WO2006027545A2 - Method - Google Patents

Abstract

We provide a method comprising modulating the activity of mTOR in a stem cell, preferably increasing the activity of mTOR to enable a stem cell to self-renew, or decreasing it to enable a stem cell to differentiate. The stem cell may be exposed to an agonist of mTOR for the former, preferably phosphatidic acid, or an antagonist of mTOR for the latter, preferably 1-butanol or rapamycin or a derivative thereof.

Description

METHOD

FIELD

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.

BACKGROUND

Stem cells, unlike differentiated cells have the capacity to divide and either self- renew or differentiate into phenotypically and functionally different daughter cells ^M. However 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. Conversely, 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).

In murine ES cells, self-renewal is maintained largely though activation of STAT3 by leukemia inhibitory factor (LIF) and PB K signaling involving Pten. Inactivation of these pathways generally leads to differentiation⁸. 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. However, such 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). 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-

77).

SUMMARY

We now demonstrate that the regulation of self-renewal versus differentiation for a stem cell is mediated through mTOR. Specifically, reduction of mTOR activity of the stem cell leads to differentiation, while maintenance of, or an increase in, mTOR activity of the stem cell promotes self-renewal. Accordingly, it is possible to influence the choice of a stem cell between these two fates by modulating the activity of mTOR.

According to a 1^st aspect of the present invention, we provide a method comprising modulating the activity of mTOR in a stem cell.

In a preferred embodiment, activity of mTOR is maintained or increased to enable a stem cell to self-renew. Preferably, 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.

Alternatively, or in addition, the stem cell is inhibited from differentiating by maintaining the activity of mTOR.

Preferably, the stem cell is exposed to an agonist of mTOR to increase the activity of mTOR in the stem cell. Preferably, the agonist comprises phosphatidic acid.

In another embodiment, activity of mTOR is decreased to enable a stem cell to differentiate. Preferably, (a) 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. Preferably, the stem cell is exposed to an antagonist of mTOR to decrease the activity of mTOR in the stem cell. Preferably, the antagonist comprises 1-butanol or rapamycin or a derivative thereof.

Preferably, 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.

Preferably, 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.

There is provided, according to a 2^nd aspect of the present invention, use of 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.

We provide, according to a 3^rd aspect of the present invention, use of 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.

Preferably, the marker of self-renewal is selected from the group consisting of: Oct4 and SSEA-I, and the marker of differentiation is selected from the group consisting of: FIk-I, Tie-2, c-kit, Brachyury, AFP, nestin and nurrl. As a 4^th aspect of the present invention, there is provided a method of identifying an agent capable of enabling self-renewal or differentiation of a stem cell, the method comprising contacting mTOR with a candidate agent and determining whether the candidate agent binds to mTOR, and optionally determining whether the activity of mTOR is thereby modulated.

We provide, according to a 5^th aspect of the present invention, a method of identifying an agent capable of enabling self-renewal of a stem cell, the method 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. Preferably, the activity is kinase activity.

In a 7^th aspect of the present invention, there is provided use of mTOR in a method of enabling self-renewal or differentiation of a stem cell.

According to an 8^th aspect of the present invention, we provide use of 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.

We provide, according to a 9^th aspect of the invention, use of an agent capable of decreasing the activity of mTOR, preferably 1-butanol or rapamycin or a derivative thereof, or an agent identified by a method according to the 4^th or 6^th aspect of the invention in a method of enabling differentiation of a stem cell.

There is provided, in accordance with a 10^th aspect of the present invention, use of 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.

We provide, according to an 11^th aspect of the present invention, a method of influencing the choice between self-renewal and differentiation by a stem cell, the method comprising modulating the activity of mTOR in the stem cell.

We provide, according to a 12^th aspect of the invention, we provide a method of determining whether a stem cell is differentiating or self-renewing, the method 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.

According to 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.

There is provided, according to a 14^th aspect of the present invention, a self- renewing cell produced or treated by a method according to the 1^st aspect of the invention.

We provide, according to a 15^th aspect of the present invention, a differented or differentiating cell produced or treated by a method according to the 1^st aspect of the invention.

According to 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. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual : Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson "Immunocytochemistry: Theory and Practice", CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D. Pound (ed); "Immunochemical Protocols, vol 80", in the series: "Methods in Molecular Biology", Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3; and The Merck Manual of Diagnosis and Therapy (17th Edition, Beers, M. H., and Berkow, R, Eds, ISBN: 0911910107, John Wiley & Sons). Each of these general texts is herein incorporated by reference. Each of these general texts is herein incorporated by reference. BRIEF DESCRIPTION OF THE FIGURES

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.

Figure 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).

Figures 2A to D show the regulation of cell cycle in RoSH cells during endothelial differentiation.

Figure 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 μg 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^χ 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.

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. One representative gene from subset A, B, C, D, E, F and G analysed by RT-PCR. PCR is performed using 10 fold serial dilution ie Ix, 1Ox and 10Ox of cDNAs. FKBP-12 is used as an internal control. 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 μg 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.

Figure 5 shows a model of proliferation and self renewal in murine ES cells. A schematic representation of LIF/Stat3 and mTOR signaling in maintaining proliferation and self-renewal of 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. Phosphorylation of 4E-BP1 causes release of eIF-4E that facilitates cap-dependent translation. Based on our observations that both LIF withdrawal and rapamycin treatment in the presence of LIF signaling decreased mTOR activity, reduced cell cycle activity and induced differentiation, part of LIF/Stat3 signaling to self-renew was routed through mTOR signaling.

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^χ 10⁵ per well in a gelatinized six-well plate and cultured for 4 days. The cells are then trypsinized, counted and replated at 2^χ 10⁵ 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 2x10⁵ 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.

DETAILED DESCRIPTION

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.

Accordingly, we broadly provide for the manipulation of the choice between maintaining self-renewal and induction of differentiation of a stem cell, by the manipulation of mTOR activity of the stem cell. Generally, in order to enable self- renewal by a stem cell, the activity of mTOR is maintained or increased; similarly, to enable differentiation by the stem cell, the activity of mTOR is decreased. Alternatively, or in addition, the stem cell may be inhibited from differentiating by maintaining the activity of mTOR.

It will be evident that 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. That is to say, 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.

Similarly, increasing mTOR activity (or maintaining an increased level of mTOR activity) in a stem cell which has committed to self-renewal, or is self-renewing, will enable it to remain in a self-renewing state. Preferably, 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. In preferred embodiments, 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.

In particular, we provide for the use of 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.

USES OF STEM CELLS

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.

For example, 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.

Thus, 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.

It will be evident that the methods and compositions described here enable the production of stem cells, which may of course be made to differentiate using methods known in the art. Thus, any uses of differentiated cells will equally attach to those stem cells for which they are progenitors. It will further be evident that 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.

Thus, we specifically provide for the use of 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. Such 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. Thus, we specifically provide for the use of phosphatidic acid in the treatment or prevention of such diseases, and in the preparation of a pharmaceutical composition for this purpose.

We therefore describe the use of mTOR in a method of enabling self-renewal of a stem cell, use of mTOR in the treatment or prevention of such diseases, mTOR for use in a method of treatment or prevention of such diseases and use of mTOR for the preparation of a pharmaceutical composition for the treatment or prevention of such diseases. USES OF DIFFERENTIATED CELLS

Differentiated cells made according to the methods described here can be used for a variety of commercially important research, diagnostic, and therapeutic purposes.

For example, 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. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogeneous cell populations are particularly suited for use in drug screening and therapeutic applications.

These and other uses of differentiated cells are described in further detail below, and elsewhere in this document. It will be evident that the uses that may be made of 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

Thus, we specifically provide for the use of 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. Such 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. Thus, we specifically provide for the use of 1-butanol or rapamycin or a derivative thereof thereof in the treatment or prevention of such diseases, and in the preparation of a pharmaceutical composition for this purpose.

Drug Screening

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.

In some applications, 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.

Particular screening applications relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook "In vitro Methods in Pharmaceutical Research", Academic Press, 1997, and U.S. Pat. No. 5,030,015), as well as the general description of drug screens elsewhere in this document. 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. In some applications, 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. [³H]thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, 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.

Tissue Regeneration

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.

For example, 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.

In another example, 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. 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.

The efficacy of 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).

Cancer

mTOR polypeptides, nucleic acids, and fragments, homologues, variants and derivatives thereof, as well as agonists and/or antagonists are suitable for treating or preventing cancer.

The terms "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. More particular examples of such 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. Further examples are 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. Further examples 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. Breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, lung cancer, malignant melanoma, leukaemia, lympyhoma, ovarian cancer, cervical cancer and biliary tract carcinoma are also included. In preferred embodiments, mTOR polypeptide, nucleic acid, and fragments, homologues, variants and derivatives thereof are used to treat T cell lymphoma, melanoma or lung cancer.

The 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. For example, drugs such as such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, and antimetabolites such as methotrexate. The term "cytotoxic agent" as used herein 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.

Also, 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. Examples of 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. No. 4,675,187), Melphalan and other related nitrogen mustards, and endocrine therapies (such as diethylstilbestrol (DES), Tamoxifen, LHRH antagonizing drugs, progestins, anti- progestins etc). DETECTION AND TREATMENT OF POORLY DIFFERENTIATED CANCERS

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.

Preferably, such tumours or cancers are mammalian, more preferably human.

When a patient presents with a poorly differentiated tumour, it is difficult without further analysis to determine the origin or source of the tumour, thus complicating the choice of treatment. Thus, for example, 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.

As described above, 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. Optionally, genetic analysis (for example, by PCR) may be used to supplement this. Once the source of the tumour is determined, the treatment regime may be chosen by the physician, e.g., by administration of a suitable drug.

Examples of poorly differentiated 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. Alternatively or in addition, 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.

MTOR

Where the term 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.

Preferably, mTOR refers to a human sequence. Thus, particular homologues encompassed by this term include human homologues, for example, accession numbers NM_004958.2, NP_004949, Hs.509145. However, the term also covers alternative peptides homologous to mTOR, such as polypeptides derived from other species, including other mammalian species. For example, 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.

Preferably, mTOR includes fragments, homologues, variants and derivatives of such a nucleotide sequence. The terms "variant", "homologue", "derivative" or "fragment" as used here include 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. Unless the context admits otherwise, references to "mTOR" include references to such variants, homologies, derivatives and fragments of mTOR. These are described in more detail below.

Preferably, the resultant nucleotide sequence encodes a polypeptide having mTOR activity, preferably having at least the same activity of the human mTOR referred to above. Preferably, 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. With respect to sequence identity (i.e. similarity), 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. These terms also encompass allelic variations of the sequences.

The following description of mTOR, referred to as FRAP, is provided from the Online Mendelian Inheritance in Man website (http://www.ncbi.nlm.nih. gov/entrez/dispomim.cgi?id=601231')

FKBP12-rapamycin associated protein (FRAP) 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. The authors stated that the binding of FRAP to FKBP12-rapamycin correlated with the ability of these ligands to inhibit cell cycle progression. Rapamycin is an efficacious anticancer agent against solid tumors. In a hypoxic environment, the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. When nutrient concentrations change, particularly those of essential amino acids, the mammalian target of rapamycin (mTOR/FRAP) functions in regulatory pathways that control ribosome biogenesis and cell growth. In bacteria, 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.

Castedo et al. (2001) delineated the apoptotic pathway resulting from human immunodeficiency virus (HIV)-I envelope glycoprotein (Env)-induced syncytia formation in vitro and in vivo. Immunohistochemical analysis demonstrated the presence of phosphorylated serl5 of p53 (191170) as well as the preapoptotic marker tissue transglutaminase (TGM2; 190196) in syncytium in the apical light zone (T-cell area) of lymph nodes, as well as in peripheral blood mononuclear cells, from HIV-I -positive but not HIV-I -negative donors. The presence of these markers correlated with viral load (HIV-I RNA levels). 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. (2001) concluded that 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. (2001) identified phosphatidic acid as a critical component of mTOR signaling. In their study, 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. (2001) concluded that their study suggested a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.

Kim et al. (2002) and Hara et al. (2002) reported that MTOR binds with RAPTOR (607130), an evolutionarily conserved protein with at least 2 roles in the MTOR pathway. Kim et al. (2002) showed that RAPTOR has a positive role in nutrient-stimulated signaling to the downstream effector S 6Kl (601684), maintenance of cell size, and MTOR protein expression. The association of RAPTOR with MTOR also negatively regulates MTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the MTOR-RAPTOR association and inhibit MTOR kinase activity. Kim et al. (2002) proposed that RAPTOR is a component of the MTOR pathway that, through its association with MTOR, regulates cell size in response to nutrient levels.

Hara et al. (2002) showed that the binding of RAPTOR to MTOR is necessary for the MTOR-catalyzed phosphorylation of 4EBP 1 (602223) in vitro and that it strongly enhances the MTOR kinase activity toward p70-alpha (S6K1). Rapamycin or amino acid withdrawal increased, whereas insulin strongly inhibited, the recovery of 4EBP 1 and RAPTOR on 7-methyl-GTP sepharose. Partial inhibition of RAPTOR expression by RNA interference reduced MTOR-catalyzed 4EBP 1 phosphorylation in vitro. RNA interference of C. elegans Raptor yielded an array of phenotypes that closely resembled those produced by inactivation of CE-Tor. Thus, the authors concluded that 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. At 25.5 degrees C, 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. Furthermore, rapamycin protects against neurodegeneration in a fly model of HD, and 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.

Moore et al. (1996) assigned the FRAP gene to Ip36 by fluorescence in situ hybridization (FISH). Lench et al. (1997) mapped the FRAP gene to Ip36.2 by FISH following radiation-hybrid mapping to that general region. Chromosome Ip36.2 is the region most consistently deleted in neuroblastomas. Given the role of PIK-related kinase proteins in DNA repair, recombination, and cell cycle checkpoints, the authors suggested that the possible role of FRAP in solid tumors with deletions at Ip36 should be investigated. Onyango et al. (1998) established the order of genes in the Ip36 region, telomere to centromere, as CDC2L1 (176873)~PTPRZ2 (604008)~ENOl (172430)-- PGD (17220O)-XBXl (604007)-FRAP2 (FRAPl)-CD30 (153243).

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. 278 (18), 15461-15464 (2003); Kimura, et al., Genes Cells 8 (1), 65-79 (2003); McMahon, et al., MoI. Cell. Biol. 22 (21), 7428-7438 (2002); Tee, et al., Proc. Natl. Acad. Sci. U.S.A. 99 (21), 13571-13576 (2002); Hudson, et al., MoI. Cell. Biol. 22 (20), 7004-7014 (2002); Choi, et al., EMBO Rep. 3 (10), 988- 994 (2002); Inoki, et al., Nat. Cell Biol. 4 (9), 648-657 (2002); Zhang, et al., J. Biol. Chem. 277 (31), 28127-28134 (2002); Castedo, et al., EMBO J. 21 (15), 4070-4080 (2002); Hara, et al., Cell 110 (2), 177-189 (2002); Kim, et al., Cell 110 (2), 163-175 (2002); Fingar, et al., Genes Dev. 16 (12), 1472-1487 (2002); Reynolds, et al., J. Biol. Chem. 277 (20), 17657-17662 (2002); Fang, et al., Science 294 (5548), 1942-1945 (2001); Dennis, et al., Science 294 (5544), 1102-1105 (2001); Onyango, et al., Genomics 50 (2), 187-198 (1998); Lench, et al., Hum. Genet. 99 (4), 547-549 (1997); Choi, et al., Science 273 (5272), 239-242 (1996); Moore, et al., Genomics 33 (2), 331-332 (1996); Chen, et al., Proc. Natl. Acad. Sci. U.S.A. 92 (11), 4947-4951 (1995); Chiu et al., Proc. Natl. Acad. Sci. U.S.A. 91 (26), 12574-12578 (1994); Brown, et al., Nature 369 (6483), 756-758 (1994).

STEM CELLS

The methods and compositions described here rely on the demonstration that activation of mTOR promotes self-renewal of stem cells, and its inactivation inhibits self- renewal and induces differentiation.

As used in this document, the term "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 Stem Cells

The term "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). Thus, the only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage.

Pluripotent Stem Cells

"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 Cells

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.

Embryonic Germ Cells

Embryonic Germ (EG) cells may be isolated from the precursor to the gonads in aborted fetuses.

Embryonic Carcinoma Cells

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. Adult Stem Cells

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

Multipotent stem cells are true stem cells but can only differentiate into a limited number of types. For example, 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 are known in the art, and 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.

In addition to morphological differences, 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). 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. (2000). PNAS 97: 11307 - 11312; Thomson J. A. et. al. (1998). Science 282: 1145-1147; Reubinoff B.E. et. al. (2000). Nature Biotechnology 18: 399-404; Henderson J.K. et. al. (2002). Stem Cells 20: 329-337; Pera M. et. al. (2000). J. Cell Science 113: 5- 10.).

SOURCES OF STEM CELLS

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. Alternatively, 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, T₃, FCS, and various tissue extracts that allow the hepatocytes to grow without malignant transformation. 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 "oχal 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). U.S. Pat. No. 5,192,553 reports methods for isolating human neonatal or fetal hematopoietic stem or progenitor 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.

Amongst 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. Non-limiting examples are primary cultures or established lines of embryonic stem cells.

Media and Feeder 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. 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. Just before use, 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 (where used) 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. 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. with 1 mL 0.5% gelatin per well overnight, and plated with 375,000 irradiated mEFs per well. 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.

Conditions for culturing other stem cells are known, and can be optimized appropriately according to the cell type. Media and culture techniques for particular cell types referred to in the previous section are provided in the references cited.

Embryonic Stem 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.

Briefly, human blastocysts are obtained from human in vivo preimplantation embryos. Alternatively, 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.

After 9 to 15 days, 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. Resulting 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

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.

Briefly, 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 mm³ 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). In an alternative approach, 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.

Ninety-six well tissue culture plates are prepared with a sub-confluent layer of feeder cells cultured for 3 days in modified EG growth medium free of LIF, bFGF or forskolin, inactivated with 5000 rad y-irradiation. Suitable feeders are STO cells (ATCC Accession No. CRL 1503). 0.2 mL of primary germ cell (PGC) suspension is added to each of the wells. The first passage is conducted after 7-10 days in EG growth medium, transferring each well to one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. The cells are cultured with daily replacement of medium until cell morphology consistent with EG cells are observed, typically after 7-30 days or 1-4 passages.

SELF-RENEWAL AND DIFFERENTIATION

Self-Renewal

According to the methods and compositions described here, 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.

For example, in the two dimensions of a standard microscopic image, 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. In general, the 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.). Other useful markers are detectable using antibodies designated Tra-1-60 and Tra-1-81 (Andrews et al., Cell Linesfrom Human Gern Cell Tumors, in E. J. Robertson, 1987, supra). 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.

Differentiation

According to the methods and compositions described here, stem cells which are differentiating display decreased or reduced mTOR activity.

They preferably display enhanced dephosphorylation of 4E-BP1 and / or S6K1. They preferably display decreased expression of Oct4 and/or SSEA-I . Preferably, expression of any one or more of FIk-I, Ti e-2 and c-kit is increased. Preferably, expression of any one or more of Brachyury, AFP, nestin and nurrl expression increased. 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. In general, 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.

In many respects, therefore, 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. For example, 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. For skeletal muscle: myoD, myogenin, and myf-5. For endothelial cells: PECAM (platelet endothelial cell adhesion molecule), FIk-I, tie-i, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC- 14.7. For smooth muscle cells: specific myosin heavy chain. For cardiomyocytes: GATA- 4, Nkx2.5, cardiac troponin I, alpha-myosin heavy chain, and ANF. For pancreatic cells, pdx and insulin secretion. For hematopoietic cells and their progenitors: GATA-I, CD34, ACl 33, .beta.-major globulin, and .beta. -major globulin like gene PHl.

Certain 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. Sequence data for the particular markers listed in this disclosure can be obtained from public databases such as GenBank (URL www.ncbi.nlm.nih.gov: 80/entrez). MTOR MODULATORS, AGONISTS AND ANTAGONISTS

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.

While any means of doing so may be used, in general, 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. Similarly, antagonists reduce the activity of the relevant protein.

In preferred embodiments, 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. Preferably, mTOR activity is assayed as described below in the section "Assays for mTOR Activity".

The term "antagonist", as used in the art, 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, however, 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). However, 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.

Where antagonists of a enzyme such as a enzyme are concerned, an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme. In addition, 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. Alternatively, or in addition, an immunoglobulin (for example, a monoclonal or polyclonal antibody) capable of binding to the enzyme may be used. 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.

Alternatively, instead of preventing the association of the components directly, 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. For example, 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.

In general, 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 (e.g., in crystalline, colloidal or other particulate form). The agent may be in the form of a monomer, dimer, oligomer, etc, or otherwise in a complex.

The terms "modulator", "antagonist" and "agent" are 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. a yeast artificial chromosome) or a part thereof, 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. 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.

In a particular embodiment, the technique of RNA interference (RNAi) 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.

The following protocol may be used: a sample of PCR product is analysed by horizontal gel electrophoresis and the DNA purified using a Qiagen QiaQuick PCR purification kit. 1 μg 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.

SPECIFIC ANTAGONISTS OF MTOR ACTIVITY

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

1-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.

Rapamycin

In a preferred embodiment, 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.

Preferably, stem cells are exposed to rapamycin and its derivatives at concentrations over InM, for example, 1OnM, 2OnM, 3OnM, 4OnM, 50 nM, 10OnM, 50OnM, lμm, lOμm, lOOμm, or more. In preferred embodiments, 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. In addition, monoacyl and diacyl derivatives of rapamycin and methods for their preparation are disclosed by Rakhit, U.S. Pat. No. 4,316,885. Furthermore, Stella et al., U.S. Pat. No. 4,650,803 disclose water soluble prodrugs of rapamycin, i.e., rapamycin derivatives including the following rapamycin prodrugs: glycinate prodrugs, propionate prodrugs and the pyrrolidino butyrate prodrugs.

The methods and 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, C₅₁ H₇₉ NOj₃, 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. the structures of the degradation products have recently been characterized. 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.

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.

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43 positions) have been shown to be useful as antifungal agents (U.S. Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin (U.S. Pat. No. 4,650,803). Recently, the numbering convention for rapamycin has been changed; therefore according to Chemical Abstracts nomenclature, the esters described above would be at the 31 -and 42-positions. Carboxylic acid esters (PCT application No. WO 92/05179), carbamates (U.S. Pat. No. 5,118,678), amide esters (U.S. Pat. No. 5,118,678), (U.S. Pat. No. 5,118,678) fluorinated esters (U.S. Pat. No. 5,100,883), acetals (U.S. Pat. No. 5,151413), silyl ethers (U.S. Pat. No. 5,120,842), bicyclic derivatives (U.S. Pat. No. 5,120,725), rapamycin dimers (U.S. Pat. No. 5,120,727) and O-aryl, O-alkyl, O-alkyenyl and 0-alkynyl derivatives (U.S. Pat. No. 5,258,389) have been described.

Rapamycin is metabolized by cytochrome P-450 3 A to at least six metabolites. During incubation with human liver and small intestinal microsomes, sirolimus was hydroxylated and demethylated and the structure of 39-O-demethyl sirolimus was identified. In bile of sirolimus-treated rats >16 hydroxylated and demethylated metabolites were detected.

In rapamycin demethylation of methoxy group at C-7 Carbon will lead to the change in the conformation of the Rapamycin due to the interaction of the released C-7 hydroxyl group with the neighbouring pyran ring system which is in equilibrium with the open form of the ring system. The C-7 hydroxyl group will also interact with the triene system and possibly alter the immunosupressive activity of rapamycin. This accounts for the degradation of rapamycin molecule and its altered activity. Structural Analogues ofRapamycin (Rapalogs)

A large number of structural variants of rapamycin have been reported, typically arising as alternative fermentation products or from synthetic efforts to improve the compound's therapeutic index as an immunosuppressive agent. Each of these may be employed in the methods and compositions described here.

For example, the extensive literature on analogs, homologs, derivatives and other compounds related structurally to rapamycin ("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. In nearly all cases, potent immunosuppressive activity is reported to accompany antifungal activity of the rapalogs. Additional historical information is presented in the background sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and 5,362,718.

Rapalogs

"Rapalogs" 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, as that term is used herein, 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.

TABLE I

WO9710502 WO9418207 WO9304680 US5527907 US5225403

WO9641807 WO9410843 WO9214737 US5484799 US5221625

WO9635423 WO9409010 WO9205179 US5457194 US5210030

WO9603430 WO9404540 US5604234 US5457182 US5208241

WO9600282 WO9402485 US5597715 US5362735 US5200411

WO9516691 WO9402137 US5583139 US5324644 US5198421

WO9515328 WO9402136 US5563172 US5318895 US5147877

WO9507468 WO9325533 US5561228 US5310903 US5140018

WO9504738 WO9318043 US5561137 US5310901 US5116756

WO9504060 WO9313663 US5541193 US5258389 US5109112

WO9425022 WO9311130 US5541189 US5252732 US5093338

WO9421644 WO9310122 US5534632 US5247076 US5091389

SPECIFIC AGONISTS OF MTOR ACTIVITY

Phosphatidic Acid

hi a preferred embodiment, 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.

Preferably, stem cells are exposed to phosphatidic acid and its derivatives at concentrations over InM, for example, 1OnM, 2OnM, 3OnM, 4OnM, 50 nM, 10OnM, 50OnM, lμm, lOμm, lOOμm, or more. In preferred embodiments, phosphatidic acid and its derivatives are used at about 5OnM. Other 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.

Stimulation of mTOR activity by phosphatic acid is described in Fang et al, Science. 2001 Nov 30;294(5548):1942-5. Park et al, J Biol Chem. 2002 Aug 30;277(35):31423-9. Epub 2002 Jun 26. This document describes the regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin, which is dependent on phosphatidic acid. Chen, Curr Top Microbiol Immunol. 2004;279:245-57 describes a direct link between mTOR and mitogenic signals which is mediated by the lipid second messenger phosphatidic acid.

Xu et al, Bioorg Med Chem Lett. 2004 Mar 22;14(6):1461-4 describe mono- and difluoromethylene phosphonate analogues of phosphatidic acid. These analogues were synthesized such that the bridging oxygen was replaced by an alpha- monofluoromethylene (-CHF-) or alpha-difluoromethylene (-CF(2)-) moiety. Most of these analogues surpassed phosphatidic acid in activating S6 kinase, a downstream target of mTOR signaling. Each of the phosphatidic acid analogues described in this document may be employed in the methods and compositions described here. Furthermore, carboxylated phosphatidic acid esters which are described in US patent 6,706,280 (Tournier) may also be used.

Other Enzymes

It is apparent to the skilled person that phosphatidic acid is synthesised by the action of the enzymes phospholipase D and diacylglycerol kinases (DGKs). Thus, phospholipase A converts lysophosphatidic acid to phosphatidic acid, while phospholipase D cleaves phospholipids to form phosphatidic acid, while diacylglycerol kinases phosphorylate diacylglycerol to phosphatidic acid. Accordingly, 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.

Nutrient Levels / Growth Factors

mTOR activity is enhanced by increased nutrient levels. Thus, as described in Schalm (PhD Thesis, supra), 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. Accordingly, 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.

ANTIBODIES

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, as used herein, 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. Especially preferred are 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. Moreover, they may be fluorescent labels or other labels which are visualisable on tissue samples.

Recombinant DNA technology may be used to improve the antibodies as described here. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, 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.

Therefore, we disclose a process for the production of 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.

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. 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.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody- producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, 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 foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; US 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incorporated herein by reference.

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.

For isolation of the antibodies, 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. If necessary and/or desired, 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.

Also included is 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. For example 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. Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 10 and 10⁷ and 10⁸ cells expressing mTOR and a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and 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. Preferably 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. After the fusion 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.

Recombinant 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. By definition such 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.

Furthermore, 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. Preferably 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.

The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, 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.

Also disclosed are 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 γl , γ2, γ3 or γ4, preferably γl or γ4. Likewise 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 λ, preferably K are also disclosed.

In another embodiment, we disclose recombinant DNAs coding for a recombinant polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule. TheDNAcodingforaneffectormoleculeisintendedtobeaDNAcodingforthe effectormoleculesusefulindiagnosticortherapeuticapplications.Thus,effector moleculeswhicharetoxinsorenzymes,especiallyenzymescapableofcatalysingthe activationofprodrugs,areparticularlyindicated.TheDNAencodingsuchaneffector moleculehasthesequenceofanaturallyoccurringenzymeortoxinencodingDNA,ora mutantthereof,andcanbepreparedbymethodswellknownintheart.

ANTI-PEPTIDEMTORANTIBODIES

Anti-peptideantibodiesmaybeproducedagainstmTORpeptidesequences.The sequenceschosenmaybebasedonthemousesequencesasfollowfromthefollowing mTORreferencesequence:

1 mlgtgpavat asaatssnvs vlqqfasglk srneetraka akelqhyvtm elremsqees

61 trfydqlnhh ifelvsssda nerkggilai asligveggn strigrfany lrnllpssdp

121 wmemaskai grlamagdtf taeyvefevk ralewlgadr negrrhaavl vlrelaisvp

181 tfffqqvqpf fdnifvavwd pkqairegav aalraclilt tqrepkemqk pqwyrhtfee

241 aekgfdetla kekgmnrddr ihgallilne lvrissmege rlreemeeit qqqlvhdkyc

301 kdlmgfgtkp rhitpftsfq avqpqqpnal vgllgysspq glmgfgtsps pakstlvesr

361 ccrdlmeekf dqvcqwvlkc rssknsliqm tilnllprla afrpsaftdt qylqdtmnhv

421 lscvkkeker taafqalgll svavrsefkv ylprvldiir aalppkdfah krqktvqvda

481 tvftcismla ramgpgiqqd ikellepmla vglspaltav lydlsrqipq lkkdiqdgll

541 kmlslvlmhk plrhpgmpkg lahqlaspgl ttlpeasdva sitlalrtlg sfefeghslt

601 qfvrhcadhf lnsehkeirm eaartcscll tpsihlisgh ahwsqtavq wadvlskll

661 wgitdpdpd irycvlasld erfdahlaqa enlqalfval ndqvfeirel aictvgrlss

721 mnpafvmpfl rkmliqilte lehsgigrik eqsarmlghl vsnaprlirp ymepilkali

781 lklkdpdpdp npgvinnvla tigelaqvsg lemrkwvdel fiiimdmlqd ssllakrqva

841 lwtlgqlvas tgywepyrk yptllevlln flkteqnqgt rreairvlgl lgaldpykhk

901 vnigmidqsr dasavslses kssqdssdys tsemlvnmgn lpldefypav smvalmrifr

961 dqslshhhtm wqaitfifk slglkcvqfl pqvmptflnv irvcdgaire flfqqlgmlv

1021 sfvkshirpy mdeivtlmre fwvmntsiqs tiillieqiv valggefkly lpqliphmlr

1081 vfmhdnsqgr ivsikllaai qlfganlddy lhlllppivk lfdapevplp srkaaletvd

1141 rltesldftd yasriihpiv rtldqspelr stamdtlssl vfqlgkkyqi fipmvnkvlv

1201 rhrinhqryd vlicrivkgy tladeeedpl iyqhrmlrss qgdalasgpv etgpmkklhv

1261 stinlqkawg aarrvskddw lewlrrlsle llkdssspsl rscwalaqay npmardlfna

1321 afvscwseln edqqdelirs ielaltsqdi aevtqtllnl aefmehsdkg plplrddngi

1381 vllgeraakc rayakalhyk elefqkgptp aileslisin nklqqpeaas gvleyamkhf

1441 geleiqatwy eklhewedal vaydkkmdtn kedpelmlgr mrclealgew gqlhqqccek

1501 wtlvndetqa kmarmaaaaa wglgqwdsme eytcmiprdt hdgafyravl alhqdlfsla

1561 qqcidkardl ldaeltamag esysraygam vschmlsele eviqyklvpe rreiirqiww

1621 erlqgcqriv edwqkilmvr slwsphedm rtwlkyaslc gksgrlalah ktlvlllgvd

1681 psrqldhplp tahpqvtyay mkninwksark idafqhmqhf vqtmqqqaqh aiatedqqhk

1741 qelhklmarc flklgewqln lqginestip kvlqyysaat ehdrswykaw hawavmnfea

1801 vlhykhqnqa rdekkklrha sganitnatt aattaasaaa atstegsnse seaesnensp

1861 tpsplqkkvt edlsktllly tvpavqgffr sislsrgnnl qdtlrvltlw fdyghwpdvn

1921 ealvegvkai qidtwlqvip qliaridtpr plvgrlihql ltdigryhpq aliypltvas

1981 kstttarhna ankilknmce hsntlvqqam mvseelirva ilwhemwheg leeasrlyfg 2041 ernvkgmfev leplhammer gpqtlketsf nqaygrdlme aqewcrkymk sgnvkdltqa 2101 wdlyyhvfrr iskqlpqlts lelqyvspkl lmcrdlelav pgtydpnqpi iriqsiapsl 2161 qvitskqrpr kltlmgsngh efvfllkghe dlrqdervmq lfglvntlla ndptslrknl 2221 siqryavipl stnsgligwv phcdtlhali rdyrekkkil lniehrimlr mapdydhltl 2281 mqkvevfeha vnntagddla kllwlkspss evwfdrrtny trslavmsmv gyilglgdrh 2341 psnlmldrls gkilhidfgd cfevamtrek fpekipfrlt rmltnamevt gldgnyrttc 2401 htvmevlreh kdsvmavlea fvydpllnwr lmdtntkgnk rsrtrtdsys agqsveildg 2461 velgepahkk agttvpesih sfigdglvkp ealnkkaiqi inrvrdkltg rdfshddtld 2521 vptqvellik qatshenlcq cyigwcpfw

Thus, 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.

IDENTIFYING MODULATORS, AGONISTS AND ANTAGONISTS

Antagonists, in particular, small molecules may be used to specifically inhibit mTOR. We therefore disclose small molecule α 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.

By "down-regulation" we include any negative effect on the behaviour being studied; this may be total, or partial. Thus, where binding is being detected, candidate antagonists are capable of reducing, ameliorating, or abolishing the binding between two entities. Preferably, 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. Thus, a candidate molecule suitable for use as an antagonist is one which is capable of reducing by 10% more the binding or other activity.

Polypeptide Binding Assays

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. Alternatively, 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.

In a preferred assay method, the polypeptide is immobilised on beads such as agarose beads. Typically this is achieved by expressing 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). As a control, 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. Again, 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. For example, the non-immobilised component may be labeled (with for example, a radioactive label, an epitope tag or an enzyme-antibody conjugate). Alternatively, binding may be determined by immunological detection techniques. For example, 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. In the case of antibodies, the final concentration used is typically from 100 to 500 μg/ml, more preferably from 200 to 300 μg/ml.

Activity Assays

Assays to detect modulators, agonists or antagonists 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. The relevant activity of mTOR (as described below) 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. Details of such screening procedures are well known in the art, and are for example described in, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9).

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. In in vitro assays, 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. Where in vitro assays are conducted, these preferably employ arrays of candidate molecules (for example, an arrayed library). In vivo assays are preferred. Preferably, therefore, the mTOR is comprised in a cell, preferably heterologously. Such a cell is preferably a transgenic cell, which has been engineered to express mTOR as described above.

Where an extract is employed, it may comprise a cytoplasmic extract or a nuclear extract, methods of preparation of which are well known in the art.

It will be appreciated that any component of a cell comprising mTOR may be employed, such as an organelle. 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.

Thus, in a specific embodiment, 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.

Alternatively or in addition to the assay methods described above, "subtractive" procedures may also be used to identify modulators, agonists or antagonists of mTOR. Under such "subtractive" procedures, 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.

Thus, for example, 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.

It will also be appreciated that the above "depletion" or "subtraction" assay may be used as a preliminary step to identify putative modulatory factors for further screening. Furthermore, or alternatively, 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.

In a preferred embodiment, a small molecule library is employed. By a "small molecule", we refer to a molecule whose molecular weight is preferably less than about 50 kDa. In particular embodiments, 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.

Alternatively or in addition, a combinatorial library, as described in further detail below, may be screened for modulators, antagonists or agonists of mTOR.

ASSAYS FOR MTOR ACTIVITY

Any of the activities of mTOR may be used as the basis of the assay.

In particular, cellular activities mediated by mTOR may be assayed to identify antagonists. For example, mToR is responsible for phosphorylating substrates including eukaryotic initiation factor 4E (eIF4E) and ribosomal S6 kinase 1 (S6K1), RNA polymerase I and eEF2 kinase. Accordingly, the effects of the putative antagonist or agonist on kinase activity mediated by mTOR one one or more of these substrates (or peptides derived from their sequences) may be assayed using for example kinase assays as known in the art.

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.

An exemplary assay for kinase activity of mTOR is described in Gary G. Chiang, Robert T. Abraham. Determination of the Catalytic Activities of mTOR and Other Members of the Phosphoinositide-3-Kinase-Related Kinase Family. Checkpoint Controls and Cancer: Volume 2: Activation and Regulation Protocols, July 2004, pps. 125-142 (ISBN: 1-59259-811-0), Volume #: 281, Series: Methods in Molecular Biology.

mTOR Kinase Assay

A further mTOR assay is disclosed in Molecular Mechanism of mTOR Downstream Signalling (PhD Thesis, S. Schalm, 17^th September 2003, Fachbereich Biologie, Chemie, Pharmazie, Freie Universitat Berlin, http://www.diss.fu- berlin.de/2003/249/index.html).

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 β- glycerophosphate, 0.2% CHAPS, 1 mM Na3 VO4, 40 mg/ml PMSF, 5 μg/ml pepstatin, 10 μg/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. Immunopreciptates are washed twice with 1 ml mTOR wash buffer A (20 mM Tris, 500 mM NaCl, 1 mM EDTA, 20 mM β-glycerophosphate, 5 mM EGTA, 1 mM DTT, 1 mM Na3 VO4.40 mg/ml PMSF, 10 μg/ml leupeptin, 5 μg/ml pepstatin, in ddH2 O, pH 7.4), once with mTOR wash buffer B (10 mM HEPES, 50 mM β-glycerophosphate, 50 mM NaCl, 1 mM DTT, 1 mM Na3 V04, 40 mg/ml PMSF, 10 μg/ml leupeptin, 5 μg/ml pepstatin, in ddH2 O, pH 7.4), and once with ST (50 mM Tris-HCl, 5 mM Tris base, 150 mM NaCl, ddtaO, pH 7.28).

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) in washed immunoprecipitates is assayed in mTOR kinase assay buffer (10 mM HEPES, 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCk, 100 μM ATP unlabeled, 10 μCi [γ- 32P] ATP (New England Nuclear), pH 7.4) for 30 min at 30⁰C. 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 3O⁰C.

mTOR Reporter Assay

Molecules and agents which activate or promote mTOR activity may be identified as follow: To screen for mTOR activating molecules, 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: To screen for mTOR inhibiting molecules, 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).

Cell Cycle Assay

Furthermore, we show that 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

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. coli and as a result the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or pVIII). Alternatively, antibody fragments are displayed externally on lambda phage capsids (phagebodies). 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.

Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al (1990) supra; Kang et al (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Natl. Acad. Sci U.S.A., 88: 10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al. (199I) J. Immunol, 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins and Winter (1992) J. Immunol, 22: 867; Marks et al, 1992, J. Biol. Chem., 267: 16007; Lerner et al (1992) Science, 258: 1313, incorporated herein by reference). Such techniques may be modified if necessary for the expression generally of polypeptide libraries.

One particularly advantageous approach has been the use of scFv phage-libraries (Bird, R.E., et al. (1988) Science 242: 423-6, Huston et al, 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary et al (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066- 1070; McCafferty et al (1990) supra; Clackson et al. (1991) supra; Marks et al (1991) supra; Chiswell et al (1992) Trends Biotech., 10: 80; Marks et al (1992) supra). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Refinements of phage display approaches are also known, for example as described in WO96/06213 and WO92/01047 (Medical Research Council et al.) and WO97/08320 (Morphosys, supra), which are incorporated herein by reference.

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. These expression systems may be used to screen a large number of different members of a library, in the order of about 10⁶ or even more. Other screening systems rely, for example, on direct chemical synthesis of library members. One early method involves the synthesis of peptides on a set of pins or rods, such as described in WO84/03564. A similar method involving peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, is described in U.S. Patent No. 4,631 ,211 and a related method is described in WO92/00091. 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. These improved bead-based methods are described in WO93/06121.

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. These methods are described in U.S. Patent No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991) Science, 251: 767; Dower and Fodor {\99\) Ann. Rep. Med. Chem., 26: 271.

Other systems for generating libraries of polypeptides or nucleotides involve the use of cell-free enzymatic machinery for the in vitro synthesis of the library members. In one method, 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 a similar way, 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. These and all the foregoing documents also are incorporated herein by reference.

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.

COMBINATORIAL LIBRARIES

Libraries, in particular, 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.

Various 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. A comprehensive review of combinatorial libraries, in particular their construction and uses is provided in Dolle and Nelson (1999), Journal of Combinatorial Chemistry, VoI 1 No 4, 235-282. Reference is also made to Combinatorial peptide library protocols (edited by Shmuel Cabilly, Totowa, N.J. : Humana Press, cl998. Methods in Molecular Biology ; v. 87). Specific combinatorial libraries and methods for their construction are disclosed in United States Patent 6,168,914 (Campbell, et al), as well as in Baldwin et al. (1995), "Synthesis of a Small Molecule Library Encoded with Molecular Tags," J. Am. Chem. Soc. 117:5588-5589, and in the references mentioned in those documents.

In a preferred embodiment, 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. For example, 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.

Further references describing chemical combinatorial libraries, their production and use include those available from the URL http://www.netsci.org/Science/Combichem/, including The Chemical Generation of Molecular Diversity. Michael R. Pavia, Sphinx Pharmaceuticals, A Division of Eli Lilly (Published July, 1995); Combinatorial Chemistry: A Strategy for the Future - MDL Information Systems discusses the role its Project Library plays in managing diversity libraries (Published July, 1995); Solid Support Combinatorial Chemistry in Lead Discovery and SAR Optimization, Adnan M. M. Mjalli and Barry E. Toyonaga, Ontogen Corporation (Published July, 1995); Non-Peptidic Bradykinin Receptor Antagonists From a Structurally Directed Non-Peptide Library. Sarvajit Chakravarty, Babu J. Mavunkel, Robin Andy, Donald J. Kyle*, Scios Nova Inc. (Published July, 1995); Combinatorial Chemistry Library Design using Pharmacophore Diversity Keith Davies and Clive Briant, Chemical Design Ltd. (Published July, 1995); A Database System for Combinatorial Synthesis Experiments - Craig James and David Weininger, Daylight Chemical Information Systems, Inc. (Published July, 1995); An Information Management Architecture for Combinatorial Chemistry, Keith Davies and Catherine White, Chemical Design Ltd. (Published July, 1995); Novel Software Tools for Addressing Chemical Diversity, R. S. Pearlman, Laboratory for Molecular Graphics and Theoretical Modeling, College of Pharmacy, University of Texas (Published June/July, 1996); Opportunities for Computational Chemists Afforded by the New Strategies in Drug Discovery: An Opinion, Yvonne Connolly Martin, Computer Assisted Molecular Design Project, Abbott Laboratories (Published June/July, 1996); Combinatorial Chemistry and Molecular Diversity Course at the University of Louisville: A Description, Arno F. Spatola, Department of Chemistry, University of Louisville (Published June/July, 1996); Chemically Generated Screening Libraries: Present and Future. Michael R. Pavia, Sphinx Pharmaceuticals, A Division of Eli Lilly (Published June/July, 1996); Chemical Strategies For Introducing Carbohydrate Molecular Diversity Into The Drug Discovery Process.. Michael J. Sofia, Transcell Technologies Inc. (Published June/July, 1996); Data Management for Combinatorial Chemistry. Maryjo Zaborowski, Chiron Corporation and Sheila H. DeWitt, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company (Published November, 1995); and The Impact of High Throughput Organic Synthesis on R&D in Bio-Based Industries, John P. Devlin (Published March, 1996).

Techniques in combinatorial chemistry are gaining wide acceptance among modern methods for the generation of new pharmaceutical leads (Gallop, M. A. et al., 1994, J. Med. Chem. 37:1233-1251; Gordon, E. M. et al., 1994, J. Med. Chem. 37:1385- 1401.). One combinatorial approach in use is based on a strategy involving the synthesis of libraries containing a different structure on each particle of the solid phase support, interaction of the library with a soluble receptor, identification of the 'bead" which interacts with the macromolecular target, and determination of the structure carried by the identified 'bead' (Lam, K. S. et al., 1991, Nature 354:82-84). An alternative to this approach is the sequential release of defined aliquots of the compounds from the solid support, with subsequent determination of activity in solution, identification of the particle from which the active compound was released, and elucidation of its structure by direct sequencing (Salmon, S. E. et al., 1993, Proc.Natl.Acad.Sci.USA 90:11708-11712), or by reading its code (Kerr, J. M. et al., 1993, J.Am.Chem.Soc. 115:2529-2531; Nikolaiev, V. et al., 1993, Pept. Res. 6:161-170; Ohlmeyer, M. H. J. et al., 1993, Proc.Natl.Acad.Sci.USA 90:10922-10926).

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).

Thus, 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 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). Commercially available libraries (e.g., from Affymetrix, ArQuIe, Neose Technologies, Sarco, Ciddco, Oxford Asymmetry, Maybridge, Aldrich, Panlabs, Pharmacopoeia, Sigma, or Tripose) may also be used with the methods described here.

In addition to libraries as described above, special libraries called diversity files can be used to assess the specificity, reliability, or reproducibility of the new methods. 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.

METHODS OF DETECTING STATE OF A STEM CELL

We demonstrate that 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. Conversely, 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.

Methods of detecting mTOR activity are known in the art, and are also described above..

Detection of Pluripotent Cells In Cell Populations

Furthermore, 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. As used herein, a "cell population" is any collection of cells which may contain one or more PGCs, ES or EG cells. Preferably, 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.

As noted hereinbefore, 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.

Preferably, 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. For example, 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. Examples of detectable gene products include bioluminescent polypeptides, such as luciferase and GFP, polypeptides detectable by specific assays, such as β-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. In a preferred aspect, the signal is detectable at the cell surface. For example, 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.

Preferred is the use of optical immunosensor technology, based on optical detection of fluorescently-labelled antibodies. 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).

EXPRESSION OF MTOR POLYPEPTIDES

In some circumstances, it may be desirable to increase the amount of mTOR polypeptide in a cell, for example a stem cell. In order to express a biologically active mTOR, 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.

Methods which are well known to those skilled in the art are used to construct expression vectors containing sequences encoding mTOR and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory Manual, ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N. Y.) and Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.).

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. The "control elements" or "regulatory sequences" 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. 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.

In bacterial systems, 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 β-galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509), and the like. pGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such 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.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel (supra) and Grant et al. (1987; Methods Enzymol. 153:516-544).

In cases where plant expression vectors are used, the expression of sequences encoding mTOR may be driven by any of a number of promoters. For example, 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. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These 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. For example, in one such system, 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. (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227.) In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, 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. (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81 :3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Thus, for example, the mTOR proteins are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize mTOR expression, 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.

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.)

In addition, 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. Such 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.

For long term, high yield production of recombinant proteins, stable expression is preferred. For example, 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. These include, but are not limited to, the 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. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); 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. 85:8047-51.) Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (Rhodes, C. A. et al. (1995) Methods MoI. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the 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. Alternatively, 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.

Alternatively, 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.

The presence of 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.

A variety of protocols for detecting and measuring the expression of mTOR, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on mTOR is preferred, but a competitive binding assay may be employed. These and other assays are well described in the art, for example, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) and in Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. 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. Alternatively, the sequences encoding mTOR, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio). 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. As will be understood by those of skill in the art, 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.). The inclusion of cleavable linker sequences, such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif), between the purification domain and the mTOR encoding sequence may be used to facilitate purification. 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. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

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.

HOST CELLS

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. Although 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.

We therefore further disclose cells comprising mTOR nucleic acid molecules or vectors. These may for example be used for expression, as described herein.

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.

Alternatively, the cell may be used in therapy for the same purposes as the mTOR polypeptide. For example, 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. Preferably 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.

In some case cells may be provided together with other material to aid the structure or function or of an implant. For example 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

We further describe 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.

Preferably, 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. For example, 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. Such 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. In general, 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. Preferably, 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).

Furthermore, 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.

Methods for generating non-human transgenic animal are known in the art, and are described in further detail in the Examples below. 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.

In an exemplary embodiment, 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. In addition, the haplotype is a significant factor.

Introduction of the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. For example, 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). Following introduction of the transgene construct into the fertilized egg, 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. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.

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. Preferably, 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.

For the purposes of this document, a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism. Generally, 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. Thus, 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. Generally, a euploid zygote is preferred. If an aneuploid zygote is obtained, then 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. In addition to similar biological considerations, 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.

Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.

Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, 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. Alternatively, the partner may be a parental line. Where 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. As set out above, 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.

It will be appreciated that it is possible to manipulate the control elements (promoters or enhancers) to regulate the spatial or temporal expression, or both, of mTOR. For example, specific control elements may be deleted from the endogenous mTOR locus so that expression is restricted to only certain tissues. Alternatively, it is possible to prepare 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. During this time, 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. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, 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).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). 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. For review see Jaenisch, R. (1988) Science 240:1468-1474.

We also provide non-human 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). Preferably, 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. For example, 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.

We also provide for a mTOR deficient transgenic non-human animal (a "mTOR knock-out" or a "mTOR null"). 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. For example, 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. Preferably, 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. Preferably, the genomic mTOR sequence which is functionally disrupted comprises a mouse mTOR genomic sequence.

Preferably, 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.

We further disclose a nucleic acid construct for functionally disrupting a mTOR gene in a host cell. 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. Additionally, 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. In a preferred embodiment, 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. Further development of 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. THERAPEUTIC PEPTIDES

The polypeptides disclosed here, for example, mTOR polypeptides, may be used therapeutically for treatment of various diseases, including cancer, in the form of peptides comprising any portion of their sequence.

Where such 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.

A number of approaches have been used to modify peptides for therapeutic application. One approach is to link the peptides or proteins to a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG) - see for example U.S. Patent Nos. 5,091,176, 5,214,131 and US 5,264,209.

Replacement of naturally-occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify the mTOR peptides.

Another approach is to use 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).

It may be desirable to use derivatives of the peptides disclosed here which are conformational^ constrained. 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 γ-lactam or other types of bridges. For example, side chains can be cyclized to the backbone so as create a L-γ- 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. Typically, 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. In general, 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. In addition, 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 N₄ 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.

Techniques for chemically synthesising therapeutic peptides are described in the above references and also reviewed by Borgia and Fields, 2000, TibTech 18, 243-251 and described in detail in the references contained therein.

PROPHYLACTIC AND THERAPEUTIC METHODS

We provide methods of treating an abnormal conditions related to both an excess of and insufficient amounts of mTOR activity. Examples of these include the mTOR associated diseases disclosed above.

If the activity of mTOR is in excess, several approaches are available. 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.

In another approach, where 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.

In still another approach, 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). Alternatively, oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; 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.

For treating abnormal conditions related to an under-expression of mTOR and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates mTOR, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of mTOR by the relevant cells in the subject.

For example, 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. For overview of 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. Such formulations 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. We further describe 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.

Preferred forms of 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. In addition, if properly formulated in enteric or encapsulated formulations, 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 μg/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. Thus, for example, 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.

PHARMACEUTICAL COMPOSITIONS

We also provide a pharmaceutical composition 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. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, 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. Alternatively, the formulation may be designed to be delivered by both routes.

Where 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.

Where appropriate, the pharmaceutical 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. For parenteral administration, the 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. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

VACCINES

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.

Since the mTOR polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for 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. Typically, 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.

In addition, if desired, 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. Examples of 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 (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

Further examples of 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, Corγnebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such 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). Typically, 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. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4⁰C, 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. For 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. These 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%. Where the vaccine composition is lyophilised, 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. 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.

ADMINISTRATION

Typically, 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.

The pharmaceutical and vaccine 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. Typically, 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. The term "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.

The term "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. Thus, whilst 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. For 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. These 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%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.

Molecule

Where the term "molecule" is 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. Similarly, 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.

Other Uses

We describe the use of inhibitors of stem cell proliferation for regulating stem cell cycle in the treatment of humans or animals having autoimmune diseases, aging, cancer, myelodysplasia, preleukemia, leukemia, psoriasis or other diseases involving hyperproliferative conditions. We also describe a method of treatment for humans or animals anticipating or having undergone exposure to chemotherapeutic agents, other agents which damage cycling stem cells, or radiation exposure. Finally, we describe methods for the improvement of the stem cell maintenance or expansion cultures for auto- and allotransplantation procedures or for gene transfer. EXAMPLES

Example 1. Materials and Methods

1.1 Cell Culture

RoSH and mouse El 4 ES cells are maintained on gelatinized culture plates as previously described¹¹'¹⁵. RoSH induced to differentiate by plating 10⁶ 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 μM, respectively. To assess the effects of rapamycin on branching, 2x10⁵ 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).

1.2 Cell Cycle Assay

To assess the rate of cell cycle, 2x10⁸ 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 lOμM dye in saline at 37°C for 15 minutes, cultured in non-differentiating conditions for 24 hours before replating 1x10⁵ cells per 3 cm tissue culture plate under non-differentiating or differentiating conditions. At 0, 24 and 48 hours, three plates of cells are harvested, fixed in 2% paraformaldehyde, and analyzed on a FACStar^¹¹⁵ (Becton Dickinson; San Jose, CA). For testing the effects of rapamycin or Gleevec, six plates are treated with or without 50 nM rapamycin or 10 μM Gleevec at 48 hours harvested 24 and 48 hours later for cellular fluorescence assay.

Assuming that each halving of cellular fluorescence represented one cell division, the number of cell cycles per 24 hours, n is calculated as follows: 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.

1.3 RNA Preparation

Total RNA are prepared using a modified method of Chomczynski and Sacchi¹⁸ as previously described¹⁹. Polysome-associated RNA is prepared using a modified protocol ²⁰. Briefly, exponentially growing cells are harvested and resuspend in 10⁸ 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 μg/ml leupeptin, 1 mg/ml pepstatin A, 100 μg/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.

1.4 RT-PCR

RT-PCR is performed as previously described ²¹. 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-3¹ and 5'- GCAACAAGGGAGGACATTAG-3', 194bp; c) AFP 5'-TCC ACG TTA GAT TCC TCC CAG-3¹ and 5'-TTG CAG CAT GCC AGA ACG ACC-3¹ and 471bp; d) nestin 5'-TGT GGG ATG ATG GCT TGA GT-3' and 5'-ACA GAA GAA AGG GGG CGT TG-3¹ and 655bp; e) nurrl 5'-GCG GAG TTG AAT GAA TGA AGA-3¹ and 5'-TGG TGG AAG TTG TGA AGG GAG-3' and 616bp; f) Oct3/4 5'-TGT GGT TCG AGT ATG GTT CTG -3' and 5'-TGG TCT ACC TCC CTT GCC TTG-3' and 317bp; g) cyclin D2 5'-GTA AGA TGC TTA CAG GAG AAC-3¹ and 5'-CCT CAC CCT CTT CCC TTA CAC -3' and 585bp; h) TSPl 5'-AGA AAG AAA ACA CAA TCA CCT-3' and 5'-ACT GGG AAG TAA AAA GCA AAA-3¹ and 514bp; i) PDGFα 5'-CTC GAA GTC AGA TCC ACA GCA-3' and 5'-GCT TCT TCC TGA CAT ACT CCA-3' and 384bp; j) PDGFRα 5'- CCA GTA GTT CCA CCT TCA TCA-3¹ and 5'-CAA GTA TCC CAG CTA TCC ACA- 3' and 275bp; k) ApoE 5'-CTG AAC CGA TTC TGG GAT TAC-3¹ and 5'-GCT CAC GGA TGG CAC TCA CAC-3¹ and 451bp; 1) fibronectin 1 5'-CGT GGG ATG TTT TGA GAC TTC-3¹ and 5'-GTA GTA AAG CGT TGG CAT GTG-3' and 484bp.

1.5 Western Blot Analysis

Standard procedures are used. Cells are lysed in RIPA buffer at 4⁰C for 30 min and then centrifuged at 14,000 rpm, 15 min. Twenty micrograms lysate are denatured and separated on 10% SDS-polyacrylamide gel before electro-blotting onto a nitrocellulose membrane. The membrane is incubated sequentially with a primary antibody, then either a HRP conjugated-secondary antibody or a biotinylated secondary antibody followed by neutroavidin-HRP, and finally, a HRP enhanced chemiluminescent substrate, ECS (Pierce, Rockford, IL). 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, PDGFα 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. After the membrane had been probed with a maximum of three primary antibodies, it is stripped and probed with rabbit anti-tsc2 polyclonal antibody (1 :200 dilution) followed by goat anti rabbit-HRP as an internal control for loading. 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). A commonly used control, β-tubulin protein level is increased during endothelial differentiation of RoSH cells.

1.6 Immunohistochemistry

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.

1.7 Microarray Analysis

Affymetrix 430A chips (~22,000 genes and ESTs) are used to analyze RNA samples in duplicates using manufacturer's protocol. The chips are analyzed by dChip ²² where the scanned images showed no artifacts and no outliers are detected. The unnormalized median intensity of the arrays is 164 (SD=I 7). The presence calls for the 8 samples are high varying between 60 to 64%. As a result of these high quality measures, we continued the subsequent analysis with all the 8 chips. Normalization is made by the smoothing spline method and model base gene expression indices are calculated. The normalized signal values as well as quality control measures are calculated.

When comparing two groups of samples to identify the genes enriched in a given phenotype, we used a two-step filtering. First, for each gene we calculated the 90% lower confidence bound (LCB) of the fold change (FC) between the experiment and the baseline. We retained only the genes with an LCB above 1.2. We then considered this reduced set and genes with a FC greater than 2.0 are called differentially expressed.

Example 2. LIF Withdrawal Induces Differentiation; Effect on mTOR

When LIF is withdrawn from the culture medium for self-renewal, murine ES cells decrease self renewal and begin to differentiate. We observed a reduction in cell cycle activity from 1.7 ± 0.12 (n=3) to 1.4 ± 0.09 (n=3) cell divisions per 24 hours (p=0.028) (Figure IA) with parallel decreases in phosphorylated Stat3, and in expression of markers associated with pluripotency e.g. Oct 4 mRNA (Figure IB) and protein (Figure 1C), and SSEA-I (Figure ID). Expression of markers specific for the three embryonic germ layers, mesoderm (brachyury), endoderm (alphafetoprotein or AFP) and ectoderm (nestin and nurrl) is induced (Figure IB). Concomitant with decreased cell cycle activity during differentiation, mTOR activity decreased as shown by progressive dephosphorylation of S6K1, and 4E-BP1. Upon dephosphorylation, 4E-BP1 transitioned from its phosphorylated β isoform to hypophosphorylated α isoform (Figure 1C).

Example 3. Rapamycin Reduces mTOR Activity, Reduces Cell Cycle Activity and Induces Differentiation in Murine ES Cells

To determine if reduced cell cycle activity and induction of differentiation during LIF withdrawal is a direct consequence of reduced mTOR activity, mES cells are treated with rapamycin, a specific inhibitor of mTOR in the presence of LIF. Rapamycin treatment caused a replication of events induced by LIF withdrawal namely a reduction in cell division to 1.2 ± 0.10 (n=3) per 24 hours (p=0.049) (Figure IA). 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).

Additionally, when 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.

Together these data suggested that inhibition of mTOR activity is sufficient to cause downregulation of cell cycle activity and initiation of differentiation whilst circumventing the consequences of LIF/STAT3 signalling. To demonstrate whether a reduction in self-renewal is sufficient for induction of differentiation, ES cells are with imatinib mesylate (Gleevec), a selective inhibitor of receptor tyrosine kinase (18). Upon Gleevec treatment, there is decreased cell cycle activity but phosphorylation of S6K and 4E-BP is not abolished and expression of nestin/nurrl was not induced. Therefore reducing cell cycle activity without influencing mTOR activity is not sufficient to induce differentiation.

Example 4. Rapamycin Reduces mTOR Activity, Reduces Cell Cycle Activity and Induces Differentiation in Mouse Embryonic Cell Line RoS H2 Cells

To extend our observations further, we investigated if mTOR is also involved in coupling self-renewal and differentiation in other cellular systems. We examined endothelial differentiation of RoSH cells, a previously described karyotypically normal mouse embryonic cell line¹¹. RoSH cells can be induced to differentiate into endothelial cells and acquire typical endothelial surface markers and morphology when plated on matrigel.

Like murine ES cells, when RoSH cells are induced to undergo endothelial differentiation, there is a concomitant reduction in cell cycle activity from 2.1 ± 0.12 (n=3) to 1.2 ± 0.13 (n=3) divisions per 24 hours (p=0.08) (Figure 2A). In addition, there is progressive dephosphorylation of 4E-BP1 and S6K1 accompanied by upregulation of endothelial specific markers such as FIk-I, Tie-2, c-kit (Figure 2B). Rapamycin treatment of RoSH cells caused inhibition of mTOR as evident by progressive dephosphorylation of 4E-BP1 and S6K1 (Figure 2C). This inhibition is sufficient to cause a significant decline in cell cycle activity from 2.4 ± 0.18 (n=3) to 1.2 ± 0.27 (n=3) divisions per 24 hours (p=0.032) (Figure 2A). Rapamycin also resulted in expression of endothelial specific markers FIk-I, Tie-2 and c-kit (Figure 2B). Furthermore, rapamycin enhanced endothelial differentiation of RoSH cells that are induced to differentiate by plating cells on matrigel. By 24 hours, significantly more branch points are generated in rapamycin-treated cells, 76.3 ± 6.9 (n=6) as compared to 38.5 ± 10.2 (n=6) in untreated cells (p<0.05) (Figure 2D). Example 5. Inhibition of mTOR Activity is Required to Induce Differentiation

The correlation of mTOR activity with downregulation of cell cycle activity and the induction of differentiation in mES cells and RoSH cells suggests that mTOR regulates the coupling between self-renewal and differentiation in stem cells. We show below that a reduction in self-renewal is not sufficient for induction of differentiation.

RoSH cells are therefore treated with imatinib mesylate (Gleevec), a selective inhibitor of receptor tyrosine kinase e.g. c-Kit and PDGFR¹². RoSH cells but not mES cells express c-Kit and PDGFR (unpublished data, Que and Lim), and are therefore sensitive to Gleevec. Upon 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).

Therefore reducing cell cycle activity without influencing mTOR activity is not sufficient to induce differentiation.

Example 6. Role of mTOR in Regulating Translation

To assess the extent of translation regulation on a genome- wide basis during endothelial differentiation, microarray analysis is performed comparing total and polysome-associated RNA from RoSH cells before and 48 hours after differentiation (Figure 3B).

Of the 414 genes that declined in total cellular transcript levels upon differentiation, surprisingly 339 had no change in translation status as assessed by their association with polysomes. Only 45 had reduced translation reflected by a decrease in their total cellular transcript levels while 30 unexpectedly had increased translation. Similarly, of the 388 genes whose total cellular transcript levels transcript levels increased upon differentiaton, 285 had no change in translation status, 20 had reduced translation and 83 had increased translation. Significantly, translation of 285 and 864 genes declined or increased respectively without changes in total transcript levels. Together, these data demonstrated that the transit from self-renewal to differentiation involved significant translation regulation consistent with mTOR-mediated regulation.

In summary, this study demonstrates that self-renewal in murine ES cells requires LIF/Stat3 and mTOR signaling (see Figure 5). Our working model postulates that part of LIF/Stat3 signaling to maintain self-renewal is routed through mTOR. As predicted, in the absence of LIF, mTOR signaling is reduced resulting in inhibition of cell cycle and induction differentiation. Similarly, these effects could be replicated if mTOR is inhibited with rapamycin in the presence of LIF/Stat3 signaling.

Therefore, coupling of self-renewal to suppression of differentiation or inhibition of self-renewal to induction of differentiation in ES cells is a specific consequence of mTOR activity that is downstream of LIF/Stat3 signaling. It also implies that translational regulation is a vital mechanism regulating the decision to self-renew or differentiate. The coupling of self-renewal and endothelial differentiation by mTOR further indicates that coupling of self-renewal and differentiation in both embryonic and somatic stem cells may be universally regulated by mTOR. We note that mTOR signaling is also important in the generation of cellular diversity during differentiation of CNS stem cells¹⁴. Our study therefore suggests that expansion or differentiation of stem cells could be easily manipulated through perturbations of mTOR activity by media supplementation with small molecules.

Example 7. Activation of mTOR Activity by Phosphatidic Acid Promotes Self- Renewal

As shown above, inhibition of mTOR signaling promotes differentiation in the presence of LIF, while mTOR activation in the presence of LIF promotes stem cell proliferation.and self renewal. Accordingly, we show in this Example that activating mTOR will reduce serum requirement of ES cell cultures, i.e., promote self-renewal. Phosphatidic acid (PA) and metabolically stabilized PA analogues (e.g. fluoromethylene phosphonate analogues of phosphatidic acid) are known to activate signaling in the mTOR (mammalian target of rapamycin) pathway (Fang, et al., Science 294, 1942-5 (Nov 30, 2001; Xu, et al. Bioorg Med Chem Lett 14, 1461-4 (Mar 22, 2004). We show that supplementation of culture media with PA reduces serum requirement of ES cell cultures.

To test the hypothesis, 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

As shown in Figure 6, reducing serum concentration in the culture media from 20 to 5% reduces the rate of proliferation. If the culture media with 5% serum is supplemented with 50 or 100 nM PA, proliferation rate is increased.

Example 8. Screening Assay for mTOR Activating Molecules

An assay to screen for mTOR activating molecule is shown shematically in Figure 7A. Molecules and agents which activate or promote mTOR activity are identified as follow:

Activation of 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)).

To screen for mTOR activating molecules, 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.

Example 9. Screening Assay for mTOR Inhibiting Molecules

An assay to screen for mTOR inhibiting molecule is shown shematically in Figure 7B. Molecules and agents which inhibit mTOR activity are identified as follow:

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. Miskimins, Cancer Cell International 3, 2 (2003); U. Gopfert, M. Kullmann, L. Hengst, Hum. MoI. Genet. 12, 1767-1779 (July 15, 2003, 2003); M. Kullmann, U. Gopfert, B. Siewe, L. Hengst, Genes Dev. 16, 3087-3099 (December 1, 2002, 2002).

To screen for mTOR inhibiting molecules, 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.

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Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments and that many modifications and additions thereto may be made within the scope of the invention. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims. Furthermore, various combinations of the features of the following dependent claims can be made with the features of the independent claims without departing from the scope of the present invention.

Claims

1. A method comprising modulating the activity of mTOR in a stem cell.

2. A method according to Claim 1 , in which activity of mTOR is maintained or increased to enable a stem cell to self-renew.

3. A method according to Claim 1 or 2, in which 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.

4. A method according to Claim 2 or 3, in which the stem cell is exposed to an agonist of mTOR to increase the activity of mTOR in the stem cell.

5. A method according to Claim 2, 3 or 4, in which the agonist comprises phosphatidic acid.

6. A method according to Claim 1, in which activity of mTOR is decreased to enable a stem cell to differentiate.

7. A method according to Claim 1 or 6, in which: (a) 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.

8. A method according to Claim 6 or 7, in which the stem cell is exposed to an antagonist of mTOR to decrease the activity of mTOR in the stem cell.

9. A method according to Claim 6, 7 or 8, in which the antagonist comprises 1- butanol or rapamycin or a derivative thereof.

10. A method according to any preceding claim, in which 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.

11. A method according to any preceding claim, in which 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.

12. Use of 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.

13. Use of 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.

14. Use according to Claim 12 or 13, in which the marker of self-renewal is selected from the group consisting of: Oct4 and SSEA-I, and the marker of differentiation is selected from the group consisting of: FIk-I, Tie-2, c-kit, Brachyury, AFP, nestin and nurrl.

15. A method of identifying an agent capable of enabling self-renewal or differentiation of a stem cell, the method comprising contacting mTOR with a candidate agent and determining whether the candidate agent binds to mTOR, and optionally determining whether the activity of mTOR is thereby modulated.

16. A method of identifying an agent capable of enabling self-renewal of a stem cell, the method comprising contacting mTOR with a candidate agent and determining whether the activity of mTOR is thereby increased.

17. 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.

18. A method according to Claim 15, 16 or 17, in which the activity is kinase activity.

19. Use of mTOR in a method of enabling self-renewal or differentiation of a stem cell.

20. Use of an agent capable of increasing the activity of mTOR, preferably phosphatidic acid or an agent identified by a method according to Claim 15, 16 or 18, in a method of enabling self-renewal of a stem cell.

21. Use of an agent capable of decreasing the activity of mTOR, preferably 1 -butanol or rapamycin or a derivative thereof, or an agent identified by a method according to Claim 15, 17 or 18 in a method of enabling differentiation of a stem cell.

22. Use of mTOR or an agent as set out in any of Claims 19 to 21, or a cell produced or treated by a method or use according to any of Claims 1 to 14 and 19 to 21, 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, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease and cancer.

23. A method of influencing the choice between self-renewal and differentiation by a stem cell, the method comprising modulating the activity of mTOR in the stem cell.

24. A method of determining whether a stem cell is differentiating or self-renewing, the method comprising detecting mTOR activity of the stem cell.

25. A cell produced or treated by a method or use according to any of Claims 1 to 14 and 19 to 23.

26. A self-renewing cell produced or treated by a method according to any of Claims 2 to 5 and 10.

27. A differented or differentiating cell produced or treated by a method according to any of Claims 6 to 9 and 11.

28. An agent identified by a method or assay according to any of Claims 15 to 18.