WO1993023533A1 - Test in vivo de medicaments anti-neoplasiques - Google Patents

Test in vivo de medicaments anti-neoplasiques Download PDF

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Publication number
WO1993023533A1
WO1993023533A1 PCT/US1993/004363 US9304363W WO9323533A1 WO 1993023533 A1 WO1993023533 A1 WO 1993023533A1 US 9304363 W US9304363 W US 9304363W WO 9323533 A1 WO9323533 A1 WO 9323533A1
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mammal
reporter
transformation
transgenic
regulatory region
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PCT/US1993/004363
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Paul J. Leibowitz
Samuel Wadsworth
Chee-Wai Woon
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Exemplar Corporation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/82—Translation products from oncogenes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to a novel, in vivo drug screen for identifying anti-neoplastic agents and to transgenic animals relating thereto.
  • Typical drug screening assays also are in vitro systems (1, 2, 3, 4, 5).
  • One recently developed ⁇ ji vitro system uses a fibroblast cell line including a transformation-sensitive promoter fused to an E. coli reporter gene (6). In an untransformed cell, the reporter gene is expressed; in a transformed cell, the reporter gene is repressed. It is suggested that this assay can be used to identify potential candidates for cancer therapy. That is, if the potential candidate is applied to a transformed cell and reverses the repression characteristic of the transformed state, then the drug possibly may work in vivo for some unidentified cancer state.
  • This system though interesting, has nearly all of the drawbacks of any other ⁇ i vitro system. It provides no information about what happens in vivo, including, for example, no information about drug availability, dosing regimen to identify and maintain active drug concentration, tissue specificity, in vivo drug modification, and age related issues. Summary of the Invention
  • an in vivo method for testing the ability of a drug to interfere with the onset or maintenance of neoplastic transformation.
  • a drug is administered to a tumor-bearing animal.
  • the animal has a transformation-sensitive reporter unit.
  • the reporter unit includes a structural gene, the expression of which is turned on or off depending upon whether cells of the animal are in a neoplastic state. It then is determined whether the drug has reversed or interfered with the effects of transformation by assaying for the expression of the structural gene.
  • This method permits detection of anti-neoplastic effects by drugs that are noncytotoxic at the doses administered.
  • the drug is administered to a novel, transgenic, nonhuman animal that has a transformation sensitive reporter unit, at least a portion of which is introduced stably into the animal, or an ancestor of the animal, preferably (but not necessarily) at an embryonic stage.
  • the transformation sensitive reporter unit includes a regulatory region that is either repressed upon neoplastic transformation of cells of the animal or activated upon such neoplastic transformation.
  • the reporter unit includes an exogenous reporter structural gene.
  • the foregoing transgenic animals are selected for or provided with a predisposition to develop neoplasia.
  • a transgenic nonhuman animal * is created, whose germ cells and/or somatic cells contain a recombinant, transformation sensitive reporter unit introduced into the animal, or an ancestor of the animal, preferably (but not necessarily) at an embryonic stage. Then, one or more of the somatic cells of the transgenic nonhuman animal are cultured.
  • Fig. l depicts a preferred, transformation-sensitive, reporter unit.
  • Fig. 2 depicts a preferred construct for creating a transgenic animal with a predisposition to develop neoplasia.
  • the invention involves a novel, living reporter system that is a model for the investigation of neoplastic transformation and the reversal thereof. It is based upon the provision of an animal that has a transformation sensitive reporter unit which produces a different signal depending upon whether or not cells of the .animal are in a neoplastic state. Preferably, the animal has the genetic trait of being predisposed to develop neoplasia. A secondary oncogenic agent or event may be presented to the animal to accelerate the induction of cellular transformation. Using the above-described animal, the neoplastic condition can be monitored by assaying for the presence or absence of the signal.
  • This provides an ideal model for drug screening, in that drugs can be administered to an animal, for example, that has already developed a tumor, and then the ability of the drug to act as an anti-neoplastic agent can be measured by detecting the ability of the drug to affect the signal from the reporter system.
  • the reporter system can be chosen so that the efficacy of the drug can be easily determined, for example, by the presence or absence of a color. A color change would occur considerably sooner than a reduction in tumor size.
  • neoplastic transformation is intended to include one or more events in a cell that initiate as well as contribute to the development of a tumor or cancerous state which may include the loss of cell growth control and redirection of the normal physiological processes of the cell.
  • hyperplasia, metaplasia, and dysplasia are commonly used to describe stages in the development of neoplastic transformation.
  • development it is meant onset, maintenance or progression,
  • This invention further provides access to obtaining information about drug availability. It can be determined if the drug reaches the tumor, the extent to which the drug penetrates the tumor, if the drug has different effects on different size tumors, if the drug has different effects on tumors at different stages of progression, and if the drug's effects depend upon the state of vascularization of the tumor. For example, differential staining patterns within a tumor (e.g., whether the borders or centers are stained) resulting from a reporter system that produces a color change if the drug reverses transformation, can provide useful information on drug delivery. Information about the tissue-specificity of a drug also can be obtained. For example, differential staining can indicate if a drug acts differently on tumors in different tissues.
  • the assay identifies a putative anti-cancer drug if the tumor changes color, even if the tumor might not have undergone a reduction in size. Once such a candidate drug is identified, the doses and/or timing of drug administration can be manipulated to achieve a desired result.
  • the invention allows an assessment of whether external conditions interfere with the efficacy of the drug. Unlike .in vitro screens, this invention permits analysis of conditions specific to live animals, e.g. , the animal's age, the animal's health, the degree of vascularization near the tumor, synergistic effects with other drugs or nutrients ingested by the animal, and the state of the animal's immune system. Moreover, the invention can permit a determination of whether the efficacy of the drug depends upon jin vivo modification of the drug.
  • the invention thus also provides novel transgenic animals.
  • animals it is meant vertebrate animals including birds, fish, amphibians and mammals.
  • Preferred animals' are mammals and particulary preferred mammals are rabbits, horses, cows, goats, sheep, dogs, cats, rodents and monkeys.
  • the animal preferably has a predisposition to develop neoplasia.
  • predisposition to develop neoplasia it is meant that the animal is in a condition where tumor development is more likely to occur, as compared to an animal of the same type which is not predisposed to develop neoplasia.
  • the predisposed condition is the result of a primary agent or event.
  • transgenic animal lines exist which have such a predisposition to develop tumors.
  • the term transgenic animal is meant to include an animal that gains new genetic information from the introduction of foreign DNA into its cells.
  • the animal may include the transgene in all of its cells including germ line cells, or in only some of its cells such as in selected somatic cells.
  • An example of an existing transgenic animal line with a predisposition to develop tumors is U.S. Patent 4,736,866, issued April 12, 1988' to Leder, the entire contents of which are incorporated herein by reference.
  • Animal lines with a predisposition to develop tumors can be created in a variety of ways, including:
  • NMU activation of a proto-oncogene by the integration of a viral genome (of a retrovirus) adjacent to the proto-oncogene;
  • sex hormones e.g. , estrogens or testosterones
  • a secondary event or agent is generally required to activate a predisposed animal to actually develop tumors.
  • agents or events are:
  • an oncogene is a gene whose product when expressed within a eukaryotic cell has the ability to transform that cell.
  • oncogenes include growth factors/mitogens, receptors, cytoplasmic or membrane associated proteins, nuclear proteins/transcription factors, and certain unclassified genes.
  • Growth factors/mitogens include hst, int2 and sis.
  • Other potential growth factors/mitogens are FGF-5, CSF-1, GM-CSF, PDGF, TGF ⁇ and EGF.
  • Receptors include protein tyrosine kina ⁇ es, e.g., erbB, fms, kit, met, ret, ros, sea, trk and neu; and non-protein kinases, e.g. , mas.
  • Cytoplasmic and membrane associated proteins include tyrosine kinases, e.g. , abl, fes, fgr, fps, lck, src and yes; G-proteins, e.g. , H-ras, K-ras, N-ras, gip and gsp; and protein serine threonine kinases, e.g.
  • Nuclear proteins/transcription factors include erbA, ets, fos, fos-B, fra-1, jun, junB, junD, myb, myc, L-myc, N-myc, rel, ski, vav, evi-1, gli-1, maf, pbx and Hox2.4.
  • Unclassified oncogenes include dbl and bcl-2.
  • Oncogenic RNA viruses include Rous Sarcoma Virus (Chicken) , Y73 Sarcoma Virus (Chicken) , Fujinami (Feline) Sarcoma Virus (Cat), Abelson Murine Leukemia Virus (Mouse), Rochester-2 Sarcoma Virus (Chicken), Gardner-Rasheed Feline Sarcoma Virus (Cat), Avian Erythroblastosis Virus (Chicken) , McDonough Feline Sarcoma Virus (Cat), Moloney Murine Sarcoma Virus (Mouse), 3611 Murine Sarcoma Virus (Mouse), Harvey Murine Sarcoma Virus (Rat and Mouse), Kirsten Murine Sarcoma Virus (Rat), Avian MC29 Myelocytomatosis Virus (Chicken), Avian Myeloblastosis Virus (Chicken), FBJ Osteosarcoma Virus (Mouse), Avian
  • a proto-oncogene is a normal cellular gene that can be activated to a dominant-acting oncogene (7).
  • Proto-oncogene ⁇ can be activated by one of the following mechanisms:
  • Over-expression e.g. , mutation in the transcriptional regulatory regions of the gene or insertion of a retroviral genome into the proto-oncogene locus.
  • An example is the- over-expression of the secreted oncogene products sis or hst (10).
  • GIP Carcinoma of ovary and adrenal gland
  • MYC Burkitt' ⁇ lymphoma Carcinoma of lung, breast and cervix
  • N-MYC Neuroblas oma small cell carcinoma of lung
  • H-RAS Carcinoma of colon, lung and pancreas; melanoma
  • K-RAS Acute myelogenous and lymphoblastic leukemia; carcinoma of thyroid; melanoma
  • N-RAS Carcinoma of genitourinary tract and thyroid; melanoma
  • Some proto-oncogenes code for receptors involved in the development of particular cell types.
  • Examples of receptors that when mutated result in oncogenesis include the erthropoietin receptor (12), the ⁇ l ⁇ adreneric receptor (13) and the EGF receptor (14).
  • Inactivation of a tumor suppressor gene can result in tumor formation. Inactivation of tumor suppressor genes can be accomplished through the following methods: a. Antisense RNA expression (This approach has been successfully used to block specific oncogene expression (15, 16, 17). b. Gene knockout by homologous recombination (18, 19, 20) c. Inactivating mutations (point mutations, deletions) introduced in vitro or by carcinogen treatment, e.g. , p53 genes (21, 22).
  • MEN-1 NF1 Neurofobromatosis type 1 FAP Carcinoma of colon MEN-1 Tumors of parathyroid, pancreas, pituitary and adrenal cortex
  • a carcinogen is an agent which has the capacity to cause changes directly or indirectly in cells that result in cellular transformation.
  • carcinogens include N-nitroso- ⁇ -methyl urea (NMU) , 7,
  • DMBA 12-dimethylbenz[a]anthracene
  • TPM tetranitromethane
  • DBACR dibenz[c,h]acridine
  • HODE 1'-hydroxy-2' ,3'-dehydroestragole
  • HOAAF N-hydroxy-2-acetylaminofluoride
  • VC vinyl carbamate
  • DN methyl (methoxymethyl) nitrosoamine
  • MCA 3-methyl-cholanthrene
  • one of the following combinations of primary and secondary agents or events can be used to improve the efficiency of tumor formation in an animal (i) oncogene synergy in spontaneous or accelerated transgenic tumor formation (23):
  • the primary and secondary agents and events which are involved in tumor formation can be introduced at various stages during animal development. As will be recognized by those of ordinary skill in the art, the choice of which stage depends at least upon the particular agent or event used and whether it is for a primary or secondary effect.
  • transformation sensitive reporter unit requires sequence of DNA that encodes for a product that can be monitored and also a sequence of DNA through which expression of the product may be regulated in response to cellular transformation.
  • Regulation of expression means regulation at the transcriptional or translational level.
  • Expression of the product of the reporter unit can be either repressed or activated in response to the transformation event.
  • Repression refers to the partial or complete inhibition of expression.
  • Activation refers to the turning on or enhancement of expression. It is important that activation or repression be readily monitored. Therefore, in preferred embodiments the reporter unit is either substantially completely repressed or substantially completely activated by transformation.
  • Monitoring expression of the product can be accomplished by histochemical, immunohistochemical, enzymatic assays or in situ hybridization methods in whole animals, tissue sections, cell homogenates or single cells.
  • the regulatory region of the transformation sensitive reporter unit is a cis-acting DNA sequence through which transcription of a gene is controlled. A function of this sequence is to be recognized by regulatory proteins.
  • a transcriptional regulatory region includes promoters and enhancers.
  • a promoter is a DNA sequence which directs the start of RNA synthesis. It is a region of DNA that is involved in binding RNA polymerase to initiate transcription. A promoter may be the target of one or many regulatory proteins. Examples of regulatory proteins are repressors and activators.
  • An enhancer is a DNA sequence that in combination with a given promoter in the presence of the appropriate protein(s) directs the increased utilization of that promoter in specific tissue(s).
  • Enhancers can act over considerable distances, at least up to several thousand base pairs, from the start point of transcription. Enhancers can function in either orientation relative to the promoter. They are position-independent in that they can function upstream or downstream relative to the promoter. A particular enhancer may be a target for tissue specific o) temporal regulation.
  • reporter units having regulatory regions that are sensitive to transformation by various oncogenes/viruses are shown in the following table.
  • reporter units having regulatory regions that are activated upon transformation include a large number of genes such as glucose transporter type I (GLUTI) (30) and multidrug resistance gene (MDRI) (31), as well as other as yet uncharacterized genes (32).
  • GLUTI glucose transporter type I
  • MDRI multidrug resistance gene
  • the transformation sensitive reporter unit contains a structural gene, a sequence of DNA that encodes for a product;
  • the structural gene can code for any RNA or polypeptide product.
  • the product may be a full length gene product, or it may be a subfragment thereof, or it may be part of a fusion product, provided that it is assayable.
  • a structural gene is meant to include sequences which include exons and introns, as well as those that include exons and some introns, or only exons.
  • Preferred reporters include exogenous structural genes so as to avoid any interference in the assays with endogenous background levels of the reporter gene product.
  • the reporter gene is a coding sequence for a protein, whether an exogenous protein or a protein the expression of which is modulated directly by the transformation state of a cell
  • an antibody specific for that protein can be used to detect expression of the protein.
  • a wide range of antibodies for cellular proteins is available from commercial suppliers such as Sigma Chemical, Boehringer Mannheim, Dako Corp.
  • reporter genes are Escherichia coli lac Z (codes for ⁇ -galactosidase) , Escherichia coli CAT (codes for chloramphenicol acetyltransferase) , Firefly luciferase (codes for luciferase) , and the proteins from the genes listed in Table 8. These reporters can be assayed histochemically, enzymatically (34), immunohistochemically (35), or by in situ hybridization (36) .
  • ⁇ -galactosidase is assayed histochemically, for example, with X-gal as a color indicator, ⁇ -galactosidase reacts with X-gal to produce a blue color which is easily visualized (33).
  • Luciferase preferably is assayed by che iluminescence (37) .
  • the proteins from the genes listed in Table 8 are assayed by antibody reaction.
  • the most preferred reporter is the Escherichia coli lacZ gene.
  • the transformation sensitive reporter unit preferably involves a transgene and may be introduced into the animal cell as an intact unit.
  • an exogenous structural gene may be reco bined with a regulatory region in vitro to form a transformation sensitive reporter unit.
  • the regulatory region may be derived from a regulatory region that is normally endogenous to the target animal, or from a regulatory region that is exogenous to the target animal.
  • the term endogenous regulatory region is a regulatory region that is normally found in the genome of the non-transgenic target animal.
  • the regulatory region is operatively coupled to the structural gene.
  • the term operatively coupled is meant to include the situation where expression of the structural gene is under the control of the regulatory region. Such control includes control by either the promoter or enhancer of the regulatory region, or by both.
  • the transformation sensitive reporter unit may be formed ii vivo as a result of recombination between a part of the unit that is introduced into the animal cell and a part of the unit that is endogenous to the cell.
  • an exogenous reporter structural gene may be introduced into the cell and undergo recombination with the animal's genome, resulting in insertion of the exogenous structural gene so that it is regulated by an endogenous regulatory region.
  • the term exogenous reporter structural gene is meant to include a gene that is not normally found in the genome of the non-transgenic animal.
  • insertion of the exogenous structural gene is recombined into a nonessential region of the genome so that the cell is not killed. Insertion may occur upstream or downstream of the transformation sensitive regulatory region, provided that the regulatory region is operatively coupled to the structural gene.
  • Insertion of either an intact transformation sensitive reporter unit, or insertion of part of a transformation sensitive reporter unit should be in a manner so as to result in stable introduction into the animal cell.
  • stable introduction it is meant that the transformation sensitive reporter unit is introduced into a recipient cell and becomes integrated into a resident chromosome or is maintained in successive generations as an autonomously replicating unit.
  • An example of an autonomously replicating unit is Bovine Papilloma Virus Vector (38).
  • a potential anti-cancer drug is screened using an animal with the following characteristics: (1) all or some of its cells express a transformation sensitive reporter unit as described above and (2) some of the same cells have undergone transformation as described above.
  • the regulatory region of the transformation sensitive reporter is repressed as a result of transformation and the reporter gene is substantially turned off so as to eliminate detectable product of the reporter gene.
  • An anti-cancer drug of the desired class will reverse the transformed state, resulting in restoration of reporter gene expression.
  • the regulatory region is activated as a result of transformation and the reporter gene is turned on. In such a situation, an anti-cancer drug will result in reversal of reporter gene expression.
  • the ability of the drug to reverse expression of the reporter gene as compared to its expression under transforming conditions, identifies the drug as an anti-cancer drug.
  • Restriction endonucleases are obtained from conventional commercial sources such as New England Biolabs (Beverly, MA.), Promega Biological Research Products (Madison, WI. ) , and Stratagene (LaJolla CA.), etc.
  • Radioactive materials are obtained from conventional commercial sources such as Dupont/ ⁇ E ⁇ or Amersham.
  • Custom-designed oligonucleotides for site-directed mutagenesis are available from any of several commercial providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Kits for carrying out site-directed mutagenesis are available from commercial suppliers such as Promega Biological Research Products, Stratagene, etc.
  • Libraries of D ⁇ A are available from commercial providers such as Stratagene, La Jolla, CA.
  • Rat 2 and ⁇ 1H 3T3 cells are available from ATCC (#CRL1764 and #CRL1658 respectively) .
  • Standard cell culture media appropriate to the cell line are obtained from conventional commercial sources such as Sigma, Gibco/BRL Murine stem cells, strain D3, were obtained from Dr. Rolf Kemler (39). Materials for D ⁇ A transfection (40) and the drug G418 for selection of stable transformants are available from Gibco/BRL,
  • various transformation sensitive promoters can be fused to a desired reporter gene using standard molecular biological manipulations (41) .
  • the ability of the promoter(s) to direct the expression of the reporter genes in these "mixed and matched" constructs are evaluated in tissue culture cells (e.g. NIH3T3, Rat2 or other cell lines) in transient and stable transfection experiments.
  • tissue culture cells e.g. NIH3T3, Rat2 or other cell lines
  • the ability of the promoter-reporter constructs to respond to cellular transformation in tissue culture cell lines also is evaluated.
  • the constructs are then introduced into animals transgenically to determine if the transcriptional characteristics are retained .in vivo in the whole animal.
  • the constructs also are evaluated for their ability to direct a predictable/reproducible pattern of tissue expression.
  • DNA can be injected into the pronucleus of the fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell following the initiation of cell division: the nucleus of a two-cell embryo for example.
  • Embryos can be infected with viruses, especially retroviruses, modified to bear transgenes.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate transgenes.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term.
  • mice suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc. Swiss Webster female mice are preferred for embryo retrieval and transfer. B6D2F 1 males can be used for mating and vasectomized Swiss Webster studs can be used to stimulate pseudopregnancy. Vasectomized mice and rats can be obtained from the supplier.
  • mice six weeks of age are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma) .
  • Females are placed with males immediately after hCG injection. Twenty-one hours after hCG, the mated females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline (DPSS) with 0.5% bovine serum albumin (BSA; Sigma) .
  • DPSS Dulbecco's phosphate buffered saline
  • BSA bovine serum albumin
  • the oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall dire cly over the oviduct. The ovarian bursa is then torn with watchmakers forceps. Embryos to be transferred are placed in DPBS and in the tip of a transfer pipet (about 10-12 embryos). The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures.
  • transgenic rats The procedure for generating transgenic rats is similar to that of mice (49). Thirty day-old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven male. At the same time, 40-80 day old females are placed in cages with vasectomized males. These will provide the foster mothers for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (C0 2 asphyxiation) and their oviducts removed, placed in DPSS with 0.5% BSA and the embryos collected.
  • the live embryos are moved to DPBS for transfer into foster mothers.
  • the foster mothers are anesthetized with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip) .
  • a dorsal midline incision is made through the skin and the ovary and oviduct are exposed by an incision through the muscle layer directly over the ovary.
  • the ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulu .
  • Approximately 10-12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the foster mothers are housed singly.
  • Methods for the culturing of ES cells and the subsequent production of transgenic animals, the introduction of DNA into ES cells by a variety of methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are well known to those of ordinary skill in the art (50). Selection of the desired clone of transgene-containing ES cells is accomplished through one of several means. Although embryonic stem cells are currently available for mice only, it is expected that similar methods and procedures as described and cited here will be effective for embryonic stem cells from different species as they become available.
  • a transgene clone is co-transfected with a gene encoding neomycin resistance.
  • the gene encoding neomycin resistance is physically linked to the transgene.
  • Transfection is carried out by any one of several methods well known to those of ordinary skill in the art (50, 51). Calcium phosphate/DNA precipitation, direct injection, and electroporation are the preferred methods.
  • cells are fed with selection medium containing 10% fetal bovine serum in DMEM supplemented with G418 (between 200 and 500 ⁇ g/ml biological weight). Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNA is extracted from drug resistant clones and Southern blotting experiments using a transgene-specific DNA probe are used to identify those clones carrying the transgene sequences. In some experiments, PCR methods are used to identify the clones of interest.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination (52).
  • Direct injection results in a high efficiency of integration. Desired clones are identified through PCR of DNA prepared from pools of injected ES cells. Positive cells within the pools are identified by PCR subsequent to cell cloning. DNA introduction by electroporation is less efficient and requires a selection step. Methods for positive selection of the recombination event (i.e. , neo resistance) and dual positive-negative selection (i.e. , neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by (52, 53), the teachings of which are incorporated herein.
  • Naturally cycling or superovulated female mice mated with males are used to harvest embryos for the implantation of ES cells. It is desirable to use the C57B strain for this purpose when using mice. Embryos of the appropriate age are recovered approximately 3.5 days after successful mating. Mated females are sacrificed by CO, asphyxiation or cervical dislocation and embryos are flushed from excised uterine horns and placed in Dulbecco's modified essential medium plus 10% calf serum for injection with ES cells. Approximately 10-20 ES cells are injected into blastocysts using a glass microneedle with an internal diameter of approximately 20 ⁇ m.
  • Embryos to Receptive Females Randomly cycling adult female mice are paired with vasectomized males. Mouse strains such as Swiss Webster, ICR or others can be used for this purpose. Recipient females are mated such that they will be at 2.5 to 3.5 days post-mating when required for implantation with blastocysts containing ES cells. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The ovaries are exposed by making an incision in the body wall directly over the oviduct and the ovary and uterus are externalized.
  • a hole is made in the uterine horn with a 25 gauge needle through which the blastocysts are transferred. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by two sutures. This procedure is repeated on the opposite side if additional transfers are to be made.
  • Tail samples (1-2 cm) are removed from three week old animals. DNA is prepared and analyzed by Southern blot or PCR to detect transgenic founder (F-) animals and their progeny (F 1 and F 2 ) . In this way, animals that have become transgenic for the desired genes are identified. Because not every transgenic animal expresses the transgene, and not all of those that do will have the expression pattern anticipated by the experimenter, it is necessary to characterize each line of transgenic animals with regard to expression of the transgene in different tissues.
  • the reporter gene is to be expressed in a particular tissue that will subsequently become neoplastic either by genetic predisposition, exposure to carcinogenic agents, infection with viruses, or other methods described in this application.
  • An organism is identified as a potential transgenic by taking a sample of the organism for DNA extraction and hybridization analysis with a probe complementary to the transgene of interest.
  • DNA extracted from the organism can be subjected to PCR analysis using PCR primers complementary to the transgene of interest.
  • the establishment of the preneoplastic state in affected tissues is determined by standard histopathological methods (63).
  • Tumor development can be induced by a number of methods, also as described above. Tumor development, of course, need not be accelerated by active intervention, but rather may occur simply as the result of a secondary event occurring with the passage of time.
  • Tumor development is established by the presence of foci of neoplastic cells or proliferative lesions in histological analysis of tissue sections.
  • Ha-ras p21 expression can be modulated iji vivo in cells in culture by exposure to antisense ras oligodeoxyribonucleotides (64).
  • the administration of antisense oligonucleotides into animals (65) has been reported to inhibit oncogene expression (66).
  • mice embryonal carcinoma 72
  • murine erythroleukemia 73
  • colon carcinoma lines 74) treatment with differentiation promoting agents like Hexamethylene bisacetamide (HMBA) (75) resulted in the reversal of the transformed phenotype and the commitment to differentiation.
  • HMBA Hexamethylene bisacetamide
  • embryonal carcinoma cells a 30 hr treatment with 5 mM HMBA produced a restoration of -Tropomyosin synthesis (72).
  • HMBA was found to be effective in producing a partial remission of some cancers (76).
  • oncogene specific inhibitors have been reported in the literature.
  • Herbimycin A which specifically inhibit the tyrosine kinase activities of the oncogenes src, yes, fps, ros, abl and erbB has been reported to be effective in reversing the transformed phenotype produced by these oncogenes as well as some human colon tumors (77, 78, 79).
  • Lovastatin which interferes with the posttranslational modification of the Ras precursor protein inhibits ras activation in mammalian cells (80).
  • tumor suppressor genes can be altered as described above.
  • Transgenic animals that have integrated the transformation sensitive promoter-reporter construct(s) will be mated with animals that are predisposed to the development of tumors (as described above) to obtain test animals with a heritable preneoplastic state that have also incorporated a transformation sensitive reporter unit in its genome.
  • assessments of efficacy and bioavailability of putative antineoplastic agents in test animals are performed following parenteral (Intraperitoneal, intramuscular, intravenous, intrathecal & subcutaneous injection), topical and oral routes of administration. Based on the pharmacokinetics profile, appropriate dosing schedules are instituted to maintain a threshold concentration (predetermined in n vitro assessment of effective concentrations). In vivo bioavailability and efficacy of the test agent(s) are evaluated by the restoration of normal reporter gene activity preferably in tissue sections, cellular homogenates or subcellular fractions of the tumor as an indicator of the reversal of the transformed state.
  • Quantitation of the expression of the reporter gene(s) is preferably made by enzymatic methods (including the use of chromogens and chemiluminescence) which measure functional activity of the reporter gene product expressed, hybridization methods that determine the steady-state levels of the reporter gene transcript, and radioimmunoassay or immunohistochemical methods that detect the level of the reporter gene product(s).
  • Tissues from embryos or adults are dissected under sterile conditions such as in a laminar flow hood with sterile instruments.
  • the tissue is chopped into small pieces with a sterile scalpel and subjected to disaggregation by trypsin digestion.
  • the disaggregated cells are cultured in a rich medium including serum appropriate to the cell.
  • the test animal is obtained by generating separately a transgenic animal that has integrated into its genome at least one copy of the transformation sensitive promoter-reporter gene construct and another transgenic animal that has sustained a genetic manipulation that renders some or all of the cells in the animal preneoplastic.
  • the test animal that combines an inherited predisposition to develop tumor in the same cells or tissues that have also incorporated a functional transformation sensitive promoter-reporter transgene is obtained by crossing the two transgenic animals derived above.
  • the regulatory region of the rat a-tropomyosin gene is fused to the bacterial B-galactosidase gene.
  • the required rat a-tropomyosin promoter is obtained from the plasmid pTMlA.
  • This plasmid contains a 6.5 kb fragment of the 5' region of the rat a-tropomyosin gene including exons 1, 2 and 3 (81, 82).
  • a 4.2 kb BamHl-Apal fragment containing the enhancer and promoter region and transcriptional start site was isolated by restriction endonuclease digestion.
  • the Apal end was then modified by conventional recombinant DNA techniques (41) using deoxyribonucleotide linkers to generate a Sail site.
  • the B-galactoside gene was isolated as a 3.7 kb Xhol-Hindlll fragment from the plasmid pCMV-B (Clontech) .
  • the a-tropomyosin-B-gal fusion construct is assembled by ligating the promoter and reporter gene fragments at the Sall/Xhol sites as shown in Figure 1 so that the reporter gene is operably linked to and whose expression is therefore placed under the control of the -Tropomyosin regulatory sequence.
  • the fusion ⁇ -Tropomyosin- ⁇ -gal construct was transfected (by Calcium Phosphate precipitation) into NIH3T3 and or Rat2 cells in culture. Clonal lines that have stably integrated the fusion reporter gene were analyzed for the expression of the B-galactosidase gene as evidence of the functionality of the ⁇ -Tropomyosin promoter in the construct. This is performed by Northern hybridization using a B-galactosidase specific probe and by histochemical assay in the presence of the chromogen X-Gal (5-bromo-4-chloro-3-indoyl-B-D-galactoside) for the expression of the B-Gal mRNA and protein respectively.
  • the sensitivity of the ⁇ -Tropomyosin regulatory region to cellular transformation will be assessed in ths clonal cell lines by the subse ⁇ uent introduction of an activated oncogene sequence for examt - the T24 Ras (83) either by conventional Calcium I tansfection or retroviral infection.
  • the alteration of expre * ion of the B-Gal reporter in these Ras transformed cells will be documented by Northern blot analysis of message expression and X-Gal staining for functional reporter protein expression.
  • the system will be further challenged by subjecting the cells to a reversion of the transformed phenotype by exposure to Ras antisense oligonucleotides at a concentration that has been previously found to inhibit H-Ras expression (84).
  • the alteration of the reporter gene expression following reversal of the transformed phenotype will be verified by measuring B-Gal mRNA in Northern hybridization and B-Galactosidase activity.
  • the ⁇ -Tropomyosin- ⁇ -Gal construct will then be introduced into animals for example by nuclear injection or ES cell technology.
  • Transgenic animals that have integrated the promoter-reporter construct into their germ line are identified by Southern hybridization and PCR techniques. The animals are bred to obtain progeny that will be examined for expression of the reporter gene in the appropriate tissues (85, 86). Tissues or whole embryos will be fixed in 0.2% glutaraldehyde/1% formaldehyde. Alternatively, adult animals will be perfused with 4% paraformaldehyde as previously described (87).
  • Expression of B-gal will be analyzed by incubation with the chromogenic substrate X-Gal which results in the deposition of blue stain. Expression of the B-gal protein can be analyzed by incubation of sectioned tissues with antibodies specific for the protein. The required antibodies can be obtained from Boehringer Mannhein.
  • a heritable preneoplastic condition is established by the mutational inactivation of p53 activity (88, 89). This is accomplished through the expression of an inactivated tumor suppressor gene product (90).
  • the p53 sequence is altered by oligonucleotide site directed mutagenesis (91) to obtain a mutant p53 that has sustained an inactivating mutation that substitutes an arginine for a histidine at position 175.
  • the mutation will be verified by DNA sequence analysis (92) . This mutation has been shown to result in an inactive p53 product with an increased efficiency for cooperation with the Ras oncogene in transforming primary rat cells in culture (93, 94).
  • the expression of the mutant p53 gene or cDNA is placed under the transcriptional control of a strong ubiquitously active promoter like the Cytomegalovirus early promoter (CMV) (95, 96) or B-actin promoter (97) .
  • CMV Cytomegalovirus early promoter
  • B-actin promoter 97
  • This is obtained by subcloning the 1.9kb Xbal fragment of the mutated human p53 cDNA (98) into a CMV vector (e.g. pCMVB) downstream from the 620bp CMV early promoter as shown in Figure 2.
  • the p53 sequence can similarly be cloned downstream from a B-actin promoter.
  • the efficiency of this construct to direct the synthesis of the mutant p53 is initially assessed by transfecting the construct into NIH3T3 or Rat2 cells in culture.
  • the stable expression of the mutant p53 in clonal cell lines is examined by both Northern blot and p53 specific immunoblot analyses.
  • the ability of the mutant p53 product to interfere with the normal tumor suppressor function in these cells is assessed for by its cooperative effect with an activated oncogene e.g. H-ras to bring about an increased frequency of foci formation. This phenomenon is well documented (25) .
  • a transgenic animal bearing the mutant p53 construct is obtained for example by nuclear injection or ES cell technology.
  • Transgenic animals that have incorporated the fusion gene construct will be identified by Southern blot or PCR analyses as previously described.
  • the expression of the mutant p53 transgene will be examined by Northern hybridization analysis of RNA from various tissues. The identity of the mutant p53 message will be further verified by RNAse protection analysis (99).
  • Other p53 mutants expressed in transgenic mice have been shown to result in the development of dysplasia in a number of tissues with a predisposed low frequency for the development of Lung, Bone and Lymphoid tumors (26) .
  • the p53 gene is inactivated by gene interruption (100) animals were also predisposed to the development of tumors.
  • Transgenic animals that have integrated the mutant p53 construct in our experiments will be examined for the development of tissue dysplasia and tumor by conventional histopathological methods (101).
  • the propensity of the cells in these animals to develop tumors at high frequency is examined by the introduction of an activated oncogene e.g. H-ras either through retroviral transduction or performing crosses with the Leder H-ras oncomouse.
  • an activated oncogene e.g. H-ras either through retroviral transduction or performing crosses with the Leder H-ras oncomouse.
  • test animal that incorporates both the transformation sensitive ⁇ -Tropomyosin-B-Galactosidase reporter construct and the mutant p53 gene is obtained by mating the two types of transgenic animals above upon attainment of sexual maturity.
  • Lovastatin (Schafer et al, Science, 1989, 245, 379-390.

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Abstract

Procédé in vivo destiné à tester la capacité d'un médicament à interférer avec le début ou le déroulement d'une transformation néoplastique. Un médicament est administré à un mammifère porteur de tumeur qui possède une unité rapporteuse sensible à la transformation. Ladite unité rapporteuse comporte un gène de structure dont l'expression est provoquée ou stoppée selon que les cellules du mammifère se trouvent ou non dans un état néoplasique. On détermine ensuite si le médicament a inversé les effets de la transformation ou interféré avec ledits effets par l'analyse de l'expression du gène de structure. Ce procédé permet la détection des effets anti-néoplasiques de médicaments qui sont non cytotoxiques aux doses administrées. Des animaux et des cultures cellulaires transgéniques sont également décrits.
PCT/US1993/004363 1992-05-08 1993-05-07 Test in vivo de medicaments anti-neoplasiques WO1993023533A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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WO1994016080A1 (fr) * 1993-01-08 1994-07-21 Exemplar Corporation Procede in vitro/in vivo d'identification de medicaments anti-neoplasiques
WO1998012337A1 (fr) * 1996-09-23 1998-03-26 Chiron Corporation Integration aleatoire et ciblee d'un promoteur pour l'analyse de genes
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO2000036106A2 (fr) * 1998-12-17 2000-06-22 Xenogen Corporation Evaluation non-invasive d'une reponse physiologique chez un mammifere

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WO1989005864A1 (fr) * 1987-12-15 1989-06-29 The Trustees Of Princeton University Systemes de test de transgeniques visant a detecter des mutagenes et des carcinogenes
WO1990004632A1 (fr) * 1988-10-21 1990-05-03 The United States Of America, Represented By The Secretary, United States Department Of Commerce Animaux transgeniques pour tester la resistance a de multiples medicaments
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1990012093A1 (fr) * 1989-03-31 1990-10-18 President And Fellows Of Harvard College Procede d'evaluation des effets in vivo d'agents biologiques sur des tumeurs
WO1990015869A1 (fr) * 1989-06-19 1990-12-27 Embryogen Corporation Systemes transgeniques d'examen de la peau
WO1991015116A1 (fr) * 1990-03-30 1991-10-17 Amrad Corporation Limited Animaux doublement transgeniques

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Publication number Priority date Publication date Assignee Title
WO1989005864A1 (fr) * 1987-12-15 1989-06-29 The Trustees Of Princeton University Systemes de test de transgeniques visant a detecter des mutagenes et des carcinogenes
WO1990004632A1 (fr) * 1988-10-21 1990-05-03 The United States Of America, Represented By The Secretary, United States Department Of Commerce Animaux transgeniques pour tester la resistance a de multiples medicaments
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1990012093A1 (fr) * 1989-03-31 1990-10-18 President And Fellows Of Harvard College Procede d'evaluation des effets in vivo d'agents biologiques sur des tumeurs
WO1990015869A1 (fr) * 1989-06-19 1990-12-27 Embryogen Corporation Systemes transgeniques d'examen de la peau
WO1991015116A1 (fr) * 1990-03-30 1991-10-17 Amrad Corporation Limited Animaux doublement transgeniques

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994016080A1 (fr) * 1993-01-08 1994-07-21 Exemplar Corporation Procede in vitro/in vivo d'identification de medicaments anti-neoplasiques
WO1998012337A1 (fr) * 1996-09-23 1998-03-26 Chiron Corporation Integration aleatoire et ciblee d'un promoteur pour l'analyse de genes
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO1998028971A3 (fr) * 1997-01-03 1998-10-29 Univ Technology Corp Toxicite de la beta-amyloide
WO2000036106A2 (fr) * 1998-12-17 2000-06-22 Xenogen Corporation Evaluation non-invasive d'une reponse physiologique chez un mammifere
WO2000036106A3 (fr) * 1998-12-17 2000-11-02 Xenogen Corp Evaluation non-invasive d'une reponse physiologique chez un mammifere
AU774183B2 (en) * 1998-12-17 2004-06-17 Xenogen Corporation Non-invasive evaluation of physiological response in a mammal
US7449615B2 (en) 1998-12-17 2008-11-11 Xenogen Corporation Non-invasive evaluation of physiological response in a transgenic mouse

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