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WO2003028443A1 - Procedes de criblage d'agents modulant des troubles degeneratifs associes aux genes bcl-2-et bim - Google Patents

Procedes de criblage d'agents modulant des troubles degeneratifs associes aux genes bcl-2-et bim Download PDF

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Publication number
WO2003028443A1
WO2003028443A1 PCT/AU2002/001325 AU0201325W WO03028443A1 WO 2003028443 A1 WO2003028443 A1 WO 2003028443A1 AU 0201325 W AU0201325 W AU 0201325W WO 03028443 A1 WO03028443 A1 WO 03028443A1
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bcl
gene
bim
endogenous
alleles
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PCT/AU2002/001325
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English (en)
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Andreas Strasser
Li-Chen Zhang
Jerry Mckee Adams
Philippe Bouillet
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The Walter And Eliza Hall Institute Of Medical Research
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Publication of WO2003028443A1 publication Critical patent/WO2003028443A1/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
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C07K14/4747—Apoptosis related proteins
    • 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
    • A01K2267/03—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
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the genetically modified animal is selected from the order Rodentia.
  • the non-human animal is a mouse.
  • a predetermined first phenotype correlates with a different copy number of a gene selected from the endogenous bcl-2 gene and the endogenous bim gene, than the copy number of that gene which correlates with the predetermined second phenotype.
  • this invention also encompasses situations in which there is non-traditional base-pairing such as Hoogsteen base pairing which has been identified in certain transfer RNA molecules and postulated to exist in a triple helix, hi the context of the definition of the term “complementary”, the terms “match” and “mismatch” as used herein refer to the hybridisation potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridise efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that hybridise less efficiently.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • RNA RNA, cRNA, cDNA or DNA.
  • the term typically refers to oligonucleotides greater than 30 nucleotides in length. Polynucleotide sequences are understood to encompass complementary strands as well as alternative backbones described herein.
  • Polypeptide , “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • “primer” is meant an oligonucleotide which, when paired with a strand of
  • selectable marker gene and “positive selection marker gene” refer to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo 1 ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Other positive selection markers are known to or are within the purview of those of ordinary skill in the art.
  • a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.
  • the term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • underscoring or italicising the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicising.
  • "bcl-2” shall mean the bcl-2 gene, whereas "Bcl-2” shall indicate the protein product of the "bcl-2” gene.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a target gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts which then anneal to form a dsRNA having homology to a target gene. Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk and
  • the promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction.
  • the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage.
  • the promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
  • Agents that may be used to enhance the activity of a BH3-only protein include any suitable BH3-only protein inducer or stabilising/activating agent which can be identified or produced by standard protocols as for example disclosed in Section 3 infra or using the transgenic non-human animal model of the invention.
  • the agent may comprise a BH3-only protein or polynucleotide encoding same. . 3. Identification of BH3-only protein modulators
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Dalton.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.
  • Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.
  • Detecting such modulation can be achieved utilising techniques including, but not restricted to, ELISA, cell-based ELISA, filter-binding ELISA, inhibition ELISA, flow cytometry including fluorescence activated cell sorting (FACS), Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin-biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
  • FACS fluorescence activated cell sorting
  • Western blots Western blots
  • immunoprecipitation slot or dot blot assays
  • a polynucleotide from which an expression product of interest is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. Further, the naturally-occurring or introduced polynucleotide may be constitutively expressed - thereby providing a model useful in screening for agents which downregulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level — or may require activation - thereby providing a model useful in screening for agents that upregulate expression of an encoded product of the sequence.
  • polynucleotide may comprise the entire coding sequence which codes for a target protein or it may comprise a portion of that coding sequence (e.g., at least a portion of a BH3 domain) or a portion that regulates expression of a product encoded by the polynucleotide (e.g., a promoter).
  • the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing, hi this regard, where only the promoter is utilised, detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, ⁇ -galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
  • GFP green fluorescent protein
  • CAT catecholamine acetyl transferase
  • These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly modulate the expression and/or activity of a target molecule according to the invention.
  • the present invention provides assays for identifying small molecules or other compounds (i.e., modulatory agents) which are capable of inducing or inhibiting the level and/or or functional activity of target molecules according to the invention.
  • the assays may be performed in vitro using non-transformed cells, immortalised cell lines, or recombinant cell lines.
  • the assays may detect the presence of increased or decreased expression of genes or production of proteins on the basis of increased or decreased mRNA expression (using, for example, the nucleic acid probes disclosed herein), increased or decreased levels of protein products (using, for example, the antigen binding molecules disclosed herein), or increased or decreased levels of expression of a reporter gene (e.g., GFP, ⁇ -galactosidase or luciferase) operably linked to a target molecule-related gene regulatory region in a recombinant construct.
  • a reporter gene e.g., GFP, ⁇ -galactosidase or luciferase
  • Target molecule related genes in vivo. These compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects.
  • these molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modelling, and other routine procedures employed in rational drug design.
  • random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a target molecule or to a functional domain thereof.
  • Identification of molecules that are able to bind to a target molecule may be accomplished by screening a peptide library with a recombinant soluble target molecule.
  • the target molecule may be purified, recombinantly expressed or synthesised by any suitable technique.
  • Such molecules may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al, (1989, supra) in particular Sections 16 and 17; Ausubel et al, (1994-1998, supra), in particular Chapters 10 and 16; and Coligan et al, (1995-1997, supra), in particular Chapters 1, 5 and 6.
  • target polypeptide expression vectors may be engineered to express a chimeric target polypeptide containing an epitope for which a commercially available antigen-binding molecule exists.
  • the epitope specific antigen-binding molecule may be tagged using methods well known in the art including labelling with enzymes, fluorescent dyes or coloured or magnetic beads.
  • the "tagged" target polypeptide conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C to allow complex formation between target polypeptide and peptide species within the library. The library is then washed to remove any unbound target polypeptide.
  • the invention also provides genetically modified, non-human animals having a partial or complete loss of function in one or both alleles of the endogenous bcl-2 gene and a partial or complete loss of function in one or both alleles of the endogenous bim gene.
  • These partial or complete loss of functions are suitably the result of an alteration to the bcl-2 gene and/or to a bim gene, which include, but are not restricted to, deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof.
  • the genetically modified animal may be either homozygous or heterozygous for the alteration.
  • a chromosomal deletion of all or part of the native bcl-2 or bim may be induced, including deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletion of a gene that activates expression of bcl-2 or bim.
  • a functional knockout may also be achieved by the introduction of an anti-sense construct that blocks expression of the native bcl-2 and/or bim genes (for example, see Li and Cohen, 1996, Cell 85: 319-329).
  • "Knockouts” also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
  • the targeting construct may contain more than one selectable marker gene.
  • the selectable marker is preferably a polynucleotide which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
  • Selectable markers may be "positive"; positive selectable markers typically are dominant selectable markers, i.e., genes which encode an enzymatic activity which can be detected in any animal, preferably mammalian, cell or cell line (including ES cells).
  • the genetically modified animals of the present invention are preferably generated by introduction of the targeting vectors into embryonal stem (ES) cells.
  • ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans, et al, 1981, Nature 292: 154-156; Bradley, et al, 1984, Nature 309: 255-258; Gossler, et al, 1986, Proc. Natl. Acad. Sci. USA 83: 9065-9069; and Robertson, et al, 1986, Nature 322: 445-448).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection using a variety of methods known to the art including electroporation, calcium phosphate co-precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated transfection. Transgenes may also be introduced into ES cells by retro virus-mediated transduction or by micro-injection. Cells are subsequently plated onto a feeder layer in an appropriate medium and those containing the transgene may be detected by employing a selective medium. Alternatively, PCR may be used to screen for ES cells which have integrated the transgene. After sufficient time for colonies to grow, they are picked and analysed for the occurrence of homologous recombination or integration of the vector.
  • This PCR technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinised, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the vector.
  • chimeric progeny can be readily detected.
  • Jaenisch (1988, Science 240: 1468-1474) The chimeric progeny are screened for the presence of the transgene and males and females having the transgene are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
  • Progeny of the genetically modified mammals may be obtained by mating the genetically modified animal with a suitable partner or by in vitro fertilisation using eggs and/or sperm obtained from the genetically modified animal. Where in vitro fertilisation is used, the fertilised embryo is implanted into a surrogate host or incubated in vitro or both.
  • the genetically modified animal may be back-crossed to a parental line, otherwise inbred or cross-bred with animals possessing other desirable genetic characteristics, hi a preferred embodiment, such other characteristics may include the partial or complete loss of function in at least one allele of a gene encoding a Bim-interacting pro-survival Bcl-2 family member including, but not limited to, Bcl-xL, Mcl-l, A-l and Bcl-w.
  • the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • bcl-2/bim knockout mice provide a means for screening test compounds beneficial for modulating Bim and/or Bcl-2 function.
  • these animals provide a means for screening compounds for the treatment or prevention in patients of conditions associated with the activation or inactivation of apoptosis.
  • agonists of Bcl-2 will test positive in a bcl-2 +/" bim +/" background if they can bring about the predetermined phenotype associated with bcl-2 + + bim " ⁇ animals (e.g., 2-5-fold elevation in blood leukocytes, lymphadenopathy, SLE-like autoimmune glomerulonephritis etc), whilst antagonists of Bcl-2 will test positive in the same background if they can lead to the predetermined phenotype associated with bcl-2 " " bim + ⁇ animals (e.g., amelioration of lymphopenia).
  • the invention also encompasses the modulatory agents obtained or identified using the animals of the present invention.
  • the agents obtained or identified by the methods broadly described in Section 3 or Section 5 supra are useful for modulating the activation or inactivation of apoptosis in various cell types including the developing kidney, lymphocytes, melanocytes and neural cells and more particularly for treating or preventing conditions associated with the activation or inactivation of apoptosis associated with degenerative disorders, including disorders characterised by inappropriate cell proliferation or inappropriate cell death or in some cases, both.
  • agonists of Bim or antagonists of Bcl-2 are useful for treatment or prophylaxis of degenerative disorders characterised by inappropriate cell proliferation such as cancer and deletion of autoreactive lymphocytes in autoimmune disease, as for example described in Section 2.
  • antagonists of Bim or agonists of Bcl-2 may be useful for treating or preventing degenerative disorders characterised by inappropriate cell death such as in chemotherapy or during HIV/AIDS or other viral infections, ischaemia or myocardial infarction, as for example described in Section 2.
  • the modulatory agent can be administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable pharmaceutically acceptable carrier. Depending on the specific conditions being treated, modulatory agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymefhylcellulose, and/or polyvinyl pyrrolidone (PVP).
  • PVP polyvinyl pyrrolidone
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilisers.
  • filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilisers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilisers may be added.
  • Dosage forms of the modulatory agents of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion.
  • Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
  • controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • Modulatory agents of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulphuric, acetic, lactic, tartaric, malic, succimc, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assavs and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilised.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1
  • Dosage amount and interval can be evaluated by routine methods in the art and may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain Bcl-2- or Bim-modulatory effects.
  • one may administer the compound in a local rather than systemic manner for example, via injection of the compound directly into a tissue often in a depot or sustained release formulation.
  • one may administer the drug in a targeted drug delivery system for example, in a liposome coated with tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the tissue.
  • mice bearing a mutant bcl— 2 allele were bred with bim " ' " animals, also backcrossed onto a C57BL/6 background.
  • the inventors were able to generate bcl-2 " ' " mice lacking one or both alleles of bim.
  • mice were backcrossed onto the C57BL/6 background for more than ten generations, bim " ' " mice of the 266Del strain (Bouillet et al, 1998) backcrossed onto C57BL/6 background for eight generations or more were used for matings.
  • Offspring were genotyped by PCR as described, and the mice that were bcl-2 + ' ⁇ bim "' " were bred with bim ' " animals to generate bcl-2 +/" &zm ⁇ /_ animals. These latter mice have been interbred to generate double homozygotes.
  • bcl-2 " " bim +/” animals were obtained by crossing bcl-2 " " bim " ' “ animals with bcl-2 + ⁇ bim “' “ or bcl-2 + ' ⁇ bim +/+ animals.
  • bcl-2 ⁇ ' ⁇ mice were obtained mostly by interbreeding heterozygotes. Some bcl-2 ⁇ ' ⁇ bim +/+ animals were also obtained from the breedings to produce double mutants.
  • the phenotype of the bcl-2 " ' " mice was essentially the same as described previously by Veis et al. (1993) and Nakayama et al. (1993).
  • the wild— type bcl-2 allele was identified using sense primer 5'-
  • mice Loss of a single bim allele in bcl-2 " " mice suffices to restore normal growth and ear length and to prevent kidney disease.
  • kidney abnormality ensues in bcl ⁇ 2 ⁇ ' ⁇ mice
  • the inventors compared the histology of kidneys from wt and bcl-2 ⁇ ' ⁇ mice from E12.5 to birth.
  • the neonatal bcl-2 " ' " kidney was much smaller than those from bcl-2*' " or wt littermates, with much fewer nephrons and smaller nephrogenic zones.
  • the inventors found that the metanephroi were already distinctly smaller than normal by E15.5, due mainly to a reduced mesenchymal area of the cortex (compare Figures 2c, d).
  • the deficit in size of the bcl-2 " ' " kidney became increasingly pronounced later in gestation, and by birth cysts were already evident.
  • Foetal liver cells from wt, bcl-2 " ' “ , bim ' “ or bcl-2 “ ' “ bim “ ' “ embryos (all Ly5.2+) were transferred into irradiated C57BL/6-Ly5.1 recipients.
  • the composition of their lymphoid organs was analysed 10-12 weeks later, using antibodies to Ly5.1 and Ly5.2 to distinguish donor-derived from host-derived leukocytes (Table 1).
  • Peripheral blood erythrocytes and leukocytes were enumerated using a ZM model Coulter counter and platelets were counted in a Sysmex NE8000 counter (TOA, Kobe, Japan).
  • Leukocytes in suspensions from spleen, lymph nodes and thymus were stained with eosin or trypan blue and counted in a hemocytometer.
  • Suspensions of cells isolated from thymus, spleen, lymph nodes, bone marrow or blood were surface stained with leukocyte-marker specific monoclonal antibodies that had been purified on protein G-Sepharose and conjugated with fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), Cy5, or biotin (Molecular Probes).
  • FITC fluorescein isothiocyanate
  • PE R-phycoerythrin
  • Cy5 Cy5
  • DMEM Dulbecco's modified Eagle's medium
  • Polycystin-1 the gene product o ⁇ PKDl, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells. Mol. Cell 6, 1267-1273.
  • Human cyclin E a new cyclin that interacts with two members of the CDC2 gene family. Cell 66, 1217-28.
  • Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice. Cell 89, 1011-1019.
  • Bim a novel member of the Bcl-2 family that promotes apoptosis. EMBO J. 17, 384-395.
  • Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.
  • C-myc-induced apoptosis in polycystic kidney disease is Bcl-2 and p53 independent. J. Exp. Med. 186, 1873-1884.
  • Bcl-2- deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.
  • the earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression. Immunity 7, 147-154.

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Abstract

L'invention concerne des procédés de criblage d'agents modulant le niveau et/ou l'activité fonctionnelle d'une protéine pro-apoptique ou modulant le niveau et/ou l'activité fonctionnelle d'une protéine de pro-survie destinée au traitement ou à la prévention de troubles dégénératifs. L'invention concerne également un modèle animal non humain génétiquement modifié s'utilisant dans lesdits procédés. Ledit modèle présente une perte totale ou partielle de fonction en au moins un allèle d'un gène pro-apoptotique, en particulier un gène bim, et une perte totale ou partielle de fonction au niveau d'au moins un allèle d'un gène de pro-survie, en particulier un gène bcl-2.
PCT/AU2002/001325 2001-09-28 2002-09-27 Procedes de criblage d'agents modulant des troubles degeneratifs associes aux genes bcl-2-et bim WO2003028443A1 (fr)

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EP2331135A1 (fr) * 2008-08-06 2011-06-15 St. Vincent's Institute Of Medical Research Procédés de traitement et de prévention de la toxicité du glucose
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US9884065B2 (en) 2011-12-13 2018-02-06 Buck Institute For Research On Aging Inhibiting activity of senescent cells using a glucocorticoid
US9901081B2 (en) 2012-08-23 2018-02-27 Buck Institute For Research On Aging Transgenic mouse for determining the role of senescent cells in cancer
US9901080B2 (en) 2012-08-23 2018-02-27 Buck Institute For Research On Aging Transgenic mouse having a transgene that converts a prodrug into a cytotoxic compound in senescent cells
US9969776B2 (en) 2007-12-20 2018-05-15 Unity Biotechnology, Inc. Drug conjugates for delivering compounds to senescent cells
US9968076B2 (en) 2011-06-21 2018-05-15 Mayo Foundation For Medical Education And Research Transgenic animals capable of being induced to delete senescent cells
US10195213B2 (en) 2015-03-13 2019-02-05 Unity Biotechnology, Inc. Chemical entities that kill senescent cells for use in treating age-related disease
US10328058B2 (en) 2014-01-28 2019-06-25 Mayo Foundation For Medical Education And Research Treating atherosclerosis by removing senescent foam cell macrophages from atherosclerotic plaques
US10378002B2 (en) 2012-04-17 2019-08-13 Unity Biotechnology, Inc. Replication conditional virus that specifically kills senescent cells
US11517572B2 (en) 2014-01-28 2022-12-06 Mayo Foundation For Medical Education And Research Killing senescent cells and treating senescence-associated conditions using a SRC inhibitor and a flavonoid

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WO2005059554A1 (fr) * 2003-12-11 2005-06-30 Institut Gustave-Roussy Proteines puma, map kinase p53 4t p38 phosphorylees comme marqueurs de l'apoptose induite par le vih-1 dans les lymphocytes t circulants dans un patient infecte
EP1542016A1 (fr) * 2003-12-11 2005-06-15 Institut Gustave Roussy PUMA, p53 phosphorylé et p38 MAPK comme marqueurs pour l'apoptose inicieé par VIH dans des cellules T circulantes d'un patient infecté
WO2005075645A1 (fr) * 2004-02-06 2005-08-18 The Walter And Eliza Hall Institute Of Medical Research Molecules therapeutiques et methodes de generation et/ou de selection de celles-ci
US10745445B2 (en) 2007-12-20 2020-08-18 Unity Biotechnology, Inc. Conjugates that are configured for targeted delivery of therapeutic compounds to senescent cells
US9969776B2 (en) 2007-12-20 2018-05-15 Unity Biotechnology, Inc. Drug conjugates for delivering compounds to senescent cells
EP2331135A1 (fr) * 2008-08-06 2011-06-15 St. Vincent's Institute Of Medical Research Procédés de traitement et de prévention de la toxicité du glucose
EP2331135A4 (fr) * 2008-08-06 2013-01-02 St Vincents Inst Med Res Procédés de traitement et de prévention de la toxicité du glucose
US9968076B2 (en) 2011-06-21 2018-05-15 Mayo Foundation For Medical Education And Research Transgenic animals capable of being induced to delete senescent cells
US10251376B2 (en) 2011-06-21 2019-04-09 Mayo Foundation For Medical Education And Research Increasing healthy lifespan and delaying progression of age-related phenotypes by selectively removing senescent cells
US9884065B2 (en) 2011-12-13 2018-02-06 Buck Institute For Research On Aging Inhibiting activity of senescent cells using a glucocorticoid
US10378002B2 (en) 2012-04-17 2019-08-13 Unity Biotechnology, Inc. Replication conditional virus that specifically kills senescent cells
US9901081B2 (en) 2012-08-23 2018-02-27 Buck Institute For Research On Aging Transgenic mouse for determining the role of senescent cells in cancer
US9901080B2 (en) 2012-08-23 2018-02-27 Buck Institute For Research On Aging Transgenic mouse having a transgene that converts a prodrug into a cytotoxic compound in senescent cells
US10655144B2 (en) 2012-08-23 2020-05-19 Buck Institute For Research On Aging Nucleic acid construct with a p16 promoter that causes a prodrug converting enzyme to be expressed specifically in senescent cells
US9855266B2 (en) 2014-01-28 2018-01-02 Unity Biotechnology, Inc. Treatment for osteoarthritis by intra-articular administration of a cis-imidazoline
US10413542B2 (en) 2014-01-28 2019-09-17 Buck Institute For Research On Aging Methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders using an inhibitor of Akt kinase
US10010546B2 (en) 2014-01-28 2018-07-03 Unity Biotechnology, Inc. Treatment of ophthalmic conditions by selectively removing senescent cells from the eye
US10130628B2 (en) 2014-01-28 2018-11-20 Unity Biotechnology, Inc. Treatment of joint pain
US10213426B2 (en) 2014-01-28 2019-02-26 Unity Biotechnology, Inc. Method of optimizing conditions for selectively removing a plurality of senescent cells from a tissue or a mixed cell population
US10258618B2 (en) 2014-01-28 2019-04-16 Unity Biotechnology, Inc. Treating pulmonary conditions by selectively removing senescent cells from the lung using an intermittent dosing regimen
US10328058B2 (en) 2014-01-28 2019-06-25 Mayo Foundation For Medical Education And Research Treating atherosclerosis by removing senescent foam cell macrophages from atherosclerotic plaques
US10328073B2 (en) 2014-01-28 2019-06-25 Unity Biotechnology, Inc. Use of sulfonamide inhibitors of BCL-2 and BCL-xL to treat ophthalmic disease by selectively removing senescent cells
US9980962B2 (en) 2014-01-28 2018-05-29 Unity Biotechnology, Inc Use of sulfonamide inhibitors of Bcl-2 to treat senescence-associated lung conditions such as pulmonary fibrosis and chronic obstructive pulmonary disease
US11963957B2 (en) 2014-01-28 2024-04-23 Mayo Foundation For Medical Education And Research Treating cardiovascular disease by selectively eliminating senescent cells
US11980616B2 (en) 2014-01-28 2024-05-14 Mayo Foundation For Medical Education And Research Treating liver disease by selectively eliminating senescent cells
US10478432B2 (en) 2014-01-28 2019-11-19 Unity Biotechnology, Inc. Compositions of matter for treatment of ophthalmic conditions by selectively removing senescent cells from the eye
US10478433B2 (en) 2014-01-28 2019-11-19 Unity Biotechnology, Inc. Unit dose of an aryl sulfonamide that is effective for treating eye disease and averting potential vision loss
US10517866B2 (en) 2014-01-28 2019-12-31 Unity Biotechnology, Inc. Removing senescent cells from a mixed cell population or tissue using a phosphoinositide 3-kinase (PI3K) inhibitor
US9993472B2 (en) 2014-01-28 2018-06-12 Unity Biotechnology, Inc. Treatment for osteoarthritis in a joint by administering a means for inhibiting MDM2
US9849128B2 (en) 2014-01-28 2017-12-26 Unity Biotechnology, Inc. Unit dose of a cis-imidazoline for treating an osteoarthritic joint by removing senescent cells
US11351167B2 (en) 2014-01-28 2022-06-07 Buck Institute For Research On Aging Treating cognitive decline and other neurodegenerative conditions by selectively removing senescent cells from neurological tissue
US11517572B2 (en) 2014-01-28 2022-12-06 Mayo Foundation For Medical Education And Research Killing senescent cells and treating senescence-associated conditions using a SRC inhibitor and a flavonoid
US10195213B2 (en) 2015-03-13 2019-02-05 Unity Biotechnology, Inc. Chemical entities that kill senescent cells for use in treating age-related disease
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