, 3T3 or the like), or yeast cell by standard recombinant DNA techniques. These recombinant cells can be used for receptor binding, signal transduction or gene expression assays. Marigo et al. (1996) Development 122: 1225-1233 illustrates a binding assay of human hedgehog to chick patched protein ectopically expressed in Xenopus ovocytes.

The assay system of Marigo et al. can be adapted to the present drug screening assays by

ectopic or endogenous expression of a smoothened protein. In the illustrated assay, the amount of smoothenedlpatched complexes can be quantitated by immunoassay or the like.

Complex formation between the smoothened polypeptide and a target molecule may bedetected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabelled, fluorescently labeled, or enzymatically labeled hedgehog polypeptides, by immunoassay, or by chromatographic detection.

Typically, for cell-free assays which utilize an extracellular or intracellular fragment of smoothened, it will be desirable to immobilize either the smoothened polypeptide or the target polypeptide to facilitate separation of complexes from uncomplexed forms of one of the proteins, as well as to accommodate automation of the assay. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/receptor (GST/receptor) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the target polypeptide, e. g. an labeled polypeptide, and the test compound and incubated under conditions conducive to complex formation, e. g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound target polypeptide, and the matrix bead-bound radiolabel determined directly (e. g. beads placed in scintillant), or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the bead, separated by SDS-PAGE gel, and the level of target polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, soluble portions of the smoothened protein can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated receptor molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e. g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

Alternatively, antibodies reactive with the smoothened but which do not interfere with, e. g., patched binding, can be derivatized to the wells of the plate and the receptor trapped in the wells by antibody conjugation. As above, preparations of a smoothened polypeptide and a test compound are incubated in the wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the smoothened or target polypeptide; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the

smoothened or target polypeptide. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein, e. g., fused with alkaline phosphatase, and the amount of fusion protein in the complex can be assessed with a chromogenic substrate of the enzyme, e. g. paranitrophenylphosphate. Likewise, a fusion protein including glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249: 7130).

For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as the anti-smoothened antibodies described herein, can be used. Alternatively, the protein to be detected in the complex can be"epitope tagged"in the form of a fusion protein which includes, in addition to the smoothened polypeptide, a second polypeptide for which antibodies are readily available (e. g. from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e. g., see Ellison et al. (1991) J Biol Chem 266: 21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharamacia, NJ).

Where the desired portion of the smoothened protein cannot be provided in soluble form, liposomal vesicles can be used to provide manipulatable and isolatable sources of the protein. For example, both authentic and recombinant forms of the smoothened protein can be reconstituted in artificial lipid vesicles (e. g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) Cell 68: 809-818; Newton et al. (1983) Biochemistry 22: 6110-6117; and Reber et al. (1987) J Biol Chem 262: 11369- 11374). Thus, in addition to cell-free assays, such as described above, the readily available source of smoothened genes provided by the subject invention also facilitates the generation of cell-based assays for identifying small molecule agonists and antagonists of smoothened activity, e. g., which can be used to mimic or inhibit the effect of hedgehog. In one embodiment, the interaction of smoothened and patched proteins in a cell or liposome is assessed.

In addition to characterizing cells that naturally express the smoothened protein, cells which have been genetically engineered to ectopically express smoothened can be utilized for drug screening assays. As an example, cells which either express low levels or lack expression of the smoothened protein, e. g. Xenopus laevis oocytes, COS cells or yeast cells, can be genetically modified using standard techniques to ectopically express the smoothened protein. (see Marigo et al., supra, for analogous expression of patched).

The resulting recombinant cells, e. g., which express a functional smoothened, can be utilized in receptor binding assays to identify agonist or antagonists its patched binding.

Binding assays can be performed using whole cells. Furthermore, the recombinant cells of the present invention can be engineered to include other heterologous genes encoding proteins involved in smoothened-dependent signal pathways. For example, the gene products of one or more of costal-2 and/orfused can be co-expressed with smoothened in the reagent cell, with assays being sensitive to the functional reconstitution of the smoothened signal transduction cascade.

Alternatively, liposomal preparations using reconstituted smoothened protein can be utilized. Smoothened protein purified from detergent extracts from both authentic and recombinant origins can be reconstituted in artificial lipid vesicles (e. g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) Cell 68: 809-818; Newton et al. (1983) Biochemistry 22: 6110-6117; and Reber et al. (1987) J Biol Chem 262: 11369-11374). The lamellar structure and size of the resulting liposomes can be characterized using electron microscopy. External orientation of the smoothened protein in the reconstituted membranes can be demonstrated, for example, by immunoelectron microscopy.

In addition to binding studies, functional assays can be used to identified modulators, i. e., agonists of smoothened activities. By detecting changes in intracellular signals, such as alterations in second messengers or gene expression in smoothened- expressing cells contacted with a test agent, candidate antagonists and antagonists of smoothened signaling can be identified (e. g., having a hedgehog-like or hedgehog- inhibitory activity).

A number of gene products have been implicated in smoothened-mediated signal transduction, including patched, the transcription factor cubitus interruptus ("Ci"or"Gli" in vertebrates), the serine/threonine kinasefused (fu) and the gene products of costal-2, and suppressor offused.

The interaction of smoothened with patched, or the disruption thereof, sets in motion a cascade involving the activation and inhibition of downstream effectors, the ultimate consequence of which is, in some instances, a detectable change in the transcription or translation of a gene. Potential transcriptional targets of smoothened signaling are the patched gene (Hidalgo and Ingham, 1990 Development 110,291-301; Marigo et al., 1996) and the vertebrate homologs of the drosophila cubitus interruptus gene, the GLI genes (Hui et al. (1994) Dev Biol 162: 402-413). Patched gene expression has been shown to be induced in cells of the limb bud and the neural plate that are responsive to Shh. (Marigo et al. (1996) PNAS, in press; Marigo et al. (1996) Development 122: 1225- 1233). The GLI genes encode putative transcription factors having zinc finger DNA

binding domains (Orenic et al. (1990) Genes & Dev 4: 1053-1067; Kinzler et al. (1990) Mol Cell Biol 10: 634-642). Transcription of the GLI gene has been reported to be upregulated in response to hedgehog in limb buds, while transcription of the GLI3 gene is downregulated in response to hedgehog induction (Marigo et al. (1996) Development 122: 1225-1233). By selecting transcriptional regulatory sequences from such target genes, e. g. from patched or GLI genes, that are responsible for the up-or down regulation of these genes in response to smoothened signaling, and operatively linking such promoters to a reporter gene, one can derive a transcription based assay which is sensitive to the ability of a specific test compound to modify smoothened signaling pathways. Expression of the reporter gene, thus, provides a valuable screening tool for the development of compounds that act as antagonists or antagonist of hedgehog, e. g., which may be useful as neuroprotective agents and the like.

Reporter gene based assays of this invention measure the end stage of the above described cascade of events, e. g., transcriptional modulation. Accordingly, in practicing one embodiment of the assay, a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on smoothened signaling. To identify potential regulatory elements responsive to smoothened signaling present in the transcriptional regulatory sequence of a target gene, nested deletions of genomic clones of the target gene can be constructed using standard techniques. See, for example, Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U. S. Patent 5,266,488; Sato et al. (1995) J Biol Chem 270: 10314-10322; and Kube et al. (1995) Cvtokine 7: 1-7. A nested set of DNA fragments from the gene's 5'-flanking region are placed upstream of a reporter gene, such as the luciferase gene, and assayed for their ability to direct reporter gene expression in smoothened expressing cells. Host cells transiently transfected with reporter gene constructs can be scored for the induction of expression of the reporter gene in the presence and absence of hedgehog to determine regulatory sequences which are responsive to smoothened-dependent signaling.

In practicing one embodiment of the assay, a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on second messengers generated by induction with hedgehog protein. Typically, the reporter gene construct will include a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the hedgehog activity, with the level of expression of the reporter gene providing the hedgehog-dependent and smoothened-dependent detection signal. The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, mRNA expression from the reporter gene may be detected using RNAse protection or RNA-based PCR, or the protein product of the reporter gene may be identified by a characteristic stain or an intrinsic activity. The amount of expression from the reporter gene is then compared to the amount of expression

in either the same cell in the absence of the test compound (or hedgehog) or it may be compared with the amount of transcription in a substantially identical cell that lacks the target receptor protein. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the signal transduction of the smoothened protein, e. g., the test compound is a potential ptc therapeutic.

As described in further detail below, in preferred embodiments the gene product of the reporter is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence. In other preferred embodiments, the reporter or marker gene provides a selective growth advantage, e. g., the reporter gene may enhance cell viability, relieve a cell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.

Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol.

Cell. Biol. 7: 725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216: 362-368).

Transcriptional control elements which may be included in a reporter gene construct include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is induced after modulation of a smoothened signal transduction pathway. The characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.

In yet other embodiments, second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium, phospholipid metabolism

or adenylate cyclase activity are quantitated, for instance, the products of phospholipid hydrolysis IP3, DAG or cAMP could be measured For example, recent studies have implicated protein kinase A (PKA) as a possible component of hedgehoglsmoothened signaling (Hammerschmidt et al. (1996) Genes & Dev 10: 647). High PKA activity has been shown to antagonize hedgehog signaling in these systems. Conversely, inhibitors of PKA will mimic and/or potentiate the action of hedgehog. Although it is unclear whether PKA acts directly downstream or in parallel with hedgehog signaling, it is possible that hedgehog signaling occurs via inhibition of PKA activity. Thus, detection of PKA activity provides a potential readout for the instant assays.

Smoothened may, under certain circumstances, stimulate the activity of phospholipases. Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. Water soluble derivatives of all three inositol lipids (IPI, IP2, IP3) can also be quantitated using radiolabelling techniques or HPLC.

The mobilization of intracellular calcium or the influx of calcium from outside the cell may also be a response to smoothened-dependent signaling, or lack there of. Calcium flux in the reagent cell can be measured using standard techniques. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca++- sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84: 45-56). As an exemplary method of Ca++ detection, cells could be loaded with the Ca++sensitive fluorescent dye fura-2 or indo-l, using standard methods, and any change in Ca++ measured using a fluorometer.

In certain embodiments of the assay, it may be desirable to screen for changes in cellular phosphorylation. As an example, the gene fused (fu) which encodes a serine/threonine kinase has been identified as a potential downstream target in smoothened signaling. (Preat et al., 1990 Nature 347,87-89; Therond et al. 1993, Mech. Dev. 44.65- 80). The ability of compounds to modulate serine/threonine kinase activation could be screened using colony immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci.

USA 81: 7426-7430) using antibodies against phosphorylated serine or threonine residues.

Reagents for performing such assays are commercially available, for example, phosphoserine and phosphothreonine specific antibodies which measure increases in phosphorylation of those residues can be purchased from commercial sources.

After identifying certain test compounds as potential modulators of one or more bioactivities of a smoothened protein, the practitioner of the subject assay will continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo.

Whether for subsequent in vivo testing, or for administration to an animal as an approved

drug, agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.

Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or inhibiting (or alternatively potentiating) proliferation of a cell, by contacting the cells with an agent which modulates smoothened-dependent signal transduction pathways. The subject method could be used to generate and/or maintain an array of different tissue both in vitro and in vivo. A "smoothened therapeutic,"whether inhibitory or potentiating with respect to modulating the activity of a smoothened protein, can be, as appropriate, any of the preparations described above, including isolated smoothened polypeptides (including both agonist and antagonist forms), gene therapy constructs, antisense molecules, peptidomimetics, or agents identified in the drug assays provided herein, e. g., which inhibit or potentiate the interactions of smoothened and patched.

The smoothened therapeutic compounds of the present invention are likely to play an important role in the modulation of cellular proliferation and maintenance of, for example, neuronal, testicular, osteogenic or chondrogenic tissues during disease states. It will also be apparent that, by transient use of modulators of smoothened activities, in vivo reformation of tissue can be accomplished, e. g. in the development and maintenance of organs such as ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes. By controlling the proliferative and differentiative potential for different cells, the subject smoothened therapeutics can be used to reform injured tissue, or to improve grafting and morphology of transplanted tissue. For instance, smoothened antagonists and agonists can be employed in a differential manner to regulate different stages of organ repair after physical, chemical or pathological insult. The present method is also applicable to cell culture techniques.

To further illustrate this aspect of the invention, in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). Once a neuronal cell has become terminally-differentiated it typically will not change to another terminally differentiated cell-type. However, neuronal cells can nevertheless readily lose their differentiated state. This is commonly observed when they are grown in culture from adult tissue, and when they form a blastema during regeneration. The present method provides a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of other trophic factors. In such embodiments of the subject method, the cultured cells can be contacted with a smoothened

therapeutic, e. g., such as an agent identified in the assays described above which potentiate smoothened-dependent hedgehog bioactivities, in order to induce neuronal differentiation (e. g. of a stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of differentiation. Alternatively, an antagonist of hedgehog induction, as certain of the smoothened homologs of the present invention are expected to be, can be used to prevent differentiation of progenitor cells in culture.

To further illustrate uses of smoothened therapeutics which may be either hedgehog agonists or antagonists, it is noted that intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J Exp Biol 123: 265-289; and Freund et al. (1985) J Neurosci 5: 603-616). Fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate behavioral defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells. The differential use of hedgehog agonists and antagonists in the culture can control the timing and type of differentiation accessible by the culture.

In addition to the implantation of cells cultured in the presence of heclgehog agonists and antagonists and other in vitro uses, yet another aspect of the present invention concerns the therapeutic application of a smoothened therapeutics to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system.

The ability of hedgehog protein to regulate neuronal differentiation during development of the nervous system and also presumably in the adult state indicates that certain of the hedgehog proteins, and accordingly smoothened therapeutic which modulate hedgehog bioactivities, can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and premature death which result from loss of differentiation in certain pathological conditions.

In light of this understanding, the present invention specifically contemplates applications of the subject smoothened therapeutics to the treatment of (prevention and/or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar

degenerations; and (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis.

Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treatable with a therapeutic regimen which includes a smoothened therapeutic that acts as a hedgehog agonist. For example, Alzheimer's disease is associated with deficits in several neurotransmitter systems, both those that project to the neocortex and those that reside with the cortex. For instance, the nucleus basalis in patients with Alzheimer's disease have been observed to have a profound (75%) loss of neurons compared to age-matched controls. Although Alzheimer's disease is by far the most common form of dementia, several other disorders can produce dementia.

Several of these are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalamus or the white matter underlying the cerebral cortex. Here, the cognitive dysfunction results from the isolation of cortical areas by the degeneration of efferents and afferents. Huntington's disease involves the degeneration of intrastraital and cortical cholinergic neurons and GABAergic neurons.

Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum. Treatment of patients suffering from such degenerative conditions can include the application of smoothened therapeutics in order to control, for example, differentiation and apoptotic events which give rise to loss of neurons (e. g. to enhance survival of existing neurons) as well as promote differentiation and repopulation by progenitor cells in the area affected.

In addition to degenerative-induced dementias, a pharmaceutical preparation of one or more of the subject smoothened therapeutics can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements. Parkinson's disease, for example, primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident. Also included are neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders. Examples include chronic atrophies such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive bulbar palsies or spinal muscular atrophies. The present method is amenable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion. For instance, a preparation of a smoothened therapeutic can used to treat a restricted form of cerebellar cortical

degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.

In an illustrative embodiment, the subject method is used to treat amyotrophic lateral sclerosis. ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex.

The therapeutic application of a smoothened therapeutic which is a hedgehog agonist can be used alone, or in conjunction with other neurotrophic factors such as CNTF, BDNF or NGF to prevent and/or reverse motor neuron degeneration in ALS patients.

Smoothened therapeutics of the present invention can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle and endocrine tissue (such as glandular tissue). For instance, the subject method can be used to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.

Furthermore, a potential role for certain of the smoothened therapeutics derives from the role of hedgehog proteins in development and maintenance of dendritic processes of axonal neurons. Potential roles for hedgehog agonists consequently include guidance for axonal projections and the ability to promote differentiation and/or maintenance of the innervating cells to their axonal processes. Accordingly, compositions comprising smoothened therapeutics which agonize hedgehog activity, may be employed to support the survival and reprojection of several types of ganglionic neurons sympathetic and sensory neurons as well as motor neurons. In particular, such therapeutic compositions may be useful in treatments designed to rescue, for example, various neurons from lesion-induced death as well as guiding reprojection of these neurons after such damage. Such diseases include, but are not limited to, CNS trauma infarction, infection (such as viral infection with varicella-zoster), metabolic disease, nutritional deficiency, toxic agents (such as cisplatin treatment).

Moreover, certain of the smoothened therapeutics (e. g., which antagonize hedgehog induction) may be useful in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

As appropriate, smoothened therapeutics can be used in nerve prostheses for the repair of central and peripheral nerve damage. In particular, where a crushed or severed axon is intubulated by use of a prosthetic device, certain of smoothened therapeutics can be added to the prosthetic device to increase the rate of growth and regeneration of the

dendridic processes. Exemplary nerve guidance channels are described in U. S. patents 5,092,871 and 4,955,892. Accordingly, a severed axonal process can be directed toward the nerve ending from which it was severed by a prosthesis nerve guide.

In another embodiment, the subject method can be used in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system.

For instance, certain of the smoothened therapeutics which induce differentiation of neuronal cells can be utilized to cause such transformed cells to become either post-mitotic or apoptotic. Treatment with a smoothened therapeutic may facilitate disruption of autocrine loops, such as TGF-P or PDGF autostimulatory loops, which are believed to be involved in the neoplastic transformation of several neuronal tumors. smoothened therapeutics may, therefore, thus be of use in the treatment of, for example, malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.

Yet another aspect of the present invention concerns the application of the discovery that hedgehog proteins are morphogenic signals involved in other vertebrate organogenic pathways in addition to neuronal differentiation as described above, having apparent roles in other endodermal patterning, as well as both mesodermal and endodermal differentiation processes. As described in the literature, Shh plays a role in proper limb growth and patterning by initiating expression of signaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in the ectoderm. Thus, it is contemplated by the invention that compositions comprising certain of the smoothened therapeutics can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non- neuronal tissue.

In one embodiment, the present invention makes use of the discovery that hedgehog proteins, such as Shh, are apparently involved in controlling the development of stem cells responsible for formation of the digestive tract, liver, lungs, and other organs which derive from the primitive gut. Shh serves as an inductive signal from the endoderm to the mesoderm, which is critical to gut morphogenesis. Therefore, for example, hedgehog agonists can be employed in the development and maintenance of an artificial liver which can have multiple metabolic functions of a normal liver. In an exemplary embodiment, a smoothened therapeutic which acts as a hedgehog agonist can be used to induce differentiation of digestive tube stem cells to form hepatocyte cultures which can be used to populate extracellular matrices, or which can be encapsulated in biocompatible polymers, to form both implantable and extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog agonists can be utilized in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to promote intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, smoothened therapeutics can be employed therapeutically to regulate such organs after physical, chemical or pathological insult. For instance, therapeutic compositions comprising hedgehog agonists can be utilized in liver repair subsequent to a partial hepatectomy. Similarly, therapeutic compositions containing hedgehog agonists can be used to promote regeneration of lung tissue in the treatment of emphysema.

In still another embodiment of the present invention, compositions comprising smoothened therapeutics can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies.

The present invention particularly contemplates the use of smoothened therapeutics which agonize a hedgehog a skeletogenic activity, such as an ability to induce chondrogenesis and/or osteogenesis. By"skeletal tissue deficiency", it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e. g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.

For instance, the present invention makes available effective therapeutic methods and compositions for restoring cartilage function to a connective tissue. Such methods are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease. The present reparative method is also useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery. The present method may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.

In one embodiment of the present invention, the subject method comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a hedgehog agonist, particularly smoothened therapeutic which agonizes Ihh activity, to generate a cartilage repair response in the connective tissue by stimulating the differentiation and/or proliferation of chondrocytes embedded in the tissue. Induction of chondrocytes by treatment with a hedgehog agonist can subsequently result in the synthesis of new cartilage matrix by the treated cells. Such connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons are particularly amenable to treatment in reconstructive and/or regenerative

therapies using the subject method. As used herein, regenerative therapies include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent. The subject method can further be used to prevent the spread of mineralisation into fibrotic tissue by maintaining a constant production of new cartilage.

In an illustrative embodiment, the subject method can be used to treat cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a smoothened, a wrist, a knuckle of either a finger or toe, or a temperomandibular joint. The treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both. To further illustrate, the subject method can be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e. g., a sports injury or excessive wear) or osteoarthritis.

An injection of a smoothened therapeutic into the joint with, for instance, an arthroscopic needle, can be used to treat the afflicted cartilage. In some instances, the injected agent can be in the form of a hydrogel or other slow release vehicle described above in order to permit a more extended and regular contact of the agent with the treated tissue.

The present invention further contemplates the use of the subject method in the field of cartilage transplantation and prosthetic device therapies. To date, the growth of new cartilage from either transplantation of autologous or allogenic cartilage has been largely unsuccessful. Problems arise, for instance, because the characteristics of cartilage and fibrocartilage varies between different tissue: such as between articular, meniscal cartilage, ligaments, and tendons, between the two ends of the same ligament or tendon, and between the superficial and deep parts of the tissue. The zonal arrangement of these tissues may reflect a gradual change in mechanical properties, and failure occurs when implanted tissue, which has not differentiated under those conditions, lacks the ability to appropriately respond. For instance, when meniscal cartilage is used to repair anterior cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue. By promoting chondrogenesis, the subject method can be used to particularly addresses this problem, by causing the implanted cells to become more adaptive to the new environment and effectively resemble hypertrophic chondrocytes of an earlier developmental stage of the tissue. Thus, the action of chondrogenesis in the implanted tissue, as provided by the subject method, and the mechanical forces on the actively remodeling tissue can synergize to produce an improved implant more suitable for the new function to which it is to be put.

In similar fashion, the subject method can be applied to enhancing both the generation of prosthetic cartilage devices and to their implantation. The need for improved treatment has motivated research aimed at creating new cartilage that is based on collagen- glycosaminoglycan templates (Stone et al. (1990) Clin Orthop Relat Red 252: 129), isolated

chondrocytes (Grande et al. (1989) J Orthop Res 7: 208; and Takigawa et al. (1987) Bone Miner 2: 449), and chondrocytes attached to natural or synthetic polymers (Walitani et al.

(1989) J Bone Jt Surg 71B: 74; Vacanti et al. (1991) Plast Reconstr Surg 88: 753; von Schroeder et al. (1991) JBiomed Mater Res 25 : 329; Freed et al. (1993) JBiomed Mater Res 27: 11; and the Vacanti et al. U. S. Patent No. 5,041,138). For example, chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers. The matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs. The cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted. One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.

In one embodiment of the subject method, the implants are contacted with a smoothened therapeutic during the culturing process, such as an Ihh agonist, in order to induce and/or maintain differentiated chondrocytes in the culture in order as to further stimulate cartilage matrix production within the implant. In such a manner, the cultured cells can be caused to maintain a phenotype typical of a chondrogenic cell (i. e. hypertrophic), and hence continue the population of the matrix and production of cartilage tissue.

In another embodiment, the implanted device is treated with a smoothened therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function. As set out above with respect to tissue transplants, the artificial transplants suffer from the same deficiency of not being derived in a setting which is comparable to the actual mechanical environment in which the matrix is implanted. The activation of the chondrocytes in the matrix by the subject method can allow the implant to acquire characteristics similar to the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhance attachment of prosthetic devices. To illustrate, the subject method can be used in the implantation of a periodontal prosthesis, wherein the treatment of the surrounding connective tissue stimulates formation of periodontal ligament about the prosthesis, as well as inhibits formation of fibrotic tissue proximate the prosthetic device.

In still further embodiments, the subject method can be employed for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient. Indian hedgehog is particularly associated with the hypertrophic chondrocytes that are ultimately replaced by osteoblasts. For instance, administration of a smoothened therapeutic of the

present invention can be employed as part of a method for treating bone loss in a subject, e. g. to prevent and/or reverse osteoporosis and other osteopenic disorders, as well as to regulate bone growth and maturation. For example, preparations comprising hedgehog agonists can be employed, for example, to induce endochondral ossification, at least so far as to facilitate the formation of cartilaginous tissue precursors to form the"model"for ossification. Therapeutic compositions of smoothened therapeutics can be supplemented, if required, with other osteoinductive factors, such as bone growth factors (e. g. TGF-ß factors, such as the bone morphogenetic factors BMP-2 and BMP-d, as well as activin), and may also include, or be administered in combination with, an inhibitor of bone resorption such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or related compounds.

However, it will be appreciated that hedgehog proteins, such as Ihh and Shh are likely to be upstream of BMPs, e. g. treatment with a hedgehog agonist will have the advantage of initiating endogenous expression of BMPs along with other factors.

In yet another embodiment, the smoothened therapeutic of the present invention can be used in the treatment of testicular cells, so as to modulate spermatogenesis. In light of the finding that hedgehog proteins are involved in the differentiation and/or proliferation and maintenance of testicular germ cells, hedgehog antagonist can be utilized to block the action of a naturally-occurring hedgehog protein. In a preferred embodiment, the smoothened therapeutic inhibits the biological activity of Dhh with respect to spermatogenesis, by competitively binding hedgehog in the testis. That is, the smoothened therapeutic can be administered as a contraceptive formulation. Alternatively, smoothened therapeutics which agonize the spermatogenic activity of Dhh can be used as fertility enhancers. In similar fashion, hedgehog agonists and antagonists are potentially useful for modulating normal ovarian function.

The source of the smoothened therapeutics to be formulated will depend on the particular form of the agent. Small organic molecules and peptidyl fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical/cosmetic usage. Products of natural extracts can be purified according to techniques known in the art.

The smoothened therapeutic formulations used in the method of the invention are most preferably applied in the form of appropriate compositions. As appropriate compositions there may be cited all compositions usually employed for systemically or locally (such as intrathecal) administering drugs. The pharmaceutically acceptable carrier should be substantially inert, so as not to act with the active component. Suitable inert carriers include water, alcohol polyethylene glycol, mineral oil or petroleum gel, propylene glycol and the like.

To prepare the pharmaceutical compositions of this invention, an effective amount of the particular. smoothened therapeutic as the active ingredient is combined in intimate

admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represents the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. In the compositions suitable for percutaneous administration. the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin.

It is especially advantageous to formulate the subject compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powders packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.

The pharmaceutical preparations of the present invention can be used, as stated above, for the many applications which can be considered cosmetic uses. Cosmetic compositions known in the art, preferably hypoallergic and pH controlled are especially preferred, and include toilet waters, packs, lotions, skin milks or milky lotions. The preparations contain, besides the smoothened therapeutic, components usually employed in such preparations. Examples of such components are oils, fats. waxes, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, perfumes, dyestuffs, lower alkanols, and the like. If desired, further ingredients may be incorporated in the compositions, e. g. anti-inflammatory agents,

antibacterials, antifungals, disinfectants, vitamins, sunscreens, antibiotics, or other anti-acne agents.

Examples of oils comprise fats and oils such as olive oil and hydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons such as liquid paraffin, ceresin, and squalane; fatty acids such as stearic acid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and esters such as isopropyl myristate, isopropyl palmitate and butyl stearate. As examples of surfactants there may be cited anionic surfactants such as sodium stearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; ampholytic surfactants such as alkylaminoethylglycine hydrochloride solutions and lecithin; and nonionic surfactants such as glycerin monostearate, sorbitan monostearate, sucrose fatty acid esters, propylene glycol monostearate, polyoxyethylene oleylether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropylene glycol (e. g. the materials sold under the trademark "Pluronic"), polyoxyethylene castor oil, and polyoxyethylene lanolin. Examples of humectants include glycerin, 1, 3-butylene glycol, and propylene glycol ; examples of lower alcohols include ethanol and isopropanol; examples of thickening agents include xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol and sodium carboxymethyl cellulose; examples of antioxidants comprise butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid and ethoxyquin; examples of chelating agents include disodium edetate and ethanehydroxy diphosphate; examples of buffers comprise citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate; and examples of preservatives are methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dehydroacetic acid, salicylic acid and benzoic acid.

For preparing ointments, creams, toilet waters, skin milks, and the like, typically from 0.01 to 10% in particular from 0.1 to 5% and more in particular from 0.2 to 2.5% of the active ingredient, e. g., of the smoothened therapeutic, will be incorporated in the compositions. In ointments or creams, the carrier for example consists of 1 to 20%, in particular 5 to 15% of a humectant, 0.1 to 10% in particular from 0.5 to 5% of a thickener and water; or said carrier may consist of 70 to 99%, in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5 to 15% of a fat; or 80 to 99.9% in particular 90 to 99% of a thickener; or 5 to 15% of a surfactant, 2-15% of a humectant, 0 to 80% of an oil, very small (< 2%) amounts of preservative, coloring agent and/or perfume, and water. In a toilet water, the carrier for example consists of 2 to 10% of a lower alcohol, 0.1 to 10% or in particular 0.5 to 1% of a surfactant, 1 to 20%, in particular 3 to 7% of a humectant, 0 to 5% of a buffer, water and small amounts (< 2%) of preservative, dyestuff and/or perfume. In a skin milk, the carrier typically consists of 10-50% of oil, 1 to 10% of surfactant, 50-80% of

water and 0 to 3% of preservative and/or perfume. In the aforementioned preparations, all % symbols refer to weight by weight percentage.

Particular compositions for use in the method of the present invention are those wherein the smoothened therapeutic is formulated in liposome-containing compositions.

Liposomes are artificial vesicles formed by amphiphatic molecules such as polar lipids, for example, phosphatidyl cholines, ethanolamines and serines, sphingomyelins, cardiolipins, plasmalogens, phosphatidic acids and cerebiosides. Liposomes are formed when suitable amphiphathic molecules are allowed to swell in water or aqueous solutions to form liquid crystals usually of multilayer structure comprised of many bilayers separated from each other by aqueous material (also referred to as coarse liposomes). Another type of liposome known to be consisting of a single bilayer encapsulating aqueous material is referred to as a unilamellar vesicle. If water-soluble materials are included in the aqueous phase during the swelling of the lipids they become entrapped in the aqueous layer between the lipid bilayers.

Water-soluble active ingredients are encapsulated in the aqueous spaces between the molecular layers. The lipid soluble active ingredient of a smoothened therapeutic, such as an organic mimetic, is predominantly incorporated into the lipid iayers, although polar head groups may protrude from the layer into the aqueous space. The encapsulation of these compounds can be achieved by a number of methods. The method most commonly used involves casting a thin film of phospholipid onto the walls of a flask by evaporation from an organic solvent. When this film is dispersed in a suitable aqueous medium, multilamellar liposomes are formed. Upon suitable sonication, the coarse liposomes form smaller similarly closed vesicles.

Water-soluble active ingredients are usually incorporated by dispersing the cast film with an aqueous solution of the compound. The unencapsulated compound is then removed by centrifugation, chromatography, dialysis or other art-known suitable procedures. The lipid-soluble active ingredient is usually incorporated by dissolving it in the organic solvent with the phospholipid prior to casting the film. If the solubility of the material in the lipid phase is not exceeded or the amount present is not in excess of that which can be bound to the lipid, liposomes prepared by the above method usually contain most of the material bound in the lipid bilayers; separation of the liposomes from unencapsulated material is not required.

A particularly convenient method for preparing liposome formulated forms of smoothened therapeutics is the method described in EP-A-253,619, incorporated herein by reference. In this method, single bilayered liposomes containing encapsulated active ingredients are prepared by dissolving the lipid component in an organic medium, injecting the organic solution of the lipid component under pressure into an aqueous component

while simultaneously mixing the organic and aqueous components with a high speed homogenizer or mixing means, whereupon the liposomes are formed spontaneously.

The single bilayered liposomes containing the encapsulated smoothened therapeutic can be employed directly or they can be employed in a suitable pharmaceutically acceptable carrier for localized administration. The viscosity of the liposomes can be increased by the addition of one or more suitable thickening agents such as, for example xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose and mixtures thereof. The aqueous component may consist of water alone or it may contain electrolytes, buffered systems and other ingredients, such as, for example, preservatives. Suitable electrolytes which can be employed include metal salts such as alkali metal and alkaline earth metal salts. The preferred metal salts are calcium chloride, sodium chloride and potassium chloride. The concentration of the electrolyte may vary from zero to 260 mM, preferably from 5 mM to 160 mM. The aqueous component is placed in a suitable vessel which can be adapted to effect homogenization by effecting great turbulence during the injection of the organic component. Homogenization of the two components can be accomplished within the vessel, or, alternatively, the aqueous and organic components may be injected separately into a mixing means which is located outside the vessel. In the latter case, the liposomes are formed in the mixing means and then transferred to another vessel for collection purpose.

The organic component consists of a suitable non-toxic, pharmaceutically acceptable solvent such as, for example ethanol, glycerol, propylene glycol and polyethylene glycol, and a suitable phospholipid which is soluble in the solvent. Suitable phospholipids which can be employed include lecithin, phosphatidylcholine, phosphatydylserine, phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholine and phospha- tidyl glycerol, for example. Other lipophilic additives may be employed in order to selectively modify the characteristics of the liposomes. Examples of such other additives include stearylamine, phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of the phospholipids may be added to the organic component. Examples of such other ingredients include tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.

Preservatives such a benzoic acid, methyl paraben and propyl paraben may also be added.

Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an hh at a particular target site.

An essential feature of certain embodiments of the implant can be the linear release of the therapeutic, which can be achieved through the manipulation of the polymer composition and form. By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled.

The selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The generation of such implants is generally known in the art. See, for example, Concise Encylopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U. S. Patent No. 4,883,666.

In another embodiment of an implant, a source of cells producing the therapeutic, e. g., secreting a soluble form of a smoothened ligand, is encapsulated in implantable hollow fibers or the like. Such fibers can be pre-spun and subsequently loaded with the cell source (Aebischer et al. U. S. Patent No. 4,892,538; Aebischer et al. U. S. Patent No. ; Hoffman et al. (1990) Expt. Neurobiol. 110: 39-44; Jaeger et al. (1990) Prog. Brain Res.

82: 41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113: 178-183), or can be co-extruded with a polymer which acts to form a polymeric coat about the cells (Lim U. S. Patent No.

4,391,909; Sefton U. S. Patent No. 4,353,888; Sugamori et al. (1989) Trans. Am. Tartit Intern. Organs 35: 791-799; Sefton et al. (1987) Siotehnol. Bioeng. 29: 1135-1143; and Aebischer et al. (1991) Biomaterials 12: 50-55).

Another aspect of the invention features transgenic non-human animals which express a heterologous smoothened gene of the present invention, and/or which have had one or more genomic smoothened genes disrupted in at least a tissue or cell-types of the animal. Accordingly, the invention features an animal model for developmental diseases, which animal has one or more smoothened allele which is mis-expressed. For example, an animal can be generated which has one or more smoothened alleles deleted or otherwise rendered inactive. Such a model can then be used to study disorders arising from mis- expressed smoothened genes, as well as for evaluating potential therapies for similar disorders.

The transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the"host cell"with respect to regulation by the smoothened protein, e. g., of cell growth, death and/or differentiation. Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described herein and those generally known in the art.

In one embodiment, the transgene construct is a knockout construct. Such transgene constructs usually are insertion-type or replacement-type constructs (Hasty et al. (1991) Mol Cell Biol 11: 4509). The transgene constructs for disruption of a smoothened gene are designed to facilitate homologous recombination with a portion of the genomic smoothened gene so as to prevent the functional expression of the endogenous smoothened gene. In preferred embodiments, the nucleotide sequence used as the knockout construct can be comprised of (1) DNA from some portion of the endogenous smoothened gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) a marker sequence which is used to detect the presence of the knockout construct in the cell.

The knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native smoothened gene. Such insertion can occur by homologous recombination, i. e., regions of the knockout construct that are homologous to the endogenous smoothened gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA. The knockout construct can comprise (1) a full or partial sequence of one or more exons and/or introns of the smoothened gene to be disrupted, (2) sequences which flank the 5'and 3'ends of the coding sequence of the smoothened gene, or (3) a combination thereof.

A preferred knockout construct will delete, by targeted homologous recombination, essential structural elements of an endogenous smoothened gene. For example, the targeting construct can recombine with the genomic smoothened gene can delete a portion of the coding sequence, and/or essential transcriptional regulatory sequences of the gene.

Alternatively, the knockout construct can be used to interrupt essential structural and/or regulatory elements of an endogenous smoothened gene by targeted insertion of a polynucleotide sequence. For instance, a knockout construct can recombine with a smoothened gene and insert a nonhomologous sequence, such as a neo expression cassette, into a structural element (e. g., an exon) and/or regulatory element (e. g., enhancer, promoter, intron splice site, polyadenylation site, etc.) to yield a targeted smoothened allele having an insertional disruption. The inserted nucleic acid can range in size from 1 nucleotide (e. g., to produce a frameshift) to several kilobases or more, and is limited only by the efficiency of the targeting technique.

Depending of the location and characteristics of the disruption, the transgene construct can be used to generate a transgenic animal in which substantially all expression of the targeted smoothened gene is inhibited in at least a portion of the animal's cells. If only regulatory elements are targeted, some low-level expression of the targeted gene may occur (i. e., the targeted allele is"leaky").

The nucleotide sequence (s) comprising the knockout construct (s) can be obtained using methods well known in the art. Such methods include, for example, screening genomic libraries with smoothened cDNA probes in order to identify the corresponding genomic smoothened gene and regulatory sequences. Alternatively, where the cDNA sequence is to be used as part of the knockout construct, the cDNA may be obtained by screening a cDNA library as set out above.

In another embodiment, the"transgenic non-human animals"of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line (s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME). Preferred strains are those with H-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/I. The line (s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i. e., obtained from animals which have one or more genes partially or completely suppressed).

In one embodiment, the transgene construct is introduced into a single stage embryo.

The zygote is the best target for micro-injection. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82: 4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.

Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.

Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from excised tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.

Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage.

During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73: 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the il4ouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82: 6927-6931; Van der Putten et al. (1985) PNAS 82: 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6: 383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298: 623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES).

ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309: 255- 258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322: 445-448). Transgenes can be efficiently introduced into the ES cells by DNA

transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240: 1468-1474.

In one embodiment, gene targeting, which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells. By targeting the smoothened gene in ES cells, these changes can be introduced into the germlines of animals to generate chimeras. The gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a smoothened locus, which which also includes an intended sequence modification to the smoothened genomic sequence (e. g., insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.

Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting a smoothened gene function through the use of a targeting transgene construct designed to undergo homologous recombination with smoothened genomic sequences. Targeting construct can be arranged so that, upon recombination with an element of a smoothened gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted smoothened gene. The inserted sequence functionally disrupts the smoothened gene, while also providing a positive selection trait.

Generally, the embryonic stem cells (ES cells) used to produce the knockout animals will be of the same species as the knockout animal to be generated. Thus for example, mouse embryonic stem cells will usually be used for generation of a smoothened- knockout mice.

Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol. Exp.

Morphol. 87: 27-45). Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is suitable for use herein. The cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRL Press. Washington. D. C.

[1987]); by Bradley et al. (1986) Current Topics in Devel. Biol. and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986]).

Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment. A preferred method of insertion is electroporation.

Each knockout construct to be inserted into the cell must first be in the linear form.

Therefore, if the knockout construct has been inserted into a vector, linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.

For insertion, the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.

If the ES cells are to be electroporated, the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct.

Screening can be accomplished using a variety of methods. Where the marker gene is an antibiotic resistance gene, the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence Alternatively, PCR can be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e. g., P-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed. One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell. All such markers are contemplated as being included within the scope of the teaching of this invention.

The knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events. The desired location of insertion is in a complementary position to the DNA sequence to be knocked out, e. g., the smoothened coding sequence, transcriptional regulatory sequence, etc. Typically, less than about 1-5 percent of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location.

To identify those ES cells with proper integration of the knockout construct, total DNA can be extracted from the ES cells using standard methods. The DNA can then be probed on a

Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with particular restriction enzyme (s). Alternatively, or additionally, the genomic DNA can be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence (i. e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size).

After suitable ES cells containing the knockout construct in the proper location have been identified, the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, the transformed ES cells can be microinjected into blastocytes.

After the ES cell has been introduced into the embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species. The stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent.

Offspring that are born to the foster mother may be screened initially for smoothened disruptants, DNA from tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals.

Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from animals that are the product of this cross, as well as animals that are known heterozygotes and wild type animals.

Other means of identifying and characterizing the knockout offspring are available.

For example, Northern blots can be used to probe the mRNA for the presence or absence of transcripts of either the smoothened gene, the marker gene, or both. In addition, Western blots can be used to assess the (loss of) level of expression of the smoothened gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the smoothened protein, or an antibody against the marker gene product, where this gene is expressed. Finally, in situ analysis (such as fixing the cells and labeling with antibody) and/or FACS (fluorescence activated cell sorting) analysis of various cells from the offspring can be conducted using suitable antibodies or smoothened ligands, e. g., hedgehog proteins, to look for the presence or absence of the knockout construct gene product.

Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways. The preferred manner of preparation is to generate a series of animals, each containing a desired transgenic phenotypes. Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct (s) and/or transgene (s).

Thus, a transgenic avian species can be generated by breeding a first transgenic bird in which the wild-type smoothened gene is disrupted with a second transgenic bird which has been engineered to express a mutant smoothened which retains most other biological functions of the receptor.

The transformed animals, their progeny, and cell lines of the present invention provide several important uses that will be readily apparent to one of ordinary skill in the art.

To illustrate, the transgenic animals and cell lines are particularly useful in screening compounds that have potential as prophylactic or therapeutic treatments of diseases such as may involve aberrant expression, or loss, of a smoothened gene, or aberrant or unwanted activation of receptor signaling. Screening for a useful drug would involve administering the candidate drug over a range of doses to the transgenic animal, and assaying at various time points for the effect (s) of the drug on the disease or disorder being evaluated. Alternatively, or additionally, the drug could be administered prior to or simultaneously with exposure to induction of the disease, if applicable.

In one embodiment, candidate compounds are screened by being administered to the transgenic animal, over a range of doses, and evaluating the animal's physiological response to the compound (s) over time. Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound.

In screening cell lines derived from the subject transgenic animals for compounds useful in treating various disorders, the test compound is added to the cell culture medium at the appropriate time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply the compound of interest to the culture medium in conjunction with co- factors that would enhance the efficacy of the compound.

Exemplification

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

In the Drosophila embryo, spatially restricted expression of the wingless (wg) signaling protein, which is essential for the normal patterning of each segment (reviewed in Klingensmith, J. & Nusse, R. (1994) Dev. Biol. 166: 3966-414), is controlled by the localized activity of hedgehog (hh) in neighboring cells (reviewed in Ingham, P. W. (1995) supra). Expression of hh is in turn maintained by wg activity, and these mutual regulatory interactions stabilize the expression domains of the two genes and hence the parasegment boundaries defined by their interfaces (reviewed in Ingham, P. W. & Martinas-Arias, A.

(1992) Cell 68 : 221-235). In the absence of either gene activity, these boundaries disappear and all patterning and polarity of the segments is lost (Fig. 1 e, f). Embryos homozygous for mutations of the smoothened (smo) locus (formerly named smooth) (Nusslein-Volhard, C. et al. (1984) Roux's Arch. Dev. Biol. 193: 267-282) exhibit phenotypes similar to those of weak alleles of hh or wg, affecting the patterning of each segment in a highly variable manner (Fig. la-c). In all cases, this phenotype is stronger at 18 ° C than at 25 ° C, suggesting that the variability may be due to the hypomorphism of each mutant smo allele.

However. such variability might also be accounted for if maternally derived smo partially compensates for the loss of zygotic gene activity. To investigate this possibility, we generated mosaic females lacking wild-type m ? o alleles in their germ line (smoglC) : smo germline clones) and analyzed the phenotype of homozygous smo embryos from such smoglC females (hereafter designated. smo-embryos). These smo-embryos display an invariant phenotype almost indistinguishable from that of hh null alleles (Fig. Id-f): thus significant amounts of smo product are contributed to the egg during oogenesis, although this contribution is neither sufficient (as smo homozygous from heterozygous females die) nor necessary (as smo heterozygotes derived from single females survive) fir normal development.

The strong similarity between the hh null and the smo phenotypes is suggestive of a role for smo in hh signaling, and consistent with this. smo-embryos lack wg transcription at their parasegment boundaries (Fig 2a, d). As wg transcription also disappears in wg mutant embryos (Bejsovec, A. & Martinez-Arias, A. (1991) Development 113: 471-485; Ingham, P. W. & Hidalgo, A. (1993) Development 117: 283-291; van den Huevel, M. et al. (1993) EMBO J 12: 5293-5302), however, this finding does not in itself exclude a role for smo in wg signaling. To address this issue directly, we examined the ability of each signal too function in the absence of smo activity by expressing either gene ectopically under the control of heterologous regulatory elements in smo-embryos. Expression of wg in alternate parasegments of a smo-embryo results in the partial restoration of posterior naked cuticle in

alternate segments (Fig. I g); by contrast, expression of hh under identical conditions has no effect on the cuticular phenotype of a smo-embryo (Fig. Ih). These findings suggest that smo is required for the activity of hh but not of wg. To confirm this inference, the expression of engrailed (en) and wg, targets of zig and hh activity respectively, were analyzed in each type of embryo. Whereas in a wild-type background, misexpression of hh induces ectopic wg expression in smo embryos, such hh misexpression has no effect on wg transcription (Fig. 2b, c) as would be expected if smo is required for hh signaling. In contrast, ectopic wg expression in smo-embryos restores the expression of en (Fig. 2g-i), confirming that wg signaling can occur in the absence of smo activity. Interestingly, however, transcription of the endogenous wg gene is not itself restored (Fig. 2e, f), implying that, contrary to previous assertions (Bejsovec, A. & Martinas-Arias. A. (1991) supra; Ingham, P. W. & Hidalgo, A. (1993) supra; van den Huevel, M. et al. (1993) supra; Hooper, J. E. (1994) Nature 372: 461-464), wg activity alone is not sufficient to maintain its own expression.

In the developing imaginal discs, expression of hh is restricted to cells of the posterior lineage compartment and, as in the embryo, its activity is required by neighboring anterior compartment cells for the transcription of other signal-encoding genes (reviewed in Blair, S. S. (1995) BioEssays 17 : 299-309). In the case of the wing disc, the principal target of hh activity is the decapentaplegic (dpp) gene, expression of which is restricted to a thin stripe of cells running along the anterior side of the compartment boundary (Raftery, L. A. et al. (1991) Development 113: 27-33) (Fig 3a). To determine whether smo is required for this hh-dependent expression of dpp, we used flippase-induced somatic recombination (Xu, T.

& Rubin, G. M. (1993) Development 117: 1223-1237) to generate mosaic imaginal discs in which small clones of cells lack wild-type smo activity. When such clones are located anterior to the stripe of dpp-expressing cells or anywhere in the posterior compartment (Fig 3a?), the expression of dpp along the border is unaffected.

Expression of dpp is lost, however, from clones of cells lacking smo activity when such clones are located within the normal dpp domain (Fig. 3b-e). Thus smo is required in a cell-autonomous manner for dpp expression. Transcription of dpp can be activated in the wing disc independently of hh by removing the activity of cAMP-dependent protein kinase (PKA), suggesting that PKA acts downstream of hh to antagonize its activity (Kalderon, D.

(1995) Curr. Biol. 5: 580-582). To determine whether such hh independent expression of dpp requires the activity of smo, we generated clones that simultaneously lack smo and the PKA catalytic subunit. Such clones express dpp at any position within the anterior compartment of the disc (Fig. 3f, g), indicating that smo is not absolutely required for dpp transcription; rather, it acts upstream of PKA to mediate activation of dpp by hh.

We mapped the smo locus more precisely by recombination analysis and by using small chromosomal deficiencies, localized it to bands 21 B7-8 on the left arm of chromosome II. Genomic PI clones (Smoller, D. A. et al. (1991) Chromasoma 100: 487- 494) covering this region were obtained and used to screen genomic DNA from newly isolated-ray-induced alleles (Fig. 1) of smo by Southern blot hybridization. We identified a fragment contained within one of the PI clones that consistently detects a novel restriction fragment in digests from the-ray-induced allele smoD16 (Fig. 4a). Northern blot analysis of RNA from adult females revealed three maternally expressed transcripts recognized by this fragment (Fig. 4c). The largest of these (4.1kilobases) shows a size alternation in RNA from smoDl 6 heterozygous females (Fig. 4b), suggesting that it may represent the smo transcript. To investigate this possibility, a genomic fragment including only this transcription unit (Fig. 4c) as transformed into flies and tested for its ability to rescue the smo phenotype. Animals homozygous for smo alleles and carrying one copy of this transgene survive to adulthood and are phenotypically wild type, confirming that this fragment includes the smo transcription unit and all sequences necessary for its expression (data not shown).

The complete nucleotide sequence of the smo transcription unit was determined by sequencing genomic and partial complementary DNA clones (Fig. 4d) to reveal the intron- exon structure shown in Fig. 4c. Conceptual translation of the sequence identified a single long open reading frame of 1,024 amino acids (Fig. 4d) preceded by a 5'untranslated leader of at least 296 nucleotides. Downstream of the translation termination codon is an untranslated region of 654 nucleotides. Hydropathy analysis of the amino acid sequence indicates a putative signal sequence followed by a further seven hydrophobic domains, each of which is long enough to span the membrane once. These features are typical of members of the large family of G-protein-coupled receptors (GPCR); searches of the databases revealed no significant homology to any well characterized GPCR. but did reveal limited homology to members of the Frizzled (Fz) family of serpentine proteins (Wang, Y. et al.

(1996) J. Biol. Chem. 271: 4468-4476) (Fig. 4d). In contrast to the latter, the predicted smo protein contains a long carboxy-terminal extension, which includes consensus target sites for PLA and G-protein-coupled-receptor kinases (Fig. 4d), both of which are involved in GPCR desensitization after agonist-induced activation (Dohlman, H. G. et al. (1991) Annal.

Rev. Biochem. 60: 653-688). On the basis of these features smo may act as a receptor for the hh protein. The involvement of both PKA and a protein with characteristics of GPCR in transduction of the Hh signal is intriguing, given the well established roles of several GPCRs in modulating adenylyl cyclase activity and hence intracellular cyclic AMP levels (Dohlman, H. G. et al. (1991) supra). We note. however, that PKA may not be directly regulated by hh activity, but rather may act in a parallel pathway to antagonize the targets of hh signaling (Kalderon, D. (1995) supra). Moreover, recent studies have emphasized the

diversity of pathways by which the activity of GPCR agonists can be transduced (Daub, H. et al. (1996) Nature 379: 557-560; Grand, R. J. A. et al. (1996) Biochem. J. 313: 353-368).

Earlier analysis has implicated another multiple membrane-spanning protein, encoded by the segment-polarity gene patched (ptc) (Nakano, Y. et al. (1989) Nature 341: 508-513; Hooper, J. & Scott, M. P. (1989) Cell 59: 751-765), in the reception of the Hh signal (Ingham, P. W. et al. (1991) supra). Although our results cast doubt on models of Ptc as the Hh receptor, we emphasize that the functional relationship between Hh, Ptc and Smo remains unclear. As Ptc has a predicted topology reminiscent of proteins involved in transport of ions and small molecule (Nakano, Y. et al. (1989) supra, Hooper, J. & Scott, M. P. (1989) supra), its activity may be regulated by Smo in a manner analogous to that of G-protein-gated ion channels (Clapham, D. E. & Neer. E. J. (1993) Nature 365: 508-513).

Epistasis analysis has, however, placed ptc upstream of smo (Hooper, J. E. (1994) supra), suggesting that there is an unprecedented relationship between these two unusual proteins, the nature of which awaits further analysis.

Utilizing the Drosophila smoothened coding sequence, we have also identified vertebrate homologs of the smoothened gene. A nearly complete chicken sequence was cloned and its sequence is given in SEQ ID No. 4 (nucleotide) and 8 (protein).

All of the above-cited references and publications are hereby incorporated by reference.