WO1999061582A2 - Animaux transgeniques a transduction du signal du 'patched' et leurs utilisations associees - Google Patents

Animaux transgeniques a transduction du signal du 'patched' et leurs utilisations associees Download PDF

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WO1999061582A2
WO1999061582A2 PCT/US1999/011983 US9911983W WO9961582A2 WO 1999061582 A2 WO1999061582 A2 WO 1999061582A2 US 9911983 W US9911983 W US 9911983W WO 9961582 A2 WO9961582 A2 WO 9961582A2
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ptc
cells
gene
cell
animal
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PCT/US1999/011983
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Ervin Epstein, Jr.
Matthew P. Scott
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The Board Of Trustees Of The Leland S. Stanford, Jr. University
The Regents Of The University Of California
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Priority to AU43215/99A priority Critical patent/AU4321599A/en
Publication of WO1999061582A2 publication Critical patent/WO1999061582A2/fr

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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • Patched is a gene that encodes a cell-surface protein that functions both in development and tumorigenesis through its ability to regulate a number of intercellular signaling molecules. Patched was originally identified in Drosophila as a segment polarity gene, one of a group of developmental genes that affect cell differentiation within the individual segments that occur in a homologous series along the anterior-posterior axis of the embryo. See Hooper, J.E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989) Nature 341:508. Patterns of expression of the vertebrate homologue of patched suggest its involvement in the development of neural tube, skeleton, limbs, craniofacial structure, and skin.
  • BCNS basal cell nevus syndrome
  • BCC basal cell carcinomas
  • One aspect of the present invention relates to transgenic non-human animals having germline and/or somatic cells in which the biological activity of one or more ptc genes are disrupted by a chromosomally incorporated transgene, in order to cause a ptc loss-of-function (ptc lo f) phenotype.
  • Another aspect of the present invention relates to transgenic non-human animals having germline and/or somatic cells in which the biological activity of one or more smoothened genes are ectopically activated, e.g., a smoothened gain-of-function (smoS°f) by a chromosomally incorporated transgene.
  • a smoothened gain-of-function e.g., a smoothened gain-of-function (smoS°f) by a chromosomally incorporated transgene.
  • Another aspect of the invention provides a method of evaluating an a test compound for its ability to alter hedgehog signal transduction pathways, e.g., to identify agents with anti-proliferative activity, or which otherwise alter the differentiation state, cause apoptosis, etc.
  • the method includes contacting a transgenic animal of the present invention, or a sample of cells from such animal, with a test agent, and determining the number of cells in the treated sample which are altered, e.g., in their phenotype, in a manner dependent upon addition of the test agent (and the presence of the »tc lof or smoothened ⁇ .
  • a statistically significant decrease in the number of transformed cells, relative to the number of transformed cells in the absense of the test agent indicates the test compound is a potential anti-proliferative agent.
  • Yet another aspect of the invention provides a method for evaluating the carcinogenic potential of an agent by (i) contacting a transgenic animal of the present invention with a test agent, and (ii) comparing the number of transformed cells in a sample from the treated animal with the number of transformed cells in a sample from an untreated transgenic animal or transgenic animal treated with a control agent. The difference in the number of transformed cells in the treated animal, relative to the number of transformed cells in the absence of treatment with a control agent, indicates the carcinogenic potential of the test compound.
  • Figure 1 Generation of the ptc mutation.
  • the ptc mutant allele was generated by homologous recombination between the KOI targeting vector and ptc. External probe A detected a 3 Eco RV polymorphism on blots and probe B detected a 5 Sac I polymorphism. Exons are numbered.
  • B Bam HI; E, Eco RI; RV, Eco RV; S, Sac I; X, Xho I; neo, neomycin resistance gene; TK, thymidine kinase gene; WT, wild type.
  • a and B Lateral views of E8.25 wild-type (WT) (A) and ptc-l- (B) embryos. The headfolds are overgrown in the mutant (white arrows) and the heart is not properly formed (red arrows).
  • C Lateral views of E8.75 ptc+l- and ptc-l- embryos stained with X-Gal (28).
  • D through G Transverse sections through E8.75 ptc+l- (D and F) and ptc-l- (E and G) embryos stained with X-Gal (D and E) or hybridized with a digoxigenin-labeled Gli probe (29) (F and G).
  • Bottle-shaped cells with basal nuclei are indicated by arrows.
  • F and G Transverse sections through E8.5 wild type (F) and ptc-l- (G) embryos hybridized with a Pax6 probe show the absence of expression in ihe ptc+l mutant.
  • H Dorsal view of E8.25 to E8.5 embryos hybridized with a Pax3 probe. Because of the kinking in the neural tube, the ptc-l- embryo is curled on itself. Weak Pax3 expression is seen in the posterior dorsal neural tube of the ptc-l- embryo (bottom, arrow).
  • Figure 4 Skeletal abnormalities and medulloblastomas in ptc+l- mice.
  • A Alcian blue and Alizarin red stained hindlimb from a ptc+l- mouse (30). The preaxial digit is duplicated (arrows).
  • B and C Dorsal views of brains from wild-type (B) and ptc+l- (C) mice. Anterior is up. In the posterior wild-type brain, the colliculi (col) are present as distinct bumps between the cortex (cor) and cerebellum (ce). In the ptc+l- mouse, a massive medulloblastoma (mb, outlined in red) grew over the colliculi and normal cerebellum, which can no longer be seen.
  • mb massive medulloblastoma
  • Figure 5 Derepression of ptc and Gli expression in medulloblastomas from ptc+l- mice.
  • a to C Semi-adjacent sections through a tumor in the cerebellum of a ptc+l- mouse hybridized with 35S- labeled probes to ptc (A), Gli (B), and Shh (C). ptc and Gli transcripts are abundant in the tumors (asterisks) compared with nearby cerebellar tissue (arrows). No Shh was detected in the tumor.
  • D ptc+l- cerebellum (ce) and tumor (mb) stained with X-Gal (28). Anterior is to the left.
  • Figure 6 shows a schematic representation of a double crossover replacement recombination event.
  • Figure 7 shows a schematic representation of an insertion crossover recombination event.
  • Basal cell nevus syndrome is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age. BCNS patients are very susceptible to the development of these tumors; in the second decade of life, large numbers appear, mainly on sun-exposed areas of the skin. This disease also causes a number of developmental abnormalities, including rib, head and face alterations, and sometimes polydactyly, syndactyly, and spina bifida. They also develop a number of tumor types in addition to BCCs: fibromas of the ovaries and heart, cysts of the skin and jaws, and in the central nervous system, medulloblastomas and meningiomas.
  • ptc may be a tumor suppressor gene.
  • the deregulation of the hedgehog signaling pathway may be a general feature of basal cell carcinomas. Consistent overexpression of human ptc mRNA has been described in tumors of familial and sporadic
  • BCCs determined by in situ hybridization. Mutations that inactivate ptc may be expected to result in overexpression of mutant Ptc, because ptc displays negative autoregulation.
  • Evaluating a chemical compound for its potential as a human therapeutic such as for use in treating or preventing basal cell carcinoma or other patched- ⁇ elated disorders, particularly those resulting from ptc lo f, smo8°f and hedgehogs 0 /, necessitates data and information about the compound's efficacy in an in vivo system.
  • the in vivo system used for data collection would be a human being; however, for ethical and pragmatic reasons, laboratory animals, and not humans, are typically used as in vivo screening systems for drug development.
  • the present invention provides a transgenic (non-human) animal in which the normal biological function of one or more tumor suppressors of the patched gene family (herein “ptc gene”) have been functionally inactivated such that, while viable at birth and into adulthood, the animal can be induced to form basal cell carcinomas at a significantly higher frequency relative to the wild-type animal, as for example, upon exposure to DNA damaging agents such as non-ionizing (e.g., UV) or ionizing radiation.
  • DNA damaging agents such as non-ionizing (e.g., UV) or ionizing radiation.
  • the heterozygous ptc knockout mice are viable at birth, but are susceptible to higher incidence of cancers when contacted with DNA damaging agents.
  • mice can be induced to form basal cell carcinomas which, histologically, are similar to BCC in humans.
  • the animals of the present invention are believed to provide the first non-human inducible BCC model.
  • the mouse model is expected to prove useful in developing new therapies for treating human skin cancer.
  • These animals can be used to test compounds for activity against BCC or other skin cancers, as well as, importantly, to test compounds for activity as preventive agents, e.g., for use in topical (sunscreens and the like) or systemic applications, which can diminish the risk of an animal developing skin cancer.
  • the loss-of-function is preferably through direct manipulation of the endogenous ptc gene.
  • the present invention also contemplates transgenic animals having inducible cancer phenotypes resulting from indirect manipulation of other genes involved in hedgehog signal transduction.
  • the transgenic animal has a mutation which, either on a conditional or constitutive basis, results in a loss- of-function of ptc gene function.
  • the present invention provides a transgenic (non-human) animal in which the normal biological function of one or more oncogenes of the smoothened gene family (herein “smo gene”) have been functionally and ectopically activated such that, while viable at birth and into adulthood, the animal can be induced to form basal cell carcinomas at a significantly higher frequency relative to the wild-type animal, as for example, upon exposure to DNA damaging agents such as ionizing radiation.
  • the gain-of- function is preferably through direct manipulation of the endogenous smo gene.
  • ectopically activated means that the expression of the smo gene is abberantly increased, and/or the activity of the smoothened protein is abberantly increased.
  • the smoS°f mouse model is expected to prove useful in developing new therapies for treating human skin cancer and other disorders resulting from abbertant activation of a hedgehog signal pathway(s).
  • the transgenic animal can be generated by any of a number methods.
  • the present invention contemplates a transgenic animal which is generated by disruption of one or more alleles of a. ptc gene, or by expression of an antisense molecule or dominant negative mutant, such that function of a ptc gene is at least partially lost in one or more cells/tissues of the animal.
  • the transgenic animal is designed to be viable into adult life, e.g., by having no more than one allele of a ptc gene constitutively inactivated at birth.
  • Full inactivation of the ptc alleles e.g., to effectively provide a homozygous loss-of-function phenotype, can be the result of inducible inactivation by genetic manipulation or by treatment with a DNA damaging agent.
  • a functional ptc gene can be constitutively disrupted by a "knock-out" mutation effecting, e.g., the transcriptional regulatory sequences, exonic sequences and/or intronic sequences of a ptc gene.
  • the mutation can be, for example, one which results in a prematurely truncated form of the protein (by introduction of a heterologous stop codon or alteration of splicing), a protein with altered hedgehog-binding or smoothened-binding activity, or complete loss of expression of the protein.
  • suitable mutations for generation of the subject ptc transgenic animals are those which have been found to occur in BCC or other tc-related disorders.
  • a ptc gene can be disrupted, such as by homologous recombination, to produce a heterozygous animal for an inactivated ptc allele.
  • the animal in the presence of a second mutagenic event which inactivates the remaining /?tc allele, the animal can develop spontaneous basal cell carcinomas or other tc-dependent lesions.
  • the animal can be treated with a DNA damaging agent, such as irradiation of the animal with ionizing radiation (such as UV) or contact with a chemical carcinogenic agent (such as psoralen, adriamycin or bleomycin).
  • a DNA damaging agent such as irradiation of the animal with ionizing radiation (such as UV) or contact with a chemical carcinogenic agent (such as psoralen, adriamycin or bleomycin).
  • inactivation of the functional ptc allele can be caused through its conditional inactivation, such as by use of an
  • the expression of a ptc gene, from one or both alleles can be disrupted in a conditional manner, e.g., by use of a conditional repressor (such as the TetR system) or other conditional element which disrupts wild-type transcription, splicing or translation of the wild-type gene.
  • a conditional repressor such as the TetR system
  • conditional expression of a dominant negative mutant of a ptc gene can be used to create a ptc loss-of-function phenotype.
  • conditional loss-of-function can itself be sufficient to effectively inactivate both alleles of a ptc gene under the induction conditions, e.g., by generation of homozygous conditional mutation, or can still require a second mutational event such as exposure to a DNA damaging agent where only one allele is disrupted.
  • the transgenic animals may be generated by aberrantly (over)expressing a recombinant hedgehog gene.
  • the function of the ptc protein is disrupted by mutation to a smoothened gene which inhibits interaction of the smoothened protein with a ptc protein.
  • a smoothened gene which inhibits interaction of the smoothened protein with a ptc protein.
  • a ptc loss-of-function phenotype can be mimicked by manipulation of other cellular genes involved in hedgehog signal transduction, as for example, glil, gli3, costal-2, fused, PKA and PKC.
  • the transgenic animals of the present invention provide a means for elucidating the molecular mechanisms involved in skin cancer, and importantly, of the role of the ptc and hedgehog proteins in the mechanism underlying proliferation, death and differentiation of skin cells.
  • a salient feature of the subject transgenic animals derives from the use of these animals in drug discovery assays.
  • heterozygote animals in which a ptc gene allele has been disrupted are viable, but develop both spontaneous and induced tumors. Accordingly, the subject transgenic animal may be used to screen for compounds which are potential anti-proliferative agents useful for preventing or treating such tumors.
  • a ptc +1- animal can be exposed to a DNA damaging agent in an amount and for a period of time sufficient for the animal to develop BCC lesions.
  • a test agent (or agents) can be administered to the animal and the ability of the compound to inhibit or reverse the growth of the lesion can be assessed, along with (optionally) the toxicity of the test agent.
  • the animal can be treated with the test compound prior to exposure to the DNA damaging agent and the ability of the test compound to inhibit formation of lesions, e.g., as a prophylactic, can be assessed after exposure to the DNA damaging agent.
  • such assays can be used to identify compounds or formulations for use as sunscreen agents.
  • Another aspect of the present invention relates to cells obtained from the transgenic animal, which can be derived in culture for a variety of uses. For instance, such cell cultures will be useful for generating drug screening assays to detect compounds which offset the effect of the transgene.
  • ptc loss-of-function refers to an aberrant modification or mutation of a ptc gene, or a decrease (or loss) in the level of expression of the gene, which results in a phenotype which resembles contacting the cell with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway.
  • the loss-of-function may include a loss of the ability of the tc gene product to regulate the level of expression of Ci genes, e.g., GUI, Gli2 and GH3.
  • smoothened gain-of-function refers to an aberrant modification or mutation of a smoothened gene (smo), or an increased level of expression of the gene, which results in a phenotype which resembles contacting the cell with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway. While not wishing to be bound by any particular theory, it is noted that ptc may not signal directly into the cell, but rather interact with smoothened, another membrane-bound protein located downstream of ptc in hedgehog signaling (Marigo et al., (1996) Nature 384: 177-179).
  • the gene smo is a segment-polarity gene required for the correct patterning of every segment in Drosophila (Alcedo et al., (1996) Cell 86: 221-232). Human homologs of smo have been identified. See, for example, Stone et al. (1996) Nature 384:129-134, and GenBank accession U84401.
  • the smoothened gene encodes an integral membrane protein with characteristics of heterotrimeric G protein-coupled receptors; i.e., 7- transmembrane regions. This protein shows homology to the Drosophila Frizzled (Fz) protein, a member of the wingless pathway. It was originally thought that smo encodes a receptor of the Hh signal.
  • allelic modification or mutation of a gene refers to such genetic lesions as, for example, deletions, substitution or addition of nucleotides to a gene, as well as gross chromosomal rearrangements of the gene and/or abnormal methylation of the gene.
  • mis-expression of a gene refers to aberrant levels of transcription of the gene relative to those levels in a normal cell under similar conditions, as well as non-wild type splicing of mRNA transcribed from the gene.
  • Basal cell carcinomas exist in a variety of clinical and histological forms such as nodular-ulcerative, superficial, pigmented, morphealike, fibroepithelioma and provoked syndrome. Basal cell carcinomas are the most common cutaneous neoplasms found in humans. The majority of the 500,000 new cases of nonmelanoma skin cancers each
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • 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
  • the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an expressible nucleotide sequence.
  • the expressible sequence includes an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • the nucleic acid is DNA or RNA.
  • Exemplary recombinant genes include nucleic acids which encode all or a portion of a ptc protein.
  • the expressible nucleotide sequence provides, upon transcription, an mRNA molecule which functions as an antisense construct, e.g. hybridizes to a ptc gene or transcript and inhibits expression of that ptc gene.
  • the term “intron” refers to a DNA sequence present in a given pt gene which is not translated into protein and is generally found between exons.
  • transgene refers to a nucleic acid sequence 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 be operably linked to 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.
  • exemplary transgenes of the present invention encode, for instance: (i) an hedgehog protein; (ii) a smoothed gene encoding a mutant which is insensitive to ptc inhibition; or (iii) an antisense transcript which inhibits expression of one or more ptc genes in the cell in which the transgene is expressed.
  • Other exemplary transgenes are directed to disrupting one or more genomic ptc genes by homologous recombination with genomic sequences of a ptc gene.
  • transgene construct refers to a nucleic acid which includes a transgene, and (optionally) such other nucleic acid sequences as transcriptionally regulatory sequence, polyadenylation sites, replication origins, marker genes, etc., which may be useful in the general manipulation of the transgene for insertion in the genome of a host organism.
  • knockout refers to partial or complete loss of expression of at least a portion of a ptc protein, or a reduction in a hedgehog responsiveness to a ptc protein, in selected cells, or all of the cells of a transgenic animal.
  • knock-in simply refers to a knockout mouse in which a functional heterologous gene is used to disrupt an endogenous ptc gene, such as the lacZ knockout gene described in the appended examples.
  • ptc knockout transgene refers to a nucleotide sequence that decreases or suppresses expression of a ptc protein encoded by endogenous DNA of the transgenic animal's cells.
  • transgene constructs can be derived to include: (i) at least one "recombination region" having a sequence that is substantially identical to or substantially complementary to a ptc gene sequence, or sequences flanking a ptc gene, present in a host cell of an intended transgene recipient, and (ii) a "replacement region" which becomes integrated into the host cell's genome.
  • recombination region refers to a segment (i.e., a portion) of a targeting transgene construct having a sequence that is substantially identical to or substantially complementary to a genomic ptc gene sequence, or sequences flanking a genomic ptc gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.
  • replacement region refers to a portion of a targeting construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.
  • transgene expression construct refers to a transgene construct which, when integrated into the host cell, results in expression of the transgene. "Expression" and
  • express will refer to the production of a transcript, e.g., as in the case of an antisense transgene, as well as the more typical meaning in the art of transcription and translation to produce a polypeptide.
  • 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. Likewise, homology can refer to structural similarities between two polypeptides, e.g. similar domains or motifs.
  • biologically active fragment refers to a nucleotide sequence that is less than the full-length genomic or cDNA nucleotide sequence of a gene, but which contains a sufficient portion of the full length coding sequence that the product of the fragment possesses at least a portion of the biological activity possessed by the gene product of the full length sequence.
  • transfection refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • Transformation 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 one of the subject ptc proteins.
  • Cells or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context. These terms include the immediate subject cell which expresses the cell-cycle regulatory protein of the present invention, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment. However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell. In the present case, such a characteristic might be the ability to produce a recombinant ptc protein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • expression vector includes plasmids, cosmids or phages capable of synthesizing the subject ptc protein encoded by the respective recombinant gene carried by the vector.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector.
  • 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, as well as polyadenylation sites, which induce or control transcription of protein (or antisense) coding sequences with which they are operably linked.
  • transcription of a recombinant ptc gene or ptc antisense construct 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 form of the regulatory protein.
  • 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 an epithelial lineage.
  • tissue-specific promoter also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • operably linked refers to the arrangement of a transcriptional regulatory element relative to other transcribable nucleic acid sequence such that the transcriptional regulatory element can regulate the rate of transcription from the transcribable sequence(s).
  • a "transgenic organisms” is any animal, preferably a non-human mammal, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by trangenic 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.
  • the transgene causes cells combines, by homologous recombination, with an endogeneous ptc gene to inhibit expression of a wild-type ptc protein, e.g., the transgene disrupts an endogenous ptc gene by recombination.
  • the "non-human animals" of the invention include vertebrates such as rodents, non-human primates, sheep, dog, cow, amphibians, birds, fish, reptiles, etc.
  • the term also includes insects, such as Drosphila spp.
  • Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse.
  • 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.
  • tissue-specific chimeric animal indicates that the transgene is present and/or expressed in some tissues but not others.
  • non-human mammal refers to all members of the class Mammalia except humans.
  • rodent refers to all members of the phylogenetic Rodentia including any and all progeny of all future generations derived therefrom.
  • murine refers to any and all members of the family Muridae.
  • founder line and “founder animal” refer to those animals that are the mature product of the embryos to which the transgene was added, i.e., those animals that grew from the embryos into which DNA was inserted, and that were implanted into one or more surrogate hosts.
  • progeny and “progeny of the transgenic animal” refer to any and all offspring of every generation subsequent to the originally transformed mammals.
  • naturally-occurring e.g., as applied a ptc gene or protein, refers to the fact that the gene or protein is identical or highly homologous with the corresponding gene or protein which exists in most animals of that species, and performs substantially the same biological function as the gene or protein occurring in such animals.
  • allelic modification or mutation of a gene refers to such genetic lesions as, for example, deletions, substitution or addition of nucleotides to a gene, as well as gross chromosomal rearrangements of the gene and/or abnormal methylation of the gene.
  • mis-expression of a gene refers to aberrant levels of transcription of the gene relative to those levels in a normal cell under similar conditions, as well as non-wild type splicing of mRNA transcribed from the gene.
  • zygote For instances, it has been known for sometime that it is possible to carry out the genetic transformation of a zygote (and the embryo and mature organism which result therefrom) by the placing or insertion of exogenous genetic material into the nucleus of the zygote or to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote.
  • the genotype of the zygote and the organism which results from a zygote will include the genotype of the exogenous genetic material.
  • the genotype of the exogenous genetic material is expressed upon the cellular division of the zygote.
  • the phenotype expression e.g., the production of a protein product or products of the exogenous genetic material, or alterations of the zygote's or organism's natural phenotype, will occur at that point of the zygote's or organism's development during which the particular exogenous genetic material is active.
  • Alterations of the expression of the phenotype include an enhancement or diminution in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype, including the addition of a new promoter and/or controller or supplementation of an existing promoter and/or controller of the phenotype.
  • the genetic transformation of various types of organisms is disclosed and described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10, 1989.
  • the genetic transformation of organisms can be used as an in vivo analysis of gene expression during differentiation and in the elimination or diminution of genetic diseases.
  • the genetic transformation of a zygote is carried out by the addition of exogenous genetic material in a manner such that the exogenous genetic material becomes part of the nucleic portion of the zygote prior to a division of the zygote. If the exogenous genetic material is added after mitosis or cell division of the zygote, the exogenous genetic material must be added to each resulting nucleus. However, there is a possibility that the exogenous genetic material may not be integrated into and become a part of the genetic material of the zygote and the organism which results therefrom. Thus, the exogenous genetic material can be added to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote, including the zygote nucleus.
  • the nucleic genetic material of the organism being transformed must be in a physical state which enables it to take up the exogenous genetic material.
  • the exogenous genetic material can be placed in the nucleus of a primordial germ cell which is diploid, e.g., a spermatogonium or oogonium.
  • the primordial germ cell is then allowed to mature to a gamete, which is then united with another gamete or source of a haploid set of chromosomes to form a zygote.
  • the exogenous genetic material can be placed in the nucleus of a mature egg. It is preferred that the egg be in a fertilized or activated (by parthenogenesis) state.
  • a complementary haploid set of chromosomes e.g., a sperm cell or polar body
  • the zygote is allowed to develop into an organism such as by implanting it in a pseudopregnant female.
  • the resulting organism is analyzed for the integration of the exogenous genetic material. If positive integration is determined, the organism can be used for the in vivo analysis of the gene expression, which expression is believed to be related to a particular genetic disease.
  • 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 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 below.
  • 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.
  • 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-2 b , H-2 d or H-21 haplotypes such as C57BL/6 or DBA/1.
  • 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) .
  • the transgene construct is introduced into a single stage embryo.
  • the zygote is the best target for micro-injection.
  • the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution.
  • 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.
  • the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.
  • ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.
  • the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
  • the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.
  • the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.
  • Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.
  • transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • 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.
  • a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
  • the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
  • the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
  • a euploid zygote is preferred.
  • the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
  • physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters.
  • the physical effects of addition must not be so great as to physically destroy the viability of the zygote.
  • the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
  • the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences. 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.
  • DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
  • 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.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the transgenic animals produced in accordance with the present invention will include exogenous genetic material.
  • the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a mutant ptc protein, an antisense transcript, or a smoothened or hedgehog mutant, or other protein in the ptc signaling pathway.
  • the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
  • 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.
  • 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 Mouse 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.
  • the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • 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.
  • Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
  • 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.
  • the gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a target ptc locus, and which also includes an intended sequence modification to the ptc 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 ptc gene function through the use of a targeting transgene construct designed to undergo homologous recombination with one ore more ptc genomic sequences.
  • the targeting construct can be arranged so that, upon recombination with an element of a ptc gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted ptc gene.
  • the inserted sequence functionally disrupts the ptc gene, while also providing a positive selection trait.
  • Exemplary ptc targeting constructs are described in more detail below. Simlar techniques can be used to develop transgenic smoS°f, hhS°fo ⁇ the like.
  • 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.
  • mouse embryonic stem cells will usually be used for generation of 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.
  • ES cell line is murine cell line D3 (American Type Culture Collection, catalog no. CKL 1934)
  • WW6 cell line utilized in the appended examples.
  • 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. LRL Press, Washington, D.C. [1987]); by Bradley et al. (1986) Current Topics in Devel Biol.
  • 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.
  • the knockout construct if the knockout construct has been inserted into a vector (described infra), 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., ⁇ -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 ptc coding sequence, transcriptional regulatory sequence, etc.
  • the ptc coding sequence e.g., the ptc coding sequence, transcriptional regulatory sequence, etc.
  • 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.
  • 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).
  • 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).
  • 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, as the appended Examples describe, the transformed ES cells can be microinjected into blastocytes.
  • the suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days.
  • the embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al. (supra).
  • preferred embryos are male.
  • the preferred embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes.
  • the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was incorporated into the developing embryo).
  • the embryo selected will carry genes for black or brown fur.
  • 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. For mice, this stage is about 2-3 days pseudopregnant.
  • Offspring that are born to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above, and in the appended examples) has been employed.
  • DNA from tail 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 mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.
  • the mosiac offspring are crossed to wild-type mates, e.g., to generate animal lines in which all of the cells of the animal are heterozygous, e.g., ptc +/- as the case may be.
  • Northern blots can be used to probe the mRNA f or the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both.
  • Western blots can be used to assess the level of expression of the gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the particular ptc protein, or an antibody against the marker gene product, where this gene is expressed.
  • in situ analysis such as fixing the cells and labeling with antibody
  • FACS fluorescence activated cell sorting
  • knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of a ptc gene can be controlled by recombinase sequences (described infra).
  • 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 mammals, each containing one of the 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) .
  • crossing and backcrossing is accomplished by mating siblings or a parental strain with an offspring, depending on the goal of each particular step in the breeding process.
  • a double knockout can be generated by injecting a single ES cell with both ptc and p53 knockout constructs, and screen for transformed cells in which both constructs integrate into the same chromosome in the same ES cell.
  • two knockout animals are generated, one containing the ptc knockout construct and one containing the p53 knockout construct. These animals can then be bred together and successively interbred and screened until an offspring is obtained that contains both knockout constructs.
  • Exemplary transgenic crosses which can made with any of the subject ptc transgenic animals include the progeny of mating with a second transgenic animal in which another tumor suppressor gene is functionally disrupted or in which an oncogene is overexpressed or has lost negative regulation (functionally overexpressed).
  • the subject ptc disruptants can be crossed with another transgenic animal (of the same species) which is disrupted at at least one locus for a tumor suppressor gene, e.g., p53, DCC, ink4, p21 ci P 1 , p27 ki P 1 , Rb, Mad and/or E2F.
  • the subject ptc disruptants can be crossed with a transgenic animal which overexpresses at least one oncogene, or for which expression and/or bioactivity is deregulated for at least one oncogene, e.g., ras, myc, cdc25A or B, Bcl-2, Bcl-6, transforming growth factors (e.g., TGF ⁇ 's, TGF ⁇ 's, etc.), neu, int-3, polyoma virus middle T antigen, SV40 large T antigen, one or both of the papillomaviral E6 and E7 proteins, CDK4, or cyclin Dl.
  • oncogene e.g., ras, myc, cdc25A or B, Bcl-2, Bcl-6, transforming growth factors (e.g., TGF ⁇ 's, TGF ⁇ 's, etc.), neu, int-3, polyoma virus middle T antigen, SV40 large T antigen, one or both
  • the second transgenic animal can be one in which developmental signals are altered by, e.g., disruption or overexpression of a differentiation factor, such as a TGF ⁇ (e.g. BMPs and the like), hedgehog, dorsalin, neurotrophic factors or the like, or the functional disruption or overexpression of a receptor or signal transduction protein involved in induction of differentiation, such as a neurotrophic factor receptor, TGF ⁇ receptors (such as the activin receptor), dpc-4, WT-1 and the like.
  • a differentiation factor such as a TGF ⁇ (e.g. BMPs and the like), hedgehog, dorsalin, neurotrophic factors or the like
  • a receptor or signal transduction protein involved in induction of differentiation such as a neurotrophic factor receptor, TGF ⁇ receptors (such as the activin receptor), dpc-4, WT-1 and the like.
  • the variety of FI x FI crosses which can be generated arises both from the effect of the transgene itself, as well as the regulation and/or pattern of defect provided by the transgene construct.
  • the crosses can be made between homozygous or heterozygous ptc transgenic animals and a second transgenic animal which can also be either homozygous or heterozygous.
  • the ptc defect of the subject transgenic animals used in the cross-breeding can be tissue-specific, developmentally specific, or ubiquitous, as can the transgenic defect of the mated second transgenic animal.
  • the transgene when under the control of a transcriptional regulatory sequence, the transgene can be regulated in tissue-specific or ubiquitous manners.
  • the regulatory element can provide for constitutive expression or inducible expression.
  • the ptc disruptant described in the appended examples can be crossed with a transgenic animal comprising an activated ras oncogene driven by the Whey acidic protein (WAP) promoter. While the ptc defect will be generalized (e.g., depending on the level of mosiasism), recombinant expression of the ras oncogene will be limited principally to the mammary epithelium of the resulting cross. Such animals can be used, for example, as models for breast cancers.
  • the ptc disruptant in place of the WAP-ras transgene, can be mated with a transgenic animal expressing an oncogene under transcriptional control of a tyrosinase promoter/enhancer element.
  • the mated transgenic animal can include such oncogenes as activated ras, cyclin Dl or the CDK4 R24C mutant under transcriptional regulation of a tyrosinase promoter.
  • Other exemplary embodiments of genetic crosses with the subject ptc transgenic animals include:
  • This transgenic strain is susceptible to the development of skin papillomas and squamous cell carcinomas upon treatment of the skin with phorbol esters (a growth promoter).
  • MMTCV mammary tumor virus
  • E ⁇ -mvc transgenic expresses c-myc under the E ⁇ enhancer promoter (an immunoglobulin promoter specifically expressed in lymphoid cells). This transgenic develops spontaneous B-cell lymphomas (Adams et al., (1985) Nature 318:533-538).
  • Cross with mTR transgenic the mouse gene encoding the RNA component of the telomerase ribonucleoprotein has been cloned (Blasio et al. (1995) Science 269: 1267-1270).
  • Transgenic mice which overexpress MTR, or which have been disrupted for MTR expression can be bred with the subject ptc transgenic animals.
  • Such genetic crosses can provide valuable information and disease models. For instance, the animals can be used to determine the effect of ptc- deficiency on tumor progression (tumors may appear earlier, or they may progress to the most malignant and invasive stages faster).
  • P C-deficiency may affect the type of tumors or their localization, and therefore they may constitute a new animal model for particular human malignancies. These animals may also constitute good animal models to assay chemotherapeutic regimes since they allow the direct comparison between various ptc + and ptc tumors phenotypes.
  • mice ptc-l gene is described in GenBank accession U46155; the mouse ptc-2 gene is described in GenBank accession AB010833; the mouse mouse Shh gene is described in GenBank accession X76290; the mouse Dhh gene is described in GenBank accession G443941; the mouse Ihh gene is described in GenBank accession mouse Ihh U85610; the rat smoothened gene is described in GenBank accession U84402; and the mouse gH3 gene is described in GenBank accession X95255.
  • Vectors used for transforming animal embryos are constructed using methods well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, plasmid and DNA and RNA purification, DNA sequencing, and the like as described, for example in Sambrook, Fritsch, and Maniatis, eds., Molecular Cloning: A Laboratory Manual., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]). Most practitioners are familiar with the standard resource materials as well as specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guideline.
  • transgene constructs of the present invention including appropriate transcriptional/ translational control signals and the desired protein or antisense-encoding nucleotide sequence. See, for example, Maniatis T., Fritsch E.F., and Sambrook J. (1989): Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and Ausubel F.M., Brent R., guitarist R.E., Moore, D.D., Seidman J.G., Smith J.A., and Struhl K. (1992): Current Protocols in Molecular Biology, John Wiley & Sons, New York.
  • coding sequence which may be provided in the transgenes of the present invention are typically operably linked to transcriptional regulatory sequences, such as promoters and/or enhancers, to regulate expression of the transgene in a particular manner.
  • transcriptional regulatory sequences such as promoters and/or enhancers
  • the useful franscriptional regulatory sequences are those that are highly regulated with respect to activity, both temporally and spatially.
  • the promoters of choice can be those that are active only in particular tissues or cell types.
  • the source of the promoter may be from any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate, or any plant. Where the promoter is obtained from a mammal, the mammal may be homologous (the same species as the mammal to be transfected) or non-homologous (a different species).
  • Promoters/enhancers which may be used to control the expression of the transgene in vivo include, but are not limited to, native ptc or smo transcriptional regulatory sequences, the human ⁇ -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) Moi 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
  • herpes simplex virus HSV
  • thymidine kinase promoter/enhancer Wagner et al. (1981) PNAS 82:3567-71
  • herpes simplex virus LAT promoter Wolfe et al. (1992) Nature Genetics, 1 :379-384.
  • the vectors useful for preparing the transgenes of this invention typically contain one or more other elements useful for optimizing expression of the fransgene in the host animal.
  • the fransgene construct may include transcription termination elements, such as to direct polyadenylation of an mRNA transcript, as well as intronic sequences.
  • the transgene can be flanked at its 3' end by SV40 sequences (SV40intron/pA) which add the transcription termination and polyadenylation signals to the transgene transcript.
  • the transgene can include intronic sequence(s) interrupting the coding sequence. In many instances, transcription of a fransgene is increased by the presence of one or more introns in the coding sequence.
  • the fransgene construct can include additional elements which facilitate its manipulation in cells (e.g., bacterial) prior to insertion in the intended recipient cell.
  • the vector may include origin of replication elements for amplification in prokaryotic cells.
  • the transgene construct can include selectable markers for isolating cells, either from the recipient animal, or generated intermediate the transgenic animal (i.e., bacterial cells used for amplifying the construct). Selectable marker genes can encode proteins necessary for the survival and/or growth of transfected cells under selective culture conditions.
  • Typical selection marker genes encode proteins that, for example: (i) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline or kanomycin for prokaryotic host cells, and neomycin, hygromycin or methotrexate for mammalian cells; or (ii) complement auxotrophic deficiencies of the cell.
  • antibiotics or other toxins e.g., ampicillin, tetracycline or kanomycin for prokaryotic host cells, and neomycin, hygromycin or methotrexate for mammalian cells
  • methotrexate for mammalian cells
  • the transcription of the fransgene produces an antisense construct.
  • antisense construct refers to in situ generation of oligonucleotide probes which specifically hybridize (e.g. bind) under cellular conditions with the cellular mRNA and/or genomic DNA encoding a ptc protein so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation of the naturally occurring ptc gene.
  • 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.
  • An antisense construct of the present invention is produced by transcription of the transgene construct in the cell to produce RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a ptc protein, or which transcript can form RNA/DNA complexes with the ptc genomic sequences.
  • the transgene gives rise to a transcriptional product which specifically hybridizes to a transcript from a naturally occurring ptc gene.
  • expression of the transgene can result in an mRNA transcript which is complementary to the mRNA transcript from a naturally occurring ptc gene.
  • Hybridization of the transgene transcript to the ptc mRNA can inhibit translation of the ptc message by, for example, disrupting the ability of ribosomes and other translational proteins to bind to the ptc transcript.
  • the hybridization by the transgene transcript may also destabilize the ptc mRNA and cause more rapid turnover than otherwise would occur.
  • Transgene constructs for expressing antisense molecules can be generated by similar techniques as described above for transgenes encoding ptc polypeptide.
  • the fransgene construct is a knockout construct.
  • Such transgene constructs usually are insertion-type or replacement-type constructs (Hasty et al.
  • the transgene constructs for disruption of a ptc gene are designed to facilitate homologous recombination with a portion of the genomic ptc gene so as to prevent the functional expression of the endogenous ptc gene.
  • the nucleotide sequence used as the knockout construct can be comprised of (1) DNA from some portion of the endogenous ptc gene (exon sequence, infron 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 ptc gene.
  • Such insertion can occur by homologous recombination, i.e., regions of the knockout construct that are homologous to the endogenous ptc 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 ptc gene to be disrupted, (2) sequences which flank the 5' and 3' ends of the coding sequence of the ptc gene, or (3) a combination thereof.
  • a preferred knockout construct will delete, by targeted homologous recombination, essential structural elements of an endogenous ptc gene.
  • the targeting construct can recombine with the genomic ptc gene in order to delete a portion of the coding sequence and/or essential franscriptional regulatory sequences of the gene.
  • the knockout construct can be used to interrupt essential structural and/or regulatory elements of an endogenous ptc gene by targeted insertion of a polynucleotide sequence.
  • a knockout construct can recombine with a ptc 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, infron splice site, polyadenylation site, etc.) to yield a targeted ptc allele having an insertional disruption.
  • a structural element e.g., an exon
  • regulatory element e.g., enhancer, promoter, infron splice site, polyadenylation site, etc.
  • 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.
  • the transgene construct can be used to generate a transgenic animal in which substantially all expression of the targeted ptc 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 ptc cDNA probes in order to identify the corresponding genomic ptc 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.
  • the knockout construct is designed to undergo 5 homologous recombination with a ptc gene.
  • Exemplary knockout constructs are provided by inclusion of a ptc cDNA, which has been mutated to give rise to a frameshift or a premature stop codon in the coding sequence.
  • the knockout fransgene can further include selectable markers, as described above.
  • the proper position for the marker gene insertion is that which will serve to prevent or l o decrease expression of the native ptc gene.
  • genetic techniques are known which allow for the expression of any of the above transgenes to be regulated via site-specific genetic manipulation in vivo.
  • genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination of a target sequence.
  • target sequence refers to a nucleotide sequence, e.g., the fransgene, that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or 0 repression of expression of a ptc gene.
  • excision of a target sequence which interferes with the expression of a recombinant ptc gene can be designed to activate expression of that gene.
  • the fransgene can be made wherein the 5 coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven franscriptional activation.
  • crelloxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of S ⁇ cch ⁇ romyces cerevisi ⁇ e (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems.
  • Cre recombinase catalyzes 5 the site-specific recombination of an intervening target sequence located between loxP sequences.
  • loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation or inactivation of expression of an ptc gene can be regulated via regulation of recombinase expression.
  • crelloxP recombinase system to regulate expression of a recombinant ptc protein requires the construction of a fransgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and the recombinant ptc genes can be provided through the construction of double transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a different fransgene, e.g., a ptc gene and a recombinase gene.
  • One advantage derived from initially constructing fransgenic animals containing a ptc-fransgene in a recombinase-mediated expressible format derives from the likelihood that certain of the ptc transgenes will, either by overexpression or disruption, be deleterious upon to the transgenic animal.
  • a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues.
  • conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the fransgene.
  • Exemplary promoters and the corresponding trans- activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the effect of a ptc transgene can remain silent into adulthood until "turned on" by the introduction of the trans-activator.
  • the fransgenic 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 gain or loss of hedgehog signal transduction, e.g., as may result from ptc lo f or smoS°f.
  • 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.
  • the drug could be administered prior to or simultaneously with exposure to induction of the disease, if applicable.
  • 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.
  • 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.
  • the transgenic animals of the present invention can also be useful in designing a therapeutic regimen aimed at preventing or curing the disease or condition.
  • the animal may be treated with a combination of a particular diet, exercise routine, radiation treatment, and/or one or more compounds or substances either prior to, or simultaneously, or after, the onset of the disease or condition.
  • Such an overall therapy or regimen might be more effective at combating the disease or condition than treatment with a compound alone.
  • Agents to be tested in the animals and cell cultures of the present invention can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly.
  • the test agent is a small organic molecule, e.g., other than a peptide, oligonucleotide, or analog thereof, having a molecular weight of less than about 2,000 daltons.
  • the transgenic animals of the present invention are useful for identifying carcinogens, and evaluating their risk to humans.
  • epidemiology and rodent bioassays are the means by which putative human carcinogens are identified. Both methods have intrinsic limitations: they are slow and expensive processes with many uncertainties.
  • the development of methods to modify specific genes in the mammalian genome has provided promising new tools for identifying carcinogens and characterizing risk.
  • Transgenic mice may provide advantages in shortening the time required for bioassays and improving the accuracy of carcinogen identification; fransgenic mice might now be included in the testing armamentarium without abandoning the two-year bioassay, the current standard. For instance, mutagenic carcinogens can be identified with increased sensitivity and specificity using homozygous ptc mice in which one allele of a ptc gene has been disrupted.
  • UV radiation mediates a number of harmful effects in the human body.
  • Chronic UV exposure is a well-recognized etiological agent for cutaneous squamous cell and basal cell carcinoma. See, for example, Elmets Pharmacology of the Skin. Boca Raton, Fla.; CRC Press, 389-416 (1992). Further, UV exposure may play a role in promoting the development of malignant melanomas. Kob, et al., Photochem. Photobiol. 31 :765-779 (1990).
  • the instant invention specifically contemplates the use of subject method to identify compounds which reduce the occurrence of basal cell carcinoma and other skin cancers resulting from exposure to solar radiation.
  • the subject method can be used to identify agents useful in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system.
  • the ptc agonists e.g., agents which reverse a ptc lo f or a smoS°f phenotype, can be utilized to cause such transformed cells to become either post- mitotic or apoptotic.
  • Such compounds may, therefore, be used as part of a treatment for, e.g., malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymomas.
  • the subject method can be used to idenitify agents useful as part of a treatment regimen for malignant medulloblastoma and other primary CNS malignant neuroectodermal tumors.
  • the subject method is used to idenitify agents useful as part of treatment program for medulloblastoma.
  • Medulloblastoma a primary brain tumor, is the most common brain tumor in children.
  • a medulloblastoma is a primitive neuroectodermal tumor arising in the posterior fossa. They account for approximately 25% of all pediatric brain tumors (Miller).
  • PNET's may arise in other areas of the brain including the pineal gland (pineoblastoma) and cerebrum. Those arising in the supratentorial region generally fare worse than their PF counterparts.
  • Medulloblastoma/PNET's are known to recur anywhere in the CNS after resection, and can even metastasize to bone. Pretreatment evaluation should therefore include an examination of the spinal cord to exclude the possibility of "dropped metastases”.
  • Gadolinium-enhanced MRI has largely replaced myelography for this purpose, and CSF cytology is obtained postoperatively as a routine procedure.
  • the subject method is used to idenitify agents useful as part of treatment program for ependymomas.
  • Ependymomas account for approximately 10% of the pediatric brain tumors in children. Grossly, they are tumors that arise from the ependymal lining of the ventricles and microscopically form rosettes, canals, and perivascular rosettes. In the CHOP series of 51 children reported with ependymomas, 3 ⁇ were histologically benign. Approximately 2/3 arose from the region of the 4th ventricle. One third presented in the supratentorial region. Age at presentation peaks between birth and 4 years, as demonstrated by SEER data as well as data from CHOP. Median age is about 5 years. Because so many children with this disease are babies, and because they often require multimodal therapy.
  • compositions comprising ptc agonists can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non-neuronal tissue.
  • the present invention makes use of the discovery that ptc and smoothened 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 mo ⁇ hogenesis. Therefore, for example, ptc agonists identified instant method can be employed for regulating the development and maintenance of an artificial liver which can have multiple metabolic functions of a normal liver.
  • the subject method can be used to regulate the proliferation and 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 extraco ⁇ oreal artificial livers.
  • the drug screening assays of the present invention can be used to idenitify agents useful in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to regulate uptake of intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue.
  • the subject method can be used to idenitify agents useful to regulate such organs after physical, chemical or pathological insult.
  • therapeutic compositions comprising ptc agonists can be utilized in liver repair subsequent to a partial hepatectomy.
  • the generation of the pancreas and small intestine from the embryonic gut depends on intercellular signalling between the endodermal and mesodermal cells of the gut.
  • the differentiation of intestinal mesoderm into smooth muscle has been suggested to depend on signals from adjacent endodermal cells.
  • One candidate mediator of endodermally derived signals in the embryonic hindgut is Sonic hedgehog. See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4.
  • the Shh gene is expressed throughout the embryonic gut endoderm with the exception of the pancreatic bud endoderm, which instead expresses high levels of the homeodomain protein Ipfl/Pdxl (insulin promoter factor 1 /pancreatic and duodenal homeobox 1), an essential regulator of early pancreatic development.
  • Ipfl/Pdxl insulin promoter factor 1 /pancreatic and duodenal homeobox 1
  • Apelqvist et al., supra have examined whether the differential expression of Shh in the embryonic gut tube controls the differentiation of the surrounding mesoderm into specialised mesoderm derivatives of the small intestine and pancreas. To test this, they used the promoter of the Ipfl/Pdxl gene to selectively express Shh in the developing pancreatic epithelium.
  • pancreatic mesoderm developed into smooth muscle and interstitial cells of Cajal, characteristic of the intestine, rather than into pancreatic mesenchyme and spleen. Also, pancreatic explants exposed to Shh underwent a similar program of intestinal differentiation. These results provide evidence that the differential expression of endodermally derived Shh controls the fate of adjacent mesoderm at different regions of the gut tube.
  • the subject ptc agonists can be used to control the regulate the proliferation and/or differentiation of pancreatic tissue both in vivo and in vitro.
  • the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival and/or affecting proliferation of pancreatic cells, by contacting the cells with the subject inhibitors.
  • the subject method could be used as part of a technique to generate and/or maintain such tissue both in vitro and in vivo.
  • modulation of the function of ptc can be employed in both cell culture and therapeutic methods involving generation and maintenance ⁇ -cells and possibly also for non-pancreatic tissue, such as in controlling the development and maintenance of tissue from the digestive tract, spleen, lungs, and other organs which derive from the primitive gut.
  • the present method can be used in the treatment of hype ⁇ lastic and neoplastic disorders effecting pancreatic tissue, particularly those characterized by aberrant proliferation of pancreatic cells.
  • pancreatic cancers are marked by abnormal proliferation of pancreatic cells which can result in alterations of insulin secretory capacity of the pancreas.
  • certain pancreatic hype ⁇ lasias such as pancreatic carcinomas, can result in hypoinsulinemia due to dysfunction of ⁇ -cells or decreased islet cell mass.
  • the subject inhibitors can be used to enhance regeneration of the tissue after anti-tumor therapy.
  • the present invention makes use of the apparent involvement of ptc and smoothened in regulating the development of pancreatic tissue.
  • the subject method can be employed therapeutically to regulate the pancreas after physical, chemical or pathological insult.
  • the subject method can be applied to to cell culture techniques, and in particular, may be employed to enhance the initial generation of prosthetic pancreatic tissue devices.
  • Manipulation of proliferation and differentiation of pancreatic tissue for example, by altering ptc activity, can provide a means for more carefully controlling the characteristics of a cultured tissue.
  • the subject method can be used to augment production of prosthetic devices which require ⁇ -islet cells, such as may be used in the encapsulation devices described in, for example, the Aebischer et al. U.S. Patent No. 4,892,538, the Aebischer et al. U.S. Patent No. 5,106,627, the Lim U.S. Patent No. 4,391,909, and the Sefton U.S. Patent No. 4,353,888.
  • Early progenitor cells to the pancreatic islets are multipotential, and apparently coactive all the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones, such as insulin, becomes restricted to the pattern of expression characteristic of mature islet cells.
  • the phenotype of mature islet cells is not stable in culture, as reappearence of embyonal traits in mature ⁇ -cells can be observed.
  • the differentiation path or proliferative index of the cells can be regulated.
  • manipulation of the differentiative state of pancreatic tissue can be utilized in conjunction with transplantation of artificial pancreas so as to promote implantation, vascularization, and in vivo differentiation and maintenance of the engrafted tissue.
  • manipulation of ptc function to affect tissue differentiation can be utilized as a means of maintaining graft viability.
  • compositions comprising ptc agonists 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 ptc agonists to regulate the rate of chondrogenesis and/or osteogenesis.
  • 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.
  • the method of the present invention can be used as part of a regimen 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.
  • the subject method comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a ptc agonist, particularly an agonist selective for Indian hedgehog signal transduction, to regulate a cartilage repair response in the connective tissue by managing the rate of differentiation and/or proliferation of chondrocytes embedded in the tissue.
  • articular cartilage 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.
  • 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 be used as part of a therapeutic intervention in the treatment of cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist, a knuckle of either a finger or toe, or a tempomandibular joint.
  • the treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both.
  • 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.
  • the subject method may be used to idenitify agents useful for injection into the joint with, for instance, an arthroscopic needle.
  • 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 to idenitify agents useful in the field of cartilage transplantation and prosthetic device therapies.
  • 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.
  • meniscal cartilage is used to repair anterior cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue.
  • the subject method can be used to particularly address this problem, by helping to adaptively control the implanted cells in the new environment and effectively resemble hyperfrophic chondrocytes of an earlier developmental stage of the tissue.
  • the subject method can be used to idenitify agents 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.
  • 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.
  • the implants are contacted with a ptc agonist during certain stages of the culturing process in order to manage the rate of differentiation of chondrocytes and the formation of hypertrophic chrondrocytes in the culture.
  • the implanted device is treated with a ptc agonist in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
  • 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 ability to regulate 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.
  • the subject method is used to enhance attachment of prosthetic devices.
  • 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.
  • the subject method can be employed as part of a regimen 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.
  • administration of a ptc agonists of the present invention can be employed as part of a method for regulating the rate of bone loss in a subject.
  • preparations comprising ptc agonists can be employed, for example, to control endochondral ossification in the formation of a "model" for ossification.
  • a ptc agonist can be used to regulate spermatogenesis.
  • the hedgehog proteins, particularly Dhh have been shown to be involved in the differentiation and/or proliferation and maintenance of testicular germ cells. Dhh expression is initiated in Sertoli cell precursors shortly after the activation of Sry and persists in the testis into the adult. Males are viable but infertile, owing to a complete absence of mature sperm. Examination of the developing testis in different genetic backgrounds suggests that Dhh regulates both early and late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol 6:298.
  • the ptc agonist can be used as a contraceptive. In similar fashion, ptc agonists of the subject method are potentially useful for modulating normal ovarian function.
  • the subject method also has wide applicability to the treatment or prophylaxis of disorders afflicting epithelial tissue, as well as in cosmetic uses.
  • the method can be characterized as including a step of administering to an animal an amount of a ptc agonist effective to alter the growth state of a treated epithelial tissue.
  • the mode of administration and dosage regimens will vary depending on the epithelial tissue(s) which is to be treated. For example, topical formulations will be preferred where the treated tissue is epidermal tissue, such as dermal or mucosal tissues.
  • a method which "promotes the healing of a wound” results in the wound healing more quickly as a result of the treatment than a similar wound heals in the absence of the treatment.
  • “Promotion of wound healing” can also mean that the method regulates the proliferation and/or growth of, inter alia, keratinocytes, or that the wound heals with less scarring, less wound contraction, less collagen deposition and more superficial surface area.
  • “promotion of wound healing” can also mean that certain methods of wound healing have improved success rates, (e.g. the take rates of skin grafts,) when used together with the method of the present invention.
  • scarring can be an important obstacle in regaining normal function and appearance of healed skin. This is particularly true when pathologic scarring such as keloids or hypertrophic scars of the hands or face causes functional disability or physical deformity. In the severest circumstances, such scarring may precipitate psychosocial distress and a life of economic deprivation.
  • Wound repair includes the stages of hemostasis, inflammation, proliferation, and remodeling. The proliferative stage involves multiplication of fibroblasts and endothelial and epithelial cells. Through the use of the subject method, the rate of proliferation of epithelial cells in and proximal to the wound can be controlled in order to accelerate closure of the wound and/or minimize the formation of scar tissue.
  • the present treatment can also be effective as part of a therapeutic regimen for treating oral and paraoral ulcers, e.g. resulting from radiation and/or chemotherapy.
  • Such ulcers commonly develop within days after chemotherapy or radiation therapy.
  • These ulcers usually begin as small, painful irregularly shaped lesions usually covered by a delicate gray necrotic membrane and surrounded by inflammatory tissue.
  • lack of treatment results in proliferation of tissue around the periphery of the lesion on an inflammatory basis.
  • the epithelium bordering the ulcer usually demonstrates proliferative activity, resulting in loss of continuity of surface epithelium.
  • a treatment for such ulcers which includes application of an ptc agonist can reduce the abnormal proliferation and differentiation of the affected epithelium, helping to reduce the severity of subsequent inflammatory events.
  • the subject method and compositions can also be used to treat wounds resulting from dermatological diseases, such as lesions resulting from autoimmune disorders such as psoriasis.
  • Atopic dermititis refers to skin trauma resulting from allergies associated with an immune response caused by allergens such as pollens, foods, dander, insect venoms and plant toxins.
  • antiproliferative preparations of ptc agonists can be used to inhibit lens epithelial cell proliferation to prevent post-operative complications of extracapsular cataract extraction.
  • Cataract is an intractable eye disease and various studies on a treatment of cataract have been made. But at present, the treatment of cataract is attained by surgical operations. Cataract surgery has been applied for a long time and various operative methods have been examined. Extracapsular lens extraction has become the method of choice for removing cataracts. The major medical advantages of this technique over intracapsular extraction are lower incidence of aphakic cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber type intraocular lenses which are now considered to be the lenses of choice in most cases.
  • a disadvantage of extracapsular cataract extraction is the high incidence of posterior lens capsule opacif ⁇ cation, often called after-cataract, which can occur in up to 50% of cases within three years after surgery.
  • After-cataract is caused by proliferation of equatorial and anterior capsule lens epithelial cells which remain after extracapsular lens extraction. These cells proliferate to cause Sommerling rings, and along with fibroblasts which also deposit and occur on the posterior capsule, cause opacification of the posterior capsule, which interferes with vision. Prevention of after-cataract would be preferable to treatment.
  • the subject method provides a means for inhibiting proliferation of the remaining lens epithelial cells.
  • such cells can be induced to remain quiescent by instilling a solution containing an ptc agonist preparation into the anterior chamber of the eye after lens removal.
  • the solution can be osmotically balanced to provide minimal effective dosage when instilled into the anterior chamber of the eye, thereby inhibiting subcapsular epithelial growth with some specificity.
  • the subject method can also be used in the treatment of corneopathies marked by corneal epithelial cell proliferation, as for example in ocular epithelial disorders such as epithelial downgrowth or squamous cell carcinomas of the ocular surface.
  • Hair is basically composed of keratin, a tough and insoluble protein; its chief strength lies in its disulphide bond of cystine.
  • Each individual hair comprises a cylindrical shaft and a root, and is contained in a follicle, a flask-like depression in the skin.
  • the bottom of the follicle contains a finger-like projection termed the papilla, which consists of connective tissue from which hair grows, and through which blood vessels supply the cells with nourishment.
  • the shaft is the part that extends outwards from the skin surface, whilst the root has been described as the buried part of the hair. The base of the root expands into the hair bulb, which rests upon the papilla.
  • Hair “growth” refers to the formation and elongation of the hair fiber by the dividing cells.
  • the common hair cycle is divided into three stages: anagen, catagen and telogen.
  • anagen the epidermal stem cells of the dermal papilla divide rapidly.
  • Daughter cells move upward and differentiate to form the concentric layers of the hair itself.
  • the transitional stage, catagen is marked by the cessation of mitosis of the stem cells in the follicle.
  • the resting stage is known as telogen, where the hair is retained within the scalp for several weeks before an emerging new hair developing below it dislodges the telogen-phase shaft from its follicle. From this model it has become clear that the larger the pool of dividing stem cells that differentiate into hair cells, the more hair growth occurs. Accordingly, methods for increasing or reducing hair growth can be carried out by potentiating or inhibiting, respectively, the proliferation of these stem cells.
  • the subject method can be employed as a way of reducing the growth of human hair as opposed to its conventional removal by cutting, shaving, or depilation.
  • the present method can be used in the treatment of trichosis characterized by abnormally rapid or dense growth of hair, e.g. hypertrichosis.
  • ptc agonists can be used to manage hirsutism, a disorder marked by abnormal hairiness.
  • the subject method can also provide a process for extending the duration of depilation.
  • a ptc agonist will often be cytostatic to epithelial cells, rather than cytotoxic, such agents can be used to protect hair follicle cells from cytotoxic agents which require progression into S-phase of the cell-cycle for efficacy, e.g. radiation-induced death.
  • Treatment by the subject method can provide protection by causing the hair follicle cells to become quiescent, e.g., by inhibiting the cells from entering S phase, and thereby preventing the follicle cells from undergoing mitotic catastrophe or programmed cell death.
  • ptc agonists can be used for patients undergoing chemo- or radiation- therapies which ordinarily result in hair loss.
  • the subject treatment can protect hair follicle cells from death which might otherwise result from activation of cell death programs.
  • the instant method can also be removed with concommitant relief of the inhibition of follicle cell proliferation.
  • the subject method can also be used in the treatment of folliculitis, such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis.
  • a cosmetic prepration of an ptc agonist can be applied topically in the treatment of pseudo folliculitis, a chronic disorder occurring most often in the submandibular region of the neck and associated with shaving, the characteristic lesions of which are erythematous papules and pustules containing buried hairs.
  • the subject method can be used to induce differentiation and/or inhibit proliferation of epithelially-derived tissue.
  • Such forms of these molecules can provide a basis for differentiation therapy for the treatment of hype ⁇ lastic and/or neoplastic conditions involving epithelial tissue.
  • such preparations can be used for the treatment of cutaneous diseases in which there is abnormal proliferation or growth of cells of the skin.
  • the pharmaceutical preparations of the invention are intended for the treatment of hype ⁇ lastic epidermal conditions, such as keratosis, as well as for the treatment of neoplastic epidermal conditions such as those characterized by a high proliferation rate for various skin cancers, as for example basal cell carcinoma or squamous cell carcinoma.
  • the subject method can also be used in the treatment of autoimmune diseases affecting the skin, in particular, of dermatological diseases involving morbid proliferation and/or keratinization of the epidermis, as for example, caused by psoriasis or atopic dermatosis.
  • psoriasis squamous cell carcinoma
  • keratoacanthoma actinic keratosis
  • psoriasis which is characterized by scaly, red, elevated plaques on the skin
  • the keratinocytes are known to proliferate much more rapidly than normal and to differentiate less completely.
  • the preparations of the present invention are suitable for the treatment of dermatological ailments linked to keratinization disorders causing abnormal proliferation of skin cells, which disorders may be marked by either inflammatory or noninflammatory components.
  • therapeutic preparations of a ptc agonist e.g., which promotes quiescense or differentiation can be used to treat varying forms of psoriasis, be they cutaneous, mucosal or ungual.
  • Psoriasis as described above, is typically characterized by epidermal keratinocytes which display marked proliferative activation and differentiation along a "regenerative" pathway.
  • Treatment with an antiproliferative embodiment of the subject method can be used to reverse the pathological epidermal activiation and can provide a basis for sustained remission of the disease.
  • keratotic lesions are also candidates for treatment with the subject method.
  • Actinic keratoses for example, are superficial inflammatory premalignant tumors arising on sun-exposed and irradiated skin. The lesions are erythematous to brown with variable scaling.
  • Current therapies include excisional and cryosurgery. These treatments are painful, however, and often produce cosmetically unacceptable scarring.
  • treatment of keratosis such as actinic keratosis, can include application, preferably topical, of a ptc agonist composition in amounts sufficient to inhibit hype ⁇ roliferation of epidermal/epidermoid cells of the lesion.
  • Acne represents yet another dermatologic ailment which may be treated by the subject method.
  • Acne vulgaris for instance, is a multifactorial disease most commonly occurring in teenagers and young adults, and is characterized by the appearance of inflammatory and noninflammatory lesions on the face and upper trunk.
  • the basic defect which gives rise to acne vulgaris is hypercornification of the duct of a hyperactive sebaceous gland. Hypercornification blocks the normal mobility of skin and follicle microorganisms, and in so doing, stimulates the release of Upases by Propinobacterium acnes and Staphylococcus epidermidis bacteria and Pitrosporum ovale, a yeast.
  • Treatment with an antiproliferative ptc agonist, particularly topical preparations, may be useful for preventing the transitional features of the ducts, e.g. hypercornification, which lead to lesion formation.
  • the subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.
  • the present invention also provides a method for treating various forms of dermatitis.
  • Dermatitis is a descriptive term referring to poorly demarcated lesions which are either pruritic, erythematous, scaley, blistered, weeping, fissured or crusted. These lesions arise from any of a wide variety of causes.
  • the most common types of dermatitis are atopic, contact and diaper dermatitis.
  • seborrheic dermatitis is a chronic, usually pruritic, dermatitis with erythema, dry, moist, or greasy scaling, and yellow crusted patches on various areas, especially the scalp, with exfoliation of an excessive amount of dry scales.
  • the subject method can also be used in the treatment of stasis dermatitis, an often chronic, usually eczematous dermatitis.
  • Actinic dermatitis is dermatitis that due to exposure to actinic radiation such as that from the sun, ultraviolet waves or x- or gamma-radiation.
  • the subject method can be used in the treatment and/or prevention of certain symptoms of dermatitis caused by unwanted proliferation of epithelial cells.
  • Such therapies for these various forms of dermatitis can also include topical and systemic corticosteroids, antipuritics, and antibiotics.
  • Ailments which may be treated by the subject method are disorders specific to non- humans, such as mange.
  • the subject method can be used in the treatment of human cancers, particularly basal cell carcinomas and other tumors of epithelial tissues such as the skin.
  • ptc agonists can be employed, in the subject method, as part of a treatment for basal cell nevus syndrome (BCNS), and other other human carcinomas, adenocarcinomas, sarcomas and the like.
  • the subject method is used as part of a treatment ot prophylaxis regimen for treating (or preventing) basal cell carcinoma.
  • the deregulation of the ptc signaling pathway may be a general feature of basal cell carcinomas caused by ptc mutations. Consistent overexpression of human ptc mRNA has been described in tumors of familial and sporadic BCCs, determined by in situ hybridization. Mutations that inactivate ptc may be expected to result in overexpression of mutant Ptc, because ptc displays negative autoregulation. Prior research demonstrates that overexpression of hedgehog proteins can also lead to tumorigenesis.
  • That sonic hedgehog (Shh) has a role in tumorigenesis in the mouse has been suggested by our research in which transgenic mice overexpressing Shh in the skin developed features of BCNS, including multiple BCC-like epidermal proliferations over the entire skin surface, after only a few days of skin development.
  • a mutation in the Shh human gene from a BCC was also described; it was suggested that Shh or other Hh genes in humans could act as dominant oncogenes in humans.
  • Sporadic ptc mutations have also been observed in BCCs from otherwise normal individuals, some of which are UV-signature mutations.
  • the subject method can also be used to treatment patients with BCNS, e.g., to prevent BCC or other effects of the disease which may be the result of ptc loss-of-function or smoothened gain-of-function.
  • Basal cell nevus syndrome is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age.
  • BCNS patients are very susceptible to the development of these tumors; in the second decade of life, large numbers appear, mainly on sun-exposed areas of the skin. This disease also causes a number of developmental abnormalities, including rib, head and face alterations, and sometimes polydactyly, syndactyly, and spina bifida.
  • fibromas of the ovaries and heart fibromas of the ovaries and heart, cysts of the skin and jaws, and in the central nervous system, medulloblastomas and meningiomas.
  • the subject method can be used to prevent or treat such tumor types.
  • Studies of BCNS patients show that they have both genomic and sporadic mutations in the ptc gene, suggesting that these mutations are the ultimate cause of this disease.
  • the present invention provides pharmaceutical preparations and methods for controlling the formation of megakaryocyte-derived cells and/or controlling the functional performance of megakaryocyte-derived cells.
  • certain of the compositions disclosed herein may be applied to the treatment or prevention of a variety hype ⁇ lastic or neoplastic conditions affecting platelets.
  • the ptc agonists for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of adminisfration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art.
  • compositions of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the ptc agonists suitable for veterinary uses, e.g., for the treatment of live stock or domestic animals, e.g., dogs.
  • 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 a ptc agonist at a particular target site.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and topical administrations are preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the adminisfration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, infravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of adminisfration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of adminisfration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the freatment, other drugs, compounds and/or materials used in combination with the particular ptc agonist employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • intravenous, infracerebroventricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • treatment is intended to encompass also prophylaxis, therapy and cure.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • micelacking ptc function By homologous recombination, part of ptc exon 1 (including the putative start codon) and all of exon 2 were replaced with lacZ and a neomycin resistance gene (Fig. 1) (13). Protein made from any alternative ATG codon would lack the first proposed transmembrane domain, flipping the orientation of the protein in the membrane.
  • Three independent embryonic stem cell clones were used to make chimeras that were bred to B6D2F1 animals to generate heterozygous mice on a mixed background. Interbreeding of heterozygotes produced no homozygous animals among 202 offspring examined.
  • Hh signaling also posttranscriptionally regulates the zinc finger protein cubitus interruptus (ci) (15).
  • Sonic hedgehog (Shh) signaling induces transcription of both ptc and a ci homolog, Gli (6, 16). Derepression of ptc and Gli in ptc-l- mice should therefore reveal where Ptc is normally active.
  • Shh induces the floor plate and motor neurons in the ventral neural tube (18). These cell types fail to form in Shh mutants (19). Large amounts of Shh produced by the notochord may induce floor plate by completely inactivating Ptc (18). If so, elimination of ptc function might cause floor plate differentiation throughout the neural tube.
  • Prospective floor plate cells transcribe the forkhead transcription factor HNF3 first and then Shh itself (18). In E8.5 ptc mutants, transcription of HNF3 and Shh was expanded dorsally (Fig. 3, A to C).
  • Ectopic Shh expression was most extensive in the anterior, where transcripts could be detected throughout the neurepithehum (Fig. 3, B and C). Cells in this region were in a single layer with basal nuclei, like floor plate cells that are normally restricted to the ventral midline (Fig. 3, D and E). Expression of the lateral neural tube marker Pax6 (20) was completely absent from ptc mutant embryos, suggesting that only ventral, and not venfrolateral, cell fates are specified (Fig. 3, F and G). In principle, dorsalizing signals from the surface ectoderm (21) could confer dorsal cell fates even in the absence of ptc function.
  • the dorsal neural tube marker Pax3 was not expressed in the anterior neural tube but was transcribed in a very small region at the dorsalmost edge of the posterior neural tube (Fig. 3, H to J).
  • erb-b3 transcription which marks migratory neural crest cells (Fig. 3K) (22) was not detected in the somites of ptc mutants (Fig. 3L).
  • BMP Bone mo ⁇ hogenetic protein
  • ptc-l- embryos cells in the anterior neural tube expressed the forebrain marker Nkx2.1 (23), and cells in the spinal cord transcribed hoxbl (24) (Fig. 3, M and N).
  • hoxbl was not transcribed in the fourth rhombomere of ptc mutants (Fig. 3N). This may reflect a transformation of hindbrain cells to floor plate, because hoxbl is excluded from the midline of wild-type embryos.
  • Nkx2.1 expression was expanded dorsally in mutants compared with wild-type embryos (Fig. 3M).
  • medulloblastomas are believed to arise from a "primitive neurectodermal" cell type (25). They are most common in children, can be metastatic or nonmetastatic, and can have glial and neuronal properties.
  • the histology of tumors from ptc+l mice was similar to that of human medulloblastoma: tumor cells were small, with dark carrot-shaped nuclei and little cytoplasm (Fig. 4, D and E), and although a subset expressed neurof ⁇ lament protein and synaptophysin (Fig.
  • medulloblastomas brains from 47 asymptomatic ptc+l- mice were randomly collected and stained with X-Gal.
  • Medulloblastomas were observed in 1 of 12 (8.3%) ptc+l mice at 5 weeks of age, 1 of 12 (8.3%) mice at 9 to 10 weeks, and 7 of 23 (30.4%) mice at 12 to 25 weeks. Tumors can therefore arise as early as 5 weeks after birth but increase in severity and frequency as the animal ages.
  • Brain tumors might arise from Ptc haploinsuff ⁇ ciency alone, from additional mutations in the second ptc allele, or from a combination of ptc mutations with mutations in other tumor suppressor loci.
  • basal cell carcinomas in ptc+l- mice, perhaps because somatic inactivation of the second ptc gene is required as it is in human basal cell carcinomas.
  • Medulloblastoma is a common childhood brain tumor and the prognosis remains grim.
  • the Hh- tc pathway may provide new diagnostic tools and new insights into tumorigenesis that can be directed toward potential therapies.
  • DNA from the ptc genomic locus was isolated from a 129SV genomic phage library (Sfratagene). Exons 1 through 15 of human PTC (1) were mapped by polymerase chain reaction (PCR) and sequencing. The 3 arm of homology was a 3.5-kb Eco RI-Bam HI fragment from the second infron that gained a Bam HI site from pBSII (Sfratagene) and was cloned into the Bam HI site of pPNT [ V. L. Tybulewicz, C. E. Crawford, P. K.
  • a cassette containing the gene for nuclear localized -galactosidase (lacZ) followed by the mPl infron and poladenylate tail was excised from pNLacF [ E. H. Mercer, G. W. Hoyle, R. P. Kapur, R. L. Brinster, R. D. Pahniter, Neuron 7, 703 (1991) ] and cloned into the Xho I site of pPNT by using Xho I and Sal I linkers.
  • the 5 arm of homology was a 6.5-kb Xho I-Nru I fragment that was cloned into the Xho I site upstream of lacZ with a Sal I linker.
  • the Nru I site is in the first ptc exon.
  • the resulting plasmid, KOI was linearized with Xho I and elecfroporated into RI embryonic stem cells that were subjected to double selection and analyzed by Southern (DNA) blot [A. L. Joyner, Gene Targeting: A Practical Approach
  • Targeted clones were expanded and used for injection into c57bl/6 blastocysts [B. Hogan, R. Beddington, F. Costantini, E.
  • Embryos were stained with X-Gal (5-bromo-4-chloro-3-indoxyl ⁇ D- galactopyranoside) [C. Bonnerot and J. Nicolas, in Guide to Techniques in Mouse Development, P. M. Wassarman and M. L. DePamphilis, Eds. (Academic Press, New York, 1993), p. 463] and then sectioned.
  • X-Gal staining of tumors in heterozygotes cerebellums were dissected, cut into four pieces along the sagittal plane, fixed briefly in 4% paraformaldehyde, and then stained.
  • a cassette containing the gene for nuclear localized [Beta]-galactosidase (lacZ) followed by the mPl infron and poladenylate tail was excised from pNLacF [E. H. Mercer, G. W. Hoyle, R. P. Kapur, R. L. Brinster, R. D. Palmiter, Neuron 7, 703 (1991)] and cloned into the Xho I site of pPNT by using Xho I and Sal I linkers.
  • the 5' arm of homology was a 6.5-kb Xho I-Nru I fragment that was cloned into the Xho I site upstream of lacZ with a Sal I linker.
  • Nra I site is in the first ptc exon.
  • the resulting plasmid, KOI was linearized with Xho I and electroporated into Rl embryonic stem cells that were subjected to double selection and analyzed by Southern (DNA) blot [A. L. Joyner, Gene Targeting: A Practical Approach (Oxford Univ. Press, New York, 1993), pp. 33-61].
  • Targeted clones were expanded and used for injection into C57BI 6 blastocysts [B. Hogan, R. Beddington, F. Costantini, E. Lacy, Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994), pp. 196-204].
  • the mouse BCC appears mo ⁇ hologically to be the equivalent of human BCC based on microscopic and histologic patterns. In human BCCs, epidermal cells proliferate and form peripheral "palisades," a columnar epithelium resembling the basal keratinocyte layer.
  • BCCs lack cell adhesion molecules that normally attach basal cells to the basement membrane zone, resulting in clefts between the basement membrane and tumor (Lever and Lever. Histopathologv of the Skin (Lippincott, Philadelphia, 1990); Miller, J. Am. Acad. Dermatol. 24, 1 (1991)). These histological features of human BCCs were also found in the epidermal proliferations of the irradiated ptc +/- transgenics.

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Abstract

L'invention porte sur des animaux transgéniques chez qui la fonction biologique normale d'un ou plusieurs suppresseurs de la famille des gènes 'patched' (dits gène ptc) ont été fonctionnellement inactivés de manière à ce que l'animal, viable à la naissance et pendant l'âge adulte, soit amené à créer des carcinomes basocellulaires à une fréquence notablement plus élevée que les animaux de type sauvage, par exemple par exposition à des agents dégradateurs de l'ADN tels que des rayonnements ionisants ou non ionisants (par exemple les UV). Ainsi que le montrent les exemples annexés, les souris fragilisées (knockout) hétérozygotes, viables à la naissance, sont susceptibles de fréquences de cancers plus élevées lorsque soumises à des agents dégradateurs de l'ADN. L'une des caractéristiques saillante desdites souris est de pouvoir être amenées à former des carcinomes basocellulaires qui, histologiquement, sont similaires aux carcinomes basocellulaires de l'homme.
PCT/US1999/011983 1998-05-29 1999-05-28 Animaux transgeniques a transduction du signal du 'patched' et leurs utilisations associees WO1999061582A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126808A3 (fr) * 2008-04-11 2011-06-09 Children's Hospital & Research Center At Oakland Modèle de cancer d'animal transgénique, et procédés d'utilisation
WO2015155490A1 (fr) 2014-04-11 2015-10-15 Centre National De La Recherche Scientifique (Cnrs) Modèle animal présentant une invalidation fonctionnelle du gène codant pour le récepteur membranaire patched ou ptc

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126808A3 (fr) * 2008-04-11 2011-06-09 Children's Hospital & Research Center At Oakland Modèle de cancer d'animal transgénique, et procédés d'utilisation
WO2015155490A1 (fr) 2014-04-11 2015-10-15 Centre National De La Recherche Scientifique (Cnrs) Modèle animal présentant une invalidation fonctionnelle du gène codant pour le récepteur membranaire patched ou ptc
FR3019712A1 (fr) * 2014-04-11 2015-10-16 Centre Nat Rech Scient Modele animal et ses utilisations

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EP1124723A4 (fr) 2002-05-15
JP2002516788A (ja) 2002-06-11
AU4321599A (en) 1999-12-13

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