WO1997038091A1 - METHODES POUR AMELIORER LA CROISSANCE ANIMALE ET LA PROLIFERATION CELLULAIRE PAR ELIMINATION DE LA p27Kip1 FONCTIONNELLE - Google Patents

METHODES POUR AMELIORER LA CROISSANCE ANIMALE ET LA PROLIFERATION CELLULAIRE PAR ELIMINATION DE LA p27Kip1 FONCTIONNELLE Download PDF

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WO1997038091A1
WO1997038091A1 PCT/US1997/005921 US9705921W WO9738091A1 WO 1997038091 A1 WO1997038091 A1 WO 1997038091A1 US 9705921 W US9705921 W US 9705921W WO 9738091 A1 WO9738091 A1 WO 9738091A1
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cells
gene
cyclin
kιpl
dependent kinase
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PCT/US1997/005921
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English (en)
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Andrew Koff
Hiroaki Kiyokawa
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Sloan-Kettering Institute For Cancer Research
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Priority to AU27257/97A priority Critical patent/AU2725797A/en
Publication of WO1997038091A1 publication Critical patent/WO1997038091A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • CDKs cyclin-dependent kinases
  • CDKs minimally contain a catalytic subunit, the CDK, and a regulatory subunit, a cyclin.
  • the activation state of these kinases ultimately determines the fate of cell proliferation during Gl; when active cells enter S-phase. Inactivation of either kinase leads to cessation of proliferation and withdrawal from the mitotic cycle.
  • the activity of either CDK4/6, CDK2, or both kinases is absent in growth arrested cells. CDC2 kinase activity appears after the Gl/S transition and is necessary for the G2/M transition.
  • Gl CDKs integrate mitogenic and anti-mitogenic signals that regulate progression through Gl to a point where further cell cycle progression continues autonomously (Pardee, 1989) . This implies a capacity to regulate CDKs in response to external factors (Peters and Herskowitz 1995) .
  • the activation of CDKs is subject to multiple levels of regulation: the synthesis of the cyclin and CDK (Ewen et al., 1993; Geng and einberg, 1993; Tanquay and Chiles, 1994), the assembly of these proteins into complexes (Serrano et al . , 1993; Guan et al . , 1996) , the activation of these complexes (Koff et al .
  • Inks specifically target the CDK4 and CDK6 kinases and bind to these proteins preventing their interaction with cyclin D (Serrano et al . , 1993; Guan et al . , 1994; Hannon and Beach, 1994; Guan et al . , 1996) .
  • Kips bind preferentially to cyclin/CDK complexes and either prevents their activation by the CDK-activating kinase (Koff et al . , 1993) , or inhibit their kinase activity (Polyak et al . , 1994b; Slingerland et al . , 1994; Toyoshima and Hunter, 1994; Lee et al. 1995; Matsuoka et al . , 1995) .
  • Kips unlike Inks, are promiscuous and interact with most Gl CDKs (Sherr and Roberts, 1995) .
  • Kip family there are three members of the Kip family: p21 (Gu et al . , 1993; Harper et al . , 1993; Xiong et al . , 1993) , p27 (Polayk et al . , 1994b; Toyoshima and Hunter, 1994) , and p57 (Lee et al . , 1995; Matsuoka et al . , 1995) . These proteins contain a conserved domain that is both necessary and sufficient for cyclin/CDK interaction and inhibition (Nakanishi et al . , 1995) . In proliferating cells, p21 and p27 associate with active CDKs (Zhang et al .
  • p27 The properties of p27 suggest that it might have an important role regulating entry into and exit from the mitotic cycle.
  • the increase in amount of p27/CDK2 complex occurs through at least one of three mechanisms dependent on the cell type and the condition leading to growth arrest: release of p27 from a sequestered state in cyclin D/CDK4 complexes (Polyak et al . , 1994) ; Reynisdotter et al .
  • p27 cDNA is sufficient to induce Gl arrest (Polyak et al . , 1994b; Toyoshima and Hunter 1994) , and in some cells induce differentiation phenotypes (Kranenberg et al . , 1995; Liu et al . , 1996) , presumably by targeting Gl cyclin-dependent kinase activity.
  • antisense vectors targeted to p27 mRNA increase the fraction of cells in S-phase (Coats et al . , 1996) . These properties suggest that p27 might function to establish an inhibitory threshold which Gl CDKs must surpass before activation and entry into S-phase. gintma y of the Invention
  • This invention provides a recombinant non-human animal lacking the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • the recombinant non-human animal may be made by altering the gene encoding p27 K ⁇ pl and introducing the altered gene into the genome of an animal.
  • the alteration comprises addition, deletion or mutation of at least one nucleotide of the gene which encodes p27 K ⁇ pl .
  • Part or all of the gene encoding for p27 K ⁇ pX may be deleted and replaced by at least one selectable marker gene .
  • This invention provides a recombinant non-human animal having an increase in the production of thymocytes as compared with the animals having the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • This invention provides a method to produce recombinant non-human animal lacking the cyclin-dependent kinase inhibitor function p27 K ⁇ pl .
  • the method comprises altering the gene encoding p27 Klpl , introducing the altered gene into the genome of the animal, identifying the altered gene encoding p27 Klpl carrying animals and interbreeding of the altered gene carrying animal to generate p27 K ⁇ pl deficient animal.
  • the gene encoding p27 K ⁇ pl is altered by insertion of a selection marker gene and a negatively selectable marker gene is then inserted adjacent to the altered gene whereby the distance between the marker gene and the altered gene is sufficient to carry homologous recombination.
  • This construct is introduced into embryonic ste cells and positively select the marker which alters the gene encoding p27 K ⁇ l and negatively select the inserted adjacent marker. This positive and negative selection scheme will ensure that the altered gene encoding p27 K ⁇ pl will integrate to the correct locus in the genome of the animal .
  • This invention further provides that the altered stem cells can be introduced by microinjection of the altered embryonic stem cell line to blastocysts.
  • the invention further provides a method for increasing the proliferation of thymic T-cells in an animal by treating the animal to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • the treatment may be performed by exposing the animal with different agents capable of inhibiting the cyclin-dependent kinase inhibitor function of the p27 K ⁇ pl .
  • the agent is an antibody or a portion of an antibody directed against p27 protein.
  • This invention also provides a 'method for increasing the proliferation of hematopoietic cells which comprises treating the hematopoietic cells to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl , thereby increasing the proliferation of the hematopoietic cells.
  • This invention also provides a method for increasing the amount of hematopoietic cells for bone marrow transplantation between a donor and a recipient comprising steps of: a) obtaining the bone marrow cells from the donor; b) treating the bone marrow cells to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl ; c) introducing the treated cells to the recipient.
  • the invention also provides a method for alleviating symptoms of AIDS patients which comprises (a) collecting peripheral lymphocytes from the patient, and (b) treating the collected lymphocytes such that after the treated lymphocytes will lack the cyclin-dependent inhibitor function of p27 K ⁇ pl .
  • a method for alleviating symptoms of AIDS patients which comprises (a) collecting peripheral lymphocytes from the patient, and (b) treating the collected lymphocytes such that after the treated lymphocytes will lack the cyclin-dependent inhibitor function of p27 K ⁇ pl .
  • only T-cells are treated.
  • only helper T-cells are treated.
  • This invention also provides a method for alleviating symptoms of AIDS patients comprising, (a) collecting multipotent cells from an AIDS patient, and (b) treating the multipotent cells with an agent to eliminate the cyclin-dependent kinase inhibition function of p27 K ⁇ pl ; and, (c) introducing the treated cells to the same AIDS patient.
  • this invention provides a method for increasing the efficacy of a cytokine to a subject comprising administering to the subject an effective amount of the cytokine in the presence of an agent which is capable ' of inhibiting the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • the cytokines which may be used in this invention include but are not limited to GM-CSF, G-CSF erythropoietin, interleukins, interferons, megakaryocyte derived growth factors and stem cell factors.
  • Figure 1 Targeted insertion of the p27 gene produces an amino-terminal truncated protein that does not inhibit Gl-CDKs.
  • the targeting construct contains a 6 kb genomic sequence encompassing both coding exons of p21 with a neo cassette introduced into the Sma I site at amino acid 42. Transcriptional direction of the neo gene is shown.
  • the thymidine kinase gene (TK) is indicated to the 3' end of the genomic sequences. Homologous recombination within the genomic sequence introduces the neo gene and eliminates the TK gene (Mansour et al., 1988) . Neo gene insertion will introduce an Eco RI site. Thus a single probe to the 5' end of the genomic sequences can be used to identify recombinant alleles at 4.0 kb and wild-type alleles at 7.5 kb.
  • Immunoprecipitates of p27 or whole cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies described to the left of the panels.
  • Antibody abbreviations are: p27-carboxyl terminal specific antibody ( ⁇ p27-CT) , p27-amino terminal specific antibody ( ⁇ p27-NT) , CDK2 and CDK4 carboxyl specific antibodies ( ⁇ CDK2 and ⁇ CDK4, respectively) , rabbit anti mouse Ig (RAM) . Markers and the migration of the Ig heavy chain are indicated.
  • mice The size of mice is a function of p27 gene copy number.
  • the mean weight of mice is represented by bars. p27+/+, filled bars; p27+/-, stippled bars; p27-/-, open bars.
  • the actual weights of individual mice are indicated by the gray dots.
  • Figure 3 There is a correlation between expression of p27 protein in an organ and the increase in weight of the organ following p27 gene disruption.
  • p27 ⁇ A mice have enlarged pituitaries which can be attributed to selective hyperplasia of the intermediate lobe.
  • p • posterior lobe; i, intermediate lobe; a, anterior lobe.
  • the intermediate lobe of 11 week old p27 "A mice is increased in size relative to the p27 * controls with little change in cellular composition.
  • Figure 5 Increased proliferation of thymocytes accounts for the increase in T-cell number.
  • Thymocytes of the p27 "A mice are susceptible to apoptosis .
  • Thymocytes were isolated from mice and exposed to either 0.25 or 5 Gray (Gy) of irradiation or to 1 mM dexamethasone as indicated below each sample. Apoptotic cells were detected by the appearance of acridine orange stained condensed or fragmented chromatin and plotted as a percent of the total cells. p27+/+, filled bars; p27-/-, open bars.
  • Vaginal smears were taken daily from p27 *A (top) or p27 "A (bottom) mice and stained with hemotoxylin and eosin. Histology is an indicator of position in estrus (Nelson et al . ,
  • mice complete estrus in four days (from left to right) passing through diestrus (6A) , proestrus (6B) , estrus (6C) and metestrus (6D) , each in a single day.
  • p27 ⁇ / ⁇ mice have a prolonged diestrus and estrus phase, and there is an increase in the amount of mucus in the smears of diestrus phase of the p27 "A mice.
  • Figure 7 p27 "A mice are deficient in corpus luteum formation.
  • Figure 8 Relationship between thymocyte number and genotype of female mice at 5, 8 and 13 weeks. Filled squares, p27+/+; Open squares, p27-/-.
  • the present invention provides a recombinant, non-human animal which comprises functionally deficiency of the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • the functional deficiency describing the p27 K ⁇ pl may occur in the DNA, RNA or protein level such that functional gene product of p27 K ⁇ pl is either not produced or deficient.
  • recombinant animals means animals produced by the recombinant DNA technologies.
  • the animals produced are genetically altered such that the kinase inhibitor function of the p27 Klpl is eliminated. It is the intention of this invention to cover animals which are genetically altered by recombinant DNA technology such that p27 ⁇ - p is either not produced or it is functionally deficient .
  • such a deficiency is created by an alteration at the DNA level.
  • the alteration may be an addition, deletion, mutation or combination thereof.
  • the alteration is created by an insertion.
  • the insertion takes place in the region responsible for the cyclm-dependent inhibitor function of p27 K ⁇ ⁇ .
  • the insertion may be achieved by inserting at least one selectable marker gene into the coding region.
  • the selectable marker genes include but are not limited to neomycin resistant gene, thymidine kinase gene, adenine phophoribosyl transferase gene, hypoxanthine-guanine phosphoribosyl transferase gene, dihydrofolate reductase gene or a combination of more than one of the preceding genes or other selectable marker genes known to an ordinary skilled in the art. These selectable marker genes will express particular phenotypes under appropriate selective conditions.
  • the recombinant non-human animal of this invention is generated by deleting part or all of the coding region of the gene encoding p27 ⁇ ipl and replaced by neomycin resistant gene.
  • selectable marker genes are examples of selectable marker genes. Some of the drug resistant genes are neomycin resistant gene and dihydrofolate reductase which can be selected by methotrexate. Other selectable marker include thymidine kinase gene, adenine phosphoribosyl transferase gene, hypoxanthine-guanine phosphoribosyl transferase gene. In a preferred embodiment, neomycin resistance gene is used for selection and to altered the gene encoding p27 K ⁇ pl .
  • the altered gene can be introduced either: (1) at an early developmental stage such that it is stably integrated into the germline and somatic cells of the animal.
  • One embodiment would be microinjection into fertilized eggs (1 cell stage) and development of transgenic founder animals using standard transgenic technology or (2) after the animal is born in which case the transgene is only introduced into somatic cells.
  • Several related embodiments would include, but not limited to, the uses of retroviruses , adenoviruses , asialoglycoprotein (ASO/PL/DNA) DNA complexes or liposomes to transfer the altered genes to somatic cells for the purpose of producing the claimed recombinant nonhuman animals. This embodiment is not limited to these methods but includes any method which transfers the altered gene to somatic cells for the purpose stated above.
  • the invention also provides a recombinant p27 K ⁇ pl deficient animals which show a significant increase in the number of granulocyte/ acrophage progenitors and mixed pluripotent progenitors in the spleen and a significantly greater number of thymocytes as compared to the wild type mouse.
  • a recombinant p27 K ⁇ pl deficient mouse it is clear that the recombinant animals of this invention may be of any animal species. Therefore, it is understood that the invention encompasses all animals.
  • the animal is a mammal. In another embodiment, the animal is selected from a group consisting of rodent, cattle, pig and sheep. In a still another embodiment, the animal is a frog. It is recently identified that a p27 Klpl analog exist in Xenopus (Shou and Dunphy, 1996) . In a separate embodiment, the animal is a fish. In a another separate embodiment, the animal is a Caenorhabdi tis el egans .
  • Another aspect of the invention involves a method to produce a recombinant p27 K ⁇ pl deficient non-human animal.
  • such animal is produced by a) altering the gene encoding p27 K ⁇ pl so that the p27 K ⁇ pl gene product is functionally deficient, b) introducing the altered gene into the genome of the animal , c) identifying the altered gene carrying animals, and d) generating altered gene carrying animals which is p27 Klpl deficient.
  • the alteration of the gene encoding p27 Kipl comprises addition, deletion or mutation, or any other methods known to those skilled in the art.
  • the DNA encoding p27 Klpl may be altered by methylation or the like to inactivate the gene expression.
  • the gene encoding p27 K ⁇ pl is cloned in a plasmid and the alteration of the gene encoding p27 Klpl is done on the cloned DNA.
  • plasmids well known to a skilled practitioner will serve this purpose.
  • the introduction of the altered gene encoding p27 K ⁇ pl into an animal comprises steps of (a) electroporating of the altered gene into embryonic stem cells, (b) culturing in vitro the treated embryonic stem cells, (c) selecting of the deficient p27 K ⁇ pl carrying embryonic stem cells and introducing the deficient p27 K ⁇ pl carrying embryonic stem cells to the blastocysts by microinjection. The blastocysts are introduced into the animal .
  • the gene encoding p27 K ⁇ pl is isolated and cloned in a plasmid and then altered by insertion of a selection marker gene.
  • a negatively selectable marker gene is then inserted adjacent to the altered gene whereby the distance between the marker gene and the altered gene is sufficient to carry out homologous recombination.
  • the plasmid containing the altered gene is used to transform an embryonic ste cell line.
  • the cells which incorporate the altered gene are positively selected for the marker in the gene encoding p27 K ⁇ pl locus and negatively selected for the inserted adjacent marker.
  • the final selected altered embryonic stem cell line is microinjected into the blastocyst .
  • the plasmid containing altered gene with the selection marker gene transformed ste cells are selected for the selectable marker phenotype.
  • the negative selection marker is the thymidine kinase gene.
  • the transfected stem cells are then positively selected with the expression of neomycin resistance phenotype and negatively selected with thymidine kinase phenotype.
  • the invention further provides a method for increasing the proliferation of thymic T-cells in an animal by treating the animal to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ p ⁇ .
  • the treatment may be performed by contacting the animal with different agents capable of inhibiting the cyclin-dependent kinase inhibitor function of the p27 K ⁇ pl .
  • the agent is an antibody or a portion of an antibody directed against p27 protein.
  • the agents may be entrapped in liposo es for efficient delivery. Methods to make liposomes are well-known in the art.
  • This invention also provides a method for increasing the proliferation of T-cells which comprises treating the T- cells to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl , thereby increasing the proliferation of the T-cells.
  • This invention also provides a method for increasing the proliferation of hematopoietic cells which comprises treating the hematopoietic cells to eliminate the cyclin-dependent kinase inhibitor function of p27 Klpl , thereby increasing the proliferation of the hematopoietic cells.
  • This invention also provides a method for increasing the amount of hematopoietic cells for bone marrow transplantation between a donor and a recipient comprising steps of: a) obtaining the bone marrow cells from the donor; b) treating the bone marrow cells to eliminate the cyclin-dependent kinase inhibitor function of p27 K ⁇ l ; c) introducing the treated cells to the recipient.
  • the invention also provides a method for alleviating symptoms of AIDS patients which comprises (a) collecting peripheral lymphocytes from the patient, and (b) treating the collected lymphocytes such that after the treated lymphocytes will lack the cyclin-dependent inhibitor function of p27 K ⁇ pl .
  • a method for alleviating symptoms of AIDS patients which comprises (a) collecting peripheral lymphocytes from the patient, and (b) treating the collected lymphocytes such that after the treated lymphocytes will lack the cyclin-dependent inhibitor function of p27 K ⁇ pl .
  • only T-cells are treated.
  • only helper T-cells are treated.
  • This invention also provides a method for alleviating symptoms of AIDS patients comprising, (a) collecting ultipotent cells from an AIDS patient, and (b) treating the multipotent cells with an agent to eliminate the cyclin-dependent kinase inhibition function of p27 K ⁇ pl ; and, (c) introducing the treated cells to the same AIDS patient.
  • the cells are treated with an agent.
  • the agent is an antisense oligonucleotide capable of inhibiting the expression of p27 K ⁇ pl .
  • the agent is a vector containing a promoter operatively linked to a cDNA which encodes an antisense mRNA capable of inhibiting the expression of p27 K ⁇ pl .
  • the above-described cells including lymphocytes, thymic T- cells, helper T-cells, hematopoietic cells and bone marrow cells may be treated with linearized vector containing the gene encoding p27 K ⁇ pl which is disrupted by a selectable marker gene.
  • Cells w:.th incorporated the vector will be selected in an appropriate selection medium.
  • the event of homologous recombination may be confirmed by the performing restriction enzyme digests.
  • the treatment is performed by contacting the above-described cells with an oligonucleotide antisense encoding the region of p27 K ⁇ pl responsible for the cyclin-dependent kinase inhibitor function of p27 Klpl .
  • Antisense DNA or RNA which can hybridize with the gene encoding p27 K ⁇ pl may be introduced into the cells to hybridize with the p27 Klpl mRNA such that no translation can occur and therefore, no functional protein is produced.
  • the methods for introduction of the antisense DNA or RNA into the cell are well known to a skilled practitioner.
  • One method is to clone and express an antisense under a promoter such that a larger amount of antisense RNA against the mRNA encoding p27 K ⁇ pl will be produced.
  • the antisense for p27 Klpl RNA produced will hybridize with the normal p27 K ⁇ pl mRNA and therefore, interfering with the expression of p27 K ⁇ pl .
  • cells are treated with chemicals or small peptides capable of preventing p27 K ⁇ pl from associating with cyclin/CDK complex.
  • the invention also provides a method for alleviating the symptoms of AIDS patients, comprising administering to the patient an effective amount of an adenovirus or any suitable vector containing a promoter operatively linked to a cDNA which encodes an antisense mRNA capable of inhibiting the expression of p27 K ⁇ pl .
  • this invention provides a method for increasing the efficacy of a cytokine to a subject comprising administering to the subject an effective amount of the cytokine in the presence of an agent which is capable of inhibiting the cyclin-dependent kinase inhibitor function of p27 K ⁇ pl .
  • the cytokines which may be used in this invention include but are not limited to GM-CSF, G-CSF erythropoietin, interleukins, interferons, megakaryocyte derived growth factors and stem cell factors such as c- kit.
  • the mouse p27 gene was isolated by screening a 129SV mouse genomic library (Stratagene) using the total coding region of mouse p27 cDNA as a probe.
  • This construct was cleaved at a Sma I site located in codon 42 of exon 1 by partial digestion, and a 1.1 kb blunt-ended Xho I-Bam HI fragment encoding pMC-lneo polyA (Stratagene) was inserted.
  • This construct was subsequently digested with Not I and Bam HI, and inserted a 1.2 kb HSV-TK gene cassette driven by the polyoma enhancer.
  • Not I-linearized targeting vector was introduced into CJ7 ES cells by electroporation (Swiatek and Gridley, 1993) . Positively transfected cells were selected in 0.5 mg/ml G418 and 0.2 mM ganciclovir, and the surviving colonies were picked, expanded and analyzed with their genotype at the p27 locus by Southern blotting. Genomic DNA was digested with Eco RI . The l.l-kb Eco RI-Bam HI fragment 5' to the vector fragment was used as a probe as shown in Fig. 1. This probe hybridizes to a 7.5 kb Eco RI fragment from the wild-type allele, and a 4.0 kb Eco RI fragment from the homologous recombinant allele (Fig. IB) .
  • p27 heterozygous ES cells (10-15 cells per blastocyst) were icroinjected into blastocyst stage C57BL/6 mouse embryos. Injected blastocysts were transplanted into uteri of pseudo pregnant C57BL/6 mice. Chimeric males were crossed to C57BL/6 females. Germline transmission of the injected ES cells monitored by detecting agouti mice among the FI offspring, and subsequent Southern blotting as described above.
  • Embryonic ' fibroblasts were prepared from embryos 13.5 days post coitus. Fibroblasts were cultured to confluence
  • p27 was immunoprecipitated from 300 ⁇ g of extract and blotted with antibodies specific for the carboxyl terminus of CDK2 and CDK4 (Santa Cruz Biotechnology) .
  • [7- 32 P]ATP, GST-Rb fusion protein, and increasing amounts of wild-type or mutant p27 protein Reactions were stopped by the addition of sample buffer and proteins separated on a 10% SDS-polyacrylamide gel. The incorporation of [ ⁇ - 32 P]ATP into GST-Rb fusion protein was measured by phosphoimager (Fuji) .
  • mice were anesthetized by intraperitoneal injection of avertin, immobilized on a cassette containing XAR film (Kodak) , and irradiated at 70 kV for 0.2 sec.
  • IGF-I was measured by a double antibody radioimmunoassay using recombinant hIGF-I (Genentech) and anti-hIGF-I serum UBK487 (National Hormone and Pituitary Program, NIH) after removal of binding proteins from a 5 ⁇ l aliqot of serum by means of a Sep-Pak C18 reverse phase cartridge (Millipore) . After obtaining all the measurements from these mice, the amount of IGF1 in p27+/+ and p27-/- mice were compared in the following ways: all sex matched, age/sex matched, and all age matched.
  • Histology- Tissues were fixed in 4% paraformaldehyde at 4°C overnight, treated stepwise with 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, histoclear, histoclear/paraffin (1:1) , and paraffin, and sectioned at 4 microns .
  • the sections were used for hematoxylin/eosin staining or immunohistochemistry as indicated in the legends to the figures.
  • mice were injected intraperitoneally (50 ⁇ g per g body weight) with bromodeoxyuridine (BrdU) (Morstyn et al . , 1983) .
  • the mice were sacrificed 2 hours later. Thymus, spleen, and in females the ovaries were isolated and prepared the tissues for sectioning.
  • the tissue sections were rehydrated and treated with 5 ⁇ g/ml proteinase K, 50 mM Tris-HCl (pH 7.5) , and 5 mM EDTA at 37°C for 10 minutes.
  • the slides were washed with PBS and then acid-treated (IN HCl) for 10 minutes at 55°C.
  • Thymocyte apoptosis was measured as described (Lowe et al., 1993; Clarke et al . , 1993) .
  • thymocytes were exposed to ⁇ -rays from a 137 Cs source at 0.1 Gy/min. Following irradiation cells were cultured in DMEM supplemented with 5% fetal calf serum for 8 hours. The cells were subsequently fixed to glass slides with acetic acid/ethanol solution (1:9) overnight.
  • RT RT
  • RT 1.5% normal goat serum (30 minutes, RT)
  • rabbit-anti hACTH sera (1:10,000, NIH)
  • monkey-anti rat GH sera (1:50,000, LAF 82469) (24 hours, 4°C)
  • biotinylated goat-anti rabbit or goat-anti monkey IgG (1:200, 30 minutes, RT; Vector)
  • Vectastain Elite ABC reagent (30 minute, RT; Vector)
  • DAB diaminobenzidine tetrahydrochloride
  • Reagents were prepared in 0.05 M Tris buffered saline (TBS, pH 7.4) containing 0.1% Triton X-100, except for ABC and DAB which were diluted in TBS alone. Replacement of the primary immune serum with normal rabbit or monkey serum abolished staining. Specificity of each antisera was confirmed in a previous study by inhibition of staining when each sera was preabsorbed with the respective antigen (1 mg/ml dilute primary antibody) for 2h at 37°C before use in the immunocytochemical procedure .
  • Spleen cells were obtained by pressing the spleen against the bottom of a tissue culture dish with a bent syringe needle. Cells were collected by centrifugation and resuspended them in 0.15M NH disciplineC1, ImM KHC0 3 , and 0. ImM EDTA (pH 7.2) to lyse erythrocytes . The remaining cells were centrifuged through FBS, washed with PBS, and resuspended in RPMI 1640 + 10% FBS, 2mM glutamine, non-essential amino acids, ImM sodium pyruvate, and 50 mM 2-mercaptoethanol .
  • p27 coding sequence was disrupted by homologous recombination using a vector containing genomic p27 isolated from a 129Sv mouse DNA library (Fig. 1A) .
  • the mouse p27 gene is encoded by two exons divided within codon 159.
  • neo neomycin-resistance gene cassette
  • TK thymidine kinase
  • ES CJ7 mouse embryonic stem
  • the cyclin/CDK inhibition domain was targeted for gene disruption rather than a complete deletion.
  • Fig. 1C immunoblotting
  • Using an antibody to either full-length p27 or the carboxyl-terminus of p27 a 20 kDa protein in p27 " fibroblasts, and a 27 kDa protein in p27 *A fibroblasts were detected. Both proteins were detected in p27 *A fibroblasts (data not shown) .
  • antibody specific to the amino-terminus of p27 recognized only the 27 kDa protein and not the 20 kDa protein (Fig.
  • RNA transcripts from the p27 mutant allele were amplified and its structure determined; the mutant transcript had the insertion of the neo gene in the antisense direction and normal splicing of the intron separating exons I and II.
  • This transcript contains a predicted open reading frame that encodes amino acid 52-198 of the p27 protein (data not shown) .
  • ⁇ 51 amino-truncated mutant of p27 protein which was called ⁇ 51.
  • the His tagged ⁇ 51 from bacteria was purified and added it to extracts of Sf9 cells coinfected with baculoviruses expressing either cyclin E and CDK2 , or cyclin D2 and CDK , and measured the Rb kinase activity of the cyclin/CDK complexes.
  • ⁇ 51 inhibited Rb-phosphorylation by these kinases less efficiently than the full length p27 (Fig. ID) .
  • ⁇ 51 interacted poorly with cyclin/CDK complexes in cells (Fig. 1C) .
  • mice lacking functional p27 are larger than normal Following weaning (day 21 to day 28) the p27 ⁇ A mice weighed 20-40% more than sex matched littermate controls.
  • a representative litter of male mice at 8 weeks of age is shown in Fig. 2A.
  • mice In 8 week old mice the difference between the populations was much greater and the smallest p27 ⁇ A mice weighed as much as the largest p27 *A mice (p ⁇ 0.01) . The mean weight and range of p27* A mice were intermediate . To determine the earliest time after birth that could be detected this difference, the mice were weighed from one week post-partum. From a total of 252 offspring, there were 21 p27 ' mice in litters where sex matched comparisons to controls could be made.
  • mice expressing GH (Palmiter et al., 1982) or IGF-I (Mathews et al. , 1988) grow larger than control mice.
  • GH secreted from the pituitary in response to hypothalamic signals, regulates postnatal body growth mainly by stimulating IGF-I expression in the peripheral tissues.
  • IGF-I insulin growth factor
  • FIG. 4B right panel
  • a marked increase in vascularity was present throughout the lobe, manifested as lakes of distended capillaries filled with red blood cells. Cellular morphology, however, was normal and no evidence of tumor formation was present.
  • the anterior lobes were even more compressed than at 11 weeks . In some animals the tissue mass was sufficiently large as to cause compression of the ventral hypothalamus .
  • the intermediate lobe contains cells that produce alpha-MSH. There was homogeneous staining with an antibody that reacts with POMC-derived peptides, including alpha-MSH, in the IL cells at 11 weeks (Fig. 4B) .
  • the staining was non-homogeneous with some of the nodular regions exhibiting intense staining while in others, staining was markedly decreased, in some, to the point of non-staining.
  • immunohistochemistry was used to detect GH.
  • the somatotropes producing GH are in the anterior lobe of the pituitary.
  • both the numbers of somatotropes and the intensity of GH staining were comparable to controls (data not shown) .
  • IGF-I is the major GH dependent post-natal growth factor. IGF-I induces cell proliferation in major body components including the bone, cartilage, and connective tissue. It was next determined if the serum level of IGF-I was perturbed in p27 ⁇ / ⁇ mice. Applicants did not find any significant difference in serum levels of IGF-I. The amount of IGF-I in 27 samples isolated from six pairs of animals as described in the methods section was measured. The level of IGF-I in sera isolated from both male and female p27 *A mice was 36+ .
  • p27 disruption increases the number of S-phase thymocytes
  • the disruption of p27 might affect the proportion of proliferating cells in a tissue before maturation. In most specialized cell types of the animal the proliferative index decreases following birth; however, thymocytes are still in the process of expansion and can be used to determine if the absence of p27 affected the number of proliferating cells.
  • An increase in the number of splenic T-cells in p27 ⁇ A mice (Table I) was detected.
  • An increase in T-cell number might occur either following antigen exposure in the periphery or during maturation in the thymus.
  • splenic T-cells were activated by cross-linking the T-cell receptor with increasing amounts of antibody to CD3. For each amount of anti-CD3 added, the activation of lymphocytes isolated from the p27 "A mouse was equivalent to the control (data not shown) .
  • CD44 * CD25 cells represent the earliest thymocyte stage examined and the biological significance of this result is not clear.
  • Thymocyte number is a function of the balance between cell proliferation and cell death. To determine if p27 affected proliferation, death, or both, these processes in the thymocytes of p27 ⁇ A mice and controls were examined. To detect thymocytes engaged in S-phase, four-week old animals were injecte with a single intraperitoneal injection of BrdU, and following dissection two hours later, the extent of BrdU incorporation into chromosomal DNA was measured by immunohistochemistry. It was found an increase in the number of BrdU-positive thymocytes in thymus from p27 " animals, in both the cortical region (Fig.
  • Thymocytes from p27 "A and control mice were exposed to either g-irradiation or dexamethasone. These treatments induced cell death in a similar percentage of p27" A and control thymocytes (Fig 5B) . Taken together these data suggest that loss of p27 might increase the proportion of cells engaged in the mitotic cycle.
  • mice lacking functional p27 are infertile
  • FSH luteinizing hormone
  • LH luteinizing hormone
  • vaginal smears taken from 8-20 week old mice showed that control mice passed though diestrus (day 1) , proestrus (day 2) , estrus (day 3) , and metestrus (day 4) in four days (Fig. 6) as described previously (Nelson et al . , 1982) .
  • all the p27 "A mice had prolonged estrus cycles, typically showing a prolongation prior to estrus and a delay in exiting estrus (Fig. 6) .
  • the most characteristic was the diestrus-proestrus like smear with numerous leukocytes and nucleated epithelial cells within abundant mucous secretion.
  • the disordered estrus cycle might reflect an underlying problem in endocrine signaling between the pituitary and ovary.
  • three-week old mice were injectd with an FSH preparation, pregnant mare serum, and forty-eight hours later with an LH substitute, human chorionic gonadotropin (hCG) and observed the effect on ovulation.
  • hCG human chorionic gonadotropin
  • the infertility of p27 "A females might be due not only to irregular ovulation, but also to defects in the ability to maintain an environment suitable to maintain pregnancy.
  • Corpus luteum formation plays an important role in maintenance of pregnancy by actively secreting progesterone and other factors.
  • Granulosa cells the major somatic cell component of follicles differentiate, by a poorly defined pathway, into progesterone producing luteal cells following ovulation.
  • p27 might have a central function to control of cell proliferation and in turn to animal growth. In animals, both complex regulatory signaling networks between cell types and intrinsic responses of cells to particular signals determine phenotype. Here it is reported that loss of p27 function as an inhibitor of Gl cyclin-dependent kinases affects the response of cells to environmental signals resulting in increased body growth. p27 disruption also appears to alter endocrine signaling by affecting the decision of the cells involved in the hypothalamic-pituitary-ovarian axis to proliferate or withdraw from the mitotic cycle.
  • p27 might be, at least in part, responsible for establishing the balance between proliferating and non-proliferating cells. For these reasons, it was decided to examine the role of p27 in proliferation and differentiation using a mouse model. Disruption of p 7 affects the manner in which cells respond to extracellular signals to proliferate or withdraw from the cell cycle As part of an inhibitory threshold, p27 might antagonize the S-phase promoting effect of CDKs and affect the decision to proliferate or withdraw from the mitotic cycle.
  • p27 affects the balance between proliferating and non-proliferating cells in the animal. There is an increase in the fraction of mitotic cells in the thymus and spleen isolated from young mice. This effect of p27 gene disruption might be largely restricted to tissues undergoing post-natal maturation and reconstitution. Further analysis during embryogenesis will be required to address this. In addition, the expression of p27 correlates with differentiation of luteal cells. A well-developed corpus luteum were unable to be detected in p27 ⁇ ⁇ mice; however, one cannot exclude the possibility that this is due to either hormonal changes, the loss of p27 in differentiating cells, or a combination of both.
  • Hyperplasia of the intermediate lobe of the pituitary suggests that p27 might maintain cells in a non-proli erative state
  • the disruption of p27 gives rise to intermediate lobe pituitary hyperplasia without evidence of adenoma (up to 30 weeks) .
  • all cells are originally alpha-MSH positive the staining in the hyperplastic nodules is variable.
  • p27 might be required to maintain these cells in a non-proliferative differentiated state.
  • the significance of hyperplasia restricted to these cells is unclear. Mice that have lost heterozygosity at the retinoblastoma (Rb) locus develop adenoma of the intermediate lobe (Jacks et al .
  • Hyperplasia rather than adenoma, might occur in p27 " mice because of either redundancy of regulation at the level of CDK activity, or the loss of the p27-inhibitory pathway might not be sufficient to obviate all the properties of Rb that make it a tumor suppressor.
  • GnRH hypothalamic gonadotropin releasing hormone
  • the LH receptor expressing granulosa cells respond to pulsatile LH secretion and ovulation follows a short time later.
  • the remnant follicle composed of granulosa cells in some undefined manner, subsequently undergoes differentiation into luteal cells that produce the estrogen and progesterone necessary to maintain pregnancy. If the oocyte is not fertilized, the corpus luteum disintegrates, and a new cycle begins.
  • the products of other endocrine networks can modulate this network. It is interpreted that the estrus phenotype of the p27-/- mouse to reflect perturbation of endocrine signaling networks between the ovary and the pituitary.
  • mice signaling between the ovary and the pituitary is compromised, however further work is required to determine the mechanism by which IL hyperplasia, the perturbed estrus cycle, and luteal cell differentiation are linked.
  • Pituitary hyperplasia might affect either GnRH, FSH, and/or LH production.
  • Hyperplasia might interfere directly or indirectly with other endocrine networks that modulate the GnRH/FSH-LH axis.
  • mice with disrupted p27 alleles reflects its function as an intracellular regulator of prolifer tion
  • GH hypothalamic growth hormone releasing hormone
  • atostatin positive and negative regulators of the somatotropes that secrete GH.
  • GH acts on liver cells to induce IGF-I production, and IGF-I increases protein synthesis and mitogenesis in target cells.
  • Transgenic mice expressing either GH or IGF-I grow to a larger size than control animals (Palmiter et al . , 1982; Mathews et al . , 1988) .
  • our data indicate the GH/IGF-I hormonal axis does not play a major role in the p27 '/ ⁇ growth phenotype.
  • p27 gene disruption results in an increase in the proportion of cycling cells and cell number.
  • p27 *A mice were significantly larger than p27 *A mice, which were in turn larger than wild-type litter mates.
  • the thymus and spleen, which in control animals express the highest amounts of p27 were significantly increased in weight whereas organs that normally express lower levels of p27 were less affected by its disruption.
  • p27 as an inhibitor of Gl CDKs, affects growth of mice by increasing the proportion of cycling cells before maturation. It is speculated that p27 is a true regulator of growth by exerting its actions on the decision of a cell to either proliferate or withdraw from the cell cycle, in response to environmental signals. In single cell eukaryotes and cells of vertebrate origin, growth is an accumulation of sufficient cell mass (Prescott, 1976) . However, growth of an animal is due, in part, to a net increase in the number of cells. Until now, there has not been a report of an intracellular regulator of animal growth. Accordingly, p27 might have a central function, albeit partially redundant, to control of cell proliferation and in turn to enhance animal growth.
  • splenocytes from mice in methylcellulose medium supplemented with a variety of cytokines were cultured.
  • Spleen cell suspensions were prepared from p27-/- or p27+/+ mice, and erythrocytes eliminated by incubation with hemolysis buffer.
  • Equal numbers (6xl0 5 ) of the resulting splenocytes were plated into a 60-mm culture dish with 3ml of alpha-minimum essential supplemented with 0.88% methylcellulose, 30% fetal calf serum, 1% bovine serum albumin, 0.
  • Conditioned medium is a source of cytokines including stem cell factor (kit-ligand) , IL-3 and GN-CSF.
  • Table 2 Comparison of thymocyte cell number in p27-/- and p27+/+.
  • CD441o 1.62 4.18 2.6 0.88 0.76 CD25 +
  • the steady-state number of thymocytes is higher in p27-/-animals than controls.
  • pl9INK4d a pl6-related inhibitor specific to CDK6 and CDK4. Mol . Biol. Cell 7, 57-70.
  • pl5INK4B is a potential effector of TGFb-induced cell cycle arrest. Nature 371, 257-261.
  • Cipl is a potent inhibitor of Gl cyclin-dependent kinases. Cell 75, 805-816.
  • p57 KIP2 a structurally distinct member of the p21 CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes & Dev. 9, 650-662. Matsushime, H., Jo, D. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J. , and Kato, J.-Y. (1994) D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol. 14 , 2066-2076.
  • p21 is a universal inhibitor of cyclin kinases. Nature 366, 701-704.

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Abstract

L'invention concerne un animal recombiné dépourvu de la fonction inhibiteur de la kinase cyclino-dépendante exercée par la p27Kip1, ainsi que le procédé pour le produire. Cette invention concerne également une méthode pour accroître la prolifération des cellules T thymiques en traitant ces dernières afin d'éliminer la fonction d'inhibition de la kinase cyclino-dépendante exercée par la p27Kip1. L'invention concerne d'autre part une méthode pour augmenter la prolifération des cellules hématopoïétiques, qui consiste à traiter ces dernières en vue d'éliminer la fonction d'inhibition de la kinase cyclino-dépendante exercée par la p27Kip1, ce qui permet d'accroître la prolifération des cellules hématopoïétiques. L'invention concerne par ailleurs une méthode pour atténuer les symptômes d'un patient atteint du SIDA, consistant à: a) recueillir les lymphocytes ou d'autres cellules chez ce patient; b) traiter les cellules recueillies afin d'éliminer la fonction d'inhibition de la kinase cyclino-dépendante exercée par la p27Kip1; et c) réintroduire les cellules traitées dans le patient atteint du SIDA.
PCT/US1997/005921 1996-04-10 1997-04-10 METHODES POUR AMELIORER LA CROISSANCE ANIMALE ET LA PROLIFERATION CELLULAIRE PAR ELIMINATION DE LA p27Kip1 FONCTIONNELLE WO1997038091A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0911634A1 (fr) * 1997-10-24 1999-04-28 Het Nederlands Kanker Instituut Regulateurs de CDK-2 et leurs utilisations pharamaceutiques
WO2001066699A2 (fr) * 2000-03-09 2001-09-13 The General Hospital Corporation P27 et p21 utilisees en therapie genique
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, 15 March 1995, Vol. 55, PIETENPOL et al., "Assignment of the Human p27Kip1 Gene to 12p13 and its Analysis in Leukemias", pages 1206-1210. *
CELL, 15 July 1994, Vol. 78, POLYAK et al., "Cloning of p27Kip1, a Cyclin-Dependent Kinase Inhibitor and a Potential Mediator of Extracellular Antimitogenic Signals", pages 59-66. *
CELL, 15 July 1994, Vol. 78, TOYOSHIMA et al., "P27, a Novel Inhibitor of G1 Cyclin-Cdk Protein Kinase Activity, is Related to p21", pages 67-74. *
MOLECULAR AND CELLULAR BIOLOGY, July 1994, Vol. 14, No. 7, FIRPO et al., "Inactivation of a Cdk2 Inhibitor During Interleukin-2 Induced Proliferation of Human T-Lymphocytes", pages 4889-4901. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0911634A1 (fr) * 1997-10-24 1999-04-28 Het Nederlands Kanker Instituut Regulateurs de CDK-2 et leurs utilisations pharamaceutiques
WO1999022240A1 (fr) * 1997-10-24 1999-05-06 Het Nederlands Kanker Instituut Utilisation pharmaceutique des regulateurs de la kinase cyclino-dependante 2 (cdk-2)
WO2001066699A2 (fr) * 2000-03-09 2001-09-13 The General Hospital Corporation P27 et p21 utilisees en therapie genique
WO2001066699A3 (fr) * 2000-03-09 2002-04-18 Gen Hospital Corp P27 et p21 utilisees en therapie genique
US7462483B2 (en) 2000-03-09 2008-12-09 The General Hospital Corporation p27 and p21 in gene therapies
US8461127B2 (en) 2000-03-09 2013-06-11 The General Hospital Corporation p27 and p21 in gene therapies
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide

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