Background of the Invention

Ciliary neurotrophic factor (CNTF), originally identified for its ability to support the survival of several types of peripheral ganglionic neurons, has also been shown to exert significant effects on other functions of the vertebrate nervous system. Other biological activities include supporting survival of sympathetic and sensory neurons (Barbin et al. J Neurochem 43:1468; Heymanns et al. PNAS 84:7758 (1987); and Lin et al. J. Bio Chem. 265:8942 (1990)), and promoting the differentiation of neuronal and glial cell types (Ernsberger et al. Neuron 2:1275 (1989); Suadat et al. J.Cell. Biol. 108:1807; Hughes et al. Nature 335:70 (1988); and Lillien et al. Neuron 1 :485 (1988)). This factor also promotes/enhances the survival of embryonic motor neurons (Arakawa et al. J Neuroscϊ) 10:3507 (1990); and Magal et al. £>ev. Brain Res. 63:141 (1991)), rescues motorneurons from both lesion-induced and naturally occurring death during development (M. Sendtner et al. Nature 345:440 (1990); M. Sendtner et al.. Nature 358:502 (1992); and Oppenheim et al. Science 251:1616 (1991), and increases the threshold of neuronal sensitivity to excitatory amino acid injury (Skaper et al. J. Neurosci Res. 33:330 (1992).

The effects of CNTF are mediated by interaction of CNTF with its receptor (S. Davis et al., Science 253:59 (1991); S.P. Squinto et al., Neuron 5:757 (1990); S. Davis and G. D. Yancopoulos, Curr. Opin. Cell Biol. 5:281 (1993)). Recent evidence suggests that CNTF first binds to its a receptor component and then sequentially associates with two structurally related β signal transducing receptor components, gpl30 and the leukemia inhibitory factor receptor β (LIFRβ). It is the final step in CNTF receptor complex formation, heterodimerization between the related β components, that apparently transduces a signal across the membrane and activates intracellular signaling (S. Davis et al.. Science 260:1805 (1993); and M. Murakami et al., Science 260:1808 (1993)). Dimerization of β components appears to provide a common mechanism by which cytokines related to CNTF (such as interleukin-6, LIF, and oncostatin M (M. Hibi et al., Cell 63:1149 (1990); T. Kishimoto et al. Science 258:593 (1992); T. Taga et al., Cell 58:573 (1989); D. Gearing and A. Bruce, New Biol. 4:61 (1992); D. Gearing et al., EMBO J. 10:2839 (1991); and D. Gearing et al.. Science 255:1434 (1992)) initiate signaling, although in the case of IL-6 this signal initiation results from homodimerization of gpl30 rather than heterodimerization between gp 130 and LIFRβ (S. Davis et al., Science 260:1805 (1993); and M. Murakami et al., Science 260:1808 (1993)). Consistent with the similarities between receptor subunits. secondary structure determination predict that all of these cytokines belong to an α-helical cytokine family. The predicted 3-

dimensional structures of the members of this family are characterized by a bundle of four anti-parallel helices.

Although it is known that activation of the receptors for CNTF and its cytokine relatives results in intracellular protein tyrosine phosphorylation and immediate early gene induction (N.Y. Ip et al., Cell 69:1121 (1992); K. Nakajima and R. Wall, Mol. Cell. Biol. 11:1409 (1991); K.A. Lord et al., Mol. Cell. Biol. 11:4371 (1991); M. Murakami et al., Proc. Natl. Acad. Sci. USA 88:11349 (1991)), the mechanisms by which signaling proceeds from the membrane to the nucleus has remained almost completely unknown.

Summary of the Invention

The present invention provides a method and composition for potentiating the efficacy of an α-helical cytokine in regulating development and/or maintenance of a cytokine-responsive cell, especially neuronal cells. The subject method stems from the finding that contacting a cell with an agent which induces upregulation of the intracellular concentration of a 91kd protein can produce enhanced responsiveness of the cell to treatment with an α-helical cytokine.

The subject method comprises administering a p91 -inducing agent in an amount sufficient to potentiate the trophic activity of an α-helical cytokine, such as CNTF or LIF, on a population of cells, and thereby enhance the ability of the cytokine to regulate differentiation of the treated cells, and/or prevent cell death. The subject method of treatment is generally applicable to cells responsive to a particular α-helical cytokine, wherein the cytokine also employs p91 as a part of its signal transduction pathway. Such α-helical cytokines can be selected, for example, based on their ability to induce activation of a cellular protein of the Janus kinase family, such as JAK1, JAK2, tyk2, or the like, which ultimately causes phosphorylation of p91. In one embodiment, the subject method can be used to treat neuronal cells, including nerve and glial cells, and prevent cell death or de-differentiation. In another aspect of the present invention, the subject method is a combinational therapy comprising administering either CNTF or LIF in conjunction with a p91-inducer in order to enhance the efficacy of the α-helical cytokine on non-neuronal cell-types.

Detailed Description of the Invention

The present invention provides a method for potentiating the efficacy of α-helical cytokines in regulating development and/or maintenance of a cytokine-responsive cell. The subject method stems from the finding that contacting a cell with an agent which induces

upregulation of the intracellular concentration of a 91kd protein can produce enhanced responsiveness of the cell to treatment with an α-helical cytokine.

In particular, it has been discovered that intracellular signaling pathways utilized by certain members of the α-helical cytokine family involve a 91kd protein, hereinafter referred to as "p91". As described below, it has been found that the intracellular concentration of p91 can be increased by contacting a cell with an inducing agent such as an interferon, and that the induction of elevated cellular levels of p91 can function to potentiate the biological activity of an α-helical cytokine by increasing the availability of, as a substrate for phosphorylation, an intracellular signaling protein involved in α-helical cytokine signal transduction. Thus, the subject method can be employed to increase the sensitivity of a target cell to treatment with an α-helical cytokine.

As used herein, the term "α-helical cytokine" refers to a distantly related family of cytokines which includes, based on secondary structure predictions, both hematopoietic and neuropoietic factors (Bazan. JF (1990) Immunol Today 11 :350-354; Bazan, JF (1991) Neuron 7:197-208; Yamamori et al. (1989) Science 246:1412-1416; and Rose et al. (1991) PNAS 88:8641-8645). Members of this family, whose structures are characterized by a bundle of four anti-parallel helices, include ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin-M, interleukin-11, and granulocyte colony stimulating factor (G-CSF). As a family, as well as individually, the α-helical cytokines can control the differentiation and proliferation of diverse cell types.

CNTF, to illustrate, has been shown to be able to act as both a differentiation factor and a survival factor, with the ability to prevent degeneration of neurons, as well as induce the differentiation of 02-A progenitor cells into type-2 astrocytes and promote the differentiation of sympathetic neurons and their precursors. For instance, while cultured sympatheitc neurons acquire adrenergic transmitter properties in the absence of added factors, the addition of CNTF to the cell culture induces differentiation of cholinergic transmitter properties (Ernsberger et al. (1989) Neuron 2:1275-1284). CNTF also appears to control aspects of neural cell fate within the developing CNS. The decision of O-2A progenitors to differentiate into oligodendrocytes or type 2 astrocytes appears to be influenced by CNTF. O-2A progenitors cultured in medium containing low serum invariably differentiates into oligodendrocytes. while CNTF treatment initiates type 2 astrocyte differentiation. In addition, CNTF has been shown to be able to rescue dopaminergic neurons and fragmented nerve cells from cell death, promote survival or retinal ganglion, and inhibit nerve damage induced by oligodentrocyte-damaging cytokines such as tumor nercrosis factor (TNF) such as which occurs in neurodegenerative disorders such as multiple sclerosis, AIDS-associated neurological disease, and neuronal injury casued by head trauma (Louis et al. (1993) Science 259:689-692). CNTF can also increase the threshold sensitivity of nerve cells to excitatory

amino acids (Skaper et al. (1992) J. Neuroscience Res 33:330-337). CNTF has been previously purified and cloned (see Collins et al. U.S. Patent Nos. 4997929, 5011914, and 5141856)

In addition to well characterized hematopoietic activities, the α-helcial cytokine LIF can exert biological effects on neural cell differentiation. For example, along with the wide range of activities associated with LIF, including the ability to promote the proliferation of hematopoietic stem cells, LIF can induce differentiation of sympathetic neurons to cholinergic phenotypes, promote the differentiation of neural crest cells into sensory neurons, and act as a survival factor for dorsal root ganglion neurons (Murphy et al. (1991) PNAS 89: 3498-3501; Yamamori et al. (1989) Science 246:1412-1416; Patterson et al. (1990) Cell 62:1035-1038; and Barlett et al. International Publication No. WO 91/14443). LIF has been previously purified and cloned (see Gearing et al. U.S. Patent No. 5187077)

One aspect of the present invention concerns a method for regulating neuronal cells comprising contacting the cells with a p91 -inducing agent, defined herein as an agent which causes an increase in the intracellular level of p91, in an amount sufficient to potentiate the trophic activity of an α-helical cytokine, such as CNTF or LIF, on a population of neuronal cells, and thereby enhance the ability of the cytokine to regulate differentiation and/or prevent cell death when used to subsequently, or simultaneously, treat the neuronal cells.

Another aspect of the present invention concerns a method for regulating non- neuronal cells in a similar combinational treatment as above, utilizing an α-helical cytokine in conjunction with a p91-inducer in order to enhance the efficacy of the cytokine on non- neuronal cell-types. For example, the subject method can be utilized to increase the potency of LIF for modifying bone cell function, such as to induce bone resorption and/or bone turnover in osteogenic therapies (Cornich et al. (1993) Endocrinology 132: 1359-1366; and Reid et al. (1990) Endocrinology 126: 1416-1420). Similarly, the adjunctive use of a p91- inducing agent in the subject method can be employed to boost the potency of an α-helical cytokine, such as LIF, in the expansion and maintenance of stem cells (especially hematopoietic).

The present method is generally applicable to many different in vitro embodiments, as for example, the maintenance of cells in culture, as well as the induction of differentiation of cultured cells along a path towards a particular phenotype. In an illustrative embodiment, the treated cells can be maintained in culture and used to provide in vitro assay systems. In another embodiment, the treated cells can be used as a source of tissue for transplantation in vivo. Intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate behavioral defects. In the

development of cell cultures for implantation, the use of a p91 -inducing agent in conjunction with an α-helical cytokine, such as CNTF, can prevent loss of differentiation, or where fetal tissue is used, especially neuronal stem cells, the combinatorial application of the two agents can be used to enhance differentiation. Stem cells useful in the present invention are generally known. Several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which can generate only one type of cell, such as sensory neurons, and likely represent committed progenitor cells. The role of the present method in culturing such stem cells can be to induce differentiation of the uncommitted progenitor and thereby give rise to a committed progenitor cell, or to cause further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally-differentiated neuronal cell. For example, the subject method can be used in vitro to induce and/or maintain the differentiation of neural crest cells into glial cells, schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, as well as peptidergic and serotonergic neurons. The present method is also amenable to direct in vivo application. As described below, the subject method can be utilized in the prophylaxis and treatment of neurodegenerative disorders associated with, for example, the progressive and persistent loss of certain neuronal cells. To illustrate, various embodiments of the invention contemplate providing combinatorial therapies which comprise an α-helical cytokine and a p91 -inducing agent administered to a patient whose nervous system has been damaged by trauma, surgery, ischemia, infection (e.g. polio or AIDS), metabolic disease, nutritional deficiency, malignancy, or toxic agents. In particular embodiments of the invention, the combinatorial therapy can be used to treat congenital conditions or neurodegenerative disorders, including, but not limited to, Alzheimer's disease, aging, peripheral neuropathies, Parkinson's disease, Huntington's chorea and diseases and disorders of motomeurons, including Amyotrophic lateral sclerosis (ALS).

In a preferred embodiment, treatment of ALS by administration of CNTF can be further augmented by pretreatment and/or concurrent treatment with IFNγ or other suitable p91 -inducing agent. CNTF, like other α-helical cytokines, is pyrogenic, a factor which can contribute to a relatively narrow safety margin for possible therapeutic effects. Accordingly, the synergistic effect of treatment with IFNγ can be used to alleviate detrimental side-effects associated with CNTF treatment by reducing the CNTF dosage regimen required to yield a particular biological effect. In similar fashion, the improved efficacy of CNTF derived from conjunctive use with a p91 -inducing agent can also help to offset a relatively short half-life of the cytokine.

The exact concentration of α-helical cytokine employed in the subject method will vary, depending on such factors as the mode of delivery, the ability to be sequestered at the

intended treatment site, the particular half-life of the cytokine, and any associated side effects which may vary from one patient to the next. In a preferred, yet optional embodiment, the concentration of α-helical cytokine used in the subject method can be in the range of, for example, lpg/ml to lOOng/ml in the tissue and bodily fluid surrounding the treatment site. For instance, where intravenous injection is utilized, the α-helical cytokine can be adminstered in the range of lOOμg/kg to lOOOmg/kg body weight. Similar considerations will also effect the level of p91 -inducing agent administered. For instance, in an exemplary embodiment, IFN-γ is administered to a patient to deliver a final concentration in the tissue and fluid surrounding the treatment site in the range of 0. lpg/ml to lOOng/ml. It is understood however, that the exact amounts appropriate for an individual can be assessed by one of ordinary skill in the art using no more than routine experimentation, including routine monitoring of treatment efficacy and side-effects.

In simial fashion, the dosage of p91-inducer may be varied depending on the agent, as well as the age and body weight of the patient, the condition of disease, and the like. In general, for interferon-γ, it will be desirable to raise the concentration in the treatment area to about 10 to about 4 x 10 ⁷ IU/ ml. A preferred range of tissue concentrations of interferon is from about 10 ³ to about 10 ⁷ IU/cc. Administration of the interferon composition by any appropriate route and incorporating any preferred vehicle may be repeated as often as deemed necessary to achieve the therapeutic goal. IFNγ from any source can be used in the present method, including IFNγ isolated from naturally-occurring sources and recombinant IFNγ. As used herein, IFNγ includes all proteins, peptides and polypeptides which are characterized by the biological activity of IFNγ, for example, natural and recombinant IFNγ or derivatives thereof. These include IFNγ- like compounds from a variety of sources such as natural IFNγ, recombinant IFNγ and synthetic IFNγ or combinations thereof. For example, IFNγ useful in the present method includes natural IFNγ produced in vitro by established or transformed cell lines and natural IFNγ produced in vitro by a variety of cells in response to interferon inducers. IFNγ useful in the present method also includes IFNγ produced by cloning and expression of various host/vector systems using recombinant DNA technology. Recombinant IFNγ is particularly useful because it is readily available and cost-effective.

As described herein, and especially detailed in the examples provided below, α- helical cytokines such as CNTF and LIF regulate gene expression by tyrosine phosphorylation of several intracellular proteins which, when phosphorylated, serve as DNA binding and transcriptional-activating factors. At least one of these intracellular proteins, p91, is normally sequestered in the cytoplasm of the cell. Upon tyrosine phosphorylation in response to treatment of the cell with an α-helical cytokine, phospho-p91 assembles with other activated proteins to form multimeric complexes, translocates to the nucleus, and

activates expression of a particular set of genes by binding to transcriptional regulatory sequences of each of the genes. p91 -inducing agents useful in the subject method are characterized as compounds which are able to elevate the intracellular concentration of p91, and thereby increase the responsiveness of a target cell to treatment with the α-helical cytokine by virtue of the concentration-dependent kinetics of a kinase(s) which phosphorylates p91 as part of the intracellular signaling of the cytokine. In addition, a suitable p91 -inducing agent may also elevate the phosphotyrosine content of p91, so as to generate a competent subunit of a multimeric transcriptional activator complex of the α-helical cytokine. In this manner, the treatment of the cell with the α-helical cytokine synergizes with the treatment of cells with the p91 -inducing agent, resulting in reduction of the effective dose of the cytokine necessary to bring about the same desired biological effect relative to a non-induced cell.

The p91 -inducing agent is selected on the basis of an ability to induce upregulation of p91 levels in a treated cell by, for example, increasing the expression of the protein, or inhibiting destruction of the protein (i.e. stabilizing). In an exemplary embodiment, the p91- inducing agent is a cytokine, defined here as those factors that bind to structurally related receptors characterized by a conserved pattern of cysteine residues as well as a "WSXWS" box (see, for example, Brazan et al. (1990) PNAS 87:6934-6938; and Stahl et al. (1993) Cell 74:587-590). Cytokines of this family include, but are not limited to, interferons, interleukins, colony stimulating factors, erythropoietin, and growth hormone. In yet another embodiment, the p91 -inducing agents is a growth factor, such as epithelial growth factor (EGF) or platelet-derived growth factor (PDGF), which induces accumulation of p91 in the cell, above the concentration in the equivalent un-induced cell, so as render the cell more sensitive to treatment with an α-helical cytokine. In one embodiment, the p91 -inducing agent is an interferon. Interferons (IFNs) comprise a class of protein factors which function within the hematopoietic network. There are two major types of interferons. IFNα/β and IFNγ. Gene activation by IFNs is dependent on the activation of transcriptional factor complexes which include a phosphorylated 91kd protein. As described in the examples below, the 91kd protein involved in signal transduction by IFNs is also involved in the biological activity of α-helical cytokines. IFNα- induced transcriptional complexes comprise a number of different phosphoproteins in addition to p91. IFNγ activated factors, on the other hand, comprises a homodimer of p91. As further described herein, pre-treating a cell with an IFN (such as IFNγ) results in induction of elevated p91 levels in the cell, and enhances the effectiveness of subsequent treatment with an α-helical cytokine such as CNTF or LIF.

Another aspect of the invention makes use of the identification of CNTF -responsive elements (CNTR-RE) capable of binding transcriptional complexes which comprise p91. The CNTF-RE. as described herein, can be used in assays to rapidly identify therapeutic agents able to potentiate the activity of CNTF, including potential p91 -inducing agents. In an illustrative embodiment, the CNTF-RE is used to construct a reporter gene using techniques well known in the art. The CNTF-RE can be employed, for instance, to drive expression of a detectable marker such as a luciferase, β-galactosidase, or chloramphenicol acetyltransferase (CAT; described in Example 7). Cells transfected with the reporter gene can be contacted with CNTF at threshold levels such that a minimal baseline of expression of the reporter gene is detectable. The cells are also contacted with a candidate agent, preferably at varying concentrations. An increase in the level of reporter gene expression, detectable by simple photometric techniques in the above embodiments, is indicative of a candidate agent able to potentiate the activity of CNTF.

The present screening assay can be carried out in a number of alternative embodiments including the use of phenotypic markers in the gene construct. For example, the gene construct can comprise a drug resistance marker such that proliferation of the cells under conditions requiring expression of the marker provides an easy measure of a potential p91 -inducing agent. The present assay system can also be assembled in vitro, taking advantage of, for example, cell lysates. Utilizing embodiments similar to those described above, the present assay is amenable to high throughput analysis. Agents to be tested in the present assay can be those produced by bacteria, yeast or other organisms (including plant), as well as those produced chemically.

In cetain embodiments of the present invention, the p91 -inducing agent can be generated as a chimeric protein comprising a moiety that binds a component of the extracellular matrix. Such a chimeric molecule can be useful in circumstances wherein diffusion of the p91 -inducing agent from a treatment site is undesirable, and will function to such an end by virtue of localizing the chimeric molecule at or proximate a treatment site. A p91 -inducing agent of this embodiment can be generated as the product of a fusion gene, or by chemical cross-linking. A number of proteins have been characterized from the extracellular matrix (ECM) of tissues that will support the localization of a chimeric inducing agent, such as IFNγ, at a target site. One example of a well characterized protein is fibronectin. Fibronectin is a large adhesive glycoprotein with multiple functional domains. Several of these domains have matrix attachment activity. For example, one of these is a single "type-Ill repeat" which contains a tetrapeptide sequence R-G-D-S (Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al. (1985) EMBO 4:1755-9). Peptides as small as pentapeptides containing these amino acids are able to support attachment to a cell through binding ECM components

(Ruoslahti et al. (1987) Science 238:491-497; Pierschbacheret al. (1987) J. Biol. Chem. 262:17294-8.: Hynes (1987) Cell 48:549-54; and Hynes (1992) Cell 69: 1 1-25). In fact, several companies have commercialized products based on this cell attachment sequence for use as reagents in cell culture and various biomaterials applications. See, for example, recent catalogs from Telios Pharmaceutical, BRL, Stratagene, Protein Polymer Technologies etc., as well as U.S. Patent Nos. 4,517,686; 4,589,881 ; 4,578,079; 4,614,517; 4,661,1 1 1; and 4,792.525.

In another embodiment, the p91 -inducing agent can comprise a recombinant intracellularly-localized protein(s) that is the product of gene therapy (in vivo) or simple transfection (in vitro). The p91 -inducing agent can be derived from, for example, receptor subunits involved in the activation of Janus kinases. Signaling by IFNs is initiated when the IFNs interact with, and cause association of, their cognate multimeric receptors. The binding of IFNs activates cytoplasmic tyrosine kinases, such as Tyk2, presumably through interactions of the kinase, or some intermediate regulatory protein, with the cytoplasmic domains of associated receptor subunits. Therefore, utilizing technology similar to that applied in creating single-chain antibodies, single-chain associated subunits, particularly cytoplasmic domains, can be generated in a cell and localized to the cytoplasmic side of the cell membrane. Such single-chain subunits are generated in the form of fusion proteins, with one of the two cytoplasmic domains utilized in the fusion protein also including its transmembrane domain. It may be necessary in some instances to introduce an unstructured polypeptide linker region between the polypeptide sequences derived from each of the intracellular domains. This linker can facilitate enhanced flexibility of the fusion protein allowing each receptor subunit domain to freely interact with the other, reduce steric hindrance between the two fragments, as well as allow appropriate folding of each fragment to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly4Ser)3 can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85:4879; and U.S. Patent No. 5,091,513. In such a manner, an artificially associated complex of the intracellular domains making up, for example, the IFNγ receptor complex can be generated at the cytoplasmic face of the cell membrane, and used to recruit and activate kinases in a manner analogous to the wild-type receptor, though as a constitutively active pathway. Thus, a cell expressing a construct of this type can be induced to sustain a higher intracellular concentration of p91 , and thereby be rendered more sensitive to treatment with an α-helical cytokine. Moreover, expression of the p91 -inducing agent from the recombinant gene construct can be controlled at least in part by a transcriptional regulator responsive to the α-helical cytokine. For

example. expression of the p91-inducer can be controlled by constructing the gene encoding the inducing agent with the CNTF-RE as a part of its regulatory sequences. Initial contact of the cell with CNTF results in expression of the p91 -inducing agent and, concurrently, upregulation of p91 levels in the cell. Subsequent or continued exposure of the p91- upregulated cell to CNTF will result in improved efficacy and therefore permit lower doses to be used. The fortitude of the self-amplification by CNTF exposure can be controlled such as by the choice of associated promoter and enhancer sequences, as well as the number of CNTF-RE sequences (i.e. repeats) used in the gene construct.

In one embodiment, the present method is applicable to cell and tissue culture techniques. Accordingly, the present invention provides a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or promoting proliferation of cultured cells responsive to an α-helical cytokine, by contacting the cells with a p91 -inducing agent, such as IFNγ, to potentiate the biological activity of an α-helical cytokine which is also applied to the cell culture. For instance, in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). Once a neuronal cell has become terminally- differentiated it typically will not change to another terminally differentiated cell-type. However, neuronal cells can nevertheless readily lose their differentiated state. This is commonly observed when they are grown in culture from adult tissue, and when they form a blastema during regeneration. The present method provides a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of other trophic factors, as well as in cell cultures designed to screen compounds for their effects on neuronal tissue.

Several α-helical cytokines, including CNTF, LIF and IL-6, have been shown to be able to induce differentiation of neuronal cells, as well as increase the survival of neuronal cells in culture. The cultured cells can be contacted with an α-helical cytokine in order to induce neuronal differentiation (e.g. of a stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of differentiation. Co-application of a p91 -inducing agent with the α-helical cytokine can be used to lower the dose of cytokine required to maintain the cells. In an illustrative embodiment of the subject method, the cultured cells can be contacted with both IFNγ and CNTF in order to induce neuronal differentiation (e.g. to a cholinergic phenotype), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of differentiation.

The source of each of the p91 -inducing agent and α-helical cytokine in the culture can be derived from, for example, a purified or semi-purified composition added directly to the cell culture media, or alternatively, released from a polymeric device which supports the growth of various neuronal cells and which has been doped with at least one of the two agents. In other embodiments, the source of the p91 -inducing agent and/or the α-helical cytokine can be a cell that is co-cultured with the intended target cell and which produces a naturally occurring or recombinant form of one of the two agents. Alternatively, the source may be the target cell itself which as been engineered, for example, to produce a recombinant p91 -inducing agent so as to render itself more susceptible to the α-helical cytokine then other cells, particularly neighboring cells, not expressing the p91 -inducing agent.

Neural cell cultures generated by the subject method can also have an ultimate use as a source of implantable cells. Intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J. Exp. Biol. 123:265-289; and Freund et al. (1985) J. Neurosci 5:603- 616). Fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate neurological defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells.

Thus, use of the present method for maintenance of neuronal cell cultures can provide a source of implantable neuronal tissue. The present method can be used in vitro to induce and/or maintain the differentiation of neural progenitor cells into glial cells, schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, as well as peptidergic and serotonergic neurons. In certain embodiments, the cultured cells can be transformed with a plasmid or vector encoding an expressible p91 -inducing activity (e.g., IFNγ or other p91- inducing cytokine, or a single-chain IFNγ receptor) such that the implanted cells can be regulated by treatment with CNTF, LIF or another α-helical cytokine, at doses below the threshold of sensitivity of the surrounding host cells.

Another objective of the present invention concerns the direct in vivo application of the present combinatorial method to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system. As set out above, the biological activity of α-helical cytokines such as CNTF and LIF includes the ability to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and death which result from loss of

differentiation in certain pathological conditions. In light of this understanding, the present invention specifically contemplates applications of the subject method to the treatment of

(prevention and/or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute. or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease. Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; and (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis.

Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treatable with a therapeutic regimen which includes a hedgehog agonist. For example, Alzheimer's disease is associated with deficits in several neuro transmitter systems, both those that project to the neocortex and those that reside with the cortex. For instance, the nucleus basalis in patients with Alzheimer's disease have been observed to have a profound (75%) loss of neurons compared to age-matched controls. Although Alzheimer's disease is by far the most common form of dementia, several other disorders can produce dementia. Several of these are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalmus or the white matter underlying the cerebral cortex. Here, the cognitive dysfunction results from the isolation of cortical areas by the degeneration of efferents and afferents. Huntington's disease involves the degeneration of intrastraital and cortical cholinergic neurons and GABAergic neurons. Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum. Treatment of patients suffering from such degenerative conditions can include the combinatorial application of a p91-inducer and an α-helical cytokine, such as CNTF or LIF, in order to control, for example, differentiation and apoptotic events which give rise to loss of neurons (e.g. to enhance survival of existing neurons) as well as promote differentiation and repopulation by progenitor cells in the area affected. In preferred embodiments, a source of one or both of the agents is stereotactically provided within or proximate the area of degeneration.

In addition to degenerative-induced dementias, the present combinatorial therapy can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements. Parkinson's disease, for example. primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism

is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident. Also included are neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders. Examples include chronic atrophies such as amyotrophic lateral sclerosis (ALS), Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive bulbar palsies or spinal muscular atrophies. The present method is amenable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion. For instance, the present combinatorial method can be used to treat a restricted form of cerebellar cortical degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.

The present combinatorial approach of providing a p91 -inducing agent and an α- helical cytokine can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle and endocrine tissue (such as glandular tissue). For instance, the subject method can be used to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.

In another illustrative embodiment, the subject method is used to treat amyotrophic lateral sclerosis. ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in spinal cord and the upper motor neurons in the cerebral cortex. Preclinical and clinical trials have demonstrated that CNTF can be useful in preventing or slowing degeneration of the primary neurons that ultimately lead to death for persons suffering from ALS. The treatment of ALS with CNTF may therefore be further augmented by treatment of the individual with a p91 -inducing agent sufficient to upregulate p91 levels and thereby potentiate the biological effects of CNTF on the CNTF- treated neuronal cells.

In addition to enhancing CNTF activity in ALS treatments, the subject method can be used to improve the efficacy of CNTF in other therapeutic applications. For instance, preclinical research indicates that CNTF may also be an effective treatment to prevent damage to peripheral nerve cells resulting from conditions such as diabetes and kidney dysfunction, and from the toxic effects of chemotherapeutic agents used to treat cancer and AIDS patients. Both the p91 -inducing agent and the α-helical cytokine, or pharmaceutically acceptable salts thereof, may be conveniently formulated for administration with a

biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredients in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein. "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of either the p91 -inducing agent or the α-helical cytokine, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on the above, the pharmaceutical formulation includes, although not exclusively, solutions or a freeze-dried powder of at least one of the p91 -inducing agent and the α-helical cytokine (such as CNTF or LIF) in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. For illustrative purposes only and without being limited by the same, possible composition of formulations which may be prepared in the form of solutions for the treatment of nervous system disorders with an α-helical cytokine are given in the della Valle U.S. Patent No. 5,218,094. In the case of freeze-dried preparations, supporting excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH. Similar solutions may also be used for the pharmaceutical compositions of the α-helical cytokine in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH. for example, neutral pH.

Methods of introducing each of the α-helical cytokine and the p91 -inducing agent at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection: intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

Methods of introduction may also be provided by rechargable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in

recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of at least one of the α-helical cytokine and the p91 -inducing agent at a particular target site. Such embodiments of the present invention can be used for the delivery of exogenously purified α- helical cytokine and p91 -inducing agent, which has been incorporated in the polymeric device, or for the delivery of at least one of the α-helical cytokine and the p91 -inducing agent produced by a cell encapsulated in the polymeric device.

An essential feature of certain embodiments of the implant is the linear release of the α-helical cytokine and the p91 -inducing agent, which can be achieved through the manipulation of the polymer composition and form. By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled. The selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666. In another embodiment of an implant, a source of cells producing one or both of the α-helical cytokine and the p91 -inducing agent, or a solution of hydrogel matrix containing a purified α-helical cytokine and/or p91 -inducing agent, is encapsulated in implantable hollow fibers. Such fibers can be pre-spun and subsequently loaded with both agents (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can be co-extruded with a polymer which acts to form a polymeric coat about a source of both agents (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotechnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55).

It is also contemplated by the present invention that CNTF-responsive genes, as well as genes responsive to other α-helical cytokines, can be identified and their gene products characterized. To illustrate, the involvement of tyrosine phosphorylated p91 and p91 related proteins in the CNTF and LIF signaling pathways has a number of significant ramifications. It provides a rationale for identifying CNTF responsive genes in neurons. As shown in Table 1 of Example 7, a number of CNTF responsive genes contain DNA sequences in their promoters that are similar to the CNTF-RE described herein. The analysis of promoters of other neuronal genes may identify novel CNTF responsive genes that encode proteins that mediate the biological effects of CNTF in the nervous system. Once isolated, the genes

regulated by CNTF can be sequenced and their distribution can be determined by in situ hybridization approaches. Function of the proteins encoded by the CNTF responsive genes can be determined by standard techniques.

For example, within the promoter of a superoxide dismutase gene, the rat SODl gene (For promoter sequence of rat SODL see Genbank accession number Z21917; for human c-fos promoter, see B. Wagner et al. EMBO J. 9:4477 (1990); for mouse c-fos. see L.A. Berkowitz et al. Mol. Cell. Biol. 9:4272 (1989); for mouse tisl l see R.N. Dubois et al. J. Biol. Chem 265:19185 (1990); for rat junB, see Z. Kawakami et al., Nuc. Acids. Res. 20:914 (1992)), we have noted a CNTF-RE like sequence. The presence of this sequence element within the SODl promoter suggests the easily testable prediction that transcription of SODl may be induced by CNTF. Interestingly, mutations of SODl have been shown to occur consistently in familial amyotrophic lateral sclerosis (D. R. Rosen et al., Nature 362:59 (1993)). a dominantly inherited degenerative motor neuron disease. Thus, one mechanism by which CNTF may protect motor neurons from degeneration could be through enhanced expression of SOD 1.

Furthermore, understanding the significance of the p91 mediated signaling pathway with respect to the biological effects of CNTF on neurons may provide novel approaches for studying the pathophysiology of human motor neuron disorders, and may also provide insight into the design of novel therapeutic strategies to combat these disorders.

Exemplification

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

To determine whether the mechanisms by which the CNTF signal is propagated utilize intermediaries in the interferon signaling pathways, we initially focused our attention on a 91kd protein (p91), which becomes tyrosine phosphorylated in the cytoplasm upon exposure to IFNα or IFNγ (C. Schindler et al. Science 257:808 (1992); K. Shuai et al. Science 258:1808 (1992)). Once phosphorylated in interferon stimulated cells, p91 translocates to the nucleus where it participates in the transcriptional activation of interferon responsive genes. IFNγ induces the binding of p91 to the GAS (K. Shuai et al. Science 258:1808 (1992)) within the promoters of IFNγ responsive genes. IFNα. on the other hand, causes the association of p91 with two newly tyrosine phosphorylated 84kd and 1 13kd proteins (p84 and pl!3

respectively) (C. Schindler et al. Science 257:808 (1992)) and with a 48kd protein to form the complex ISGF3, which then binds a distinct promoter site upstream of IFNα responsive genes (X. Fu et al. Proc. Natl. Acad. Sci. USA 89:7840 (1992); D. E. Levy et al. Genes Dev. 2:383 (1988)).

Example 1 We investigated if a protein related or identical to p91 is expressed in the cell line SK-N-MC, which is a human neuroblastoma cell line that expresses functional CNTF receptors (S. Davis et al., Science 253:59 (1991); S.P. Squinto et al., Neuron 5:757 (1990); S. Davis and G. D. Yancopoulos, Curr. Opin. Cell Biol. 5:281 (1993)). CNTF induces transcription of the immediate early gene c-fos in SK-N-MC cells indicating that the intracellular mechanisms that transduce the CNTF signal from the receptor to the nucleus are intact. Anti-p91C antibodies, which are directed against the carboxyl terminus of p91 and recognize HeLa cell p91 but not p84 (K. Shuai et al. Science 258:1808 (1992)), immunoprecipitated a 91kd protein from ³⁵ S-methionine labeled extracts of SK-N-MC cells. This protein comigrated with p91 immunoprecipitated from HeLa cells with the anti-p91C antibodies. Gel slices containing radioactively labeled 91kd protein from SK-N-MC cells and HeLa cells were subjected to partial digestion with V8 protease (D.W. Cleveland et al. J. Biol. Chem. 252:1102 (1977)). Analysis of the resulting peptides revealed an identical pattern for the two proteins indicating that the 91kd proteins in HeLa and SK-N-MC cells are closely related or identical.

To determine whether p91 from SK-N-MC cells becomes modified upon exposure to CNTF, protein immunoblotting was carried out with antibodies to p91 which recognize p91 and p84. as they are directed against amino acids 598 to 705 of p91 which are shared with p84 (K. Shuai et al. Science 258:1808 (1992)). In extracts from untreated SK-N-MC cells, anti-p91 antibodies recognized p91 as well as p84. However, in extracts obtained from cells treated with CNTF (100 ng/ml), an additional band that migrates more slowly than p91 was detected. This band comigrates with the newly phosphorylated p91 detected in extracts of IFNα treated HeLa cells, suggesting that CNTF induces a similar modification of p91. The kinetics of the CNTF induced modification of p91 were rapid and transient. Newly modified p91 was detected within 1 minute, reached maximum levels within 15 minutes, and disappeared by 60 minutes. The induction of the p91 modification occurred with as little as 1 ng/ml of CNTF, which is within the physiological range of CNTF response in biological assays (P. Masiakowski et al., J. Neurochem 57:1003 (1991)). Induction of the p91 modification upon treatment with CNTF correlated with the expression of functional CNTF receptors, since the modification was detected in SK-N-MC cells and MAH-B2 cells, both of which express the CNTF receptor, but not in HeLa or 3T3 cells, which do not express this

receptor (S. Davis et al.. Science 253:59 (1991); S.P. Squinto et al., Neuron 5:757 (1990); S. Davis and G. D. Yancopoulos, Curr. Opin. Cell Biol. 5:281 (1993)). In SK-N-MC cells pretreated with IFNγ for 18 hours, levels of p91 were significantly increased and the CNTF stimulated modification of p91 was more easily detected. In making this observation, untreated SK-N-MC cells, as well as CNTF-treated SK-N-MC cells (lOOng/ml) were compared with cells pretreated with IFN-γ (2ng/ml) and then exposed to CNTF (lOOng/ml). In the IFN-γ treated cells, a dramatic increase in the overall p91 signal, as well as the phospho-p91 signal, was observed relative to the untreated and CNTF(only)-treated cells.

Example 2

To investigate the nature of the CNTF induced modification of p91 in SK-N-MC cells, phosphotyrosine immunoblotting of immunoprecipitated p91 was carried out. In anti-p91C immunoprecipitates of extracts from untreated SK-N-MC cells, antiphosphotyrosine antibodies did not recognize any proteins within the 91kd MW range. However, in anti-p91C immunoprecipitates of CNTF treated cells, a phosphotyrosine containing protein was detected that migrates with the same mobility as the CNTF induced modified p91. The levels of p91 in untreated and CNTF stimulated cells were similar as assessed by anti-p91 immunoblots and anti-p91C immunoprecipitates of cells labeled with ³⁵ S-methionine. Taken together, these data suggest that the CNTF induced modification of p91 is due to phosphorylation of this protein on tyrosine residues.

To confirm that p91 becomes phosphorylated on tyrosine when SK-N-MC cells are exposed to CNTF, these cells were labelled in vivo with ³² P-orthophosphate and were then immunoprecipitated with the anti-p91C antibodies. Although phosphorylated p91 was immunoprecipitated from untreated cells, CNTF caused a 4 fold increase in ³² P phosphate incorporation into the immunoprecipitated protein, which was accompanied by the appearance of an immunoprecipitated band of slower electrophoretic mobility. To analyze directly which amino acids on p91 were phosphorylated, the immunoprecipitated p91 bands were excised and subjected to phosphoamino acid analysis (W.J. Boyle et al. Meth. Enz. 201 :110 (1991)). p91 from untreated cells contained only phosphoserine, whereas p91 from stimulated cells contained phosphotyrosine in addition to phosphoserine. Taken together, these experiments indicate that CNTF induces the rapid tyrosine phosphorylation of p91 in SK-N-MC cells.

Example 3

We next considered the possibility that while CNTF and the interferons are similar in their ability to stimulate tyrosine phosphorylation of p91, there might be aspects of the CNTF signaling pathway that would distinguish it from the IFNα and IFNγ pathways. In contrast to

IFNα. which stimulates the tyrosine phosphorylation of p84 and pi 13 in addition to p91 (C.

Schindler et al. Science 257:808 (1992)), thereby promoting the formation of the protein complex ISGF3a. CNTF failed to stimulate the phosphorylation of pll3 or to induce ISGF3a complex formation. Instead, the induction of p91 tyrosine phosphorylation that occurs upon exposure to CNTF is accompanied by the tyrosine phosphorylation of two p91 related proteins. A close examination of phosphotyrosine blots of anti-p91C immunoprecipitates revealed that CNTF induced not only the tyrosine phosphorylation of p91, but also the tyrosine phosphorylation of two other proteins that are slightly smaller than p91, p88 and p89. Each of these proteins is believed to be members of a family of related transcription factors, inasmuch as two different antibodies that recognize distinct regions of p91 also recognize p88 and p89 in immunoprecipitation and phosphotyrosine immunoblotting experiments. Antibodies directed against the NH ₂ terminus of p91 (anti-p91N) recognized p88 and p89 even more effectively than p91, suggesting that p88 and p89 are not simply degradation products of p91. A further indication that p88 and p89 are not likely to be degradation products of p91 comes from the finding that tyrosine phosphorylation of these two proteins is not detected in cells treated with IFNα or IFNγ. Since CNTF but not IFNα or IFNγ induces the tyrosine phosphorylation of p88 or p89, we conclude that the intracellular mechanisms by which the CNTF signal is propagated are partly distinct from the IFNγ and IFNα signaling pathways.

Example 4 We also found that tyrosine phosphorylation of p91 and the p91 related proteins was also stimulated in SK-N-MC cells by activation of the LIF receptor, which shares its signal transducing subunits with the CNTF receptor (S. Davis et al., Science 260:1805 (1993); and M. Murakami et al., Science 260:1808 (1993); N.Y. Ip et al., Cell 69:1121 (1992)). However, exposure of SK-N-MC cells to basic fibroblast growth factor (bFGF), which interacts with a receptor tyrosine kinase (P. L. Lee et al. Science 245 57 (1989)) that is structurally unrelated to the CNTF and LIF receptors, was ineffective at inducing tyrosine phosphorylation of p91 or the p91 related proteins. The presence of functional FGF receptors on SK-N-MC cells was demonstrated by the ability of bFGF to stimulate c-fos transcription in these cells. CNTF, LIF conditioned medium, and bFGF were all found to activate c-fos mRNA within minutes of treatment. In addition to bFGF, the neurotrophin NGF was also found to be ineffective in stimulating tyrosine phosphorylation of p91 or the p91 related proteins in the pheochromocytoma cell line PC 12, as determined by immunoprecipitation and phosphotyrosine immunoblotting experiments. While we cannot rule out the possibility that bFGF and NGF induce p91 tyrosine phosphorylation below the level of detection of the immunoblotting and immunoprecipitation assays, it is possible that CNTF and LIF are distinct among the neurotrophic factors in their ability to stimulate the rapid and robust tyrosine phosphorylation of p91 and the p91 related proteins.

Example 5

To determine whether the CNTF-induced phosphorylation of p91 is correlated with functional activation of this protein, we investigated the effect of CNTF on p91 subcellular localization and DNA binding activity. Immunofluorescence staining with anti-p91C antibodies revealed that p91 is diffusely localized within the cytoplasm and to a lesser extent in the nucleus of untreated SK-N-MC cells. However, after treatment with CNTF for 10 minutes, p91 was detected predominantly in the nucleus. The redistribution of p91 observed by immunofluorescence was confirmed by a comparison of anti-p91 immunoblots of whole cell and nuclear extracts. In extracts obtained from SK-N-MC cells pretreated with IFNγ for 18 hours and then treated briefly with CNTF, the unmodified and tyrosine phosphorylated forms of p91 were detected in both whole cell and nuclear extracts. However, the ratio of the inducibly phosphorylated form of p91 to the unmodified form was much greater in the nuclear extracts than in whole cell extracts. In addition, the overall level of p91 in the nuclear extract was increased in CNTF treated cells when compared to untreated cells. When SK-N-MC cells were fractionated into cytoplasmic and nuclear components, p91 was found to be predominantly in the cytoplasmic fraction prior to CNTF treatment. Following CNTF treatment, the level of p91 was increased in the nuclear fraction due to the appearance of the tyrosine phosphorylated p91 in this fraction. In contrast to p91, the transcription factor cyclic AMP response element binding protein, CREB, was found exclusively in the nuclear fraction before and after CNTF stimulation, confirming the integrity of the subcellular fractions. Taken together with the immunofluorescence results, these subcellular fractionation experiments indicate that CNTF treatment induces p91 translocation to the nucleus.

Example 6 The observation that CNTF treatment of SK-N-MC cells stimulates the translocation of p91 to the nucleus is consistent with the idea that p91 plays a role in CNTF induced changes in gene expression. To investigate this possibility, we examined the effect of CNTF treatment on the DNA binding properties of p91. Tyrosine phosphorylated p91 has been previously shown to recognize the DNA sequence 5'-TTCCNNNAA-3', termed GAS, present within promoters of interferon responsive genes (K. Shuai et al. Science 258:1808 (1992); D. J. Lew et al. Mol. Cell Biol. 11 :182 (1991); T. Decker et al. EMBO J. 10:927 (1991); R. N. Pearse et al. Proc. Natl. Acad. Sci. USA 90:4314 (1993)). Using a DNA mobility shift assay ( D. E. Levy et al. Genes Dev. 3:1362 (1989)), we tested nuclear extracts prepared from SK-N-MC cells for the presence of a factor that interacts with representative GAS sequences. A double stranded oligonucleotide, which we have termed a CNTF response element (CNTF-RE; see below) was incubated with nuclear extracts from SK-N-MC cells. When nuclear extracts were prepared from SK-N-MC cells pretreated with IFNγ followed by brief exposure to CNTF, a DNA protein complex, termed "Complex I" was detected. This complex

was not detected in the absence of CNTF. Complex I formation was effectively competed by the inclusion of an excess of unlabeled CNTF-RE DNA or a GAS sequence in the reaction mix. Complex I comigrated on a non-denaturing polyacrylamide gel with a gamma activated factor (GAF) DNA complex detected when nuclear extracts of IFNγ treated U937 cells were incubated with the CNTF-RE. This suggested that the CNTF stimulated complex I and GAF might be composed of the same or related proteins. Indeed, complex I formation was prevented by addition to the reaction mix of either of two antibodies to p91, anti-p91C or anti-p91N. The anti-p91C antibodies displayed greater effectiveness than anti-p91N antibodies in preventing complex I formation. Taken together, these data indicate that complex I is composed primarily of p91.

We also tested nuclear extracts from SK-N-MC cells not pretreated with IFNγ for the ability to form DNA complexes with a GAS DNA sequence. In this case, CNTF treatment of SK-N-MC cells induced the formation of complex I but also of two additional complexes. II and III. which migrate more slowly than complex I. While complexes II and III were of lower intensity than complex I in IFNγ pretreated SK-N-MC cells, they were clearly detected with longer autoradiographic exposure. Just as in IFNγ pretreated SK-N-MC cells, in naive SK-N-MC cells complex I induced by CNTF was found to be composed primarily of p91, since it comigrated on a non-denaturing polyacrylamide gel with the GAF DNA complex from U937 cells, and its formation was inhibited by anti-p91 and anti-p91C antibodies. Complex II also contains p91, as its formation was prevented by anti-p91 anti-p91C antibodies. Complex III does not appear to contain p91, since the anti-p91 and anti-p91C antibodies, which prevent complex I formation effectively, failed to prevent complex III formation. However, complex III appears to contain one or more p91 related proteins, since its formation was effectively inhibited by the addition of anti-p91N antibodies to the binding reaction. The proteins present in complex III may be related to the p91 related proteins, p88 and p89, since the anti-p91N antibodies recognized these two proteins effectively by immunoprecipitation and phosphotyrosine immunoblotting. It is noteworthy that complexes II and III are detected more readily in nuclear extracts from naive SK-N-MC cells exposed to CNTF than in extracts of IFNγ pretreated SK-N-MC cells treated with CNTF. This may be due to the differential ability of IFNγ pretreatment to enhance levels of p91 relative to the p91 related proteins. Due to its enhanced level, p91 might be forming complexes with target DNA sequences more effectively than the p91 related proteins in IFNγ pretreated SK-N-MC cells. Nevertheless, the finding that in naive SK-N-MC cells CNTF induces the binding of p91 as well as p91 related proteins to the GAS revealed a distinguishing feature of the CNTF signaling pathway, since IFNα and IFNγ have been found to trigger p91 binding but not binding of the p91 related proteins.

Example 7

The observation that CNTF treatment of SK-N-MC cells leads to the enhanced interaction of p91 and p91 related proteins with specific DNA sequences suggested that these proteins might be key regulators of changes in gene expression that are triggered by CNTF. Consistent with this possibility is the finding that DNA sequence elements similar to the consensus DNA sequence required for p91 binding are present upstream of a number of genes previously found to be induced by CNTF (Table 1 below). Therefore, the ability of p91 binding sites to confer CNTF responsiveness to a non-responsive reporter gene was examined. SK-N-MC cells were transfected with a reporter gene containing -71 to +109 of the mouse c-fos gene fused to the bacterial chloramphenicol acetyltransferase gene (-71fosCAT). To assess transfection efficiency, the human α-globin gene, driven by the simian virus 40 early promoter, was cotransfected. Expression of the transfected genes and the endogenous human c-fos gene was deterrnined by a ribonuclease protection assay (M. Shenget al. Science 252:1427 (1991); M. Sheng et al. Neuron 4:571 (1990)). After normalization to the level of α-globin message, the -71fosCAT gene was found to be expressed at the same low levels both before and after CNTF treatment. However, exposure to CNTF resulted in a 2-fold induction of the construct GAS/-71fosCAT, in which two copies of a palindromic GAS site were inserted immediately upstream of nucleotide -71 in the -71fosCAT gene. Two copies of another p91 binding DNA sequence were particularly effective at conferring a CNTF response when inserted upstream of nucleotide -71 in the -71fosCAT gene, resulting in an 8.5-fold induction of fosCAT transcription. Since this particular p91 binding DNA sequence is very effective in conferring CNTF responsiveness, we have termed it a CNTF response element, CNTF-RE. Taken together with the protein phosphorylation and DNA binding studies described above, these gene expression experiments suggest that in untreated SK-N-MC cells, the CNTF-RE is not occupied, and that CNTF treatment induces the tyrosine phosphorylation of p91 and the p91 related proteins, with subsequent translocation and binding of these proteins to the CNTF-RE. This then leads to activation of transcription of genes containing this DNA element within their promoters. Table 1

DNA sequences resembling the CNTF-RE core in promoters of CNTF responsive genes Gene Sequence

Human c-fos H___CCGTCAΔ SEQ ID No. 1

Mouse c-fos IXCCCGTCAA SEQ ID No. 1

Mouse tis 11 ZICCTAAGAΔ SEQ ID No.2

Rat junB* HCCGGG SEQ ID No.3

Rat SOD- 1 * _H___-_TTGA__. SEQ ID No.4

CNTF-RE (core) HCCCCGAΔ SEQ ID No.5 potential CNTF responsive genes.

All of the above-cited references and publications are hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific methods, assays and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

SEQUENCE LISTING

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