WO2006027545A2 - Procede - Google Patents

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WO2006027545A2
WO2006027545A2 PCT/GB2005/003206 GB2005003206W WO2006027545A2 WO 2006027545 A2 WO2006027545 A2 WO 2006027545A2 GB 2005003206 W GB2005003206 W GB 2005003206W WO 2006027545 A2 WO2006027545 A2 WO 2006027545A2
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mtor
cells
cell
activity
stem cell
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PCT/GB2005/003206
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WO2006027545A3 (fr
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Sai Kiang Lim
Jian Wen Que
Reida Menshawe El Oakley
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Agency For Science, Technology And Research
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/04—Immunosuppressors, e.g. cyclosporin, tacrolimus
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • the present invention relates to the fields of development, cell biology, molecular biology and genetics. More particularly, the invention relates to a polypeptide which is involved in the switch between the cell fates of self-renewal and differentiation, as well as methods involving such.
  • Stem cells unlike differentiated cells have the capacity to divide and either self- renew or differentiate into phenotypically and functionally different daughter cells M .
  • cessation of self-renewal in stem cells does not always lead to differentiation nor is it sufficient to induce differentiation 5 .
  • Stem cells that cease self-renewal without activating a differentiation program either enter quiescence or undergo apoptosis.
  • a differentiating stem cell must cease self-renewal to activate a differentiation program. Therefore, induction of differentiation must be co-ordinated with some aspects of cell cycle control to inhibit self-renewal. For example, enhancement of self-renewal in Pten-/- murine ES cells is associated with reduced capacity for differentiation (6, 7).
  • LIF leukemia inhibitory factor
  • PB K PB K signaling involving Pten. Inactivation of these pathways generally leads to differentiation 8 . Additionally, LIF signaling can activate PB K while PB K is known to phosphorylate STAT3 which is essential in the maintenance of self-renewal in ES cells.
  • crosstalk between LIF and PB K signaling has not been shown to be important in maintaining self-renewal or inducing differentiation of murine ES cells (see Figure 5).
  • mTOR The molecular target of rapamycin (mTOR) has been proposed as a therapeutic target against cancer (Mita, et al., Cancer Biol Ther. 2003 Jul-Aug;2(4 Suppl 1):S169-
  • a method comprising modulating the activity of mTOR in a stem cell.
  • activity of mTOR is maintained or increased to enable a stem cell to self-renew.
  • the stem cell is in a self-renewing state and activity of mTOR is maintained or increased in the stem cell to maintain a self-renewing state.
  • the stem cell is inhibited from differentiating by maintaining the activity of mTOR.
  • the stem cell is exposed to an agonist of mTOR to increase the activity of mTOR in the stem cell.
  • the agonist comprises phosphatidic acid.
  • activity of mTOR is decreased to enable a stem cell to differentiate.
  • the stem cell is in a self-renewing state and activity of mTOR is decreased to enable the stem cell to differentiate, or (b) the stem cell is in a differentiating state and activity of mTOR is decreased in the stem cell to maintain a differentiating state.
  • the stem cell is exposed to an antagonist of mTOR to decrease the activity of mTOR in the stem cell.
  • the antagonist comprises 1-butanol or rapamycin or a derivative thereof.
  • a stem cell in a self-renewing state is characterised by at least one of the following features: (a) decreased dephosphorylation of 4E-BP1 and/or S6K1; (b) increased expression of Oct4 and/or SSEA-I; (c) decreased expression of any one or more of FIk-I, Tie-2 and c-kit; (d) decreased expression of any one or more of Brachyury, AFP, nestin and nurrl ; (e) a shortened cell cycle, compared to a stem cell in a differentiating state.
  • a stem cell in a differentiating state is characterised by at least one of the following features: (a) increased dephosphorylation of 4E-BP1 and/or S6K1; (b) decreased expression of Oct4 and/or SSEA-I; (c) increased expression of any one or more of FIk-I, Tie-2 and c-kit; (d) increased expression of any one or more of Brachyury, AFP, nestin and nurrl ; (e) a lengthened cell cycle, compared to a stem cell in a self- renewing state.
  • a molecule capable of increasing mTOR activity in a method of promoting the expression by a stem cell of a marker of self-renewal, optionally together with decreasing the expression of a marker of differentiation.
  • a molecule capable of decreasing mTOR activity in a method of promoting the expression by a stem cell of a marker of differentiation, optionally together with decreasing the expression of a marker of self-renewal.
  • the marker of self-renewal is selected from the group consisting of: Oct4 and SSEA-I
  • the marker of differentiation is selected from the group consisting of: FIk-I, Tie-2, c-kit, Brachyury, AFP, nestin and nurrl.
  • a method of identifying an agent capable of enabling self-renewal of a stem cell comprising contacting mTOR with a candidate agent and determining whether the activity of mTOR is thereby increased.
  • the present invention in a 6 th aspect, provides a method of identifying an agent capable of enabling differentiation of a stem cell, the method comprising contacting mTOR with a candidate agent and determining whether the activity of mTOR is thereby decreased.
  • the activity is kinase activity.
  • mTOR in a method of enabling self-renewal or differentiation of a stem cell.
  • an agent capable of increasing the activity of mTOR preferably phosphatidic acid or an agent identified by a method according to the 4 th or 5 th aspect of the invention in a method of enabling self-renewal of a stem cell.
  • mTOR or an agent as set out in any of 7 th to 9 th aspects of the invention, for the treatment of, or the preparation of a pharmaceutical composition for the treatment of, any one of the following: a disease treatable by regenerative therapy, cardiac failure, bone marrow disease, skin disease, bums, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease and cancer.
  • a method of determining whether a stem cell is differentiating or self-renewing comprising detecting mTOR activity of the stem cell, in which a high mTOR activity indicates that the stem cell is self-renewing, and a low mTOR activity indicates that the stem cell is differentiating.
  • a 13 th aspect of the present invention we provide a cell produced or treated by a method or use according to the 1 st to 3 rd and 7 th to 11 th aspects of the invention.
  • a self- renewing cell produced or treated by a method according to the 1 st aspect of the invention.
  • a 16 th aspect of the present invention we provide an agent identified by a method or assay according to the 4 th , 5 th or 6 th aspect of the invention.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch.
  • Figures IA to D show the regulation of cell cycle in mES cells during differentiation.
  • Figure IA Cell cycle activity is measured in mouse El 4 ES cells (i) before and after induction of differentiation by LIF withdrawal or (ii) with and without rapamycin treatment in the presence of LIF as described in Methods.
  • Figure IB Gene expression in mouse El 4 ES cells is analysed by RT-PCR at different times after LIF withdrawal or rapamycin treatment in the presence of LIF.
  • FIG. 1C Western blot analysis of Oct4 and Nestin, and 4E-BP and phosphorylated S6K (pS6K) levels during differentiation or rapamycin treatment.
  • TSC2 protein level is used as an internal control.
  • Figure ID E14 ES cells are stained for SSEA-I by immunohistochemistry at different times after LIF withdrawal or rapamycin treatment in the presence of LIF, and counterstained with propidium iodide (PI).
  • FIGS. 2A to D show the regulation of cell cycle in RoSH cells during endothelial differentiation.
  • FIG. 2A Cell cycle activity is measured for undifferentiated RoSH cells (Undif), RoSH cells 48 hours after induction of differentiation (Dif), rapamycin-treated undifferentiated cells (R undif) and rapamycin-treated, differentiated cells (R dif).
  • Figure 2B Changes in endothelial markers during differentiation or rapamycin treatment are monitored by western blot analysis using 20 ⁇ g cell lysates. TSC2 protein level is used as an internal control.
  • Figure 2C Phosphorylation of 4E-BP and S6K during induction of differentiation or rapamycin treatment is monitored by western blot analysis.
  • Figure 2D Effects of rapamycin on branching during endothelial differentiation by determining the average number of branch points with > 3 branches in three random low power fields (10 ⁇ magnification). The averages from two independent experiments are plotted and error bars represent SEM. Representative untreated and treated cultures at 10x magnification are shown on the right.
  • Figures 3 A and B show the inhibition of cell proliferation is not sufficient to induce differentiation. Effects of Gleevec on cell cycle activity of RoSH cells (Figure 3A) and endothelial differentiation and mTOR activity by western blot assay ( Figure 3B).
  • Figures 4A to C show microarray analysis of total cellular and polysome associated RNA from RoSH cells before and 48 hours after differentiation.
  • Figure 4A Venn diagram representation of relative RNA transcript distribution.
  • Up in UTR vs DTR and Down in UTR vs DTR refers to transcripts that are present at higher/lower level, respectively in undifferentiated total RNA relative to that in differentiated total RNA;
  • Up in UPR vs DPR and Down in UTR vs DTR refers to transcripts that are present at higher/lower level, respectively in undifferentiated pplysome-associated RNA relative to that in differentiated pplysome-associated RNA.
  • A, B, C, D, E, F and G refer to subsets of transcripts.
  • FIG. 4A The microarray analysis data in Figure 4A is verified by RT-PCR and western blot analysis. The results are shown in Figures 4B and 4C.
  • Reference cDNA (Ref) is cDNA prepared using pooled RNA from adult and fetal mouse tissues.
  • Four of the eight representative genes are further analysed at the protein level by western blot assay using 20 ⁇ g cell lysates from different times after induction of differentiation. TSC2 protein level is used as an internal control. The averages from two independent experiments are plotted and error bars represent SEM.
  • FIG. 5 shows a model of proliferation and self renewal in murine ES cells.
  • LIF/Stat3 signaling is activated by binding of LIF to LIF receptor (LIFR) and co-receptor, gpl30 that then phosphorylate Stat3.
  • mTOR is activated by either growth factors, mitogens and hormones through PI3 K signaling or extracellular amino acids through unknown mechanisms.
  • Activated mTOR phosphorylates both S6K1 and 4E-BP1.
  • Activated S6K1 in turn phosphorylates ribosomal protein S6 and increases translation of 5' TOP-containing mRNAs.
  • Figure 6 is a graph showing increase in proliferation of stem cells by phosphatidic acid.
  • Mouse E14 ES cells are grown in DMEM + LIF + 20% FCS on gelatinized coated plates. They are plated at 2 ⁇ 10 5 per well in a gelatinized six-well plate and cultured for 4 days. The cells are then trypsinized, counted and replated at 2 ⁇ 10 5 per well in a gelatinized six-well plate with i) DMEM + LIF + 20% FCS, ii) DMEM + LIF + 5% FCS, iii) DMEM + LIF + 5% FCS + 50 nM PA and iv) DMEM + LIF + 5% FCS + 100 nM PA. After 4 days, each well is trypsinized, the cells counted, replated at 2x10 5 per well in a gelatinized six-well plate with the respective culture media. These are repeated at day 8 and day 12.
  • Figure 7A is a schematic diagram of an assay to screen and identify an mTOR activator.
  • Figure 7B is a schematic diagram of an assay to screen and identify an mTOR inhibitor.
  • This invention is based on the demonstration that mTOR regulates the choice of a stem cell between the different paths which the stem cell can potentially take when it exits self-renewal, namely, differentiation, apoptosis or self-renewal.
  • the Examples demonstrate that, for both murine ES cells and RoSH (mouse endothelial progenitor cell line) cells, inhibition of mTOR activity of stem cells by for example rapamycin reduces cell cycle activity and initiates differentiation. Expression of self-renewal markers is decreased, and expression of differentiation markers is increased. mTOR activity is reduced during leukaemia inhibitory factor (LIF) withdrawal-induced differentiation of both types of cells.
  • LIF leukaemia inhibitory factor
  • the activity of mTOR is maintained or increased; similarly, to enable differentiation by the stem cell, the activity of mTOR is decreased.
  • the stem cell may be inhibited from differentiating by maintaining the activity of mTOR.
  • manipulation of mTOR activity may be used to cause a stem cell to enter de novo a different pathway of self-renewal, or differentiation. That is to say, a stem cell which is in the process of differentiating may be caused to remain in the self-renewal pathway by maintenance of, or preventing a decrease in its basal mTOR activity Conversely, down-regulation of mTOR activity of a stem cell which is in the process of, or committed to, self-renewal may cause the stem cell to differentiate instead. Furthermore, in addition to changing the pathway of the stem cell, a change in mTOR activity may be used to strengthen the commitment or choice of a stem cell fate.
  • a stem cell which is in the process of exiting self-renewal may be biased towards differentiation and not other cellular fates e.g. apoptosis or quiescence by decreasing or maintaining a decreased level of mTOR activity of the stem cell.
  • the level of mTOR is decreased or increased, to such an extent (or maintained at that level) so that the stem cell remains in a differentiating (or self-renewing state, as the case may be) even if the stem cell is exposed to signals which would otherwise cause self-renewal or differentiation to occur.
  • Detection of mTOR activity may also be used to determine the status of a stem cell, i.e., whether it is in the process of, or committed to, self-renewing or differentiating.
  • Stem cells and differentiated cells made according to the methods and compositions described here may be employed for a variety of purposes, including medical treatment, as described in further detail below.
  • Any means for increasing and decreasing mTOR activity may be used, including both direct and indirect modulation. These may include for example, modulating the expression of an endogenous mTOR gene at the transcriptional, translational or post- translational level, such as modulating the persistence or breakdown of mTOR messenger RNA, modulating the persistence or breakdown of mTOR protein, etc. They may also include modulation of the activity of mTOR protein, such as by use of agonists or antagonists of mTOR. Furthermore, the expression and/or activity of inhibitors or activators of mTOR, which will typically be polypeptides, may be modulated to modulate mTOR activity. These are described in further detail below.
  • the activity of mTOR is reduced by 10%, 20%, 30%, 40%, 50% or 60% or more to effect differentiation of a stem cell. In highly preferred embodiments, mTOR activity is reduced by about 40% in order to allow differentiation to take place. In such preferred embodiments, the activity of mTOR as assayed in the "mTOR kinase assay" described below is reduced by the requisite amounts. Alternatively, or in addition, the transcript level of mTOR (measured for example by quantitation using hybridisation and autoradiography) is reduced.
  • agonists and antagonists of mTOR each of which are known in the art, and set out below.
  • Such agonists and antagonists of mTOR may furthermore be identified by screens and assays, also described in detail below.
  • Stem cells made according to the methods described here can be used for a variety of commercially important research, diagnostic, and therapeutic purposes. These uses are generally well known in the art, but will be described briefly here.
  • stem cells may be used to generate cell populations for regenerative therapy, for example by ex vivo expansion or direct administration of stem cells into a patient. They may also be used for the repopulation of damaged tissue following trauma.
  • hematopoietic stem cells may be used for bone marrow replacement, while cardiac stem cells may be used for cardiac failure patients.
  • Stem cells comprising skin progenitor cells may be employed for growing sking grafts for patients and endothelial progenitor cells for endothelization of artificial prosthetics such as stents or artificial hearts
  • Embryonic stem cells and their tissue stem cell derivatives may be used for the treatment of degenerative diseases such as diabetes, Alzheimer's disease, Parkinson's disease, etc.
  • Stem cells for example, made by the methods and compositions described here, may be used as pprogenitors for NK or dendritic cells for immunotherapy for cancer.
  • stem cells which may of course be made to differentiate using methods known in the art.
  • any uses of differentiated cells will equally attach to those stem cells for which they are progenitors.
  • the uses that may be made of stem cells described above and elsewhere in this document attach equally to molecules capable of making or maintaining such stem cells, for example, agonists of mTOR activity such as phosphatidic acid. Such uses also attach of course to mTOR itself.
  • mTOR as well as a molecule capable of increasing mTOR activity, such as phosphatidic acid, to enable self-renewal of a stem cell.
  • mTOR and such molecules may be used for the preparation of a pharmaceutical composition for the treatment of disease.
  • disease may comprise a disease treatable by regenerative therapy, including cardiac failure, bone marrow disease, skin disease, bums, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease, etc and cancer.
  • phosphatidic acid in the treatment or prevention of such diseases, and in the preparation of a pharmaceutical composition for this purpose.
  • Differentiated cells made according to the methods described here can be used for a variety of commercially important research, diagnostic, and therapeutic purposes.
  • populations of undifferentiated cells may be used to prepare antibodies and cDNA libraries that are specific for the differentiated phenotype.
  • General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.); Current Protocols in Immunology (Coligan et al., eds.); and Methods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags GmbH).
  • General techniques involved in preparation of mRNA and cDNA libraries are described in RNA Methodologies: A Laboratory Guide for Isolation and Characterization (R. E.
  • differentiated cells attach equally to molecules capable of causing or maintaining such differentiation, for example, antagonists of mTOR activity such as 1-butanol or rapamycin or a derivative thereof
  • mTOR as well as a molecule capable of decreasing mTOR activity, such as 1-butanol or rapamycin or a derivative thereof to enable differentiation of a stem cell.
  • mTOR and such molecules may be used for the preparation of a pharmaceutical composition for the treatment of disease.
  • disease may comprise a disease treatable by regenerative therapy, including cardiac failure, bone marrow disease, skin disease, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease, etc and cancer.
  • regenerative therapy including cardiac failure, bone marrow disease, skin disease, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease, etc and cancer.
  • Differentiated cells made according to the methods described here may also be used to screen for factors (such as solvents, small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of differentiated cells.
  • factors such as solvents, small molecule drugs, peptides, polynucleotides, and the like
  • environmental conditions such as culture conditions or manipulation
  • differentiated cells are used to screen factors that promote maturation, or promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation factors or growth factors are tested by adding them to differentiated cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.
  • Assessment of the activity of candidate pharmaceutical compounds generally involves combining the differentiated cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.
  • the screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects.
  • Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug—drug interaction effects.
  • compounds are screened initially for potential toxicity (Castell et al., pp. 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997). Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair.
  • [ 3 H]thymidine or BrdU incorporation is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (PP 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997) for further elaboration.
  • Differentiated cells may also be used for tissue reconstitution or regeneration in a human patient in need thereof.
  • the cells are administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.
  • the methods and compositions described here may be used to modulate the differentiation of stem cells.
  • Differentiated cells may be used for tissue engineering, such as for the growing of skin grafts.
  • Modulation of stem cell differentiation may be used for the bioengineering of artificial organs or tissues, or for prosthetics, such as stents.
  • neural stem cells are transplanted directly into parenchymal or intrathecal sites of the central nervous system, according to the disease being treated. Grafts are done using single cell suspension or small aggregates at a density of 25,000- 500,000 cells per .mu.L (U.S. Pat. No. 5,968,829). The efficacy of neural cell transplants can be assessed in a rat model for acutely injured spinal cord as described by McDonald et al. (Nat. Med. 5:1410, 1999.
  • a successful transplant will show transplant-derived cells present in the lesion 2-5 weeks later, differentiated into astrocytes, oligodendrocytes, and/or neurons, and migrating along the cord from the lesioned end, and an improvement in gate, coordination, and weight-bearing.
  • Certain neural progenitor cells are designed for treatment of acute or chronic damage to the nervous system. For example, excitotoxicity has been implicated in a variety of conditions including epilepsy, stroke, ischemia, Huntington's disease, Parkinson's disease and Alzheimer's disease.
  • Certain differentiated cells as made according to the methods described here may also be appropriate for treating dysmyelinating disorders, such as Pelizaeus-Merzbacher disease, multiple sclerosis, leukodystrophies, neuritis and neuropathies.
  • dysmyelinating disorders such as Pelizaeus-Merzbacher disease, multiple sclerosis, leukodystrophies, neuritis and neuropathies.
  • Appropriate for these purposes are cell cultures enriched in oligodendrocytes or oligodendrocyte precursors to promote remyelination.
  • Hepatocytes and hepatocyte precursors prepared using our methods can be assessed in animal models for ability to repair liver damage.
  • One such example is damage caused by intraperitoneal injection of D-galactosamine (Dabeva et al., Am. J. Pathol. 143:1606, 1993).
  • Efficacy of treatment can be determined by immunohistochemical staining for liver cell markers, microscopic determination of whether canalicular structures form in growing tissue, and the ability of the treatment to restore synthesis of liver-specific proteins.
  • Liver cells can be used in therapy by direct administration, or as part of a bioassist device that provides temporary liver function while the subject's liver tissue regenerates itself following fulminant hepatic failure.
  • cardiomyocytes prepared according to the methods described here can be assessed in animal models for cardiac cryoinjury, which causes 55% of the left ventricular wall tissue to become scar tissue without treatment (Li et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai et al., J. Thorac. Cardiovasc. Surg. 118:715, 1999).
  • Successful treatment will reduce the area of the scar, limit scar expansion, and improve heart function as determined by systolic, diastolic, and developed pressure.
  • Cardiac injury can also be modeled using an embolization coil in the distal portion of the left anterior descending artery (Watanabe et al., Cell Transplant. 7:239, 1998), and efficacy of treatment can be evaluated by histology and cardiac function. Cardiomyocyte preparations can be used in therapy to regenerate cardiac muscle and treat insufficient cardiac function (U.S. Pat. No. 5,919,449 and WO 99/03973).
  • mTOR polypeptides are suitable for treating or preventing cancer.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • glial cell tumors such as glioblastoma and neurofibromatosis
  • cervical cancer ovarian cancer
  • liver cancer bladder cancer
  • hepatoma hepatoma
  • breast cancer colon cancer
  • colorectal cancer endometrial carcinoma
  • salivary gland carcinoma salivary gland carcinoma
  • kidney cancer renal cancer
  • prostate cancer prostate cancer
  • vulval cancer thyroid cancer
  • hepatic carcinoma various types of head and neck cancer.
  • solid tumor cancer including colon cancer, breast cancer, lung cancer and prostrate cancer
  • hematopoietic malignancies including leukemias and lymphomas
  • Hodgkin's disease aplastic anemia
  • skin cancer and familiar adenomatous polyposis.
  • brain neoplasms include brain neoplasms, colorectal neoplasms, breast neoplasms, cervix neoplasms, eye neoplasms, liver neoplasms, lung neoplasms, pancreatic neoplasms, ovarian neoplasms, prostatic neoplasms, skin neoplasms, testicular neoplasms, neoplasms, bone neoplasms, trophoblastic neoplasms, fallopian tube neoplasms, rectal neoplasms, colonic neoplasms, kidney neoplasms, stomach neoplasms, and parathyroid neoplasms.
  • mTOR polypeptide, nucleic acid, and fragments, homologues, variants and derivatives thereof are used to treat T cell lymphoma, melanoma or lung cancer.
  • mTOR polypeptides and nucleic acids, etc, as described here, may also be used in combination with anticancer agents such as endostatin and angiostatin or cytotoxic agents or chemotherapeutic agent.
  • anticancer agents such as endostatin and angiostatin or cytotoxic agents or chemotherapeutic agent.
  • drugs such as such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, and antimetabolites such as methotrexate.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. I, Y, Pr), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • the term includes oncogene product/tyrosine kinase inhibitors, such as the bicyclic ansamycins disclosed in WO 94/22867; 1 ,2-bis(arylamino) benzoic acid derivatives disclosed in EP 600832; 6,7-diamino-phthalazin-l-one derivatives disclosed in EP 600831 ; 4,5-bis(arylamino)-phthalimide derivatives as disclosed in EP 516598; or peptides which inhibit binding of a tyrosine kinase to a SH2-containing substrate protein (see WO 94/07913, for example).
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil (5-FU), Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, VP-16, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Nicotinamide, Esperamicins (see U.S. Pat.
  • the methods described here may also be used for, or to aid, the diagnosis or treatment, or both, of tumours, in particular, poorly differentiated tumours or cancers. Poorly differentiated tumours or cancers are otherwise known as poorly differentiated carcinoma (PDC) of unknown primary site.
  • PDC poorly differentiated carcinoma
  • tumours or cancers are mammalian, more preferably human.
  • the poorly differentiated tumor may be of epithelial, hematopoietic, neuroendocrine, or neuroectodermal origin (i.e., melanoma); each of these may predicate a different treatment regime. Issues with poorly differentiated cancers are described in detail in Hainsworth et al, 1987, J Clin Oncol 5(8):1275-80.
  • the methods described here may be used to cause the differentiation of stem cells, including poorly differentiated tumour cells.
  • Immunohistochemical staining for markers may be used to determine the source or origin of the tumour; thus, for example, staining for keratins, leukocyte common antigen (LCA), or S-100, a neuroectodermal antigen, may be used to identify a melanoma.
  • genetic analysis for example, by PCR
  • the treatment regime may be chosen by the physician, e.g., by administration of a suitable drug.
  • tumours and cancers suitable for use with the methods and compositions described here include, but are not limited to, testis tumor, colon tumor, stomach, germ cell tumors, choriocarcinoma, lung, large cell carcinoma, uterus, and leiomyosarcoma.
  • mTOR associated diseases include any one or more of diseases of the following tissues, including: adenocarcinoma;; adrenal cortico adenoma for cushing's syndrome; cervical carcinoma; Crohn's disease; embryonal carcinoma; endometrium adenocarcinoma; epithelioid carcinoma; poorly- differentiated adenocarcinomas; acute myelogenous leukemia; chronic myelogenous leukemia; hepatocellular carcinoma; hypernephroma; insulinoma; iris; kidney tumor; large cell carcinoma; large cell carcinoma, undifferentiated; leiomyosarcoma; lung focal fibrosis; metastatic chondrosarcoma; mucoepidermoid carcinoma; neuroblastoma; papillary serous ovarian metastasis; parathyroid; pheochromocytoma; prostate tumor; serous papillary tumor; squamous cell carcinoma, preferably poorly differentiated.
  • tissues including: adenocarcinom
  • mTOR is used in this document, it should be taken to refer a polypeptide sequence having the accession number NM_004958.2, P42345 or NP_004949, more particularly NM_004958.2.
  • mTOR refers to a human sequence.
  • particular homologues encompassed by this term include human homologues, for example, accession numbers NM_004958.2, NP_004949, Hs.509145.
  • the term also covers alternative peptides homologous to mTOR, such as polypeptides derived from other species, including other mammalian species.
  • mouse homologues of mTOR having accession number NM_020009.1, NP_064393, Mm .21158 , Q9JLN9, AAF73196 and AFl 52838 are included.
  • Bovine and rat homologues of mTOR are also known (accession numbers NM_174319 and NM_019906 respectively).
  • mTOR is also known as FKBP12-Rapamycin Complex- Associated Protein 1, FRAPl, FK506-Binding Protein 12-Rapamycin Complex- Associated Protein 1, FRAP, FRAP2, Mammalian Target Of Rapamycin and RAFTl.
  • mTOR includes fragments, homologues, variants and derivatives of such a nucleotide sequence.
  • variant includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence of a mTOR nucleotide sequence.
  • references to "mTOR” include references to such variants, homologies, derivatives and fragments of mTOR. These are described in more detail below.
  • the resultant nucleotide sequence encodes a polypeptide having mTOR activity, preferably having at least the same activity of the human mTOR referred to above.
  • the term "homologue” is intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has mTOR activity.
  • sequence identity i.e. similarity
  • sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
  • FRAP mTOR
  • FKBP12-rapamycin associated protein is one of a family of proteins involved in cell cycle progression, DNA recombination, and DNA damage detection. In rat, it is a 245-kD protein (symbolized RAFTl) with significant homology to the Saccharomyces cerevisiae protein TORI and has been shown to associate with the immunophilin FKBP12 (186945) in a rapamycin-dependent fashion (Sabatini et al., 1994). Brown et al. (1994) noted that the FKBP12-rapamycin complex was known to inhibit progression through the Gl cell cycle stage by interfering with mitogenic signaling pathways involved in Gl progression in several cell types, as well as in yeast.
  • Rapamycin is an efficacious anticancer agent against solid tumors.
  • the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients.
  • mTOR/FRAP mammalian target of rapamycin
  • ribosome biogenesis is independently regulated by amino acids and ATP.
  • Dennis et al. (2001) demonstrated that the human mTOR pathway is influenced by the intracellular concentration of ATP, independent of the abundance of amino acids, and that mTOR/FRAP itself is an ATP sensor.
  • Quantitative immunoblot analysis showed that phosphorylation of serl5 of p53 in response to HIV-I Env is mediated by FRAP and not by other phosphatidylinositol kinase-related kinases, and it is accompanied by downregulation of protein phosphatase 2 A (see 176915). The phosphorylation is significantly inhibited by rapamycin.
  • Immunofluorescence microscopy indicated that FRAP is enriched in syncytial nuclei and that the nuclear accumulation precedes the phosphorylation of serl5 of p53. Castedo et al.
  • HIV-I Env-induced syncytium formation leads to apoptosis via a pathway that involves phosphorylation of serl5 of p53 by FRAP, followed by activation of BAX (600040), mitochondrial membrane permeabilization, release of cytochrome C, and caspase activation.
  • Fang et al. identified phosphatidic acid as a critical component of mTOR signaling.
  • mitogenic stimulation of mammalian cells led to a phospholipase D-dependent accumulation of cellular phosphatidic acid, which was required for activation of mTOR downstream effectors.
  • Phosphatidic acid directly interacted with the domain in mTOR that is targeted by rapamycin, and this interaction was positively correlated with mTOR's ability to activate downstream effectors.
  • the involvement of phosphatidic acid in mTOR signaling reveals an important function of this lipid in signal transduction and protein synthesis, as well as a direct link between mTOR and mitogens.
  • Fang et al. concluded that their study suggested a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.
  • RAPTOR is an essential scaffold for the MTOR-catalyzed phosphorylation of 4EBP 1 and mediates TOR action in vivo.
  • Vellai et al. (2003) demonstrated that TOR deficiency in C. elegans more than doubles its natural life span.
  • the absence of Let363/TOR activity caused developmental arrest at the L3 larval stage.
  • the mean life span of Let363 mutants was 25 days compared with a life span of 10 days in wildtype worms.
  • Huntington disease (HD; 143100) is an inherited neurodegenerative disorder caused by a polyglutamine tract expansion in which expanded polyglutamine proteins accumulate abnormally in intracellular aggregates.
  • Ravikumar et al. (2004) showed that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice, and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of HD, and inhibition of autophagy has converse effects.
  • mTOR mammalian target of rapamycin
  • rapamycin protects against neurodegeneration in a fly model of HD
  • the rapamycin analog CCI-779 improved performance on 4 different behavioral tasks and decreased aggregate formation in a mouse model of HD.
  • the data provided proof of principle for the potential of inducing autophagy to treat HD.
  • mTOR is described in detail in Beugnet, et al. J. Biol. Chem. 278 (42), 40717- 40722 (2003); Kristof, et al., J. Biol. Chem. 278 (36), 33637-33644 (2003); Chen,Y., et al., Oncogene 22 (25), 3937-3942 (2003); Gararni, et al., MoI. Cell 11 (6), 1457-1466 (2003); Nojima, et al., J. Biol. Chem.
  • stem cell refers to a cell that on division faces two developmental options: the daughter cells can be identical to the original cell (self-renewal) or they may be the progenitors of more specialised cell types (differentiation). The stem cell is therefore capable of adopting one or other pathway (a further pathway exists in which one of each cell type can be formed). Stem cells are therefore cells which are not terminally differentiated and are able to produce cells of other types. Stem cells as referred to in this document may include totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • totipotent cell refers to a cell which has the potential to become any cell type in the adult body, or any cell of the extraembryonic membranes (e.g., placenta).
  • the only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage.
  • Pluripotent stem cells are true stem cells, with the potential to make any differentiated cell in the body. However, they cannot contribute to making the extraembryonic membranes which are derived from the trophoblast. Several types of pluripotent stem cells have been found.
  • Embryonic Stem (ES) cells may be isolated from the inner cell mass (ICM) of the blastocyst, which is the stage of embryonic development when implantation occurs.
  • ICM inner cell mass
  • Embryonic Germ (EG) cells may be isolated from the precursor to the gonads in aborted fetuses.
  • Embryonic Carcinoma (EC) cells may be isolated from teratocarcinomas, a tumor that occasionally occurs in a gonad of a fetus. Unlike the first two, they are usually aneuploid. All three of these types of pluripotent stem cells can only be isolated from embryonic or fetal tissue and can be grown in culture. Methods are known in the art which prevent these pluripotent cells from differentiating.
  • EC Embryonic Carcinoma
  • Adult stem cells comprise a wide variety of types including neuronal, skin and the blood forming stem cells which are the active component in bone marrow transplantation. These latter stem cell types are also the principal feature of umbilical cord-derived stem cells. Adult stem cells can mature both in the laboratory and in the body into functional, more specialised cell types although the exact number of cell types is limited by the type of stem cell chosen.
  • Multipotent stem cells are true stem cells but can only differentiate into a limited number of types.
  • the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells.
  • Multipotent stem cells are found in adult animals. It is thought that every organ in the body (brain, liver) contains them where they can replace dead or damaged cells.
  • Methods of characterising stem cells include the use of standard assay methods such as clonal assay, flow cytometry, long-term culture and molecular biological techniques e.g. PCR, RT-PCR and Southern blotting.
  • standard assay methods such as clonal assay, flow cytometry, long-term culture and molecular biological techniques e.g. PCR, RT-PCR and Southern blotting.
  • human and murine pluripotent stem cells differ in their expression of a number of cell surface antigens (stem cell markers).
  • Antibodies for the identification of stem cell markers including the Stage-Specific Embryonic Antigens 1 and 4 (SSEA-I and SSEA-4) and Tumor Rejection Antigen 1-60 and 1-81 (TRA- 1-60, TRA- 1-81) may be obtained commercially, for example from Chemicon International, Inc (Temecula, CA, USA).
  • SSEA-I and SSEA-4 Stage-Specific Embryonic Antigens 1 and 4
  • TRA- 1-60, TRA- 1-81 Tumor Rejection Antigen 1-60 and 1-81
  • the immunological detection of these antigens using monoclonal antibodies has been widely used to characterize pluripotent stem cells (Shamblott MJ. et. al. (1998) PNAS 95: 13726-13731; Schuldiner M. et. al.
  • Stem cells of various types which may include the following non-limiting examples, may be used in the methods and compositions described here.
  • U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtained from brain tissue.
  • U.S. Pat. No. 5,766,948 reports producing neuroblasts from newborn cerebral hemispheres.
  • U.S. Pat. Nos. 5,654,183 and 5,849,553 report the use of mammalian neural crest stem cells.
  • U.S. Pat. No. 6,040,180 reports in vitro generation of differentiated neurons from cultures of mammalian multipotential CNS stem cells.
  • WO 98/50526 and WO 99/01159 report generation and isolation of neuroepithelial stem cells, oligodendrocyte- astrocyte precursors, and lineage-restricted neuronal precursors.
  • U.S. Pat. No. 5,968,829 reports neural stem cells obtained from embryonic forebrain and cultured with a medium comprising glucose, transferrin, insulin, selenium, progesterone, and several other growth factors.
  • Primary liver cell cultures can be obtained from human biopsy or surgically excised tissue by perfusion with an appropriate combination of collagenase and hyaluronidase.
  • EP 0953 633 Al reports isolating liver cells by preparing minced human liver tissue, resuspending concentrated tissue cells in a growth medium and expanding the cells in culture.
  • the growth medium comprises glucose, insulin, transferrin, T 3 , FCS, and various tissue extracts that allow the hepatocytes to grow without malignant transformation.
  • liver parenchymal cells The cells in the liver are thought to contain specialized cells including liver parenchymal cells, Kupffer cells, sinusoidal endothelium, and bile duct epithelium, and also precursor cells (referred to as "hepatoblasts" or “o ⁇ al cells") that have the capacity to differentiate into both mature hepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaro et al., Cancer Res. 58:514, 1998).
  • hepatoblasts precursor cells
  • U.S. Pat. No. 5,716,827 reports human hematopoietic cells that are Thy-1 positive progenitors, and appropriate growth media to regenerate them in vitro.
  • U.S. Pat. No. 5,635,387 reports a method and device for culturing human hematopoietic cells and their precursors.
  • U.S. Pat. No. 6,015,554 describes a method of reconstituting human lymphoid and dendritic cells.
  • U.S. Pat. No. 5,486,359 reports homogeneous populations of human mesenchymal stem cells that can differentiate into cells of more than one connective tissue type, such as bone, cartilage, tendon, ligament, and dermis. They are obtained from bone marrow or periosteum. Also reported are culture conditions used to expand mesenchymal stem cells.
  • WO 99/01145 reports human mesenchymal stem cells isolated from peripheral blood of individuals treated with growth factors such as G-CSF or GM- CSF.
  • WO 00/53795 reports adipose-derived stem cells and lattices, substantially free of adipocytes and red cells. These cells reportedly can be expanded and cultured to produce hormones and conditioned culture media.
  • Stem cells of any vertebrate species can be used. Included are stem cells from humans; as well as non-human primates, domestic animals, livestock, and other non-human mammals.
  • the stem cells suitable for use in this invention are primate pluripotent stem (pPS) cells derived from tissue formed after gestation, such as a blastocyst, or fetal or embryonic tissue taken any time during gestation.
  • pPS pluripotent stem
  • Non-limiting examples are primary cultures or established lines of embryonic stem cells.
  • Media for isolating and propagating pPS cells can have any of several different formulas, as long as the cells obtained have the desired characteristics, and can be propagated further. Suitable sources are as follows: Dulbecco's modified Eagles medium (DMEM), Gibco#l 1965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco#l 0829-018; 200 mM L-glutamine, Gibco#l 5039-027; non-essential amino acid solution, Gibco 11140-050; beta-mercaptoethanol, Sigma#M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco#l 3256-029.
  • DMEM Dulbecco's modified Eagles medium
  • KO DMEM Knockout Dulbecco's modified Eagles medium
  • Gibco#l 0829-018 200 mM L-glutamine, Gibco#l 5039-027; non-essential amino acid solution, Gibco 11140-0
  • Exemplary serum- containing ES medium is made with 80% DMEM (typically KO DMEM), 20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol.
  • the medium is filtered and stored at 4 degrees C for no longer than 2 weeks.
  • Serum-free ES medium is made with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol.
  • An effective serum replacement is Gibco#l 0828-028.
  • the medium is filtered and stored at 4 degrees C for no longer than 2 weeks.
  • human bFGF is added to a final concentration of 4 ng/mL (Bodnar et al., Geron Corp, International Patent Publication WO 99/20741).
  • Feeder cells are propagated in mEF medium, containing 90% DMEM (Gibco#l 1965-092), 10% FBS (Hyclone#30071-03), and 2 mM glutamine.
  • mEFs are propagated in Tl 50 flasks (Coming#430825), splitting the cells 1:2 every other day with trypsin, keeping the cells subconfluent.
  • Tl 50 flasks Coming#430825), splitting the cells 1:2 every other day with trypsin, keeping the cells subconfluent.
  • To prepare the feeder cell layer cells are irradiated at a dose to inhibit proliferation but permit synthesis of important factors that support hES cells (.about.4000 rads gamma irradiation).
  • Six-well culture plates (such as Falcon#304) are coated by incubation at 37 degrees C.
  • Feeder cell layers are typically used 5 h to 4 days after plating. The medium is replaced with fresh hES medium just before seeding pPS cells.
  • Embryonic stem cells can be isolated from blastocysts of members of the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995).
  • Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.
  • human blastocysts are obtained from human in vivo preimplantation embryos.
  • in vitro fertilized (FVF) embryos can be used, or one cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989).
  • Human embryos are cultured to the blastocyst stage in Gl .2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998).
  • Blastocysts that develop are selected for ES cell isolation. The zona pellucida is removed from blastocysts by brief exposure to pronase (Sigma).
  • the inner cell masses are isolated by immunosurgery, in which blastocysts are exposed to a 1 :50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, then washed for 5 minutes three times in DMEM, and exposed to a 1 :5 dilution of Guinea pig complement (Gibco) for 3 minutes (see Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.
  • ICM inner cell mass
  • inner cell mass-derived outgrowths are dissociated into clumps either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium.
  • Dissociated cells are replated on mEF feeder layers in fresh ES medium, and observed for colony formation. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated.
  • ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli.
  • ES cells are then routinely split every 1-2 weeks by brief trypsinization, exposure to Dulbecco's PBS (without calcium or magnesium and with 2 mM EDTA), exposure to type IV collagenase (.about.200 U/mL; Gibco) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells are optimal.
  • Embryonic Germ Cells are then routinely split every 1-2 weeks by brief trypsinization, exposure to Dulbecco's PBS (without calcium or magnesium and with 2 mM EDTA), exposure to type IV collagenase (.about.200 U/mL; Gibco) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells are optimal.
  • Human Embryonic Germ (hEG) cells can be prepared from primordial germ cells present in human fetal material taken about 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.
  • genital ridges are rinsed with isotonic buffer, then placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cut into ⁇ 1 mm 3 chunks.
  • the tissue is then pipetted through a 100/.mu.L tip to further disaggregate the cells. It is incubated at 37 degrees C. for about 5 min, then about 3.5 mL EG growth medium is added.
  • EG growth medium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM sodium bicarbonate; 15% ES qualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/ml human recombinant basic fibroblast growth factor (bFGF, Genzyme); and 10 .mu.M forskolin (in 10% DMSO).
  • EG cells are isolated using hyaluronidase/collagenase/DNAse.
  • Gonadal anlagen or genital ridges with mesenteries are dissected from fetal material, the genital ridges are rinsed in PBS, then placed in 0.1 ml HCD digestion solution (0.01% hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all from Sigma prepared in EG growth medium). Tissue is minced and incubated 1 h or overnight at 37 degrees C, resuspended in 1-3 mL of EG growth medium, and plated onto a feeder layer.
  • HCD digestion solution 0.01% hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all from Sigma prepared in EG growth medium.
  • stem cells which are exiting self-renewal and entering differentiation display reduced mTOR activity.
  • Stem cells which are self-renewing may be identified by various means known in the art, for example, morphology, immunohistochemistry, molecular biology, etc. Such stem cells preferably display increased expression of Oct4 and/or SSEA-I. Preferably, expression of any one or more of FIk-I, Tie-2 and c-kit is decreased. Stem cells which are self-renewing preferably display a shortened cell cycle compared to stem cells which are not self-renewing.
  • human ES cells display high nuclear/cytoplasmic ratios in the plane of the image, prominent nucleoli, and compact colony formation with poorly discemable cell junctions.
  • Cell lines can be karyotyped using a standard G-banding technique (available at many clinical diagnostics labs that provides routine karyotyping services, such as the Cytogenetics Lab at Oakland Calif.) and compared to published human karyotypes.
  • hES and hEG cells may also be characterized by expressed cell markers.
  • tissue-specific markers discussed in this disclosure can be detected using a suitable immunological technique—such as flow cytometry for membrane-bound markers, immunohistochemistry for intracellular markers, and enzyme-linked immunoassay, for markers secreted into the medium.
  • the expression of protein markers can also be detected at the mRNA level by reverse transcriptase-PCR using marker-specific primers. See U.S. Pat. No. 5,843,780 for further details.
  • Stage-specific embryonic antigens (SSEA) are characteristic of certain embryonic cell types. Antibodies for SSEA markers are available from the Developmental Studies Hybridoma Bank (Bethesda Md.).
  • hES cells are typically SSEA-I negative and SSEA-4 positive.
  • hEG cells are typically SSEA-I positive.
  • Differentiation of pPS cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression and increased expression of SSEA-I .
  • pPS cells can also be characterized by the presence of alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.).
  • Embryonic stem cells are also typically telomerase positive and OCT-4 positive.
  • Telomerase activity can be determined using TRAP activity assay (Kim et al., Science 266:2011, 1997), using a commercially available kit (TRAPeze.RTM. XK Telomerase Detection Kit, Cat. s7707; Intergen Co., Purchase N. Y.; or TeloTAGGG.TM. Telomerase PCR ELISA plus, Cat. 2,013,89; Roche Diagnostics, Indianapolis).
  • hTERT expression can also be evaluated at the mRNA level by RT-PCR.
  • the LightCycler TeloTAGGG.TM. hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics) is available commercially for research purposes.
  • stem cells which are differentiating display decreased or reduced mTOR activity.
  • Stem cells which are self-renewing preferably display a lenghtened cell cycle compared to stem cells which are not self-renewing. Differentiating stem cells, i.e., cells which have started to, or are committed to a pathway of differentiation can be characterized according to a number of phenotypic criteria, including in particular transcript changes.
  • the criteria include but are not limited to characterization of morphological features, detection or quantitation of expressed cell markers and enzymatic activity, gene expression and determination of the functional properties of the cells in vivo.
  • differentiating stem cells will have one or more features of the cell type which is the final product of the differentiation process, i.e., the differentiated cell. For example, if the target cell type is a muscle cell, a stem cell which is in the process of differentiating to such a cell will have for example a feature of myosin expression.
  • the criteria will depend on the fate of the differentiating stem cell, and a general description of various cell types is provided below.
  • Markers of interest for differentiated or differentiating neural cells include beta- tubulin EIII or neurofilament, characteristic of neurons; glial fibrillary acidic protein (GFAP), present in astrocytes; galactocerebroside (GaIC) or myelin basic protein (MBP); characteristic of oligodendrocytes; OCT-4, characteristic of undifferentiated hES cells; nestin, characteristic of neural precursors and other cells.
  • A2B5 and NCAM are characteristic of glial progenitors and neural progenitors, respectively.
  • Cells can also be tested for secretion of characteristic biologically active substances.
  • GABA- secreting neurons can be identified by production of glutamic acid decarboxylase or GABA.
  • Dopaminergic neurons can be identified by production of dopa decarboxylase, dopamine, or tyrosine hydroxylase.
  • Markers of interest for differentiated or differentiating liver cells include alpha- fetoprotein (liver progenitors); albumin, .alphaj -antitrypsin, glucose-6-phosphatase, cytochrome p450 activity, transferrin, asialoglycoprotein receptor, and glycogen storage (hepatocytes); CK7, CKl 9, and gamma-glutamyl transferase (bile epithelium). It has been reported that hepatocyte differentiation requires the transcription factor BNF-4 alpha (Li et al., Genes Dev. 14:464, 2000).
  • Markers independent of HNF-4 alpha expression include alphaj -antitrypsin, alpha-fetoprotein, apoE, glucokinase, insulin growth factors 1 and 2, IGF-I receptor, insulin receptor, and leptin. Markers dependent on HNF-4 alpha expression include albumin, apoAI, apoAII, apoB, apoCIII, apoCII, aldolase B, phenylalanine hydroxylase, L-type fatty acid binding protein, transferrin, retinol binding protein, and erythropoietin (EPO).
  • Cell types in mixed cell populations derived from pPS cells can be recognized by characteristic morphology and the markers they express.
  • skeletal muscle myoD, myogenin, and myf-5.
  • endothelial cells PECAM (platelet endothelial cell adhesion molecule), FIk-I, tie-i, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC- 14.7.
  • smooth muscle cells specific myosin heavy chain.
  • cardiomyocytes GATA- 4, Nkx2.5, cardiac troponin I, alpha-myosin heavy chain, and ANF.
  • pancreatic cells pdx and insulin secretion.
  • hematopoietic cells and their progenitors GATA-I, CD34, ACl 33, .beta.-major globulin, and .beta. -major globulin like gene PHl.
  • tissue-specific markers listed in this disclosure or known in the art can be detected by immunological techniques-such as flow immunocytochemistry for cell- surface markers, immunohistochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • the expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods.
  • RT-PCR reverse transcriptase initiated polymerase chain reaction
  • the methods and compositions described here rely, in some embodiments, on blocking, reducing, or decreasing the activity of mTOR protein. In other embodiments, the activity of mTOR is increased, heightened, up-regulated, etc. Such modulation of mTOR activity may be used to influence whether the stem cell differentiates or self renews.
  • the methods employ modulators of mTOR activity or expression.
  • Agents which are capable of increasing the activity of mTOR protein are referred to as agonists of that activity.
  • antagonists reduce the activity of the relevant protein.
  • agonists of mTOR activity have the ability to increase a relevant activity of mTOR, for example, kinase activity, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.
  • Antagonists of mTOR activity on the other, preferably have the ability to reduce its activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.
  • mTOR activity is assayed as described below in the section "Assays for mTOR Activity".
  • antagonist is generally taken to refer to a compound which binds to an enzyme and inhibits the activity of the enzyme.
  • the term as used here is intended to refer broadly to any agent which inhibits the activity of a molecule, not necessarily by binding to it. Accordingly, it includes agents which affect the expression of an mTOR protein, or the biosynthesis of a regulatory molecule, or the expression of modulators of the activity of mTOR.
  • the specific activity which is inhibited may be any activity which is exhibited by, or characteristic of, the enzyme or molecule, for example, any activity of mTOR as the case may be, for example, a kinase activity.
  • the kinase activity may comprise the ability to phosphorylate one or either of S6K1 and/or 4E/BP1.
  • the antagonist may bind to and compete for one or more sites on the relevant molecule preferably, the catalytic site of the enzyme. Preferably, such binding blocks the interaction between the molecule and another entity (for example, the interaction between a enzyme and its substrate).
  • the antagonist need not necessarily bind directly to a catalytic site, and may bind for example to an adjacent site, another protein (for example, a protein which is complexed with the enzyme) or other entity on or in the cell, so long as its binding reduces the activity of the enzyme or molecule.
  • an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme.
  • whole or fragments of a substrate generated natively or by peptide synthesis may be used to compete with the substrate for binding sites on the enzyme.
  • an immunoglobulin for example, a monoclonal or polyclonal antibody
  • the antagonist may also include a peptide or other small molecule which is capable of interfering with the binding interaction.
  • Other examples of antagonists are set forth in greater detail below, and will also be apparent to the skilled person.
  • Non-functional homologues of a mTOR may also be tested for inhibition of mTOR activity as they may compete with the wild type protein for binding to other components of the cell machinery whilst being incapable of the normal functions of the protein. Alternatively, they may block the function of the protein bound to the cell machinery.
  • Such non-functional homologues may include naturally occurring mutants and modified sequences or fragments thereof.
  • the substance may suppress the biologically available amount of a mTOR. This may be by inhibiting expression of the component, for example at the level of transcription, transcript stability, translation or post-translational stability.
  • An example of such a substance would be antisense RNA or double-stranded interfering RNA sequences which suppresses the amount of mRNA biosynthesis. Blocking the activity of an inhibitor of the mTOR protein may therefore also be achieved by reducing the level of expression of the protein or an inhibitor in the cell.
  • the cell may be treated with antisense compounds, for example oligonucleotides having sequences specific to the mTOR mRNA.
  • the level of expression of pathogenic forms of adhesion proteins may also be regulated this way.
  • agonists, antagonists and modulators comprise agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate.
  • An agent may be in solution or in suspension (
  • modulator is also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signalling molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g.
  • RNA including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate.
  • PNA peptide nucleic acid
  • Small molecules including inorganic and organic chemicals, which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented, are also included.
  • Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
  • RNA interference may be used to abolish or knock out or reduce gene activity, for example, mTOR activity.
  • the overall strategy is to prepare double stranded RNA (dsRNA) specific to each gene of interest and to transfect this into a cell of interest to inhibit the expression of the particular gene.
  • dsRNA double stranded RNA
  • a sample of PCR product is analysed by horizontal gel electrophoresis and the DNA purified using a Qiagen QiaQuick PCR purification kit.
  • 1 ⁇ g of DNA is used as the template in the preparation of gene specific single stranded RNA using the Ambion T7 Megascript kit.
  • Single stranded RNA is produced from both strands of the template and is purified and immediately annealed by heating to 90 degrees C for 15 mins followed by gradual cooling to room temperature overnight.
  • a sample of the dsRNA is analysed by horizontal gel electrophoresis, and introduced into the relevant cell by conventional means.
  • Any agent which is capable of reducing mTOR activity or expression, as described above, may be used as an antagonist of mTOR for the purposes fo reducing its activity.
  • butanol is an inhibitor of mTOR activity, as described in Kam and Exton, FASEB J. 2004 Feb;18(2):311-9 and Fang et al., Science 294:1942-1945. Butanol may therefore be used in the methods and compositions described here as an agent capable of reducing mTOR activity.
  • an agent capable of reducing mTOR activity comprises rapamycin and its derivatives. Rapamycin and such derivatives are therefore provided as specific antagonists of mTOR activity.
  • stem cells are exposed to rapamycin and its derivatives at concentrations over InM, for example, 1OnM, 2OnM, 3OnM, 4OnM, 50 nM, 10OnM, 50OnM, l ⁇ m, lO ⁇ m, lOO ⁇ m, or more.
  • rapamycin and its derivatives are used at about 5OnM.
  • Rapamycin is an antifungal antibiotic which is extractable from a streptomycete, e.g., Streptomyces hygroscopicus.
  • Methods for the preparation of rapamycin are disclosed in Sehgal et al., U.S. Pat. Nos. 3,929,992, and 3,993,749.
  • monoacyl and diacyl derivatives of rapamycin and methods for their preparation are disclosed by Rakhit, U.S. Pat. No. 4,316,885.
  • rapamycin derivatives including the following rapamycin prodrugs: glycinate prodrugs, propionate prodrugs and the pyrrolidino butyrate prodrugs.
  • compositions described here include the use of natural and synthetic rapamycin, genetically engineered rapamycin and all derivatives and prodrugs of rapamycin, such as described in the aforementioned U.S. patents, U.S. Pat. Nos. 3,929,992; 3,993,749; 4,316,885; and 4,650,803, the contents of which are hereby incorporated by reference.
  • Rapamycin known as sirolimusis, is a 31-membered macrolide lactone, C 51 H 79 NOj 3 , with a molecular mass of 913.6 Da. In solution, sirolimus forms two conformational trans-, cis-isomers with a ratio of 4:1 (chloroform) due to hindered rotation around the pipecolic acid amide bond. It is sparingly soluble in water, aliphatic hydrocarbons and diethyl ether, whereas it is soluble in alcohols, halogenated hydrocarbons and dimethyl sulfoxide. Rapamycin is unstable in solution and degrades in plasma and low-, and neuteral-pH buffers at 37 degrees C with half-life of ⁇ 10 h.
  • Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus, which was found to have antifungal activity, particularly against Candida albicans, both in vitro and in vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No. 3,929,992; and U.S. Pat. No. 3,993,749].
  • Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination with picibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumor activity.
  • R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the formation of IgE-like antibodies.
  • rapamycin The immunosuppressive effects of rapamycin have been disclosed in FASEB 3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules, also have been shown to be effective as immunosuppressive agents, therefore useful in preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183 (1978)]. Although it shares structural homology with the immunosuppressant tacrolimus and binds to the same intracellular binding protein in lymphocytes, rapamycin inhibits S6p70-kinase and therefore has a mechanism of immunosuppressive action distinct from that of tacrolimus.
  • Rapamycin was found to prolong graft survival of different transplants in several species alone or in combination with other immunosupressants. In animal models its spectrum of toxic effects is different from that of cyclosporin or FK-506., comprising impairment of glucose homeostasis, stomach, ulceration, weight loss and thrombocytopenia, although no nephrotoxicity has been detected. Rapamycin Prodrugs Rapamycin Dialdebydes
  • Rapamycin prodrugs such as rapamycin dialdehydes described in United States Patent 6,680,330 (Zhu, et al) may be employed in the methods and compositions described here.
  • Rapamycin is metabolized by cytochrome P-450 3 A to at least six metabolites.
  • sirolimus was hydroxylated and demethylated and the structure of 39-O-demethyl sirolimus was identified.
  • bile of sirolimus-treated rats >16 hydroxylated and demethylated metabolites were detected.
  • rapalogs include among others variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at Cl 3, C43 and/or C28; reduction, elimination or derivatization of the ketone at C 14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.
  • Japanese Patent Application Lailogs as that term is used herein denotes a class of compounds comprising the various analogs, homologs and derivatives of rapamycin and other compounds related structurally to rapamycin.
  • "Rapalogs” include compounds other than rapamycin (or those rapamycin derivatives modified in comparison to rapamycin only with respect to saturation of one or more of the carbon-carbon double bonds at the 1, 2, 3, 4 or 5, 6 positions) which comprise the substructure shown in Formula I, bearing any number of a variety of substituents, and optionally unsaturated at one or more carbon—carbon bonds unless specified to the contrary herein.
  • Rapalogs include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at Cl 3, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5- membered prolyl ring; and elimination, derivatization or replacement of one or more substituents of the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted or unsubstituted cyclopentyl ring.
  • Rapalogs do not include rapamycin itself, and preferably do not contain an oxygen bridge between Cl and C30.
  • Illustrative examples of rapalogs are disclosed in the documents listed in Table I.
  • Examples of rapalogs modified at C7 are shown in Table II.
  • an agent capable of increasing mTOR activity comprises phosphatidic acid and its derivatives, phosphatidic acid and such derivatives are therefore provided as specific agonists of mTOR activity.
  • stem cells are exposed to phosphatidic acid and its derivatives at concentrations over InM, for example, 1OnM, 2OnM, 3OnM, 4OnM, 50 nM, 10OnM, 50OnM, l ⁇ m, lO ⁇ m, lOO ⁇ m, or more.
  • phosphatidic acid and its derivatives are used at about 5OnM.
  • Suitable agonists of mTOR include lysophosphatidic acid or 1-acyl-sn- glycerol-3-phosphate (LPA), 1-alkyl-lysophosphatidic acid or alkenyl-ether lysophosphatidic acid, sphingolipid analogues, sphingosine-1 -phosphate, cyclic phosphatidic acid, pyrophosphatidic acid, diacylglycerol pyrophosphate, lysobisphosphatidic acid, lysobisphosphatidic acid or bis(monoacylglycerol)phosphate and semilysobisphosphatidic acid.
  • LPA 1-acyl-sn- glycerol-3-phosphate
  • sphingolipid analogues sphingosine-1 -phosphate
  • cyclic phosphatidic acid pyrophosphatidic acid, diacylglycerol pyrophosphate
  • lysobisphosphatidic acid lysobisphosphatidic
  • phosphatidic acid is synthesised by the action of the enzymes phospholipase D and diacylglycerol kinases (DGKs).
  • phospholipase A converts lysophosphatidic acid to phosphatidic acid
  • phospholipase D cleaves phospholipids to form phosphatidic acid
  • diacylglycerol kinases phosphorylate diacylglycerol to phosphatidic acid.
  • any agent capable of increasing the activity of phospholipase D or any other relevant synthesis enzyme may be used in place of, or to supplement the activity of, phosphatidic acid or that enzyme for the purposes of infleuncing, commiting, or forcing the stem cell to adopt a self-renewing state. It will of course be possible to use additional amounts of phospholipase D or the enzyme itself, to increase the activity of this enzyme in the stem cell.
  • mTOR activity is enhanced by increased nutrient levels.
  • TOR-dependent growth regulation in S.cerevisiae is dependent on nitrogen, carbohydrate and amino acid levels rather than mitogen inputs, while TOR signaling in multicellular organisms is more complex where cell growth and proliferation are regulated by the integration of both nutrients and growth factors.
  • any agent which is capable of increasing nutrient levels and/or growth factors in the environment of a stem cell may be used instead of, or to supplement the activity of, phosphatidic acid for the purposes of infleuncing, commiting, or forcing the stem cell to adopt a self-renewing state.
  • Specific antagonists of mTOR which may be used to regulate the activity of these proteins (for example, for methods of treating or preventing diseases such as cancer) may include antibodies against the protein(s).
  • Antibodies refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab' and F(ab') 2 , monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR- grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such as Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
  • the antibodies according described here are especially indicated for the detection of PGCs and other pluripotent cells, such as ES or EG cells. Accordingly, they may be altered antibodies comprising an effector protein such as a label.
  • labels which allow the imaging of the distribution of the antibody in vivo or in vitro.
  • Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within an embryo or a cell mass.
  • radioactive labels such as metal particles
  • they may be fluorescent labels or other labels which are visualisable on tissue samples.
  • chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications.
  • immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [EP 0 239 400].
  • Antibodies may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.
  • an antibody comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said antibody protein, and isolating said protein.
  • a host e.g. E. coli or a mammalian cell
  • a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said antibody protein, and isolating said protein.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium
  • a mammalian serum e.g. foetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells
  • feeder cells such as normal mouse peritoneal exudate cells, sple
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
  • the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody- producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody- producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • the cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of PGCs or other pluripotent cells, such as ES or EG cells, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with mTOR, or fragments thereof, or with Protein-A.
  • Hybridoma cells secreting the monoclonal antibodies are also provided.
  • Preferred hybridoma cells are genetically stable, secrete monoclonal antibodies of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.
  • a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to mTOR characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with a one or more mTOR polypeptides, or antigenic fragments thereof; antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • spleen cells of Balb/c mice immunised with mTOR are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Agl4, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
  • spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol.
  • a fusion promoter preferably polyethylene glycol.
  • the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000.
  • the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
  • DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to mTOR as described hereinbefore are also disclosed.
  • DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
  • DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to mTOR can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
  • a mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids.
  • said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody.
  • Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s).
  • Such a mutant sequence is also a degenerated sequence.
  • Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded.
  • Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
  • mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
  • the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
  • recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed to mTOR fused to a human constant domain g, for example ⁇ l , ⁇ 2, ⁇ 3 or ⁇ 4, preferably ⁇ l or ⁇ 4.
  • recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to mTOR fused to a human constant domain K or ⁇ , preferably K are also disclosed.
  • TheDNAencodingsuchaneffector molecule hasthesequenceofanaturallyoccurringenzymeortoxinencodingDNA,ora mutantthereof,andcanbepreparedbymethodswellknownintheart.
  • Anti-peptideantibodies maybeproducedagainstmTORpeptidesequences.
  • preferred anti-peptide antibodies may be raised from any one or more of the following sequences: amino acids 22-139; amino acids 647-907; amino acids 937-1140; amino acids 1382-1982; amino acids 2019-2112; or amino acids 2181-2549.
  • Corresponding sequences from human mTOR may be chosen for use in eliciting anti-peptide antibodies from immunised animals.
  • Antibodies may be produced by injection into rabbits, and other conventional means, as described in for example, Harlow and Lane (supra).
  • Antibodies are checked by Elisa assay and by Western blotting, and used for immunostaining as described in the Examples.
  • Antagonists in particular, small molecules may be used to specifically inhibit mTOR.
  • small molecules ⁇ mTOR inhibitors as well as assays for screening for these.
  • Antagonists of mTOR kinase are screened by detecting modulation, preferably down regulation, of binding or other activity.
  • down-regulation we include any negative effect on the behaviour being studied; this may be total, or partial.
  • candidate antagonists are capable of reducing, ameliorating, or abolishing the binding between two entities.
  • the down-regulation of binding (or any other activity) achieved by the candidate molecule is at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, or more compared to binding (or which ever activity) in the absence of the candidate molecule.
  • a candidate molecule suitable for use as an antagonist is one which is capable of reducing by 10% more the binding or other activity.
  • Modulators, agonists and antagonists of mTOR activity or expression may be identified by any means known in the art. Putative such molecules may be identified by their binding to mTOR, in an assay which detects binding between mTOR and the putative molecule.
  • One type of assay for identifying substances that bind to a polypeptide involves contacting a polypeptide, which is immobilised on a solid support, with a non- immobilised candidate substance determining whether and/or to what extent the polypeptide and candidate substance bind to each other.
  • the candidate substance may be immobilised and the polypeptide non-immobilised. This may be used to detect substances capable of binding to mTOR polypeptides, or fragments, homologues, variants or derivatives thereof.
  • the polypeptide is immobilised on beads such as agarose beads.
  • beads such as agarose beads.
  • the mTOR polypeptide, or a fragment, homologue, variant or derivative thereof as a GST-fusion protein in bacteria, yeast or higher eukaryotic cell lines and purifying the GST-fusion protein from crude cell extracts using glutathione-agarose beads (Smith and Johnson, 1988).
  • binding of the candidate substance, which is not a GST-fusion protein, to the immobilised polypeptide is determined in the absence of the polypeptide.
  • the binding of the candidate substance to the immobilised polypeptide is then determined.
  • This type of assay is known in the art as a GST pulldown assay.
  • the candidate substance may be immobilised and the polypeptide non-immobilised. It is also possible to perform this type of assay using different affinity purification systems for immobilising one of the components, for example Ni-NTA agarose and histidine-tagged components.
  • Binding of the mTOR polypeptide, or a fragment, homologue, variant or derivative thereof to the candidate substance may be determined by a variety of methods well-known in the art.
  • the non-immobilised component may be labeled (with for example, a radioactive label, an epitope tag or an enzyme-antibody conjugate).
  • binding may be determined by immunological detection techniques.
  • the reaction mixture can be Western blotted and the blot probed with an antibody that detects the non-immobilised component. ELISA techniques may also be used.
  • Candidate substances are typically added to a final concentration of from 1 to 1000 nmol/ml, more preferably from 1 to 100 nmol/ml.
  • the final concentration used is typically from 100 to 500 ⁇ g/ml, more preferably from 200 to 300 ⁇ g/ml.
  • Assays to detect modulators typically involve detecting modulation of any activity of mTOR, preferably kinase activity, in the presence, optionally together with detection of modulation of activity in the absence, of a candidate molecule.
  • the assays involve contacting a candidate molecule (e.g., in the form of a library) with mTOR whether in the form of a polypeptide, a nucleic acid encoding the polypeptide, or a cell, organelle, extract, or other material comprising such, with a candidate modulator.
  • a candidate modulator e.g., in the form of a library
  • the relevant activity of mTOR may be detected, to establish whether the presence of the candidate modulator has any effect.
  • Promoter binding assays to detect candidate modulators which bind to and/or affect the transcription or expression of mTOR may also be used.
  • Candidate modulators may then be chosen for further study, or isolated for use.
  • the screening methods described here preferably employ in vivo assays, although they may be configured for in vitro use.
  • In vivo assays generally involve exposing a cell comprising mTOR to the candidate molecule.
  • mTOR is exposed to the candidate molecule, optionally in the presence of other components, such as crude or semi-purified cell extract, or purified proteins.
  • these preferably employ arrays of candidate molecules (for example, an arrayed library).
  • the mTOR is comprised in a cell, preferably heterologously.
  • a cell is preferably a transgenic cell, which has been engineered to express mTOR as described above.
  • an extract it may comprise a cytoplasmic extract or a nuclear extract, methods of preparation of which are well known in the art.
  • a preferred embodiment utilises a cytoplasmic or nuclear preparation, e.g., comprising a cell nucleus which comprises mTOR as described. See Zhang, et al, Predominant Nuclear Localization of Mammalian Target of Rapamycin in Normal and Malignant Cells in Culture. J. Biol. Chem., JuI 2002; 277: 28127 - 28134.
  • the nuclear preparation may comprise one or more nuclei, which may be permeabilised or semi-permeabilised, by detergent treatment, for example.
  • an assay format may include the following: a multiwell microtitre plate is set up to include one or more cells expressing mTOR in each well; individual candidate molecules, or pools of candidate molecules, derived for example from a library, may be added to individual wells and modulation of mTOR activity measured. Where pools are used, these may be subdivided in to further pools and tested in the same manner. mTOR activity, for example, kinase activity, is then assayed.
  • “subtractive” procedures may also be used to identify modulators, agonists or antagonists of mTOR.
  • a plurality of molecules is provided, which comprises one or more candidate molecules capable of functioning as a modulator (e.g., cell extract, nuclear extract, library of molecules, etc), and one or more components is removed, depleted or subtracted from the plurality of molecules.
  • the "subtracted” extract, etc is then assayed for activity, by exposure to a cell comprising mTOR (or a component thereof) as described.
  • an 'immunodepletion' assay may be conducted to identify such modulators as follows.
  • a cytoplasmic or nuclear extract may be prepared from a pluripotent cell, for example, a pluripotent EG/ES cell.
  • the extract may be depleted or fractionated to remove putative modulators, such as by use of immunodepletion with appropriate antibodies. If the extract is depleted of a modulator, it will lose the ability to affect mTOR function or activity or expression.
  • a series of subtractions and/or depletions may be required to identify the modulators, agonists or antagonists.
  • the above “depletion” or “subtraction” assay may be used as a preliminary step to identify putative modulatory factors for further screening.
  • the “depletion” or “subtraction” assay may be used to confirm the modulatory activity of a molecule identified by other means (for example, a "positive” screen as described elsewhere in this document) as a putative modulator.
  • Candidate molecules subjected to the assay and which are found to be of interest may be isolated and further studied. Methods of isolation of molecules of interest will depend on the type of molecule employed, whether it is in the form of a library, how many candidate molecules are being tested at any one time, whether a batch procedure is being followed, etc.
  • the candidate molecules may be provided in the form of a library. In a preferred embodiment, more than one candidate molecule is screened simultaneously.
  • a library of candidate molecules may be generated, for example, a small molecule library, a polypeptide library, a nucleic acid library, a library of compounds (such as a combinatorial library), a library of antisense molecules such as antisense DNA or antisense RNA, an antibody library etc, by means known in the art.
  • Such libraries are suitable for high-throughput screening.
  • Different cells comprising mTOR may be exposed to individual members of the library, and effect on the stem cell determined.
  • Array technology may be employed for this purpose.
  • the cells may be spatially separated, for example, in wells of a microtitre plate.
  • a small molecule library is employed.
  • a small molecule we refer to a molecule whose molecular weight is preferably less than about 50 kDa.
  • a small molecule has a molecular weight preferably less than about 30 kDa, more preferably less than about 15 kDa, most preferably less than 10 kDa or so.
  • Libraries of such small molecules, here referred to as “small molecule libraries” may contain polypeptides, small peptides, for example, peptides of 20 amino acids or fewer, for example, 15, 10 or 5 amino acids, simple compounds, etc.
  • a combinatorial library may be screened for modulators, antagonists or agonists of mTOR.
  • mTOR Any of the activities of mTOR may be used as the basis of the assay.
  • mToR is responsible for phosphorylating substrates including eukaryotic initiation factor 4E (eIF4E) and ribosomal S6 kinase 1 (S6K1), RNA polymerase I and eEF2 kinase.
  • substrates including eukaryotic initiation factor 4E (eIF4E) and ribosomal S6 kinase 1 (S6K1), RNA polymerase I and eEF2 kinase.
  • Such assays may employ 4E-BP1 and/or S6K1 as substrates, or use peptides from these polypeptides as substrates.
  • mTOR is known to phosphorylate 4E-BP1 at Thr37 and Thr46 and S6K1 at Thr389 (Schalm SS, Fingar DC, Sabatini DM, Blenis J. Curr Biol. 2003 May 13;13(10):797-806; Schalm SS, Blenis J. Curr Biol. 2002 Apr 16;12(8):632- 9.), and accordingly peptide substrates containing these positions may be generated using known peptide synthesis methods.
  • Cells are grown for 48 hours in DMEM containing 10% FBS, and lysed in lysis buffer B (40 mM HEPES, 120 mM NaCl, 50 mM NaF, 1 mM EDTA, 50 mM ⁇ - glycerophosphate, 0.2% CHAPS, 1 mM Na3 VO4, 40 mg/ml PMSF, 5 ⁇ g/ml pepstatin, 10 ⁇ g/ml leupeptin, 1 mM DTT, ddH2, O, pH 7.5).
  • lysis buffer B 40 mg/ml PMSF, 5 ⁇ g/ml pepstatin, 10 ⁇ g/ml leupeptin, 1 mM DTT, ddH2, O, pH 7.5.
  • One third of total cell lysate from a 150-mm plate is incubated with an anti mTOR-antibody (e.g., Bethyl, Inc, Texas USA) for 2 h, followed by another hour of incubation with protein-G-Sepharose beads.
  • an anti mTOR-antibody e.g., Bethyl, Inc, Texas USA
  • Immunopreciptates are washed twice with 1 ml mTOR wash buffer A (20 mM Tris, 500 mM NaCl, 1 mM EDTA, 20 mM ⁇ -glycerophosphate, 5 mM EGTA, 1 mM DTT, 1 mM Na3 VO4.40 mg/ml PMSF, 10 ⁇ g/ml leupeptin, 5 ⁇ g/ml pepstatin, in ddH2 O, pH 7.4), once with mTOR wash buffer B (10 mM HEPES, 50 mM ⁇ -glycerophosphate, 50 mM NaCl, 1 mM DTT, 1 mM Na3 V04, 40 mg/ml PMSF, 10 ⁇ g/ml leupeptin, 5 ⁇ g/ml pepstatin, in ddH2 O, pH 7.4), and once with ST (50 mM Tris-HCl, 5 mM Tris base, 150 mM NaC
  • kinase assays towards recombinant GST-4E-BP1 WT or GST-4E-BP1 Fl 14A i.e., human 4E-BP1 subcloned into pGEX-2T/GST, Pharmacia
  • washed immunoprecipitates is assayed in mTOR kinase assay buffer (10 mM HEPES, 50 mM NaCl, 50 mM ⁇ -glycerophosphate, 10 mM MnCk, 100 ⁇ M ATP unlabeled, 10 ⁇ Ci [ ⁇ - 32P] ATP (New England Nuclear), pH 7.4) for 30 min at 30 0 C.
  • the reaction is separated by 12% SDS-PAGE and 32P incorporated into GST-4E-BP1 is assessed by autoradiography and quantified by phosphoimaging (BioRad).
  • One kinase unit is defined by the amount of kinase ie protein required to catalyze the transfer of 1 pmol of phosphate to the substrate per reaction volume in one minute at 3O 0 C.
  • Molecules and agents which activate or promote mTOR activity may be identified as follow:
  • a hybrid gene encoding for a mRNA with a 5'UTR derived from a TOP mRNA e.g. L5 ribosomal protein mRNA and coding region from a reporter gene e.g. GFP or luciferase is transfected into mammalian cells.
  • the cells are either serum starved or rapamycin-treated to shut off translation of the reporter.
  • Cells are exposed to a candidate molecule or a member of a library. Addition of an mTOR activating molecule will upregulate translation of the reporter (see Figure 8 A and Example 8)
  • Molecules and agents which inhibit mTOR activity are identified as follow:
  • a hybrid gene encoding a mRNA with a 5'UTR derived from mRNAs whose translation is upregulated when cap-mediated translation is inhibited e.g. p27Kipl mRNA and coding region from a reporter gene e.g. GFP or luciferase is transfected into mammalian cells .
  • the cells are either serum starved or rapamycin-treated to turn on translation of the reporter. Then serum will be added or rapamycin removed to activate mTOR and turn off translation of reporter.
  • Cells are exposed to a candidate molecule or a member of a library. When the reporter is off, mTOR inhibiting molecule will be added to upregulate translation of the reporter (see Figure 8B and Example 9).
  • mTOR activity is capable of lengthening cell cycle times; accordingly, the cell cycle period maybe assayed in the presence and absence of a candidate molecule to identify antagonists or agonists of mTOR activity.
  • Libraries of candidate molecules such as libraries of polypeptides or nucleic acids, may be employed in the screens described here. Such libraries are exposed to a stem cell and their effect, if any, on the choice of the stem cell between self-renewal and differentiation determined.
  • Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
  • Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith (1990 supra), have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • the nucleotide sequences encoding the V H and V L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
  • phage-based display systems An advantage of phage- based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • Alternative library selection technologies include bacteriophage lambda expression systems, which may be screened directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et al (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87; Mullinax et al (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095; Persson et al (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and are of use in the methods and compositions described here.
  • a significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide, so as to facilitate identification of the amino acid sequence of each library member.
  • a unique identifier tag such as an oligonucleotide
  • Another chemical synthesis method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface in a manner that places each distinct library member (e.g., unique peptide sequence) at a discrete, predefined location in the array.
  • the identity of each library member is determined by its spatial location in the array.
  • the locations in the array where binding interactions between a predetermined molecule (e.g., a receptor) and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location.
  • RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818).
  • a similar technique may be used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843).
  • in vitro translation can be used to synthesise polypeptides as a method for generating large libraries.
  • These methods which generally comprise stabilised polysome complexes, are described further in WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
  • Alternative display systems which are not phage-based, such as those disclosed in WO95/22625 and WO95/11922 (Affymax) use the polysomes to display polypeptides for selection.
  • the library may in particular comprise a library of zinc fingers; zinc fingers are known in the art and act as transcription factors. Suitable zinc finger libraries are disclosed in, for example, WO 96/06166 and WO 98/53057. Construction of zinc finger libraries may utilise rules for determining interaction with specific DNA sequences, as disclosed in for example WO 98/53058 and WO 98/53060. Zinc fingers capable of interacting specifically with methylated DNA are disclosed in WO 99/47656. The above zinc finger libraries may be immobilised in the form of an array, for example as disclosed in WO 01/25417. Accordingly, preferred molecules capable of altering the potency of a cell include zinc fingers.
  • libraries of candidate molecules may suitably be in the form of combinatorial libraries (also known as combinatorial chemical libraries).
  • a "combinatorial library”, as the term is used in this document, is a collection of multiple species of chemical compounds that consist of randomly selected subunits. Combinatorial libraries may be screened for molecules which are capable of changing the choice by a stem cell between the pathways of self-renewal and differentiation.
  • combinatorial libraries of chemical compounds are currently available, including libraries active against proteolytic and non-proteolytic enzymes, libraries of agonists and antagonists of G-protein coupled receptors (GPCRs), libraries active against non-GPCR targets (e.g., integrins, ion channels, domain interactions, nuclear receptors, and transcription factors) and libraries of whole-cell oncology and anti-infective targets, among others.
  • GPCRs G-protein coupled receptors
  • non-GPCR targets e.g., integrins, ion channels, domain interactions, nuclear receptors, and transcription factors
  • libraries of whole-cell oncology and anti-infective targets among others.
  • the combinatorial library which is screened is one which is designed to potentially include molecules which interact with a component of the cell to influence gene expression.
  • combinatorial libraries against chromatin structural proteins may be screened.
  • Other libraries which are useful for this embodiment include combinatorial libraries against histone modification enzymes (e.g., histone acetylation or histone metylation enzymes), or DNA modification, for example, DNA methylation or demethylation.
  • Soluble random combinatorial libraries may be synthesized using a simple principle for the generation of equimolar mixtures of peptides which was first described by Furka (Furka, A. et al., 1988, Xth International Symposium on Medicinal Chemistry, Budapest 1988; Furka, A. et al., 1988, 14th International Congress of Biochemistry, Prague 1988; Furka, A. et al., 1991, Int. J. Peptide Protein Res. 37:487-493). The construction of soluble libraries for iterative screening has also been described (Houghten, R. A. et al.1991, Nature 354:84-86). K. S. Lam disclosed the novel and unexpectedly powerful technique of using insoluble random combinatorial libraries.
  • Lam synthesized random combinatorial libraries on solid phase supports so that each support had a test compound of uniform molecular structure, and screened the libraries without prior removal of the test compounds from the support by solid phase binding protocols (Lam, K. S. et al., 1991, Nature 354:82-84).
  • a library of candidate molecules may be a synthetic combinatorial library (e.g., a combinatorial chemical library), a cellular extract, a bodily fluid (e.g., urine, blood, tears, sweat, or saliva), or other mixture of synthetic or natural products (e.g., a library of small molecules or a fermentation mixture).
  • a synthetic combinatorial library e.g., a combinatorial chemical library
  • a cellular extract e.g., a cellular extract
  • a bodily fluid e.g., urine, blood, tears, sweat, or saliva
  • other mixture of synthetic or natural products e.g., a library of small molecules or a fermentation mixture.
  • a library of molecules may include, for example, amino acids, oligopeptides, polypeptides, proteins, or fragments of peptides or proteins; nucleic acids (e.g., antisense; DNA; RNA; or peptide nucleic acids, PNA); aptamers; or carbohydrates or polysaccharides.
  • Each member of the library can be singular or can be a part of a mixture (e.g., a compressed library).
  • the library may contain purified compounds or can be "dirty" (i.e., containing a significant quantity of impurities).
  • Diversity files contain a large number of compounds (e.g., 1000 or more small molecules) representative of many classes of compounds that could potentially result in nonspecific detection in an assay. Diversity files are commercially available or can also be assembled from individual compounds commercially available from the vendors listed above.
  • mTOR activity is linked to the status of a stem cell. Therefore, we provide methods of determining the status of a stem cell, i.e., whether it is differentiating or self-renewing, as well as methods of identifying differentiating stem cells and self-renewing stem cells by detecting the activity of mTOR in the stem cell.
  • a method of detecting a differentiating stem cell includes a step of detecting a high level of mTOR activity.
  • a method of detecting a self-renewing stem cell includes a step of detecting a low level of mTOR activity. Assays for mTOR activity are described elsewhere in this document.
  • polynucleotide probes or antibodies as described here may be used for the detection of the likely pathway of pluripotent cells such as primordial germ cells (PGCs), stem cells such as embryonic stem (ES) and embryonic germ (EG) cells in cell populations, i.e, whether differentiating or self-renewing.
  • PGCs primordial germ cells
  • ES embryonic stem
  • EG embryonic germ
  • a "cell population” is any collection of cells which may contain one or more PGCs, ES or EG cells.
  • the collection of cells does not consist solely of PGCs, but comprises at least one other cell type.
  • Cell populations comprise embryos and embryo tissue, but also adult tissues and tissues grown in culture and cell preparations derived from any of the foregoing.
  • Polynucleotides as described here may be used for detection of mTOR transcripts in PGCs or other pluripotent cells, such as ES or EG cells, by nucleic acid hybridisation techniques. Such techniques include PCR, in which primers are hybridised to mTOR transcripts and used to amplify the transcripts, to provide a detectable signal; and hybridisation of labelled probes, in which probes specific for an unique sequence in the mTOR transcript are used to detect the transcript in the target cells. Where mTOR transcript level is high, this may indicate that the stem cell is self-renewing; where the transcript level is low, this may indicate that the stem cell is differentiating.
  • probes may be labelled with radioactive, radioopaque, fluorescent or other labels, as is known in the art.
  • Antibodies may be used for the same purpose by detecting protein levels of mTOR. Particularly indicated are immunostaining and FACS techniques. Suitable fluorophores are known in the art, and include chemical fluorophores and fluorescent polypeptides, such as GFP and mutants thereof (see WO 97/28261). Chemical fluorophores may be attached to immunoglobulin molecules by incorporating binding sites therefor into the immunoglobulin molecule during the synthesis thereof.
  • the fluorophore is a fluorescent protein, which is advantageously GFP or a mutant thereof.
  • GFP and its mutants may be synthesised together with the immunoglobulin or target molecule by expression therewith as a fusion polypeptide, according to methods well known in the art.
  • a transcription unit may be constructed as an in-frame fusion of the desired GFP and the immunoglobulin or target, and inserted into a vector as described above, using conventional PCR cloning and ligation techniques.
  • Antibodies may be labelled with any label capable of generating a signal.
  • the signal may be any detectable signal, such as the induction of the expression of a detectable gene product.
  • detectable gene products include bioluminescent polypeptides, such as luciferase and GFP, polypeptides detectable by specific assays, such as ⁇ -galactosidase and CAT, and polypeptides which modulate the growth characteristics of the host cell, such as enzymes required for metabolism such as HIS3, or antibiotic resistance genes such as G418.
  • the signal is detectable at the cell surface.
  • the signal may be a luminescent or fluorescent signal, which is detectable from outside the cell and allows cell sorting by FACS or other optical sorting techniques.
  • Immunosensors are biochemical detectors comprising an antigen or antibody species coupled to a signal transducer which detects the binding of the complementary species (Rabbany et ah, 1994 Crit Rev Biomed Eng 22:307-346; Morgan et ah, 1996 Clin Chem 42:193-209). Examples of such complementary species include the antigen Zif 268 and the anti-Zif 268 antibody. Immunosensors produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood (Robinson 1991 Biosens Bioelectron 6:183-191). The sensitivity of immunosensors makes them ideal for situations requiring speed and accuracy (Rabbany et ah, 1994 Crit Rev Biomed Eng 22:307-346).
  • Detection techniques employed by immunosensors include electrochemical, piezoelectric or optical detection of the immunointeraction (Ghindilis et al, 1998 Biosens Bioelectron 1:113-131).
  • An indirect immunosensor uses a separate labelled species that is detected after binding by, for example, fluorescence or luminescence (Morgan et al., 1996 Clin Chem 42:193-209).
  • Direct immunosensors detect the binding by a change in potential difference, current, resistance, mass, heat or optical properties (Morgan et al, 1996 Clin Chem 42:193-209). Indirect immunosensors may encounter fewer problems due to non-specific binding (Attridge et al., 1991 Biosens Bioelecton 6:201-214; Morgan et al, 1996 Clin Chem 42:193-209).
  • mTOR polypeptide in a cell, for example a stem cell.
  • a nucleotide sequence encoding mTOR or a homologue, variant, or derivative thereof is inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding mTOR. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Any suitable host cell may be employed.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors e.g., insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • control elements are those non-translated regions of the vector (i.e., enhancers, promoters, and 5' and 3' untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (GIBCO/BRL), and the like, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (GIBCO/BRL), and the like, may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding mTOR, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • a number of expression vectors may be selected depending upon the use intended for mTOR. For example, when large quantities of mTOR are needed for the induction of antibodies or for over-expression in a target cell, vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding mTOR may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • the expression of sequences encoding mTOR may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.).
  • An insect system may also be used to express mTOR.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding mTOR may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of mTOR will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which mTOR may be expressed.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding mTOR may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing mTOR in infected host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • the mTOR proteins are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
  • HEK293 human embryonic kidney 293
  • adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the mTOR cDNA prior to insertion into a pCDN or pCDNA3 vector.
  • the cells are transfected with mTOR cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
  • HEK293 or CHO cells transfected with the vector alone serve as negative controls.
  • To isolate cell lines stably expressing mTOR about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resistant clones analyzed.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding mTOR. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding mTOR and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used, such as those described in the literature. (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125- 162.)
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post- translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding, and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • cell lines capable of stably expressing mTOR can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells maybe proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase genes (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in tk ' or apr " cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. MoI. Biol. 150:1-14); and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding mTOR is inserted within a marker gene sequence, transformed cells containing sequences encoding mTOR can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding mTOR under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells which contain the nucleic acid sequence encoding mTOR and express mTOR may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
  • polynucleotide sequences encoding mTOR can be detected by DNA--DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding mTOR.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding mTOR to detect transformants containing DNA or RNA encoding mTOR.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding mTOR include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding mTOR, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding mTOR may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be located in the cell membrane, secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode mTOR may be designed to contain signal sequences which direct secretion of mTOR through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding mTOR to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif)
  • One such expression vector provides for expression of a fusion protein containing mTOR and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMIAC; described in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase cleavage site provides a means for purifying mTOR from the fusion protein.
  • IMIAC immobilized metal ion affinity chromatography
  • Fragments of mTOR may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 43 IA peptide synthesizer (Perkin Elmer). Various fragments of mTOR may be synthesized separately and then combined to produce the full length molecule.
  • Vectors and polynucleotides or nucleic acids comprising or encoding mTOR nucleic acids, fragments, homologues, variants or derivatives thereof may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the polypeptides encoded by the polynucleotides.
  • the polypeptides may be produced using prokaryotic cells as host cells, it is preferred to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells.
  • Vectors/polynucleotides may be introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation.
  • retroviruses such as retroviruses, herpes simplex viruses and adenoviruses
  • a cell capable of expressing a mTOR polypeptide described here can be cultured and used to provide the mTOR polypeptide, which can then be purified.
  • the cell may be used in therapy for the same purposes as the mTOR polypeptide.
  • cells may be provided from a patient (e.g. via a biopsy), transfected with a nucleic acid molecule or vector and, if desired, cultured in vitro, prior to being returned to the patient (e.g. by injection).
  • the cells can then produce the mTOR polypeptide in vivo.
  • the cells comprise a regulatable promoter enabling transcription to be controlled via administration of one or more regulator molecules. If desired, the promoter may be tissue specific.
  • Expression is not however essential since the cells may be provided simply for maintaining a given nucleic acid sequence, for replicating the sequence, for manipulating it, etc.
  • Such cells may be provided in any appropriate form. For example, they may be provided in isolated form, in culture, in stored form, etc. Storage may, for example, involve cryopreservation, buffering, sterile conditions, etc.
  • Such cells may be provided by gene cloning techniques, by stem cell technology or by any other means. They may be part of a tissue or an organ, which may itself be provided in any of the forms discussed above. The cell, tissue or organ may be stored and used later for implantation, if desired. Techniques for providing tissues or organs, include stem cell technology, the provision of cells tissues or organs from transgenic animals, retroviral and non-retroviral techniques for introducing nucleic acids, etc.
  • cells may be provided together with other material to aid the structure or function or of an implant.
  • scaffolds may be provided to hold cells in position, to provide mechanical strength, etc.
  • These may be in the form of matrixes of biodegradable or non-biodegradable material.
  • WO95/01810 describes various materials that can be used for this purpose.
  • transgenic animals capable of expressing natural or recombinant mTOR, or a homologue, variant or derivative, at elevated or reduced levels compared to the normal expression level. Included are transgenic animals ("mTOR knockouf's) which do not express functional mTOR, as the case may be.
  • the mTOR knockouts may arise as a result of functional disruption of the mTOR gene or any portion of that gene, including one or more loss of function mutations, including a deletion or replacement, of the mTOR gene.
  • the mutations include single point mutations, and may target coding or non-coding regions of mTOR.
  • such a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent. Most preferably the transgenic animal is a mouse or a rat. Such transgenic animals may be used in screening procedures to identify agonists and/or antagonists of mTOR, as well as to test for their efficacy as treatments for diseases in vivo.
  • mice which are null for mTOR may be used for various purposes.
  • transgenic animals that have been engineered to be deficient in the production of mTOR may be used in assays to identify agonists and/or antagonists of mTOR.
  • One assay is designed to evaluate a potential drug (aa candidate ligand or compound) to determine if it produces a physiological response in the absence mTOR. This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response.
  • Tissues derived from the mTOR knockout animals may be used in binding assays to determine whether the potential drug (a candidate ligand or compound) binds to mTOR.
  • assays can be conducted by obtaining a first mTOR preparation from the transgenic animal engineered to be deficient in mTOR production and a second mTOR preparation from a source known to bind any identified ligands or compounds.
  • the first and second preparations will be similar in all respects except for the source from which they are obtained. For example, if brain tissue from a transgenic animal (such as described above and below) is used in an assay, comparable brain tissue from a normal (wild type) animal is used as the source of the second preparation.
  • Each of the preparations is incubated with a ligand known to bind to mTOR, both alone and in the presence of the candidate ligand or compound.
  • the candidate ligand or compound will be examined at several different concentrations. The extent to which binding by the known ligand is displaced by the test compound is determined for both the first and second preparations.
  • Tissues derived from transgenic animals may be used in assays directly or the tissues may be processed to isolate mTOR proteins, which are themselves used in the assays.
  • a preferred transgenic animal is the mouse.
  • the ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described in further detail above).
  • antagonists of mTOR may be identified by administering candidate compounds, etc, to wild type animals expressing functional mTOR, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of mTOR function.
  • Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
  • the transgenic non-human animals described here are produced by introducing transgenes into the germline of the non-human animal.
  • Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
  • the specific line(s) of any animal used to produce transgenic animals are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
  • the haplotype is a significant factor.
  • the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • the mTOR transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
  • the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is also included.
  • the progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.
  • the litters of transgenically altered mammals can be assayed after birth for the incorporation of the construct into the genome of the offspring.
  • this assay is accomplished by hybridizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny.
  • Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity.
  • a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
  • the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
  • the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
  • a euploid zygote is preferred.
  • the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
  • physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters.
  • the physical effects of addition must not be so great as to physically destroy the viability of the zygote.
  • the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
  • the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. There will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
  • exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
  • Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
  • Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
  • DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
  • Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the transgenic animals produced in accordance the methods described here will include exogenous genetic material.
  • the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a mTOR protein. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
  • control elements promoters or enhancers
  • specific control elements may be deleted from the endogenous mTOR locus so that expression is restricted to only certain tissues.
  • transgenes which only contain one, some, or more, of the control elements. Transgenic animals made this way for mTOR and having properties of ectopic expression, temporally or spatially, or both, will be useful for investigation of mTOR gene function.
  • Retroviral infection can also be used to introduce transgene into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
  • the founder may contain various retroviral insertions of the trans gene at different positions in the genome which generally will segregate in the offspring, hi addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
  • Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
  • transgenic animals where the transgenic animal is characterized by having an altered mTOR gene, preferably as described above, as models for mTOR function, as the case may be.
  • Alterations to the gene include 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 transgenic animals may be either homozygous or heterozygous for the alteration.
  • the animals and cells derived therefrom are useful for screening biologically active agents that may modulate mTOR function.
  • the screening methods are of particular use for determining the specificity and action of potential therapies for mTOR associated diseases, as described above.
  • the animals are useful as a model to investigate the role of mTOR proteins in the body.
  • Another aspect pertains to a transgenic animal having a functionally disrupted endogenous mTOR gene but which also carries in its genome, and expresses, a transgene encoding a heterologous mTOR protein (i.e., a mTOR gene from another species).
  • a transgene encoding a heterologous mTOR protein (i.e., a mTOR gene from another species).
  • the animal is a mouse and the heterologous mTOR is a human mTOR.
  • An animal, or cell lines derived from such an animal, which has been reconstituted with human mTOR, can be used to identify agents that inhibit human mTOR in vivo and in vitro.
  • a stimulus that induces signalling through human mTOR can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured.
  • An agent that inhibits human mTOR in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.
  • mTOR knock-out or a "mTOR null”
  • mTOR null a mTOR deficient transgenic non-human animal
  • Such an animal is one which expresses lowered or no mTOR activity, preferably as a result of an endogenous mTOR genomic sequence being disrupted or deleted.
  • the endogenous mTOR genomic sequence may be replaced by a null allele, which may comprise non-functional portions of the wild-type mTOR sequence.
  • the endogenous mTOR genomic sequence may be replaced by an allele of mTOR comprising a disrupting sequence which may comprise heterologous sequences, for example, reporter sequences and/or selectable markers.
  • the endogenous mTOR genomic sequence in a mTOR knock-out mouse is replaced by an allele of mTOR in which one or more, preferably all, of the coding sequences is replaced by such a disrupting sequence, preferably a lacZ sequence and a neomycin resistance sequence.
  • the genomic mTOR sequence which is functionally disrupted comprises a mouse mTOR genomic sequence.
  • such an animal expresses no mTOR activity. More preferably, the animal expresses no activity of the mTOR protein shown in the sequence listings.
  • mTOR knock-outs may be generated by various means known in the art, as described in further detail below.
  • the nucleic acid construct comprises: a) a non-homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first mTOR gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second mTOR gene sequence, the second mTOR gene sequence having a location downstream of the first mTOR gene sequence in a naturally occurring endogenous mTOR gene.
  • the first and second homology regions are of sufficient length for homologous recombination between the nucleic acid construct and an endogenous mTOR gene in a host cell when the nucleic acid molecule is introduced into the host cell.
  • the non-homologous replacement portion comprises an expression reporter, preferably including lacZ and a positive selection expression cassette, preferably including a neomycin phosphotransferase gene operatively linked to a regulatory element(s).
  • Another aspect pertains to recombinant vectors into which the nucleic acid construct described above has been incorporated.
  • Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous mTOR gene of the host cell, resulting in functional disruption of the endogenous mTOR gene.
  • the host cell can be a mammalian cell that normally expresses mTOR from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell.
  • an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous mTOR gene produces a transgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the mTOR gene disruption in their genome. Animals that carry the mTOR gene disruption in their germline can then be selected and bred to produce animals having the mTOR gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the mTOR gene disruption.
  • polypeptides disclosed here may be used therapeutically for treatment of various diseases, including cancer, in the form of peptides comprising any portion of their sequence.
  • mTOR peptides are used therapeutically, it is preferred to use peptides that do not consist solely of naturally-occurring amino acids but which have been modified, for example to reduce immunogenicity, to increase circulatory half-life in the body of the patient, to enhance bio-availability and/or to enhance efficacy and/or specificity.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • bi-functional crosslinkers such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-(2 pyridyldithio) propionamido] hexanoate, and sulfosuccinimidyl 6-[3-(2 pyridyldithio) propionamido]hexanoate (see US Patent 5,580,853).
  • Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide.
  • Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.
  • the active conformation of the peptide maybe stabilised by a covalent modification, such as cyclization or by incorporation of ⁇ -lactam or other types of bridges.
  • side chains can be cyclized to the backbone so as create a L- ⁇ - lactam moiety on each side of the interaction site. See, generally, Hruby et al., "Applications of Synthetic Peptides,” in Synthetic Peptides: A User's Guide: 259-345 (W. H. Freeman & Co. 1992).
  • Cyclization also can be achieved, for example, by formation of cystine bridges, coupling of amino and carboxy terminal groups of respective terminal amino acids, or coupling of the amino group of a Lys residue or a related homologue with a carboxy group of Asp, GIu or a related homologue. Coupling of the .alpha-amino group of a polypeptide with the epsilon-amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken. See Wood and Wetzel, 1992, Int'l J. Peptide Protein Res. 39, 533-39.
  • Another approach described in US 5,891,418 is to include a metal-ion complexing backbone in the peptide structure.
  • the preferred metal-peptide backbone is based on the requisite number of particular co-ordinating groups required by the co ⁇ ordination sphere of a given complexing metal ion.
  • most of the metal ions that may prove useful have a co-ordination number of four to six.
  • the nature of the co ⁇ ordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulfur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities.
  • the peptide chain or individual amino acids can be chemically altered to include a co-ordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino.
  • the peptide construct can be either linear or cyclic, however a linear construct is typically preferred.
  • One example of a small linear peptide is Gly-Gly- Gly-Gly which has four nitrogen atoms (an N 4 complexation system) in the back bone that can complex to a metal ion with a co-ordination number of four.
  • a further technique for improving the properties of therapeutic peptides is to use non-peptide peptidomimetics.
  • a wide variety of useful techniques may be used to elucidating the precise structure of a peptide. These techniques include amino acid sequencing, x-ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, computer-assisted molecular modelling, peptide mapping, and combinations thereof. Structural analysis of a peptide generally provides a large body of data which comprise the amino acid sequence of the peptide as well as the three- dimensional positioning of its atomic components. From this information, non-peptide peptidomimetics may be designed that have the required chemical functionalities for therapeutic activity but are more stable, for example less susceptible to biological degradation. An example of this approach is provided in US 5,811,512.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of a relevant molecule to the mTOR, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • mTOR act by binding a ligand.
  • soluble forms of mTOR polypeptides still capable of binding the ligand in competition with endogenous mTOR may be administered.
  • Typical embodiments of such competitors comprise fragments of the mTOR polypeptide.
  • expression of the gene encoding endogenous mTOR can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FIa. (1988).
  • oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 3:173; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • a polynucleotide as described in this document may be engineered for expression in a replication defective retroviral vector.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a mTOR polypeptide such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). FORMULATION AND ADMINISTRATION
  • Peptides and polypeptides such as the mTOR peptides and polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art.
  • pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions.
  • Polypeptides and other compounds may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localize, in the form of salves, pastes, gels and the like.
  • the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • compositions comprising administering a therapeutically effective amount of the polypeptide, polynucleotide, peptide, vector or antibody (such as a mTOR polypeptide, etc) and optionally a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition as described here may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • Another embodiment relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the mTOR polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from mTOR associated disease.
  • Yet another embodiment relates to a method of inducing immunological response in a mammal which comprises delivering a mTOR polypeptide via a vector directing expression of a mTOR polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further embodiment relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a mTOR polypeptide wherein the composition comprises a mTOR polypeptide or mTOR gene.
  • the vaccine formulation may further comprise a suitable carrier.
  • the mTOR polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • Vaccines may be prepared from one or more polypeptides or peptides as described here.
  • the preparation of vaccines which contain an immunogenic polypeptide(s) or peptide(s) as active ingredient(s), is known to one skilled in the art.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-( 1 ' -2 ' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (C
  • adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Cor ⁇ nebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • aluminum hydroxide aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide
  • adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
  • adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
  • the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al 2 O 3 basis).
  • the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
  • the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4 0 C, or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
  • the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer
  • Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
  • enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
  • the polypeptides described here may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl amine, 2-ethylamino ethanol, histidine and procaine.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl amine, 2-ethylamino ethanol, histidine and procaine.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • compositions as disclosed here may be administered by direct injection.
  • the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • each protein maybe administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the term "administered” includes delivery by viral or non-viral techniques.
  • Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
  • Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
  • administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
  • co-administered means that the site and time of administration of each of for example, the polypeptide and an additional entity such as adjuvant are such that the necessary modulation of the immune system is achieved.
  • the polypeptide and the adjuvant may be administered at the same moment in time and at the same site, there may be advantages in administering the polypeptide at a different time and to a different site from the adjuvant.
  • the polypeptide and adjuvant may even be delivered in the same delivery vehicle - and the polypeptide and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled.
  • the mTOR polypeptide, polynucleotide, peptide, nucleotide, antibody etc and optionally an adjuvant may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
  • the vaccine composition and pharmaceutical compositions described here may be administered by a number of different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra-vaginal, urethral or ocular administration.
  • the vaccines and pharmaceutical compositions described here may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, may be 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
  • the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
  • molecules are used, it should be taken to include reference to atoms.
  • the molecules identified by the assays and methods described here may vary in nature, but in general will comprise small molecules, simple compounds, hormones, signalling molecules, nucleic acids, antisense nucleic acids such as antisense DNA and antisense RNA, polypeptides, transcription factors, etc.
  • candidate molecules may comprise any of the above. The possible nature of such molecules is described in further detail below, but in a highly preferred embodiment, the molecules comprise nucleic acids, genes, polypeptides or proteins.
  • RoSH and mouse El 4 ES cells are maintained on gelatinized culture plates as previously described 11 ' 15 .
  • RoSH induced to differentiate by plating 10 6 cells per 6 cm matri gel-coated tissue culture dish as previously described 16 .
  • Mouse E14 ES cells are induced to differentiate by LIF -withdrawal and plating on bacterial Petri dishes 17 .
  • the working concentration of rapamycin (Sigma, St. Louis, MO) and Gleevec (Novartis, Basel, Switerland) is 50 nM and 10 ⁇ M, respectively.
  • 2x10 5 cells per well are plated on a matrigel-coated 6-well tissue culture dish and cultured for six hours. Triplicate wells are then treated with or without 5OnM rapamycin. Twenty-four hours later, branching is quantitated by determining the average number of branch points with > 3 branches in three random low power fields (1Ox magnification).
  • 2x10 8 RoSH or ES cells are pre-labeled with a cell-permeable fluorescent dye CFDA (Molecular Probe, Eugene, Or) by incubating the cells in 2ml of lO ⁇ M dye in saline at 37°C for 15 minutes, cultured in non-differentiating conditions for 24 hours before replating 1x10 5 cells per 3 cm tissue culture plate under non-differentiating or differentiating conditions.
  • CFDA cell-permeable fluorescent dye
  • n (Ig F/F n )/lg 2 where F is initial average cellular fluorescence and F n is the average cellular fluorescence after 24 hours.
  • Total RNA are prepared using a modified method of Chomczynski and Sacchi 18 as previously described 19 .
  • Polysome-associated RNA is prepared using a modified protocol 20 . Briefly, exponentially growing cells are harvested and resuspend in 10 8 cells per ml buffer (10 mM Tris— Cl, pH 7.6, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol (DTT), 10% glycerol, 1 ⁇ g/ml leupeptin, 1 mg/ml pepstatin A, 100 ⁇ g/ml phenylmethylsulfonyl fluoride). The cells are homogenized in a prechilled Dounce homogenizer sitting in an ice-water bath.
  • the cell lysates are centrifuged at 9000 rpm for 10 min.
  • the supernatant is layered over a cushion of 30% (w/v) sucrose in lysis buffer and centrifuge at 130,000g at 2°C for 2.5 h.
  • the polysome pellet is resuspended in acid-guanidinium thiocyanate buffer and RNA is purified over a CsCl gradient.
  • Primer set for amplification of the following genes and the expected amplified cDNA fragment size are: a) FKBP 12 5'- CAC GGG GAT GCT TGA AGA TGG-3' and 5'- GTC TAT ACA AAG GGT GGT GGG-3' and 371bp; b) Brachyury 5 '-G A AGCC AAGG AC AG AG AG AC-3 1 and 5'- GCAACAAGGGAGGACATTAG-3', 194bp; c) AFP 5'-TCC ACG TTA GAT TCC TCC CAG-3 1 and 5'-TTG CAG CAT GCC AGA ACG ACC-3 1 and 471bp; d) nestin 5'-TGT GGG ATG ATG GCT TGA GT-3' and 5'-ACA GAA GAA AGG GGG CGT TG-3 1 and 655bp; e) nurrl 5'-GCG GAG TTG AAT GAA TGA A
  • Each membrane is probed using three primary antibodies and the membrane is stripped with stripping buffer consisting of 2% SDS, 100 mM beta- mercaptoethanol and 50 mM Tris (pH 6.8) between each primary antibody.
  • the primary antibodies used are rabbit anti-Tie2, phospho S6K1, Oct 4, PDGFA, PDGF ⁇ polyclonal antibody, goat anti-(4E-BPl) polyclonal antibody from Santa Cruz Biotechnology, CA, and mAb nestin and TSPl from Chemicon, CA.
  • Antibodies from Santa Cruz Biotechnology are used at 1 :500 dilution and the rest are used at 1 :200 dilution.
  • the secondary antibodies are HRP-conjugated goat anti-rabbit rabbit anti-goat and rabbit anti- mouse.
  • tuberin or tsc2 is a gene product of tuberous sclerosis complex 2 (TSC2) and its total protein level is not modulated during differentiation of RoSH cells (unpublished data).
  • TSC2 tuberous sclerosis complex 2
  • ⁇ -tubulin protein level is increased during endothelial differentiation of RoSH cells.
  • Immunofluorescence staining is performed using standard procedures. The cells on culture slide chambers are fixed in 4% paraformaldehyde. After blocking with 5% goat serum and 0.05% Tween-20 in PBS for 1 hour at room temperature, the cells are probed with 1 :30 dilution of anti-SSEA-1 monoclonal antibody (MC-480, Developmental Studies Hybridoma Bank, Iowa City, IA) or 1:30 mouse serum, followed by incubation with biotinylated goat anti-mouse antibody, and then avidin-FITC. The cells are counterstained with propidium iodide before analysis by confocal microscopy.
  • mES cells are treated with rapamycin, a specific inhibitor of mTOR in the presence of LIF.
  • Rapamycin also caused progressive dephosphorylation of 4E-BP1 and S6K1 (FigurelC) with decreased expression of markers associated with pluripotency e.g. Oct 4 mRNA ( Figure IB) and protein (Figure 1C), SSEA-I ( Figure ID)., and increased expression of markers associated with differentiation e.g. Brachyury, AFP, nestin and nurrl ( Figure IB). Since murine ES cells are treated with rapamycin in the presence of LIF, phosphorylation of Stat3 is not abolished (Figure 1C).
  • siRNAs are used to maintain mTOR mRNA at 40-80% normal level over a 96 hour period, there is a progressive decline in luciferase activity from an OCT4-luciferase reporter gene to 20-40% of normal level. Endogenous Oct4 mRNA level declines while that of brachyury, AFP and nestin increases, indicating that the siRNA-treated ES cells are differentiating.
  • RoSH cells a previously described karyotypically normal mouse embryonic cell line 11 .
  • RoSH cells can be induced to differentiate into endothelial cells and acquire typical endothelial surface markers and morphology when plated on matrigel.
  • RoSH cells are therefore treated with imatinib mesylate (Gleevec), a selective inhibitor of receptor tyrosine kinase e.g. c-Kit and PDGFR 12 .
  • RoSH cells but not mES cells express c-Kit and PDGFR (unpublished data, Que and Lim), and are therefore sensitive to Gleevec.
  • Gleevec treatment there is decreased cell cycle activity but phosphorylation of S6K and 4E-BP is not abolished and expression of Tie-2 protein endothelial specific marker is not induced (Figure 3A).
  • PA Phosphatidic acid
  • PA analogues e.g. fluoromethylene phosphonate analogues of phosphatidic acid
  • mouse El 4 ES cells which are normally maintained in DMEM + LIF + 20% fetal calf serum (FCS) on gelatinized coated plates are cultured in 5% FCS with different concentrations of PA. Their proliferation rate (ratio of cell number at day 4 to cell number at day 1) is measured over a period of twelve days
  • mTOR upregulates cap-mediated translation of a class of mRNA known as TOP mRNAs (or mRNAs with a tract of pyrimidines at the 5' end) and a few other classes of mRNAs e.g. c-myc (I. B. Rosenwald, Oncogene 23, 3230-47 (Apr 19, 2004; C. G. Proud, Curr Top Microbiol Immunol 279, 215-44 (2004)).
  • TOP mRNAs or mRNAs with a tract of pyrimidines at the 5' end
  • c-myc I. B. Rosenwald, Oncogene 23, 3230-47 (Apr 19, 2004; C. G. Proud, Curr Top Microbiol Immunol 279, 215-44 (2004).
  • a hybrid gene encoding for a mRNA with a 5'UTR derived from a TOP mRNA e.g. L5 ribosomal protein mRNA and coding region from a reporter gene e.g. GFP or luciferase is transfected into mammalian cells.
  • the cells are either serum starved or rapamycin-treated to shut off translation of the reporter.
  • Cells are exposed to a candidate molecule or a member of a library. Addition of an mTOR activating molecule will upregulate translation of the reporter.
  • p27Kipl Inhibition of cap-mediated translation by mTOR induces translation of p27Kipl (H. W. Lee, K. O. Nam, S. J. Park, B. S. Kwon, Eur J Immunol 33, 2133-41 (Aug, 2003); K. A. Martin et al., Am J Physiol Cell Physiol 286, C507-17 (Mar, 2004)).
  • p27Kipl is translationally upregulated in a cap-independent manner when cap-mediated translation is inhibited (A. Vidal, S. S. Millard, J. P. Miller, A. Koff, J Biol Chem 277, 16433-40 (May 10, 2002); H. Jiang, J. Coleman, R. Miskimins, W. K.
  • a hybrid gene encoding a mRNA with a 5'UTR derived from mRNAs whose translation is upregulated when cap-mediated translation is inhibited e.g. p27Kipl mRNA and coding region from a reporter gene e.g. GFP or luciferase is transfected into mammalian cells .
  • the cells are either serum starved or rapamycin-treated to turn on translation of the reporter. Then serum will be added or rapamycin removed to activate mTOR and turn off translation of reporter.
  • Robertson, E. J. in Teratocarcinomas and embryonic stem cells a practical approach, (ed. Robertson, E. J.) 71-112 (IRL Press Limited, Oxford, 1987). 18. Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156-159 (1987).
  • bin AH A. et al. Expression of major HDL-associated antioxidant PON-I is gender dependent and regulated during inflammation. Free Radic Biol Med 34, 824-9. (2003).
  • rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin MoI. Cell. Biol. 22 (21), 7428-7438 (2002)
  • Tuberous sclerosis complex- 1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling Proc. Natl. Acad. Sci. U.S.A. 99 (21), 13571-13576 (2002)
  • Inoki,K., Li,Y., Zhu,T., Wu,J. and Guan.K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling Nat. Cell Biol. 4 (9), 648-657 (2002)
  • FKBP- rapamycin associated protein maps to chromosomal band Ip36.2 Hum. Genet. 99 (4), 547-549 (1997)
  • RAFTl a mammalian protein that binds to FKBP 12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78: 35-43, 1994.

Abstract

L'invention concerne un procédé consistant à moduler l'activité de mTOR dans une cellule souche, et de préférence à accroître l'activité de mTOR afin de permettre l'auto-régénération de la cellule souche, ou à réduire l'activité de mTOR de manière à permettre la différenciation de la cellule souche. Dans le premier cas, la cellule souche peut être exposée à un agoniste de mTOR, de préférence à l'acide phosphatidique, et dans le second cas la cellule souche peut être exposée à un antagoniste de mTOR, de préférence du 1-butanol, ou de la rapamycine ou un dérivé de celle-ci.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009017269A1 (fr) * 2007-08-01 2009-02-05 Korea Research Institute Of Bioscience And Biotechnology Procédé de différenciation de cellules souches embryonnaires humaines en une lignée ostéoblastique
US8263403B2 (en) 2007-04-23 2012-09-11 Stowers Institute For Medical Research Methods and compositions for stem cell self-renewal
WO2013106547A1 (fr) 2012-01-10 2013-07-18 President And Fellows Of Harvard College Composés promoteurs de réplication des cellules bêta et leurs procédés d'utilisation
US9423402B2 (en) 2011-11-30 2016-08-23 Singapore Health Services Pte. Ltd. GM1 ganglioside to annexin V microparticle polypeptide ratio for biological monitoring
EP3406248A4 (fr) * 2016-01-20 2019-09-04 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Composition de composé à petite molécule qui induit efficacement la différenciation de cellules souches multipotentes humaines en cellules myocardiques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106244522A (zh) * 2016-08-12 2016-12-21 浙江译美生物科技有限公司 一种干细胞培养体系及其培养方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002080975A1 (fr) * 2001-04-06 2002-10-17 Wyeth Combinaisons antineoplasiques telles que la rapamycine associee a la gemcitabine ou au fluorouracile
WO2004089369A2 (fr) * 2003-04-11 2004-10-21 Cambridge University Technical Services Limited Methodes et moyens permettant de traiter des troubles de conformation des proteines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002080975A1 (fr) * 2001-04-06 2002-10-17 Wyeth Combinaisons antineoplasiques telles que la rapamycine associee a la gemcitabine ou au fluorouracile
WO2004089369A2 (fr) * 2003-04-11 2004-10-21 Cambridge University Technical Services Limited Methodes et moyens permettant de traiter des troubles de conformation des proteines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DAS A V ET AL: "C - Kit receptor plays an important role in the maintenance of the adult neural stem cells: interaction with Notch signaling." SOCIETY FOR NEUROSCIENCE ABSTRACT VIEWER AND ITINERARY PLANNER, vol. 2003, 2003, pages Abstract No. 452.12 URL-http://sf, XP009060950 & 33RD ANNUAL MEETING OF THE SOCIETY OF NEUROSCIENCE; NEW ORLEANS, LA, USA; NOVEMBER 08-12, 2003 *
FENG L X ET AL: "Stem cell factor/c-kit up-regulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia." THE JOURNAL OF BIOLOGICAL CHEMISTRY. 18 AUG 2000, vol. 275, no. 33, 18 August 2000 (2000-08-18), pages 25572-25576, XP002365724 ISSN: 0021-9258 *
MITA MONICA M ET AL: "The molecular target of rapamycin (mTOR) as a therapeutic target against cancer." CANCER BIOLOGY & THERAPY. 2003 JUL-AUG, vol. 2, no. 4 Suppl 1, July 2003 (2003-07), pages S169-S177, XP009060677 ISSN: 1538-4047 cited in the application *
MURAKAMI MIREI ET AL: "mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells." MOLECULAR AND CELLULAR BIOLOGY. AUG 2004, vol. 24, no. 15, August 2004 (2004-08), pages 6710-6718, XP002365723 ISSN: 0270-7306 *
RAJAN PRITHI ET AL: "BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells." THE JOURNAL OF CELL BIOLOGY. 9 JUN 2003, vol. 161, no. 5, 9 June 2003 (2003-06-09), pages 911-921, XP002365725 ISSN: 0021-9525 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8263403B2 (en) 2007-04-23 2012-09-11 Stowers Institute For Medical Research Methods and compositions for stem cell self-renewal
US8476071B2 (en) 2007-04-23 2013-07-02 Stowers Institute For Medical Research Methods and compositions for stem cell self-renewal
EP2798956A1 (fr) 2007-04-23 2014-11-05 Stowers Institute for Medical Research Procedes et compositions pour auto-renouvellement de cellules souches hematopoeitiques
US9771559B2 (en) 2007-04-23 2017-09-26 Stowers Institute For Medical Research Methods and compositions for stem cell self-renewal
US10787643B2 (en) 2007-04-23 2020-09-29 Stowers Institute For Medical Research Methods, kits, and compositions for stem cell self-renewal
WO2009017269A1 (fr) * 2007-08-01 2009-02-05 Korea Research Institute Of Bioscience And Biotechnology Procédé de différenciation de cellules souches embryonnaires humaines en une lignée ostéoblastique
US8093050B2 (en) 2007-08-01 2012-01-10 Korea Research Institute Of Bioscience And Biotechnology mTOR inhibtors and mTOR signaling pathway inhibitors induce differentiation of human embryonic stem cells into the osteoblastic lineage
US9423402B2 (en) 2011-11-30 2016-08-23 Singapore Health Services Pte. Ltd. GM1 ganglioside to annexin V microparticle polypeptide ratio for biological monitoring
US9977032B2 (en) 2011-11-30 2018-05-22 Agency For Science, Technology And Research Microparticle fractionation
US10481166B2 (en) 2011-11-30 2019-11-19 Singapore Health Services Pte. Ltd. Microparticle fractionation
WO2013106547A1 (fr) 2012-01-10 2013-07-18 President And Fellows Of Harvard College Composés promoteurs de réplication des cellules bêta et leurs procédés d'utilisation
EP3406248A4 (fr) * 2016-01-20 2019-09-04 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Composition de composé à petite molécule qui induit efficacement la différenciation de cellules souches multipotentes humaines en cellules myocardiques

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