WO2009086425A1 - Methods for reprogramming cells to a pluripotent state and therapeutic applications related thereto - Google Patents

Abstract

The present invention provides methods for reprogramming cells to a pluripotent state, either in vitro or in vivo, and the application of the methods in stem cell-based therapies, for example, autologous cell therapies and/or in vivo reprogramming of cells. The methods generally involve the induction of pluripotency by modulating the expression and/or activity of one or more pluripotency factors selected from, for example, Sox-2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, and the like, or substrates, cofactors and/or downstream effectors thereof.

Description

METHODS FOR REPROGRAMMING CELLS TO A PLURIPOTENT STATE AND THERAPEUTIC APPLICATIONS RELATED THERETO

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates generally to methods for reprogramming cells to a pluripotent state, either in vitro or in vivo, and the application of the methods in stem cell-based therapies, for example, autologous cell therapies and/or in vivo reprogramming of cells. DESCRIPTION OF THE RELATED ART

Stem cells are primitive undifferentiated cells having the capacity to mature into other cell types, for example, brain, muscle, liver, skin, neural and blood cells. Stem cells are typically classified as either embryonic stem cells, or adult tissue derived-stem cells, depending on the source of the tissue from which they are derived. Pluripotent stem cells are undifferentiated cells having the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm). Adult progenitor cells are adult stem cells which can give rise to a limited number of particular types of cells.

Because stem cells have the potential of developing into a specific types of cells and can proliferate indefinitely, they hold particular, but so far unrealized, potential in the context of therapeutic applications such as organs repair and replacement, cell therapies for degenerative diseases, gene therapy, and toxicology testing of new drugs.

For example, replacement therapy using stem cells could dramatically change the prognosis of many untreatable diseases. For example, many neurological diseases, such as disorders of the brain, spinal cord, peripheral nerves and muscles, are characterized by the sudden or gradual death of brain or muscle cells. These diseases which include stroke, head and spinal cord trauma, Alzheimer's Disease, Parkinson's Disease, Multiple sclerosis, Amyotrophic lateral sclerosis (ALS), genetic enzyme deficiencies such as Gaucher disease, Muscular dystrophy and others could potentially be treated using stem cell replacement therapy. However, available sources of stem cells useful for experimental and therapeutic applications have been limited and controversial. Further, although ES cells represent promising donor sources for cell transplantation therapies, they face immune rejection after transplantation. In addition, there are a number of controversial ethical issues relating to the use human embryos as a stem cell source. Thus there is a significant need for identifying approaches by which pluripotent stem cells can be directly derived from a patients somatic cells. The present invention addresses these needs and offers other related advantages.

SUMMARY OF THE INVENTION

Methods for the epigenetic reprogramming of cells to a pluripotent state offers a readily available and non-controversial source of autologous stem cells that can be used for customized transplantation therapy and other important therapeutic applications. Therefore, according to one aspect of the present invention, there is provided a method for reprogramming a cell to a pluripotent state comprising the steps of: (a) providing a population of cells; (b) treating the population of cells to effect chromatin remodeling; (c) treating the population of cells to induce pluripotency by modulating the expression and/or activity of at least two, at least three or at least four pluripotency factors selected from Sox-2, c-Myc, Oct3/4, Klf4, Lin28 and Nanog, or substrate(s), cofactor(s) or downstream effector(s) thereof; and (d) selecting for cells having a pluripotent phenotype.

Each of the pluripotency factors employed in the methods of the invention may be introduced into the cells using known techniques, or, alternatively, certain of the pluripotency factor may be introduce into the cells while others may be expressed endogenous Iy in the cell population selected for induction of pluripotency. The population of cells selected for induction of pluripotency may be homogeneous or heterogeneous and may comprise essentially any suitable cell type of interest, including adult and non-adult cells. In certain illustrative embodiments, the population of cells comprises adult stem cells, such as adult bone marrow cells. In other illustrative embodiments, the population of cells comprises cord cells, placenta cells or cord blood cells. The step of effecting chromatin remodeling can be carried out using any suitable technique known and available in the art. For example, in certain illustrative embodiments, chromatin remodeling is effected by treatment with one or more HDAC inhibitors, HAT activators, histone demethylase inhibitors and/or histone methyltransferase inhibitors. In a more particular embodiment, chromatin remodeling is effected by treatment with one or more HDAC inhibitor selected from butyrate, suberoylanilide hydroxamic acid, trichostatin A, chlamydocin, cyclic tetrapeptide trapoxin A and/or apicidin. In another particular embodiment, chromatin remodeling is effected by treatment with one or more histone demethylase inhibitor selected from trans-2-phenyl cyclopropylamine and/or peptide-based inhibitors.

In order to induce pluripotency in at least a subset of the cells contained within the population of cells to be induced, the population of cells is treated in a manner such that the expression and/or activity of the desired pluripotency factors is desirably modulated. For example, in one illustrative embodiment, one or more of the pluripotency factors, or substrate(s), cofactor(s) or downstream effector(s) thereof, are introduced into the population of cells by a transient introduction technique, e.g., by transient transfection. In another illustrative embodiment, one or more of the pluripotency factors, or substrate(s), cofactor(s) or downstream effector(s) thereof, are introduced into the population of cells by viral tranduction. In another illustrative embodiment, one or more of the pluripotency factors, or substrate(s), cofactor(s) or downstream effector(s) thereof, are introduced into the population of cells by microinjection. In yet another illustrative embodiment, the step of treating the population of cells to induce pluripotency comprises a step of contacting the population of cells with a small molecule, protein, peptide, sugar, or other compound, agent or treatment that effects modulation of one or more of the pluripotency factors, or substrate(s), cofactor(s) or downstream effector(s) thereof.

In addition to the pluripotency factors noted above, it may be desired in certain embodiments to further include a step of modulating the expression and/or activity of one or more additional factors selected from STAT-3, Rex-1, B-Myb, Foxd3, Gbx2, UTFl, Fgf4, Pern, Sail 4, Zic3 and/or Zfx, or substrate(s), cofactor(s) or downstream effector(s) thereof. Following treatment of the cells as noted above, the induced pluripotent cells contained within the population of treated cells may be selected on the basis of appropriate morphological, molecular and/or biochemical features. For example, in certain embodiments, the induced cells having a pluripotent phenotype may be selected by monitoring one or more cellular marker selected from SSEl, SSEA3, SSEA4,

TRA1-60/81, TRA2-54, GCTM-2, TG343, TG30, CD9, CD133/prominin, OCT4,

Nanog and/or Sox2. In other embodiments, the induced cells having a pluripotent phenotype may be selected for by contacting the cells with a Gl /S phase inhibitor or an epigenetic modulator that selectively inhibits the growth of somatic cells relative to pluripotent cells.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

As noted above, the present invention relates to methods of generating pluripotent stem cells and multipotent progenitor cells and related therapeutic applications involving same. More particularly, the present invention relates to methods for reprogramming cells to a pluripotent state, in vivo or ex vivo, by modulating specific cellular pathways of cells, either directly or indirectly, using polynucleotide-, polypeptide- and/or small molecule-based approaches. For example, according to one aspect of the invention, there are provided methods for reprogramming mature, fully differentiated cells into pluripotent stem cells or multipotent progenitor cells. The methods of the invention generally involve one or more steps of (1) chromatin remodeling, (2) induction of pluripotency, (3) selection of pluripotent cells and (4) amplification of pluripotent cells, wherein the steps may occur simultaneously or sequentially.

A starting population of cells may be derived from essentially any suitable source, and may be heterogeneous or homogeneous. In certain embodiments, the cells to be treated according to the invention are adult cells, including essentially any accessible adult cell types. In other embodiments, the cells used according to the invention are adult stem cell populations, including bone marrow, or non-adult cells such as cord and placenta cells and cord blood cells. In still other embodiments, the cells treated according to the invention include any type of adult cell that can give rise to a cancer or which can be transformed in cell culture. These cells are going to have a lower level of silencing in the important loci. In a further aspect of the invention, the induced pluripotent stem cells or multipotent progenitor cells produce according to the invention may be reprogrammed into mature cell types of interest using, for example, chemically defined media, conditions, or other manipulations, and such cells may be used for cell-based therapeutic applications. For example, in one embodiment, the methods of the invention are used in the generation of beating cardiomyocytes from pluripotent cells and the cells may be used in autologous cardiac cell therapies. In another embodiment, the methods are used in the generation of pancreatic beta-islets from pluripotent cells and the cells may be used in autologous cell therapy for Type I diabetes. In yet another embodiment, the methods are used in the generation of hematopoietic stem cells from pluripotent cells and the cells may be used in autologous hematopoietic transplants. Still further therapeutic applications according to the invention, include, e.g., modulation of adipogenesis as a treatment for metabolic disease, modulation of the proliferation and/or survival of dopanminergic neurons in the treatment of Parkinson's disease, modulation of the proliferation and/or survival of motor neurons in the treatment of ALS, etc.

1. EPIGENETIC MODULATION: CHROMATIN REMODELING

Prior to induction of pluripotency according to the invention, chromatin structure will generally require opening up, via chromatin remodeling, in order to unsilence genetic loci. This can be achieved by essentially any process known to effect chromatin remodeling, including, for example, the use of HDAC inhibitors, HAT activators, histone demethylase inhibitor, histone methyltransferase inhibitors, and the like, as further discussed below.

Chromatin accessibility plays a key role in the transcriptional regulation of cell-type specific gene expression. In eukaryotic cells, DNA is wrapped around core histones and form nucleosomes that fold into higher-order chromatin structures. DNA is packaged into nucleosomes, repeating complexes in chromatin, composed of approximately 146 base pairs of 2 superhelical turns of DNA wrapped around an octomer of pairs of histones H4, H3, H2a and H2b.

Histone acetyltransferase (HAT) and histone deacetylase (HDAC) regulate transcription by selectively acylating or deacylating the ε amino group of lysine residues located in the NH2 terminal tails of core histones. The anionic phosphate backbone of DNA strongly interacts with the cationic lysine residues of histone proteins, resulting in a condensed chromatin structure. In addition, lysine acetylation attenuates these ionic interactions, relieving the highly condensed structure of chromatin (Science, 293, 2001, 1074-1080; PNAS, 2004, 101, 47, 16659).

Further, ES cell chromatin is characterized by an overall decondensed structure, active histone marks, and a large fraction of only loosely bound proteins notably histones (BMC Developmental biology, 2007, 7:46). A. HDAC inhibitors

One illustrative approach for effecting chromatin remodeling includes the use of compounds, agents and/or other treatments that modulate the expression and/or activity of one or more HDAC enzymes, either directly or indirectly, or that modify the expression and/or activity of one or more HDAC substrates or co factors related thereto.

For example, HDAC inhibitors can induce an open chromatin conformation through the accumulation of acetylated histones, facilitating the transcription of numerous regulatory genes. There are 4 classes of HDAC enzymes. Class I, II, and IV share sequence and structural homology within their catalytic domains and share a related catalytic mechanism that does not require a co-factor, but does require a zinc (Zn) metal ion. In contrast, class III (sirtuins) do not share sequence or structural homology with the other HDAC families and use a distinct catalytic mechanism that is dependant on the oxidized form of nicotinamide adenine dinucleotide (NAD+) as a co- factor. Sirtuins have been linked to counteracting age associated diseases such as type II diabetes, obesity and neurodegenerative diseases (Oncogene, 2007, 26, 5528).

Illustrative proteins that are non-histone substrates of HDACs and that may be targeted in order to effect chromatin remodeling include, for example, DNA binding transcription factors (e.g.,p53, c-myc, AML-I, BCL-6, E2F1, E2F2, E2F3, GATA-I, GATA-2, GATA-3, GATA-4, YYl, NF-kb, MEF-2, CREB, HIF-lα, BETA-2, POP-I, IRF -2, IRF-7, SRY, EKLF), steroid receptors (e.g., androgen receptor, estrogen receptor alpha, glucocorticoid receptor), transcription co-regulators (e.g., Rb, DEK, MSL-3, HMGI(Y)/HMGA1, CtBP2, PGC-lalpha), signaling mediators (e.g., STAT-3, Smad-7, beta catenin, IRS-I), DNA repair enzymes (e.g., KU70, WRN, TDG, NEIL2, FENl), nuclear import proteins (Rchl, importin-alpha7),chaperone proteins (e.g., HSP90), structural proteins (e.g., alpha-tubulin), inflammation mediators (e.g., HMGBl) and/or viral proteins (e.g., ElA, L-HDAg, SHDAg, T-antigen, HIV tat).

Particular illustrative examples of HDAC inhibitors include, for example, butyrate; suberoylanilide hydroxamic acid (SAHA a.k.a. Vorinostat); Trichostatin A; Chlamydocin; cyclic tetrapeptide trapoxin A; and the natural product Apicidin. B. HA T activators

Chromatin remodeling may also be effected with compounds, agents and/or other treatments that modulate the expression and/or activity of histone acetyltransferases (HATs), either directly or indirectly, or that modulate the expression and/or activity of substrates or co factors thereof.

HATs are multiprotein complexes that recruit other HATs, coactivators, or corepressors. HATs can be divided into several families, including GCN5/PCAF, p300/CBP, TAFII250, and SRC. HATs do not bind directly to DNA but are recruited to sites in the promoter region by transcription factors. Histones and transcription factors such as p53, E2F1, and GATAl are known to be substrates for HATs. (The Cancer Journal, 13,1, 2007, 23). Other non histone HAT substrates include, for example, Sin Ip, HMG- 17, EKLF, TFIIEbeta, and TFIIF.

C. Histone demethylase inhibitor

Chromatin remodeling may also be effected with compounds, agents and/or other treatments that modulate the expression and/or activity of histone demethylases, either directly or indirectly, or that modulate the expression and/or activity of substrates or cofactors thereof.

With the discovery of demethylases, including lysine-specifϊc demethylase 1 (LSDl) and the JmjC domain-containing demethylases, there is now recognition that lysine methylation is a dynamic protein modification. LSDl belongs to the amine oxidase superfamily, members of which are flavin enzymes that utilize oxygen and generate hydrogen peroxide. The LSDl -catalyzed reaction converts mono- and dimethyllysine 4 of histone H3 to demethylated products. In a complex with CoREST, LSDl can efficiently demethylate histones in nucleosomes. LSDl serves as a transcriptional repressor since methylation of histone H3 can activate gene expression. LSDl is a 100 kDa protein which contains two domains, SWIRM and amine oxidase. Inhibitors of LSDl may be useful biological tools and have therapeutic properties in the treatment of diseases involving abnormal epigenetic regulation, such as cancer (Biochemistry, 2007, 46, 23, 6897 and Biochemistry, 2007, 46, 14, 4410). Illustrative examples of inhibitors of histone demethylase include trans-2- phenyl cyclopropylamine, which is an irreversible inhibitor of LSDl. Peptides-based inhibitors may also be used.

D. Histone methyltransferase inhibitor: Chromatin remodeling may also be effected with compounds, agents and/or other treatments that modulate the expression and/or activity of histone methyltransferases, either directly or indirectly, or that modulate the expression and/or activity of substrates or co factors thereof.

Links between histone methylation and DNA methylation have been demonstrated in Neurospora crassa and in plants, and experimental evidence has shown that histone methylation may be a prerequisite for DNA methylation and transcriptional silencing in Neurospora and Arabidopsis . There are also reports that DNA methylation may trigger H3-K9 methylation in Arabidopsis, suggesting interplay between histone and DNA methylation in maintaining the silent status of the chromatin. Lysine methylation in mammals occurs at the amino group of the lysine residue. The lysine residues are mono-, di-, or trimethylated. Several histone H3-K9 specific methyltransferase such as Clr4 in yeast, Suv39h in mammals, a homologue to Drosophila Su(var)3-9, SETDB 1/ESET, and G9a have been identified (J Biol Chem, 2004, 279, 51, 53248). 2. INDUCTION OF PLURIPOTENCY (CELL REPROGRAMMING)

Following one or more desired treatments to effect chromatin remodeling, as illustratively discussed above, the cells may be reprogrammed to a pluripotent state via modulation of the expression and/or activity of at least two, at least three or at least four pluripotency factors selected from Sox-2, c-Myc, Oct3/4, Klf4, Lin28 and Nanog, and/or via modulation of their respective pathways, either directly or indirectly.

In certain embodiments, the cells being reprogrammed to a pluripotent state endogenously express one or more pluripotency factor, e.g., neural cells, which express c-myc and sox-2, or spermatogonal cells, which express mRNA for c-myc, sox-2, oct4 and nanog (but which don't express the Sox-2 protein). In certain other embodiments, in addition to modulating the pluripotency factors noted above, the expression and/or activity of one or more additional pluripotency factors is modulated, wherein the additional pluripotency factor is selected from STAT-3, Rex-1 (MoI Cell Biol, 13, 2919-2928 (1993)), B-Myb (J Biol Chem, 274, 28067 (1999)), Foxd3 (J Biol Chem, 271, 23126 (1996)), Gbx2 (Genomics, 46, 223-233, 1997), UTFl (Embo J, 17, 2019-2032 (1998)), Fgf4 (MoI Cell Biol, 17, 6321-6329 (1997)), Pern (Dev Biol, 210, 481-496 (1999)), Sail 4 (Nat Cell Biol, 8, 1114 (2006)), Zic3 (MoI Biol Cell, 18, 1348 (2007)) and/or Zfx (Cell, 129, 345-357 (2007)).

Pluripotency factors, or substrates or cofactors or downstream effectors thereof, can be introduced into cells using, for example, transient methods, e.g. protein transduction, microinjection, non-integrating gene delivery, mRNA transduction, etc., or any other suitable technique. Alternatively, or in addition, pluripotency factors can be exogenous molecules contacted with or otherwise introduced into cells (e.g., small molecules, proteins, peptides, sugars, etc) which modulate the factors themselves and/or the signaling pathways within which the pluripotency factors act.

Other relevant pluripotency factors which may be used, separately or in conjunction with those noted above, can include essentially any other factors capable of modulating pathways involved in induction and/or maintenance of pluripotency. For example, in certain embodiments, a further pluripotency factor used according to the invention can include any compound, agent or other treatment that modulates MAPK (e.g., inhibitors), FGF, PI3K (e.g., activation via RAF GAP inhibitor), MEK, ERiC, GSK3 (e.g., inhibition), wnt, hedgehog and/or notch activators, as well as growth factors and/or cytokines implicated in embryonic stem cell self-renewal, including peptide derivatives.

3. SELECTION OF PLURIPOTENT CELLS

Following induction of pluripotency, cells in which reprogramming has been effected can be selected based on relevant and detectable morphological, molecular and/or biochemical changes, and the like. For example, ES cell colony morphology is well described in the literature, as are cell surface markers and these can be monitored separately or in combination. In certain embodiments, any of a number of cell surface and/or intracellular markers may be used for monitoring pluripotency, including SSEl, SSEA3/4; TRAl- 60/81; TRA2-54, GCTM-2, TG343, TG30, CD9, CD133/prominin, OCT4, Nanog and/or Sox2. In other embodiments, selection is achieved using a drug which takes advantage of differences between ES cells and somatic cells, e.g. a Gl/S phase inhibitor that will halt the growth (or kill) somatic cells, but to which ES cells are resistant. These may include, for example, MAPK pathway inhibitors. Alternatively, in other embodiments, epigenetic modulators may be used for selection of cells since normal cells die or stop growing in response to such modulators, but ES cells are resistant to such treatments.

In still other embodiments, the selection process can include the monitoring of enzymatic activities, such as AP and/or telomerase activities.

4. STEM CELL PLURIPOTENCY AND SELF RENEWAL (AMPLIFICATION)

Following selection of pluripotent cells, the cells may be amplified and maintained under suitable conditions well know in the art. Numerous pathways and/or factors important for the maintenance and/or self-renewal of stem cells may be modulated, illustrative examples of which are discussed below.

A. Wnt pathway (pluripotencv and self renewal) Wnt proteins are secreted cystein-rich proteins and about 20 have been identified in mammals. Several pathways exist through which Wnt proteins can elicit cell responses. For example, the Wnt pathway which involves beta-catenin has been shown to control the specification, maintenance and activation of stem cells. Further, Wnt signaling pathways have been implicated in the maintenance of ES-cell pluripotency.

For example, Wnt3a activity can contribute to the self-renewal of ES cells, and its activation can sustain the expression of the pluripotent stage-specific transcription factors Oct 4 and Nanog. (J Cell Sci, 120, 55-65 and Stem Cells, 20, 284, 2002). Activation of the pathway leads to inhibition of GSK3, subsequent nuclear accumulation of beta catenin and the expression of target genes. In addition, activation of the Wnt canonical pathway maintains the undifferentiated phenotype in both mouse and human ES cells, and sustains expression of the pluripotent state specific transcription factors Oct4, REX-I and Nanog (Nature Med, 10, 55-63, 2004).

Illustrative inhibitors of GSK3 include the natural products kenpaullone. azakenpaulione, hymenialdisinc, TWSl 19, and 3-(l-(3-Hydroxypropyl)-lH- ρyrrolo[2,3-b]pyridm-3-yl]-4-pyrazin-2-y!-pyrrole-2,5-dione,

B. Notch pathway (pluripotencv and maintenance)

Notch signaling controls selective cell-fate determination in a variety of tissues. The canonical Notch signaling pathway specifically regulates cell-fate decisions through close-range cell-cell interactions, and in both murine somatic and IxES cells, the cytoplasmic signals induced by Notch activation are opposed by a control mechanism that involves the ρ38 mitogen-activated protein kinase (Nature, 442, 823-826, 2006).

Inhibition of MEK/ERK by the MEK inhibitor PD098059 also inhibits differentiation and maintains ES-cell self-renewal in culture.

C. LIF (maintenance)

Fetal calf serum (FCS) and LlF are generally required for the maintenance of undifferentiated mES-cell lines in vitro (Nature, 1988, 336, 688-690): however, LIF is not necessary for the maintenance of hES cells. LIF is a soluble glycoprotein of the interleukin (IL)-6 family of cytokines and acts via a membrane bound gρ!30 signaling complex to control signal transduction and activation of transcription (STAT) signaling. One specific phosphorylation target for this signaling cascade is c-Myc, which is critical for LIF regulation of mES cells (Development, 2005, 885-896). In addition to the pathway leading to STAT3 nuclear translocation, the intracellular domains of the LIFR-gpl30 heterodimer can, on binding LIF, recruit the non receptor tyrosine kinase Janus (JAK) and the antiphosphotyrosine immunoreactive kinase (TIK) and activate other pathways. The treatment of ESCs with LIF also induces the phosphorylation of extracellular signal-regulated protein kinases, ERKl andERK2, and increases mitogen- activated protein kinase (MAPK) activity. Other members in this family of cytokines, including ΪL-6, IL-1 1, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1, all show similar properties with respect to the maintenance of the pluripotency of mES cells. Importantly, the absence of SL-6 family members, the removal of mouse embryonic fibroblasts (MEFs), or the inactivation of STAT3 (a downstream signaling molecule of the gpl30 signaling complex) promote ES cells to differentiate spontaneously in vitro (J Cell Biol, 1997, 138, 1207). LIF, when applied to serum-free ES-celi cultures, is however insufficient to maintain pluripotency, and other factors generally need to also be used in conjunction with LSF. D. TGF-betα (self renewal)

Members of the transforming growth factor-beta (TGF-β) superfamily play important roles in the biology of epiblasts and ES cells. This family, which is composed of nearly 30 members, including activin. Nodal, and BMPs, elicit their responses through a variety of cell surface receptors that activate Smad protein signaling cascades. In combination with LlF, BMPs sustain self-renewal, multi-lineage differentiation, chimera colonization, and germ-line transmission properties. An important contribution of BMP is to induce the expression of Id genes via activation of Smads 1. 5, or 8. The forced expression of Id genes frees ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Blockade of lineage specific transcription factors by Id proteins furthermore permits the self-renewal response to LIF/STAT3 signaling. Activin-Nodal signaling is, however, mediated primarily via Smads 2 and 3, and recent results have suggested that activin-Nodal-TGFβ signaling, but not BMP signaling, is indispensable for ES-cell propagation (Biochem Biophys Res Commun, 343,159-166,2006; Cell research, 2007, 17:42-49). E. FGF signaling Pathway

Autocrine FGF signaling has also been shown to be important in human ES cells and these also express FGF2, 13, and 19 which are down-regulated upon induction of differentiation. While other pathways may as well be FGF-dependent in hES cells,

FGF2 has been shown to activate the ERK/MAPK signaling cascade. (BMC Developmental biology, 2007, 7:46). F. PBK/ A KT Signaling Pathway

PBKs are a family of lipid kinases, whose products, phosphoinositide 3,4- bisphosphate (PI(3,4)P2) and phosphoinositide 3,4,5-trisphosphate (PI(3,4,5)P3) act as intracellular second messengers. Members of the three distinct classes of PBKs have been implicated in the regulation of an array of physiological processes, notably the control of proliferation, cell survival, cell migration, and trafficking. Members of the class IA family of PBKs, comprising a regulatory subunit (typically 85 or 55 kDa) and a 110 kDa catalytic subunit are known to be activated via gpl30, the signaling component of the LIF receptor. The role of phosphoinositide signaling in ES cells has been shown in reports implicating PBKs in the control of ES cell proliferation. PBKs are also involved in regulation of self-renewal of murine ES cells. Using both pharmacological and molecular tools, it has been demonstrated that PBK signaling is required for efficient self-renewal in the presence of LIF (J Biol Chem, 279, 46, 2004, 48063). Loss of self renewal upon inhibition of PBK signaling is associated with an increase in ERK phosphorylation, which appears to play a functional role in this response. Additional evidence further supports the involvement of PBK (J Biol Chem, 282, 9, 6265, 2007). The downstream molecular mechanisms that contribute to the ability of PBKs to regulate pluripotency of ES cells in murines was studied and it was shown that inhibition of PBK activity with either pharmacological or genetic tools resulted in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels.

G. Grb2/MEK pathway

A sodium vanadate-induced tyrosine phosphorylation signal to repress Nanog in murines has been shown to be transmitted via Grb2 (MoI Cell Biol, 2006, 26, 20, 7539). Grb2 is an adaptor molecule with an SH2 domain that specifically binds to a peptide motif containing a phosphotyrosine. This motif links Grb2 to downstream signaling cascades, in particular to the Sos/Ras/Raf/Mek/Erk pathway. Among the various kinase inhibitors tested, only the Mek inhibitor selectively blocked the effects of sodium vanadate on Nanog repression. Moreover, transfection of a constitutively active form of Mek mutant repressed Nanog and led to primitive endoderm differentiation.

Illustrative inhibitors of MEK include flavone PD98059. PD-325901, ARRY-142886, ARRY-438168, UOl 26 H. PBK/AKT;MAPK/ERK;

Large-scale transcriptional comparison of the hE S-NCLl line derived from a day 8 embryo with Hl line derived from a day 5 embryo (WiCeIl Inc.) showed that only 0.52% of the transcripts analysed varied significantly between the two cell lines. This is within the variability range that has been reported when hESC derived from days 5-6 embryos have been compared with each oilier. This implies that transcriptional differences between the cell lines are likely to reflect their genetic profile rather than the embryonic stage from which they were derived. Bioinformatic analysis of expression changes observed when these cells were induced to differentiate as embryo id bodies suggested that many of the downregulated genes were components of signal transduction networks. Subsequent analysis using western blotting, flow cytometry and antibody arrays implicated components of the PBEL' AKT kinase, MAPK/ERK and NFkb pathways and confirmed that these components are decreased upon differentiation. Disruption of these pathways in isolation using specific inhibitors resulted in loss of piuripotency and/or loss of viability, confirming the importance of such signaling pathways in embryonic stem cells. (Mum an Molecular Genetics, 2006, 15, 11, 18940).

POLYPEPTIDES

As noted above, the present invention, in certain aspects, provides methods for inducing, modulating and/or maintaining piuripotency by administering polypeptide-based piuripotency factors {e.g., Sox-2, c-Myc, Oct3/4, Klf4, Lin28,

Nanog, etc.), or by administering polynucleotides encoding such polypeptides, using techniques known and available in the art.

As used herein, the terms "polypeptide" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g. , they may comprise a full length protein sequence or a fragment of a full length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. Polypeptides of the invention may be prepared using any of a variety of well known recombinant and/or synthetic techniques, illustrative examples of which are further discussed below.

The present invention, in certain embodiments, employs active fragments of a polypeptide described herein (e.g. Sox-2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, etc., or a substrate, co factor and/or downstream effector thereof), for example, comprising at least about 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, etc., contiguous amino acids, or more, including all intermediate lengths, of a polypeptide described herein. In a preferred embodiment, the fragment or combination of fragments employed retain the ability to modulate, induce and/or maintain pluripotency when used in the methods described herein.

In another aspect, the present invention employs variants of the polypeptide compositions described herein (e.g. Sox-2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, etc., or a substrate, co factor and/or downstream effector thereof). Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein. . In a preferred embodiment, the variant or combination of variants employed retain the ability to induce pluripotency as described herein. In another aspect, the present invention employs polypeptide variants which exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to the corresponding region of a wild-type mammalian polypeptide used according to the present disclosure. A polypeptide variant may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences used in the methods of the invention and evaluating their effects using any of a number of techniques well known in the art.

In certain embodiments, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with an ability to modulate, induce and/or maintain pluripotency as described herein. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide of the invention, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence, e.g., according to Table 1.

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that generally defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the polypeptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said polypeptides without appreciable loss of their desired activity.

TABLE 1

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU Amino Acids Codons

Aspartic acid Asp D GAC GAU

Glutamic acid GIu GAA GAG

Phenylalanine Phe UUC UUU

Glycine GIy G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro CCA CCC CCG CCU

Glutamine GIn Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser AGC AGU UCA UCC UCG UCU

Threonine Thr ACA ACC ACG ACU

Valine VaI V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). For example, it is known that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those within +1 are particularly preferred, and those within +0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity .

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on secondary structure and hydropathic nature of the polypeptide.

Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide {e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region. When comparing polypeptide sequences, two sequences are said to be

"identical" if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) MoI. Biol. Evol. 4:406- 425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Nat 'I Acad., Sci. USA 50:726-730.

Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Nat 'I Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

In one illustrative approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

In certain embodiments of the invention, there are provided fusion polypeptides, and polynucleotides encoding fusion polypeptides. Fusion polypeptide and fusion proteins refer to a polypeptide of the invention that has been covalently linked, either directly or via an amino acid linker, to one or more heterologous polypeptide sequences (fusion partners). The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C- terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order. The fusion partner may be designed and included for essentially any desired purpose provided they do not adversely effect the desired activity of the polypeptide. For example, in one embodiment, a fusion protein may be designed to encode multiple pluripotency factors as described herein, from a single transcript. In another embodiment, a fusion partner comprises a sequence that assists in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein. Fusion proteins may generally be prepared using standard techniques. For example, DNA sequences encoding the polypeptide components of a desired fusion may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures, if desired. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Certain peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain GIy, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et ah, Gene 40:39 46 (1985); Murphy et ah, Proc. Natl. Acad. Sci. USA SJ:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides.

Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

In general, polypeptides and fusion polypeptides (as well as their encoding polynucleotides) are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

POL YNUCLEOTIDES

The present invention also provides isolated polynucleotides that encode a polypeptides of the invention and that is employed in the modulation, induction and/or maintenance of pluripotency as described herein {e.g. Sox-2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, etc., or a substrate, cofactor and/or downstream effector thereof), as well as compositions comprising such polynucleotides.

As used herein, the terms "DNA" and "polynucleotide" and "nucleic acid" refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms "DNA segment" and "polynucleotide" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

As will be understood by those skilled in the art, the polynucleotide sequences of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may be single- stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide of the invention or a portion thereof) or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the angiostatic activity of the encoded polypeptide is not substantially diminished relative to the unmodified polypeptide. The effect on the angiostatic activity of the encoded polypeptide may generally be assessed as described herein.

In additional embodiments, the present invention provides isolated polynucleotides comprising various lengths of contiguous stretches of sequence identical to or complementary to a polynucleotide encoding a polypeptide as described herein. For example, polynucleotides are provided by this invention that encode at least about 100, 150, 200, 250, 300, 350, or 400, or more, more contiguous amino acid residues of a polypeptide of the invention, as well as all intermediate lengths. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. The polynucleotides of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention, for example polynucleotides that are optimized for human and/or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison). Polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well established techniques known and available in the art. For example, polynucleotide sequences which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a truncated tyrosyl-tRNA synthetase polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non- naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be 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 may be used to construct expression vectors containing sequences encoding a polypeptide of interest 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 et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. 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; tobacco mosaic virus, TMV) or with bacterial expression vectors {e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 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 PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest 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 & Schuster, J. Biol. Chem. 264:5503 5509 (1989)); 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 et al. (supra) and Grant et ah, Methods Enzymol. 755:516-544 (1987).

In cases where plant expression vectors are used, the expression of sequences encoding polypeptides 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, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al, EMBO J. 5:1671- 1680 (1984); Broglie et al, Science 224:838-843 (1984); and Winter et al, Results Probl Cell Differ. 77:85-105 (1991)). 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, e.g., Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest. 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 the polypeptide 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 the polypeptide-encoding sequence 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 the polypeptide of interest may be expressed (Engelhard et al, Proc. Natl. Acad. Sci. U.S.A. 91:1224-1221 (1994)).

In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest 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 the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:1655-1659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, 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 portion 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 which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. et al, Results Probl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability to modulate the 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 such as CHO, HeLa, MDCK, HEK293, and Wl 38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, 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 generally preferred. For example, cell lines which stably express a polynucleotide of interest may 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 1-2 days in an enriched media before they are 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 may be 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 (Wigler et al., Cell ll'lΥh-Υhl (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. MoI. Biol. 150:1- 14 (1981)); and als or pat, which 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 & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 55:8047-51 (1988)). 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, being widely 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 et al, Methods MoI Biol. 55:121-131 (1995)). A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). These and other assays are described, among other places, in Hampton et al , Serological Methods, a Laboratory Manual (1990) and Maddox et al , J. Exp. Med. 755:1211-1216 (1983).

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 include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions 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. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be 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 of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.

In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifϊeld, J. Am. Chem. Soc. 55:2149-2154 (1963)). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

IN VIVO POLYNUCLEOTIDE DELIVERY TECHNIQUES

In certain embodiments, it will be preferred to deliver one or more pluripotency factors to a cell using a viral vector or other in vivo polynucleotide delivery technique. This may be achieved using any of a variety or well-known approaches, several of which are outlined below for purposes of illustration.

1. ADENOVIRUS

One illustrative method for in vivo delivery of one or more nucleic acid sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form of an adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus & Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (ElA and ElB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5 '-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and pro virus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure. Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et ah, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et ah, 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately 5.5 kB of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vera cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication- defective adenovirus vector for use in the present invention, since Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10^π plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet & Perricaudet, 1991; Stratford-Perricaudet et al, 1990; Rich et al, 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al. , 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle ef α/., 1993). 2. RETROVIRUSES

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas & Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975). Another approach designed to allow specific targeting of retrovirus vectors is based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification permits the specific infection of hepatocytes via sialoglycoprotein receptors. A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989).

3. ADENO-ASSOCIATED VIRUSES

AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replications is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VPl, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP 1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three viral promoters have been identified and named p5, pi 9, and p40, according to their map position. Transcription from p5 and pl9 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat & Muzyczka, 1984).

There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4.5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited for delivering the antisense constructs of the present invention.

AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.

4. OTHER VIRAL VECTORS AS EXPRESSION CONSTRUCTS

Other viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al, 1988; Horwich et α/., 1990).

With the recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. (1991) introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991). 5. NON-VIRAL VECTORS

In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. In certain embodiments of the invention, the expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty & Reshef (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.

FORMULA TIONAND ADMINISTRA TION

The compositions of the invention may comprise one or more polypeptides, polynucleotides, vectors comprising same, etc., as described herein, formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely effect the angiostatic effects desired to be achieved.

In the pharmaceutical compositions of the invention, formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g. , oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation. In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to a subject. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards. Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, polynucleotides, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al. , 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety). In certain embodiments, the delivery may occur by use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle or the like. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMSWE CLAIM:

1. A method for reprogramming a cell to a pluripotent state comprising the steps of:

(a) providing a population of cells;

(b) treating the population of cells to effect chromatin remodeling;

(c) treating the population of cells to induce pluripotency by modulating the expression and/or activity of at least three pluripotency factors selected from Sox-2, c- Myc, Oct3/4, Klf4, Lin28 and Nanog, or a substrate, cofactor or downstream effector thereof; and

(d) selecting for cells having a pluripotent phenotype.

2. The method of claim 1, wherein the cells endogenous Iy express one or more of the pluripotency factors selected from Sox-2, c-Myc, Oct3/4, Klf4, Lin28 and Nanog, or a substrate, cofactor or downstream effector thereof.

3. The method of claim 1, wherein the population of cells comprises adult cells.

4. The method of claim 1, wherein the population of cells comprises adult stem cells.

5. The method of claim 1, wherein the population of cells comprises adult bone marrow cells.

6. The method of claim 1, wherein the population of cells comprises non- adult cells.

7. The method of claim 1, wherein the population of cells comprises cord cells, placenta cells or cord blood cells.

8. The method of claim 1, wherein chromatin remodeling is effected by treatment with one or more HDAC inhibitors, HAT activators, histone demethylase inhibitors and/or histone methyltransferase inhibitors.

9. The method of claim 1, wherein chromatin remodeling is effected by treatment with one or more HDAC inhibitor selected from butyrate, suberoylanilide hydroxamic acid, trichostatin A, chlamydocin, cyclic tetrapeptide trapoxin A and/or apicidin.

10. The method of claim 1, wherein chromatin remodeling is effected by treatment with one or more histone demethylase inhibitor selected from trans-2 -phenyl cyclopropylamine and/or peptide-based inhibitors.

11. The method of claim 1 , wherein the step of treating the population of cells to induce pluripotency comprises a step of transiently transfecting at least one pluripotency factor, or a substrate, cofactor or downstream effector thereof.

12. The method of claim 1, wherein the step of treating the population of cells to induce pluripotency comprises a step of viral transduction of at least one pluripotency factor, or a substrate, cofactor or downstream effector thereof.

13. The method of claim 1, wherein the step of treating the population of cells to induce pluripotency comprises a step of microinjection of at least one pluripotency factor, or a substrate, cofactor or downstream effector thereof.

14. The method of claim 1, wherein the step of treating the population of cells to induce pluripotency comprises a step of contacting the population of cells with a small molecule, protein, peptide, sugar, or other compound, agent or treatment that effects modulation of at least one pluripotency factor, or a substrate, cofactor or downstream effector thereof.

15. The method of claim 1, wherein the step of inducing pluripotency further comprises a step of modulating the expression and/or activity of one or more additional factors selected from STAT-3, Rex-1, B-Myb, Foxd3, Gbx2, UTFl, Fgf4, Pern, Sail 4, Zic3 and/or Zfx, or a substrate, cofactor or downstream effector thereof.

16. The method of claim 1, wherein the step of selecting for cells having a pluripotent phenotype comprises monitoring a cellular marker selected from SSEl, SSEA3, SSEA4, TRA1-60/81, TRA2-54, GCTM-2, TG343, TG30, CD9, CD133/prominin, OCT4, Nanog and/or Sox2.

17. The method of claim 1, wherein the step of selecting for cells having a pluripotent phenotype comprises a step of contacting the cells with a Gl /S phase inhibitor or an epigenetic modulator that selectively inhibits the growth of somatic cells relative to pluripotent cells.

18. The method of claim 1, wherein the method further comprises amplifying the pluripotent cells selected in step (d).