WO2002094842A2 - Control of myogenesis by modulation of p38 map kinase activity - Google Patents

Abstract

Systems, devices, and methods are disclosed in which p38 MAPK activity modulators promote myogenesis, suppress development of other cell types, or suppress myocyte proliferation for treatment or diagnosis of medical conditions, tissue repair, or creation of experimental models.

Description

CONTROL OF MYOGENESIS BY MODULATION OF P38 MAP KINASE ACTIVITY

Background

Mitogen-activated protein kinases (MAPKs) are components of several important signaling pathways in eukaryotic cells, including the extracellular signal- regulated kinases (ERK1 and -2), the Jun-N-terminal kinases (JNK1, -2, and -3), and the p38 isoforms (alpha, beta, gamma, and delta) (Obata et al. 2000; Chang and Karin 2001; Chen and Cobb 2001; Kyriakis and Avruch 2001; Pearson et al. 2001). Recent studies have begun to identify functions for some of these MAPK pathways in developmental programs. Of particular interest, p38 MAPK appears to play a critical role in myogenic differentiation or "myogenesis."

Myogenesis is the formation of muscle cells or fibers. It is controlled by an intricate network of intracellular and extracellular cues. Among these cues are the myogenic regulatory factors (MRFs), and the myocyte enhancer factors (MEFs) (Perry and Rudnick 2000). In mammals, the MRF group includes MyoD, myogenin, Myf5, and MRF4/herculin/Myf6. All of the MRFs are expressed solely in skeletal muscle, becoming sequentially activated throughout myogenesis. These factors are nuclear phosphoproteins that transactivate muscle-specific genes containing one or more DNA binding sites with the general consensus sequence CANNTG, or E-box motifs. The MRFs bind to these E-box motifs as heterodimers with the ubiquitously expressed E- type bHLH transcription factors such as El 2/47, ITF-2, and HEB.

The second major group of myogenic factors — the MEF2 family — also includes four factors, MEF2A, B, C, and D which contain both a MADS-box domain and a MEF2 domain (Naya and Olson 1999). These factors bind to A+T rich MEF2 sites which co-exist within the promoters of muscle-specific genes with E boxes. In this respect, the MRFs and the MEF2 factors synergistically activate muscle-specific promoters, to induce the formation of muscle. The roles of the MRFs and MEFs in myogenesis have been studied extensively.

Moreover, there have been several extracellular factors found to modulate the expression of the MRFs and/or MEF2 factors, in turn influencing myogenesis. Of these factors, insulin and insulin-like growth factor positively regulate muscle differentiation, whereas basic FGF-2 and TGF-beta, have negative influences on muscle differentiation. Though much is known about the downstream components, namely the MRFs and the MEF2 factors, the pathways upstream of these components and downstream of the extracellular factors that modulate them are less well defined. p38 MAPK has been implicated in the myogenesis signaling pathway upstream of the MRFs and MEFs. Therefore, modulation of p38 MAPK activity may facilitate control of myogenesis.

Systems and methods including the control of cell differentiation by modulation of p38 MAPK have commercial utility in the diagnosis, treatment, and prevention of diseases or conditions that are caused or contributed to by diseased or defective muscle tissue, and in the production of muscle cell lines for experimental purposes.

Summary

In an embodiment, a system for implanting muscle tissue has a plurality of myogenic precursor cells and an effective amount of a p38 mitogen activated protein kinase (MAPK) inhibitor to stimulate myogenesis in the plurality of cells.

In another embodiment, a method of stimulating myogenesis includes delivering an effective amount of a p38 MAPK inhibitor to cells capable of differentiating into muscle cells.

In yet another embodiment, a method for treating or preventing a disease or condition that is caused or contributed to by diseased or defective muscle tissue includes providing a plurality of myogenic precursor cells, delivering an effective amount of a p38 MAPK inhibitor to the plurality of cells to stimulate myogenesis, and introducing the plurality of cells into a subject.

In still another embodiment, a method for treating or preventing a disease or condition that is caused or contributed to by diseased or defective muscle tissue includes providing a plurality of myogenic precursor cells, introducing the plurality of cells into a subject, and delivering an effective amount of a p38 MAPK inhibitor to the plurality of cells to stimulate myogenesis.

In an embodiment, the disease or condition may comprise at least one of a congenital heart defect, damage to a fetus caused by a teratogen, a fetal muscular defect, muscle tissue death, muscle tissue excision, muscle atrophy, muscle cell tumor, muscle cell cancer, muscle overgrowth, muscle denervation, or fϊbrodysplasia ossificans progressiva. Brief Description of Figures

FIG. 1 shows representative results of an experiment demonstrating the effects of a p38 MAPK inhibitor on chondrogenesis and myogenesis. FIGs. 2A, 2B, and 2C show representative results of reporter gene activity following treatment of cells with a p38 MAPK inhibitor.

FIG. 2D shows representative results of Northern blot analysis of cells treated or not treated with a p38 MAPK inhibitor.

FIG. 3 shows representative results of an experiment demonstrating the effects of a p38 MAPK inhibitor on chondrogenesis and myogenesis.

FIG. 4 shows representative results of an experiment demonstrating the effects of a p38 MAPK inhibitor on myogenesis during the first twenty four hours after treatment of cells with the inhibitor.

FIG. 5 shows representative results of an experiment demonstrating the effects of BMP-4, Noggin, and a p38 MAPK inhibitor alone and in combination, on chondrogenesis and myogenesis.

Detailed Description of the Invention

1. General

The present invention is based at least in part on the finding that, contrary to what had been thought previously, inhibition of p38 MAPK activity results in enhanced myogenesis of myogenic precursors.

Indeed, previously, reports in the art indicated that activation of p38 MAPK stimulates myogenesis (e.g., Cuenda and Cohen 1999; Zetser et al. 1999; Li et al. 2000; Puri et al. 2000; Wu et al. 2000; Conejo et al. 2001; Gallea et al. 2001). From these and other studies, it was believed that the mitogen-activated protein kinase (MAPK) p38 is required for muscle differentiation and that its inhibition interfered with myogenesis. The disparity of these reports in the art and the findings in the present invention may exist because the prior reports described the control of myogenesis in myogenic cell lines — in particular, the mouse C2C12 and rat L6 lines, both of which are thought to be derivatives of satellite cells from adult muscle fibers. Earlier studies did not report effects of p38 MAPK inhibition in primary cultures of myogenic progenitors or precursors.

Experiments forming the basis of the present invention demonstrated that inhibition of p38 attenuates chondrogenesis substantially but contrary to other studies, potently and dramatically enhances myogenesis. Experiments reported herein (see Examples) demonstrated this result in primary limb mesenchymal structures from embryonic mice. This induction occurs in the appropriate environment, established, for example, in the limb mesenchyme. Myogenesis of G8 myoblasts is not affected by p38 MAPK inhibitors when cultured alone, but is enhanced substantially when co-cultured in primary limb bud cultures. While confirming earlier results that indicate a role for p38 in chondrogenesis, these studies indicate that a loss in p38 activity is myogenic. Without wanting to be limited to a particular mechanism of action, the discrepancy between our results and previous results may be explained by the late effects that p38 inhibition appears to have in primary cultures, that is, as a modulator of aggregation and polarization of terminally differentiated myocytes. Thus, our results serve not only to highlight the primary limb mesenchymal culture system as perhaps a more relevant system to study muscle formation with respect to normal embryonic development, but most importantly, to provide methods and compositions for modulating myogenesis.

The progression of cells through the myogenic program is regulated by the sequential expression of various MRFs. In turn, the activity of these MRFs is modulated by various extracellular signaling pathways, adding an additional level of control to the coordination of the myogenic phenotype. In the first stage of skeletal muscle formation, mesodermal progenitors are specified to the myogenic lineage. These specified cells then exit the cell cycle to differentiate into myocytes. Myocyte differentiation is accompanied by the acquisition of a bipolar phenotype, alignment and eventual fusion to form the myotubes of mature muscle fibers. The signals regulating these latter stages of muscle formation are less well understood. Herein we provide evidence that signaling through the MAPK p38 pathway has at least a critical role in regulating the expression of a bipolar myocyte phenotype and in their alignment. Several recent reports have demonstrated that activation of the p38 MAPK signaling pathway is required at several stages of myogenesis (Cuenda and Cohen 1999; Zetser et al. 1999; Li et al. 2000; Puri et al. 2000; Wu et al. 2000; Conejo et al. 2001; Gallea et al. 2001). Using specific inhibitors, similar to the ones employed herein, these reports demonstrate that p38 inhibition results in a loss of the myogenic phenotype and the downregulation of genes associated with myogenesis. At first glance, these findings appear to directly contradict our results obtained using primary mesenchymal cultures. However, a number of possibilities can explain this disparity, the most obvious being that different experimental models were employed. Our studies relied on primary limb mesenchymal cultures while the other studies mostly involved satellite-derived cells such as the C2C12 mouse cell line and the L6 rat cells. Given that these cells are derived from muscle satellite cells, they would have originated in the hematopoietic compartment, unlike embryonic limb myoblasts which have a somitic origin. In this respect, the different origins of these cell populations may contribute to the differences in the cellular response to various external stimuli. Furthermore, satellite cells are important in muscle repair, a program that differs from the embryonic myogenic program (Seale and Rudnicki 2000; Seale et al. 2000). Set aside from these differences in origin, are differences in the cell conditions under which these results were obtained. The primary mesenchymal cultures contain a variety of cell types that are not of the myogenic lineage, and thus the factors present in these cultures are more likely reflective of in vivo conditions. In contrast, many critical factors, normally present in vivo are absent from established clonal cell lines such as C2C12 and L6. The importance of environmental cues in modulating the response to p38 MAPK inhibitors, such as SB202190, is further strengthened by the observation that when G8 cells are co-cultured with primary limb mesenchyme cells they behave differently in the presence of p38 MAPK inhibitors than when cultured alone.

2. Definitions

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. As used herein, the terms "myogenic progenitor" and "myogenic precursor" refer to any cell that is capable of becoming confined to the muscle fate or giving rise to a descendent cell confined to the muscle fate.

As used herein, the terms "p38," "p38 mitogen activated protein kinase," and "p38 MAPK" refer to members of the p38 MAPK family, including but not limited to p38 alpha, p38 beta, p38 gamma and p38 delta, their isoforms (Kumar et al, Biochem. Biophys. Res. Commun., 1997, 235, 533) and other members of the p38 MAPK family of proteins whether they function as p38 MAP kinases per se or not. The nucleotide and amino acid sequences of preferred p38 MAPK are provided in US Pat. No. 6,140,124. p38 is a 41 kD protein containing 360 amino acids which is activated by heat shock, hyperosmolar medium, IL-lor LPS endotoxin (Han J et al (1994) Science 265:808-811) produced by invading gram-negative bacteria. p38 is activated by dual phosphorylation at Thrl80 and Tyrl82 within the motif Thr-Gly-Tyr and once activated, p38 phosphorylates MBP and EGF-R and to a lesser extent IkB, but not cytoplasmic phospholipase A2, c-Myc nor c-Jun (Davis R (1994) TIBS 19:470-473). p38 is phosphorylated by MKK, which exists as isoforms MKK3, MKK6, and MKK4 (including MKK4-α, -β, and -γ). These kinases have serine, threonine, and tyrosine kinase activity, and specifically phorphorylate the human MAP kinase p38 at Thrl80 and Tyr 182. The amino acid and nucleotide sequences of MKK3, MKK4, and MKK6 are set forth in published PCT application having publication No. WO 96/36642 by Davis et al. P38 activity is regulated by the mitogen-induced dual specificity phosphatases MKP1 and PAC1 (Davis R (1994) TIBS 19:470-473).

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents can be evaluated for potential activity by inclusion in assays described, for example, herein below.

The term "agonist", as used herein, is meant to refer to an agent that mimics or upregulates (e.g. potentiates or supplements) p38. A p38 agonist can be a wild-type p38 protein or derivative thereof having at least one biological activity of p38.

"Antagonist" as used herein is meant to refer to an agent that downregulates (e.g. suppresses or inhibits) a p38 bioactivity. A p38 antagonist can be a compound which inhibits or decreases the interaction between a p38 protein and another molecule, e.g., a kinase that phosphorylates p38, or a polypeptide that is phosphorylated by p38. An antagonist can also be a compound that downregulates expression of a p38 gene or which reduces the amount of p38 protein present. A p38 antagonist can be a dominant negative form of a p38 polypeptide. The p38 antagonist can also be a nucleic acid encoding a dominant negative form of a p38 polypeptide, a p38 antisense nucleic acid, or a ribozyme capable of interacting specifically with a p38 RNA. Yet other p38 antagonists are molecules which bind to a p38 polypeptide and inhibit its action. Yet other p38 antagonists include antibodies interacting specifically with an epitope of an p38 molecule, and inhibit or decrease its biological activity. In yet another preferred embodiment, a p38 antagonist is a small molecule, such as a molecule capable of inhibiting the interaction between a p38 polypeptide and a polyptide with which it interacts and/or binding to the catalytic site of the enzyme.

The term "p38 therapeutic" refers to a compound which increases or decreases a biological activity of p38. "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term "compounds of the invention" refers, for example, to small molecules, peptides, polypeptides, nucleic acids, which can be used according to the method of the invention, e.g., to modulate myogenesis.

A "delivery complex" shall mean a targeting means (e.g. a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular or nuclear uptake by a target cell). Examples of targeting means include: sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) or target cell specific binding agents (e.g. ligands recognized by target cell specific receptors). Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form.

The term "modulation" as used herein refers to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating)) and downregulation (i.e. inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)).

The "non-human animals" of the invention include mammalians such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

As used herein, the term "promoter" means a DNA sequence that regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in cells. The term encompasses "tissue specific" promoters, i.e. promoters, which effect expression of the selected DNA sequence only in specific cells (e.g. cells of a specific tissue). The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well. The term also encompasses non-tissue specific promoters and promoters that constitutively express or that are inducible (i.e. expression levels can be controlled).

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product.

The term "recombinant protein" refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally, DNA encoding a p38 polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. A "direct inhibitor" of a kinase, e.g., p38, is an inhibitor which interacts with the kinase or binding partner thereof or with a nucleic acid encoding the kinase.

An "indirect inhibitor" of a kinase, e.g., p38, is an inhibitor which interacts upstream or downstream of the kinase in the regulatory pathway and which does not interacts with the kinase or binding partner thereof or with a nucleic acid encoding the kinase.

The terms "induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g., which denote quantitative differences between two states, refer to at least statistically significant differences between the two states. For example, "an amount of an agent effective to inhibit activation of p38" means that the activation state of p38 in cells treated with the agent will be at least statistically significantly different from that in untreated cells. ^~"

An "inhibitor" of a kinase is any molecule which decreases the activity of the kinase or decreases the protein level of the kinase. Thus, a kinase inhibitor can be a small molecule which decreases activity of the kinase, e.g., by interfering with interaction of the kinase with another molecule, e.g., its substrate. It can also be a small molecule which decreases expression of the gene encoding the kinase. An inhibitor can also be an antisense nucleic acid, a ribozyme, an antibody, a dominant negative mutant of the kinase, or a phosphatase.

"Small molecule" as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.

Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.

Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate p38.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of p38 polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the p38 polypeptide is disrupted. As used herein, the term "transgene" means a nucleic acid sequence (encoding, e.g., a p38 polypeptides, or an antisense transcript thereto) which has been introduced into a cell. A transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can also be present in a cell in the form of an episome. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

The term "treating" or "treatment" of a subject having a disease or disorder refers to the improvement of at least one symptom of the disease or disorder.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. 3. Therapeutics of the invention

Modulation of p38 MAPK activity may be accomplished at the DNA, RNA, and protein levels. Inhibitors of p38 MAPK protein include SB202190, SB203580, and others cited, for example, in US Patents Nos. 6,087,496, 6,184,226, and 6,147,080. p38 MAPK may also be antagonized by administration of homologous antisense oligonucleotides to disrupt p38 RNA processing and translation. In addition, dominant-negative variants of p38 MAPK DNA may be introduced into target cells to overwhelm endogenous p38 activity. Such an introduction may be accomplished with a vector, possibly a retro viral vector. The vector may be designed to provide permanent or transient expression of an inhibitor. Any of the foregoing types of inhibitors may be designed to be activated only upon receiving a signal. Such a signal might be, for example, a chemical or pharmaceutical compound administered to the target cells, in the vicinity of the target cells, or to the subject containing the target cells, by any route of administration known to practitioners of ordinary skill in the art.

Preferred inhibitors of p38 include inhibitors of the alpha and beta isoforms of p38, e.g., SB202190. In certain embodiments of the invention, it may be desirable to use inhibitors which inhibit all isoforms of p38 (alpha, beta, gamma and delta); or inhibitors which are specific for one or more of the isoforms, e.g., an inhibitor that is specific for the alpha, beta, gamma or delta isoform. Assays described herein can be used to determine which specificity may be preferable. In addition, assays can also be used to identify compounds which do not significantly affect other events in the cell.

3.1. Small molecule inhibitors of p38 p38 MAPK inhibitors include SB202190 and SB203580, as described herein;

PD169316 as described in Journal of Clinical Investigation, 106(5):681-8, September 2000; SKF86002 as described in Cancer Research, 60(17):4873-80, September 1, 2000; Quinazoline derivatives as described by Chakravarty et al. in US Pat. No 6,184,226; pyrido [1,2-c] pyrimidin-3-one or 1,2-dihydro-pyrido [1,2-c] pyrimidin-3- one derivatives as described by Bemis et al. in US Pat. No 6,147,080; pyrazole derivatives as described by Anantanarayan et al in US Pat. No. 6,087,496 and by Hanson et al in US Pat. No. 6,087,381; omega-carboxy aryl substituted diphenyl ureas as described by Riedl et al. in PCT application WO 00/41698; heteroalkylamino- substituted bicyclic nitrogen heterocycles as described by Dunn et al. in PCT application WO 01/29042; substituted azoles as described in PCT application WO 00/63204; substituted imidazoles as described by Dinner et al in PCT application WO 98/07425; 1,3-Thiazole compounds substituted by optionally substituted pyridyl at the 5-position as described in PCT application WO 01/10865; substituted pyrazoles as described in PCT application WO 00/75131; aryl and heteroaryl substitued heterocyclic urea as described Dumas et al. in PCT application WO 99/32110; symmetrical and unsymmetrical diphenyl ureas as described in PCT application WO 99/32463; substituted heterocyclic ureas as described in PCT application WO 99/32111; aryl ureas as described in PCT application WO 98/52558; 1,5-diaryl substituted pyrazoles as described in PCT application WO 99/58523; pyrazole derivatives as described in PCT application WO 98/52941; indole-type derivatives as described in PCT application WO 00/71535; alkylamino substituted bicyclic nitrogen heterocycles as described in PCT application WO 01/29041; 3-carboxyindole piperidine amide containing compounds as described in PCT application WO 98/28292 and WO 98/06715; 5- pyridil-l,3-azole compounds as described in PCT application WO 00/64894; pyrimidinylimidazole compounds as described in Bioorganic & Medicinal Chemistry Letters, 8(22):3111-6, 1998 Nov 17; and aminopyrazole derivatives as described in PCT application WO 00/39116.

3.2. Antisense. ribozymes. and triplex techniques

Set forth below is a description on how to design and prepare antisense and ribozymes, for modulating the activity of MAP kinase pathways, in particular, to modulate the p38 pathway. The nucleotide and amino acid sequences of the subject p38 polypeptides and kinases regulating p38 are known in the art. Although in a preferred embodiment, the target of the antisense is p38, the discussion below applies to the design of antisense and triplex molecules targeting any MAP kinase or even other polypeptide. An exemplary p38 antisense molecule is described in US Pat. No. 6,140,124.

As used herein, "antisense" therapy refers to administration or in situ generation of oligonucleotide molecules or their derivatives which specifically hybridize (e.g., bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a member of the MAP kinase pathways, e.g., p38, or protein regulating such, so as to inhibit expression of the member of the MAP kinase pathway or protein regulating such, e.g., by inhibiting transcription and/or translation. The protein against which an antisense molecule is prepared is termed herein "target protein" and the gene encoding the target protein is referred to as the "target gene. The binding of the antisense molecule may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a target protein. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a target gene. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Nan der Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. With respect to antisense DΝA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target nucleotide sequence of interest, are preferred.

Antisense approaches involve the design of oligonucleotides (either DΝA or RΝA) that are complementary to a target mR A. The antisense oligonucleotides will bind to the target mRΝA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. In the case of double-stranded antisense nucleic acids, a single strand of the duplex DΝA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RΝA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the mRΝA, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotides complementary to either the 5' or 3' untranslated, non- coding regions of a gene of interest could be used in an antisense approach to inhibit translation of endogenous mRNA of interest. Oligonucleotides complementary to the 5' untranslated region of the mRNA preferably should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the invention. Whether designed to hybridize to the 5', 3' or coding region of the target mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably less that about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988), hybridization-triggered cleavage agents. (See, e.g., ICrol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' -methoxy carboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5- methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-like backbone. Such molecules are termed peptide nucleic acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is an oc-anomeric oligonucleotide. An -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate olgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. In another embodiment, the antisense molecule is stablilized by the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' of the molecule.

While antisense nucleotides complementary to the coding region of the target gene can be used, those complementary to the transcribed untranslated region and to the region comprising the initiating methionine are most preferred.

The antisense molecules can be delivered to cells which express the target gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

However, it may be difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation on endogenous mRNAs in certain instances. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target transcripts and thereby prevent translation of the target mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave mRNA transcripts of interest, e.g., MAPK mRNA transcripts, can also be used to prevent translation of target mRNA and expression of target proteins (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al, 1990, Science 247:1222-1225 and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3\ The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. There are a number of potential hammerhead ribozyme cleavage sites within the nucleotide sequence of human target. Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al, 1986, Nature, 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in a MAPKgene.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express the target gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination. (E.g., see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al, 1989 Cell 5:313-321; each of which is incorporated by reference herein in its entirety). For example, a mutant, nonfunctional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express target in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive MAPK (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body. (See generally, Helene, C. 1991, Anticancer Drug Des., 6(6):569-84; Helene, C, et al, 1992, Ann. N.Y. Acad. Sci., 660:27-36; and Maher, L.J., 1992, Bioassays 14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription are preferably single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Ribozyme and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, ribozymes and triplex molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, ribozyme and triplex molecule constructs that encode ribozymes or triplex molecules constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. These techniques are further described herein in relation to antisense molecules.

Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. 3.3 Antibodies for use in the invention

Antibodies binding specifically to MAP kinases, e.g., p38, can be used to inhibit activation of MAP kinases according to the methods of the invention. Antibodies can also be used for detecting MAP kinases, e.g., p38, and for use in assays for isolating compounds which inhibit the activity of MAP kinases.

Antibodies, including anti-p38 antibodies can be prepared according to methods known in the art. For example, by using immunogens derived from a MAP kinase protein, anti-protein anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a mammalian target polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a target protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of a p38 of a mammal.

Following immunization of an animal with an antigenic preparation of a target polypeptide, anti- target polypeptide antisera can be obtained and, if desired, polyclonal anti- target polypeptide antibodies isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a mammalian target polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells. The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with a mammalian MAP kinase, e.g., a p38 polypeptide.

Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific, single-chain, and chimeric, and humanized molecules having affinity for an MAP kinase conferred by at least one CDR region of the antibody. Other preferred antibody molecules include intracellular antibodies, e.g., single chain antibodies. Such antibodies can, e.g., inhibit the activity of a p38 kinase or upstream or downstream kinase in their pathways. Production and use of such antibodies is known in the art, as well as gene therapy methods for administering to a subject construct(s) encoding such.

3.4. Methods for identifying modulators of the kinase pathways

Modulators of kinase pathways and in particular, of a p38 polypeptide, can be identified in cell based assays or in in vitro assays. In a preferred embodiment, a modulator is identified by screening for compounds which are capable of inhibiting the interaction between a kinase, e.g., p38, and a protein interacting with it (referred to as "binding partner"), such as a substrate of a protein acting upstream of the kinase, and which e.g., phosphorylates or dephosphorylates the kinase. Alternatively, an in vitro kinase assay comprising a kinase and a substrate or upstream kinase, can be performed and test compounds added to the reaction. Such a reaction can be performed as described in the Examples.

In addition, cell free assays can be used to identify compounds which are capable of interacting with a kinase or binding partner, to thereby modify the activity of the kinase or binding partner. Such a compound can, e.g., modify the structure of a kinase or binding partner and thereby effect its activity. Accordingly, one exemplary screening assay of the present invention includes the steps of contacting a kinase or functional fragment thereof or a kinase binding partner with a test compound or library of test compounds and detecting the formation of complexes. For detection purposes, the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker. Interaction of a test compound with a kinase or fragment thereof or kinase binding partner can then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction. An interaction between molecules can also be identified by using real-time BIA

(Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants. In one embodiment, a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the kinase, functional fragment thereof, or binding partner is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia. Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a kinase or portion thereof, (ii) a kinase binding partner (e.g., substrate or directly upstream kinase), and (iii) a test compound; and (b) detecting interaction of the kinase and the kinase binding protein. The kinase and kinase binding partner can be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein. A statistically significant change (potentiation or inhibition) in the interaction of the kinase and kinase binding protein in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of kinase bioactivity for the test compound. The compounds of this assay can be contacted simultaneously. Alternatively, a kinase can first be contacted with a test compound for an appropriate amount of time, following which the kinase binding partner is added to the reaction mixture. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified kinase or binding partner is added to a composition containing the kinase binding partner or kinase, and the formation of a complex is quantitated in the absence of the test compound.

Complex formation between a kinase and a binding partner may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled kinases or binding partners, by immunoassay, or by chromatographic detection.

Typically, it will be desirable to immobilize either the kinase or its binding partner to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of kinase to a binding partner, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/kinase (GST/kinase) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an 35§_ι_abeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase or binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.

Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either the kinase or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated kinase molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the kinase can be derivatized to the wells of the plate, and the kinase trapped in the wells by antibody conjugation. As above, preparations of a binding protein and a test compound are incubated in the kinase presenting wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the kinase and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the binding partner. To illustrate, the binding partner can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydro chloride or 4-chloro-l-napthol. Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1- chloro-2,4-dinitrobenzene (Habig et al (1974) JBiol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as anti-p38 antibodies, can be used. Alternatively, the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the kinase sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, NJ).

Other assays for identifying kinase, e.g., p38, modulators include cell based assays. In an exemplary embodiment, a cell expressing a kinase of interest, e.g., p38, is incubated with a test compound, and the activity of the kinase is measured, e.g., by measuring p38 phosphorylation or phosphorylation of a p38 substrate. Detection can be done on isolated protein or on the cell.

In certain embodiments, the kinase inhibitors are derivatives of MAP kinases, e.g., p38, such as dominant negative mutants. Although a prefered dominant negative mutant is a dominant negative mutant of the MAP kinase p38, the assay set forth below applies to any MAP kinase. Mutants can be obtained by screening libraries of MAP kinase analogs, such as MAP kinases having amino acid substitutions. In one embodiment, the variegated library of kinase variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential kinase sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of kinase sequences therein.

There are many ways by which such libraries of potential kinase homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential kinase sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3^rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for a kinase clone in order to generate a variegated population of kinase fragments for screening and subsequent selection of inhibitors such as dominant negative forms of the kinase. A variety of techniques are known in the art for generating such libraries, including chemical synthesis. In one embodiment, a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of an kinase coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with S 1 nuclease; and (v) ligating the resulting fragment library into an expression vector. By this exemplary method, an expression library can be derived which codes for N- terminal, C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate kinase sequences created by combinatorial mutagenesis techniques.

Combinatorial mutagenesis has a potential to generate very large libraries of mutant proteins, e.g., in the order of 10^6 molecules. Combinatorial libraries of this size may be technically challenging to screen even with high throughput screening assays. To overcome this problem, a new technique has been developed recently, recrusive ensemble mutagenesis (REM), which allows one to avoid the very high proportion of non-functional proteins in a random library and simply enhances the frequency of functional proteins, thus decreasing the complexity required to achieve a useful sampling of sequence space. REM is an algorithm which enhances the frequency of functional mutants in a library when an appropriate selection or screening method is employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fi'om Nature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering 6(3):327-331).

The invention also provides for reduction of the kinase proteins to generate mimetics, e.g., peptide or non-pepide agents, such as small molecules, which are able to disrupt binding of a kinase of the present invention with a molecule, a substrate. Thus, such mutagenic techniques as described above are also useful to map the determinants of the kinases which participate in protein-protein interactions involved in, for example, binding of the subject kinases to a substrate. To illustrate, the critical residues of a subject kinases which are involved in molecular recognition of its binding partner, e.g., substrate, can be determined and used to generate kinase derived peptidomimetics or small molecules which competitively inhibit binding of the authentic kinase with that moiety. By employing, for example, scanning mutagenesis to map the amino acid residues of the subject kinase proteins which are involved in binding other proteins, peptidomimetic compounds can be generated which mimic those residues of the kinase which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of a kinase. For instance, non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9 American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1 :1231), and b-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71). Assays for testing the activity of the compounds of the various combinatorial libraries, including kinase assays are described herein. Cell based assays are described, e.g., in the Examples.

4. Exemplary diseases that can be treated according to the invention

The invention provides methods for treating diseases or conditions which would benefit from a stimulation or alternatively an inhibition of myogenesis, i.e., the production of essentially differentiated muscle cells. Accordingly, in one embodiment, the invention provides a method for treating a disease or condition that would benefit from stimulation of production of muscle cells, such as conditions characterized by a degeneration of muscle cells, e.g., certain myopathies. In a preferred method of the invention, such diseases or conditions are treated by administering to a subject suffering from the disease a pharmaceutically effective amount of an inhibitor of a p38 polypeptide. The invention also includes a use of a pharmaceutically effective amount of an inhibitor of a p38 polypeptide to treat a disease or condition that would benefit from stimulation of production of muscle cells. The invention further includes a use of a pharmaceutically effective amount of an inhibitor of a p38 polypeptide for the manufacture of a medicament to treat a disease or condition that would benefit from stimulation of production of muscle cells. Exemplary inhibitors are described further herein.

In another embodiment, the invention provides methods for treating a disease or condition which would benefit from a reduction or inhibition of myogenesis, such as by administering to a subject suffering from such a disease or condition a pharmaceutically efficient amount of a stimulator of p38. The invention also includes a use of a pharmaceutically effective amount of a stimulator of p38 to treat a disease or condition that would benefit from a reduction or inhibition of myogenesis. The invention further includes a use of a pharmaceutically effective amount of a stimulator of p38 for the manufacture of a medicament to treat a disease or condition that would benefit from a reduction or inhibition of myogenesis. Exemplary stimulators of p38 include p38 agonists.

Modulation of p38 MAPK activity may facilitate a wide variety of disease treatment methods, assay systems, diagnostic systems, experimentation systems, tissue repair devices, and other systems, devices, and methods.

Disease treatment methods may be directed to promoting the differentiation of muscle progenitor cells into functioning muscle (myogenesis), to suppressing development of other cell types such as chondrocytes, to the halting or reversal of muscle cell division or hyperplasia, or to any combination of these or other effects. The following examples are provided for illustrative purposes only and are not intended to be limiting in any way. As one example, these methods and systems may be directed to correction of congenital heart defects. Congenital heart defects as a disease class are responsible for considerable morbidity and mortality in fetuses, newborns, children, and even adults. Congenital defects include abnormalities in the formation of distinct chambers in the heart, positioning of cardiac structural elements, and functional capacity of the heart. Examples of congenital heart defects include hypoplastic left heart syndrome, atrial and ventricular septal defects, tetralogy of Fallot, persistent truncus arteriosus, transposition of the great vessels, and many others. Failure of adequate muscle growth is often a characteristic of such defects. Therefore, such defects may be corrected by employing a system or method whereby myocytes are provided for introduction into the defect and are caused to differentiate and/or undergo maturation into functioning muscle by administration of a p38 MAPK inhibitor. Alternatively, myocytes may be treated with p38 MAPK inhibitor before introduction into the defect or before introduction into the fetus, newborn, child, or adult, to promote myogenesis, thereby providing cells that may be more likely to survive and function immediately upon introduction in vivo. Myocytes treated with p38 MAPK inhibitor may also facilitate repair of damage to a fetus caused by any teratogen affecting muscle development, or of repair of any other muscular defect, for example, a diaphragmatic hernia or cleft palate.

Similarly, systems, devices, and methods for promoting myogenesis by p38

MAPK inhibition may provide differentiated, functional muscle for integration into any other compartment or location in the body. For example, muscle mass lost as a result of, e.g., tissue death, muscle atrophy, surgical excision or resection, or for any reason, might be replaced by introduction of muscle derived from p38 MAPK inhibitor-treated myocytes. A medical device or delivery system may be deployed for introducing the myocytes alone or in combination with other agents active upon the myocytes or in combination with supporting structures for arranging the myocytes. Such treatment may be carried out ex vivo, before implantation, or in vivo, after implantation, to promote growth and differentiation of muscle tissue exceeding the muscle development possible in the absence of a p38 MAPK inhibitor. As an example, a tissue scaffold may be provided having a substrate composed of collagen or some other substance known in the art to provide cellular support and a plurality of myogenic precursor cells seeded in or on the substrate. The precursors may be stimulated to proliferate using, e.g., BMP-4 or any other known growth or proliferative factor for myogenic progenitors. Once a population of myocytes is obtained that is adequate for restoration of the lost muscle function, the myocytes may then be treated with a p38 MAPK inhibitor to cause myogenesis and thus drive the myocytes to exit the cell cycle, enlarge, polarize, align, aggregate, fuse, form myotubes, muscle fibers, and ultimately, fully functional muscle. The scaffold is introduced into the body at some stage of myocyte development, either before or after treatment with a p38 MAPK inhibitor. In an embodiment, the scaffold may be an implantable prosthetic, such as a vascular graft, within which muscle is stimulated to develop by administration of a p38 MAPK inhibitor so as to recapitulate the muscle tone of an artery, urethra, bladder, diaphragm, or any other muscular structure, conduit, or member. Alternatively, a tissue scaffold may be provided having a population of myocytes not requiring proliferation.

Damage caused to muscle may also be treated by p38 MAPK inhibitors. For example, these systems and methods may be used to treat muscle tissue killed during a myocardial infarction ("heart attack"). Surrounding the area of infarction is a penumbra of injured tissue that may go on to die or to recover. Treatment of injured tissue with a p38 MAPK inhibitor may help the tissue recover by stimulating myogenesis. Alternatively, a p38 MAPK inhibitor may stimulate surviving cells surrounding an infarcted area to "fill in" the damaged area with functional muscle tissue, thereby restoring at least part of the heart's former function. In another alternative, a tissue scaffold as described above may be deployed in the wall of the heart to repair or replace damaged tissue following an infarction.

Inhibitors of p38 MAPK may also facilitate treatment of muscle cell tumors, cancers, and other muscle overgrowth conditions by causing responsive cells to exit the cell replication cycle and begin a terminal differentiation program. Thus, devices, systems, and methods for suppressing cell replication by inhibition of p38 MAPK may enable halting or reversing the growth of muscle tumors such as rhabdomyomas, rhabdomyosarcomas, leiomyomas, leiomyosarcomas, or other conditions of muscle hyperplasia or overgrowth, such as may result from excesses of various growth factors or derangement of cell replication cues. In an embodiment, a p38 MAPK inhibitor could be delivered to tumor cells, through any route of administration familiar to practitioners of ordinary skill in the art. The p38 MAPK inhibitor would then slow or halt the cell division characteristic of such neoplasms and instead promote terminal differentiation of the tumor cells, possibly causing reversion of the neoplastic mass to a normal phenotype. A device or method according to such an embodiment may thus serve as a primary antitumor treatment, in concert with some other antitumor treatment, or as an adjuvant or adjunct therapy.

As another example, the systems and methods described herein may be employed to treat muscles affected by denervating processes. Following denervation, muscles typically undergo atrophy, a process in which they diminish in size and lose functional capacity. This is believed to occur because the muscle no longer receives stimulation to maintain its load-bearing structural components. This condition may be avoided only if innervation is re-established to the affected muscle or external stimulation is applied to the muscle that mimics neural stimulation. If these events do not occur, atrophy may become irreversible. However, reinnervation is a very slow and imprecise healing process that rarely, if ever, restores full function, while external stimulation, for example by a transcutaneous (needle) electrical muscle stimulator, is a cumbersome, incapacitating, and often painful process. p38 MAPK inhibitors may provide a new therapy for preventing or reversing atrophy. An inhibitor may be applied to an affected muscle by any of the routes described above. The inliibitor may restore the stimulation to the muscle to maintain or increase its load-bearing structural elements. In some disease states, such as Bell's Palsy, following which muscle function is only partially or imprecisely restored, treatment with a p38 MAPK inhibitor may compensate for these deficits. Alternatively, a tissue scaffold as described above may be deployed to rebulk or replace an atrophied muscle to which innervation is only partly restored to which innervation has been restored after atrophy has become irreversible.

Damage to established muscle occurring as a result of various disease states may also be repaired, ameliorated, or compensated for by treatments outlined as above. For example, fibrodysplasia ossificans progressiva (FOP) is a condition, at present untreatable and incurable, characterized by intermittent and progressive ectopic ossification of muscle and other tissues through an endochondral process. FOP may result in severe morbidity and mortality by causing ankylosis of joints such as those of the jaw, extremities, and spine, predisposition to pneumonia by causing fixation of the lungs to the chest wall, and confinement to a wheelchair at an early age due to "freezing" of the lower extremity joints. p38 MAPK inhibitors may provide a therapy or cure of FOP by promoting myogenesis and suppressing chondrogenesis. p38 MAPK inhibitor may be delivered to affected tissue through any of the routes described above The inhibitor may abrogate the pathogenic process in FOP that begins with chrondogenesis and proceeds to ossification by suppressing the differentiation of chondrocytes. Other diseases, characterized by muscle atrophy or weakness, may also be amenable to treatment by p38 MAPK inhibitors. The inliibitor may facilitate myogenesis in atrophied or quiescent muscle, or could be employed in a tissue scaffold as described above to replace damaged tissue. Examples of such diseases include the muscular dystrophies, Neu-Laxova syndrome, the spinal muscular atrophies, Wieacker syndrome, Charcot-Marie-Tooth disease, and central core disease of muscle.

Other diseases characterized by ectopic or otherwise inappropriate cell proliferation may be treated by p38 MAPK inhibitors. An inliibitor may antagonize development of typically affected cell lineages. Examples of such diseases include scleroderma, ataxia-telangiectasia, and neoplasms of cell types other than muscle. p38 MAPK inhibitors may also facilitate functional assays of muscle cells and other cell types. For example, a muscle tissue sample could be tested for its degree of hypertrophy or inherent developmental capacity by treating it with an inhibitor and measuring the amount, duration, and/or functional qualities of any resulting myogenesis. Such a system may also be employed to test the myogenic capacity of sample following administration of a drug. In an embodiment, such a system may facilitate diagnosis of a disease state that changes the myogenic potential of a muscle. p38 MAPK inhibitors as disclosed herein may also provide standards to which new candidate agonists or inhibitors may be compared for efficacy. p38 MAPK inhibitors may also facilitate generation of model systems for performing experiments on muscle cells. Currently in the art, muscle cultures are very difficult to maintain beyond a few days because myogenesis fails in vitro. This makes prolonged testing of a given culture impossible and also makes experiments on muscle particularly difficult because cultures must be continuously and frequently replaced. An inhibitor such as those disclosed herein may enable the creation of large numbers of muscle cell cultures with greater longevity. Primary muscle cell explants, such as those described herein, or muscle stem cells may be obtained, grown in vitro to the desired density, and then simulated to develop by a p38 MAPK inhibitor. Alternatively, existing cultures could be stimulated to continue myogenesis by administration of an inliibitor. Treatment by an inhibitor may also be combined with manipulations well known in the art for immortalizing cell lines, such as by transformation with the large T antigen. This may result in the creation of an immortal myocyte cell line in which myogenesis is readily inducible. Such applications may enable further studies in the generation of in- vitro muscle preparation for implantation, tissue engineering of muscle and other such bioorganic prostheses, and facilitate further elucidation of muscle structural biology and molecular mechanics.

The methods of the invention can be used for treating muscular diseases resulting from a defect in a protein associated with myocytes, e.g, dystrophin or sarcoglycans. Dystrophin abnormalities are responsible for both the milder Becker's Muscular Dystrophy (BMD) and the severe Duchenne's Muscular Dystrophy (DMD). In BMD dystrophin is made, but it is abnormal in either size and/or amount. The patient is mild to moderately weak. In DMD no protein is made and the patient is wheelchair-bound by age 13 and usually dies by age 20.

Another type of dystrophy that can be treated according to the methods of the invention includes congenital muscular dystrophy (CMD), a very disabling muscle disease of early clinical onset, is the most frequent cause of severe neonatal hypotonia. Its manifestations are noticed at birth or in the first months of life and consist of muscle hypotonia, often associated with delayed motor milestones, severe and early contractures and joint deformities. Serum creatine kinase is raised, up to 30 times the normal values, in the early stage of the disease, and then rapidly decreases. The histological changes in the muscle biopsies consist of large variation in the size of muscle fibers, a few necrotic and regenerating fibers, marked increase in endomysial collagen tissue, and no specific ultrastructural features. The diagnosis of CMD has been based on the clinical picture and the morphological changes in the muscle biopsy, but it cannot be made with certainty, as other muscle disorders may present with similar clinico-pathological features. Within the group of diseases classified as CMD, various forms have been individualized. The two more common forms are the occidental and the Japanese, the latter being associated with severe mental disturbances, and usually referred to as Fukuyama congenital muscular dystrophy (FCMD). Other muscular dystrophies within the scope of the invention include limb- girdle muscular dystrophy (LGMD), which represents a clinically and genetically heterogeneous class of disorders. These dystrophies are inherited as either autosomal dominant or recessive traits. An autosomal dominant form, LGMD 1 A, was mapped to 5q31-q33 (Speer, M. C. et al, Am. J. Hum. Genet. 50:1211, 1992; Yamaoka, L. Y. et al., Neuromusc. Disord.4:471, 1994), while six genes involved in the autosomal recessive forms were mapped to 15ql5.1 (LGMD2A)(Beckmann, J. S. et al., C. R. Acad. Sci. Paris 312:141, 1991), 2pl6-pl3 (LGMD2B)(Bashir, R. et al., Hum. Mol. Genet. 3:455, 1994), 13ql2 (LGMD2C)(Ben Othmane, K. et al., Nature Genet. 2:315, 1992; Azibi, K. et al., Hum. Mol. Genet. 2: 1423, 1993), 17ql2-q21.33 (LGMD2D)(Roberds, S. L. et al, Cell 78:625, 1994; McNally, E. M., et. al, Proc. Nat. Acad. Sci. U. S. A. 91 :9690, 1994), 4ql2 (LGlMD2E)(Lim, L. E., et. al, Nat. Genet. 11 :257, 1994; Bonnemann, C. G. et al. Nat. Genet. 11 :266, 1995), and most recently to 5q33-q34 (LGMD2F) (Passos-Bueno, M. R., et. al, Hum. Mol. Genet. 5:815, 1996). Patients with LGMD2C, 2D and 2E have a deficiency of components of the sarcoglycan complex resulting from mutations in the genes encoding gamma -, alpha -, and beta -sarcoglycan, respectively. The gene responsible for LGMD2A has been identified as the muscle-specific calpain, whereas the genes responsible for LGMD 1 A, 2B and 2F are still unknown. Yet other types of muscular dystrophies that can be treated according to the methods of the invention include Welander distal myopathy (WDM), which is an autosomal dominant myopathy with late-adult onset characterized by slow progression of distal muscle weakness. The disorder is considered a model disease for hereditary distal myopathies. The disease is linked to chromosome 2pl3. Another muscular dystrophy is Miyoshi myopathya, which is a distal muscular dystrophy that is caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF (Weiler et al. (1999) Hum Mol Genet 8: 871-7). Yet other dystrophies include Hereditary Distal Myopathy, Benign Congenital Hypotonia, Central Core disease, Nemaline Myopathy, and Myotubular (centronuclear) myopathy. Other diseases that can be treated or prevented according to the methods of the invention include those characterized by tissue atrophy, e.g., muscle atrophy, other than muscle atrophy resulting from muscular dystrophies, provided that the atrophy is stopped or slowed down upon treatment with a therapeutic of the invention. Furthermore, the invention also provides methods for reversing tissue atrophies, e.g., muscle atrophies. This can be achieved, e.g., by providing to the atrophied tissue a therapeutic of the invention, such as an inhibitor of p38. Muscle atrophies can result from denervation (loss of contact by the muscle with its nerve) due to nerve trauma; degenerative, metabolic or inflammatory neuropathy (e.g., GuillianBarre syndrome), peripheral neuropathy, or damage to nerves caused by environmental toxins or drugs. In another embodiment, the muscle atrophy results from denervation due to a motor neuronopathy. Such motor neuronopathies include, but are not limited to: adult motor neuron disease, including Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease); infantile and juvenile spinal muscular atrophies, and autoimmune motor neuropathy with multifocal conduction block. In another embodiment, the muscle atrophy results from chronic disuse. Such disuse atrophy may stem from conditions including, but not limited to: paralysis due to stroke, spinal cord injury; skeletal immobilization due to trauma (such as fracture, sprain or dislocation) or prolonged bed rest. In yet another embodiment, the muscle atrophy results from metabolic stress or nutritional insufficiency, including, but not limited to, the cachexia of cancer and other chronic illnesses, fasting or rhabdomyolysis, endocrine disorders such as, but not limited to, disorders of the thyroid gland and diabetes.

Since muscle tissue atrophy and necrosis are often accompanied by fibrosis of the affected tissue, the reversal or the inhibition of atrophy or necrosis can also result in an inhibition or reversal of fibrosis. In addition, the therapeutics of the invention may be of use in the treatment of acquired (toxic or inflammatory) myopathies. Myopathies which occur as a consequence of an inflammatory disease of muscle, include, but not limited to polymyositis and dermatomyositis. Toxic myopathies may be due to agents, including, but are not limited to adiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol, hydroxychloroquine, organophosphates, perihexiline, and vincristine.

Neuromuscular dystrophies within the scope of the invention include myotonic dystrophy. Myotonic dystrophy (DM; or Steinert's disease) is an autosomal dominant neuromuscular disease which is the most common form of muscular dystrophy affecting adults. The clinical picture in DM is well established but exceptionally variable (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co., London, 1989). Although generally considered a disease of muscle, with myotonia, progressive weakness and wasting, DM is characterized by abnormalities in a variety of other systems. DM patients often suffer from cardiac conduction defects, smooth muscle involvement, hypersomnia, cataracts, abnormal glucose response, and, in males, premature balding and testicular atrophy (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co., London, 1989). The mildest form, which is occasionally difficult to diagnose, is seen in middle or old age and is characterized by cataracts with little or no muscle involvement. The classical form, showing myotonia and muscle weakness, most frequently has onset in early adult life and in adolescence. The most severe form, which occurs congenitally, is associated with generalized muscular hypoplasia, mental retardation, and high neonatal mortality. This disease and the gene affected is further described in U.S. Patent No. 5,955,265.

Another neuromuscular disease is spinal muscular atrophy ("SMA"), which is the second most common neuromuscular disease in children after Duchenne muscular dystrophy. SMA refers to a debilitating neuromuscular disorder which primarily affects infants and young children. This disorder is caused by degeneration of the lower motor neurons, also known as the anterior horn cells of the spinal cord. Normal lower motor neurons stimulate muscles to contract. Neuronal degeneration reduces stimulation which causes muscle tissue to atrophy (see, e.g., U.S. patent No. 5,882,868).

The above-described muscular dystrophies and myopathies are skeletal muscle disorders. However, the invention also pertains to disorders of smooth muscles, e.g., cardiac myopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy and restrictive cardiomyopathy. At least certain smooth muscles, e.g., cardiac muscle, are rich in sarcoglycans. Mutations in sarcoglycans can result in sarcolemmal instability at the myocardial level (see, e.g., Melacini (1999) Muscle Nerve 22: 473). For example, animal models in which a sarcoglycan is mutated show cardiac creatine kinase elevation. In particular, it has been shown that delta-sarcoglycan (Sgcd) null mice develop cardiomyopathy with focal areas of necrosis as the histological hallmark in cardiac and skeletal muscle. The animals also showed an absence of the sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiac membranes. Loss of vascular smooth muscle SG-SSPN complex was associated with irregularities of the coronary vasculature. Thus, disruption of the SG-SSPN complex in vascular smooth muscle perturbs vascular function, which initiates cardiomyopathy and exacerbates muscular dystrophy (Coral-Vazquez et al. (1999) Cell 98: 465).

Therapeutics of the invention can also be used to treat or prevent cardiomyopathy, e.g., dilated cardiomyopathy, of viral origin, e.g., resulting from an enterovirus infection, e.g., a Coxsackievirus B3. It has been shown that purified

Coxsackievirus protease 2A cleaves dystrophin in vitro and during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function (Badorff et al. (1999) Nat Med 5: 320. Thus, cardiomyopathy could be prevented or reversed by administration of a therapeutic of the invention to a subject having been infected with a virus causing cardiomyopathy, e.g., by disruption of dystrophin or a protein associated therewith.

5. Pharmaceutical compositions

Pharmaceutical compositions of the invention include either small molecules, polypeptides, nucleic acids or other. Generally, the following guidelines apply to administration of the compounds of the invention. In certain embodiments, the therapy is conducted by gene therapy, e.g., by admininstering to a subject in need thereof a pharmaceutically effective amount of an expression vector encoding a protein or an RNA which modulates, e.g., inhibits or at least reduces the activity of a p38 protein. In one embodiment, the protein is a dominant negative mutant of an enzyme, e.g., a dominant negative mutant of a p38. The protein can also be an intracellular antibody, e.g., a single chain antibody. In yet other embodiments, the expression vector encodes antisense RNA.

In a preferred embodiment, the nucleic acid encoding the protein or RNA is operably linked to all necessary transcriptional and translational regulatory elements, such as a promoter, enhancer and polyadenylation sequence. Regulatory sequences are art-recognized and are described, e.g., in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). In a preferred embodiment, the promoter is a constitutive promoter, e.g., a strong viral promoter, e.g., CMV promoter. The promoter can also be cell- or tissue-specific, that permits substantial transcription of the DNA only in predetermined cells, e.g., precursors of myocytes, e.g., mesenchymal cells, or myocytes. The promoter can also be an inducible promoter, e.g., a metallothionein promoter. Other inducible promoters include those that are controlled by the inducible binding, or activation, of a transcription factor, e.g., as described in U.S. patent Nos. 5,869,337 and 5,830,462 by Crabtree et al., describing small molecule inducible gene expression (a genetic switch); International patent applications PCT/US94/01617, PCT/US95/10591 , PCT/US96/09948 and the like, as well as in other heterologous transcription systems such as those involving tetracyclin-based regulation reported by Bujard et al., generally referred to as an allosteric "off-switch" described by Gossen and Bujard (Proc. Natl. Acad. Sci. U.S.A. (1992) 89:5547) and in U.S. Patents 5,464,758; 5,650,298; and 5,589,362 by Bujard et al. Other inducible transcription systems involve steroid or other hormone-based regulation.

The nucleic acid encoding the RNA or protein of interest and optionally regulatory elements may be present in a plasmid or a vector, e.g., an expression vector. Any means for the introduction of these polynucleotides into mammals, human or non- human, may be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient. In one embodiment of the invention, the DNA constructs are delivered to cells by transfection, i.e., by delivery of "naked" DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA construct may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al, Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al, Nat Genet. 5:135-142, 1993 and U.S. patent No. 5,679,647 by Carson et al. Colloidal dispersion systems.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject.

In a preferred method of the invention, the DNA constructs are delivered using viral vectors. The transgene may be incorporated into any of a variety of viral vectors useful in gene therapy, such as recombinant retroviruses, adenovirus, adeno-associated virus (AAV), and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. While various viral vectors may be used in the practice of this invention, AAV- and adenovirus-based approaches are of particular interest. Such vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. Viral vectors are abundantly described in the art and are available from the American Type Culture Collection, Rockville, Maryland, or by request from a number of commercial and academic sources. A preferred mode of delivering DNA to mesenchymal or precursor of or muscle cells include using recombinant adeno-associated virus vectors, such as those described in U.S. Patent No. 5,858,351. Alternatively, genes have been delivered to muscle by direct injection of plasmid DNA, such as described by Wolff et al. (1990) Science 247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science 254:1507-1509. However, this mode of administration generally results in sustained but generally low levels of expression. Low but sustained expression levels may be effective in certain situations, such as for providing immunity.are expected to be effective for practicing the methods of the invention.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. In one embodiment of the invention an inhibitor of p38 is administered at a location where one desires to stimulate myogenesis, e.g., a tissue comprising precurors of myocytes, such as mesenchymal cells or myoblasts. The therapeutic can also be administered at the site of degeneration of muscle cells. For example, an inhibitor of p38 can be administered by injection into the site, or by topical administration. For such therapy, the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

In clinical settings, a gene delivery system for the therapeutic gene encoding a p38 polypeptide or antagonist thereof of the invention can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994) PNAS 91 : 3054-3057). A gene encoding a p38 can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115). The pharmaceutical preparation of the gene therapy construct or compound of the invention can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

6. Diagnostic assays

The invention also provides diagnostic assays for determining whether a subject has or is likely to develop a disease or condition that is associated with an abnormal muscle tone, e.g., characterized by excessive myocytes or by a lack thereof. In a preferred embodiment, the method comprises obtaining a biopsy from a subject and determining the level of p38 polypeptide or mRNA or its biological activity (e.g., by determining its state of phosphorylation, or its ability to phosphorylate a substrate).

7. Other utilities for the invention

Modulators of p38 can also be used as a supplement to a cell or tissue culture (e.g., system for growing organs), such as to induce myogenesis or to prevent it. The amount of compound to be added to the cultures can be determined in small scale experiments, by, e.g., incubating the cells or organs with increasing amounts of a specific compound of the invention. Preferred cells include eukaryotic cells, e.g., muscle cells or mesenchymal cells. Other preferred tissues include atrophic tissue. Thus, such tissue can be incubated in vitro with an effective amount of a compound of the invention to reverse tissue atrophy. In one embodiment, atrophic tissue is obtained from as subject, the tissue is cultured ex vivo with a compound of the invention in an amount and for a time sufficient to reverse the tissue atrophy, and the tissue can then be readminstered to the same or a different subject.

Alternatively, the compounds of the invention can be added to in vitro cultures of cells or tissue obtained from a subject having a muscular dystrophy, or other disease that can be treated with a compound of the invention, to improve their growth or survival in vitro. The ability to maintain cells, such muscle cells from subjects having a muscular dystrophy or other disease, is useful, for, e.g., developing therapeutics for treating the disease.

8. Kits of the invention The invention provides kits for diagnostic tests or therapeutic purposes.

The materials for performing the diagnostic assays of the present invention can be made available in a kit and sold, for example, to hospitals, clinics and doctors. A kit for detecting altered expression and/or localization of p38, for example, can contain a reagent such as antibody binding to p38, and, if desired, a labeled second antibody, a suitable solution such as a buffer for performing, for example, an immunohistochemical reaction and a known control sample for comparison to the test sample.

A kit for detecting altered expression of mRNA encoding p38 in a sample obtained from an individual, e.g., an individual suspected of being predisposed to a disorder, e.g., a muscular disorder, also can be prepared. Such a kit can contain, for example one or more of the following reagents: a reagent such as an oligonucleotide probe that hybridizes to mRNA encoding p38, suitable solutions for extracting mRNA from a tissue sample or for performing the hybridization reaction and a control mRNA sample for comparison to the test sample, and a series of control mRNA samples useful, for example, for constructing a standard curve.

Such diagnostic assay kits are particularly useful because the kits can contain a predetermined amount of a reagent that can be contacted with a test sample under standardized conditions to obtain an optimal level of specific binding of the reagent to the sample. The availability of standardized methods for identifying an individual predisposed to a disorder, e.g., muscular dystrophy will allow for greater accuracy and precision of the diagnostic methods.

Kits for therapeutic purposes include, e.g., a modulator of p38, for example in a pharmaceutical composition, and optionally a method of administration of the modulator or p38.

Kits for therapeutic or preventive purposes can include a therapeutic and optionally a method for administering the therapeutic or buffer necessary for solubilizing the therapeutic.

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications) as cited throughout this application are hereby expressly incorporated by reference.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2^nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Examples

1. Inhibition of p38 MAPK attenuates chondrogenesis while promoting myogenesis. Previous studies using chick distal limb mesenchyme have demonstrated an approximate 30% reduction in chondrogenesis in response to the p38 inhibitor SB203580 (Yoon et al. 2000). Using the same concentration (10 μM) of a different p38 inhibitor with similar activities, SB202190 (4-(4-fluorophenyl)-2-2(4- hydroxyphenyl)-5-(4-pyridyl)-imidazole, Calbiochem) we have demonstrated a decrease in chondrogenesis in cultures of mouse El 1.5 whole-limb mesenchyme (FIG 1). Limb mesenchymal cells were plated at high density and cultures were fixed and stained with alcian blue (bright field) to visualize cartilage and immunofluorescence with a MF20 mouse antibody to examine myocytes. In untreated cultures (left panels) cartilage nodule formation increases over time, whereas MF20 staining declines such that there are very few MF20-positive cells observed by day 10. Exposure of limb mesenchymal cultures to 10 μM SB202190 (right panels) inhibits cartilage nodule formation, whereas there is a substantial increase in MF20-positive cells in comparison to control cultures. At later stages the SB202190-treated cultures exhibit extensive myocyte fusion accompanied by the organization of the myocytes into parallel arrays. The scale bars are as follows: bright field, 1 mm; fluorescence images, 0.5 mm.

This attenuation, however, was more pronounced than that described in chick mesenchyme, and was later shown to be induced to a similar extent by SB203580. Interestingly, the reduction of chondrogenesis in response to these inhibitors was accompanied by a very substantial increase in the formation of cells that immunoreact with MF20 (Figure 1). MF20 is an antibody that reacts with myosin heavy chain (MyHc) of both cardiac and skeletal muscle. Though most studies carried out focused on the effects of 10 mM SB202190, concentrations as low as 1 μM elicited qualitatively similar responses, though to a lesser extent (data not shown). At these concentrations (1-10 μm), SB202190 and SB203580 selectively inhibit the and β isoforms of p38, leaving the other two isoforms (γ and δ) fully active (Wilson et al. 1997; Young et al. 1997; Wang et al. 1998). Thus, p38 and β either alone or in combination, appear to have an inhibitory effect on myogenesis. 2. A loss in p38 activity results in detectable changes in chondrocytic and myocytic markers.

To examine further the effects of p38 inhibition on chondrogenesis and myogenesis, luciferase reporter assays were carried out to follow the activation of a cartilage- specific col II reporter (pGL3-4X48Luc) and the muscle-specific reporters: pGL2- E4Luc (an E-box reporter), pGL3-myogeninLuc (a myogenin reporter), and pGL3-c- actinLuc (a cardiac actin reporter). Addition of SB202190 to cultures transfected with a col II reporter gene led to a dose-dependent decrease in reporter gene activity (FIG. 2A). In contrast, the activity of a transfected E-box-luciferase (pGL2-E4) reporter was increased in response to increasing concentrations of SB202190. A modest increase was also observed in the activities of reporters containing the myogenin promoter (pGL3 -myogenin) and the cardiac actin promoter (pGL3-c-actin) (FIG. 2B). When co- transfected with Gal4-Mef2A or Gal4-Mef2C, the activity of a Gal4 reporter was, in both instances, enhanced by the p38 inhibitor (FIG. 2C). Northern analysis was carried out to follow endogenous expression of genes associated with chondrogenesis (col II) or myogenesis (myogenin and Mef2c) using total RNA isolated from both treated and untreated limb mesenchymal cultures (FIG. 2D). The normal cline in col II expression is blocked by SB202190, whereas myogenin, present at low levels early on, decreases in expression in the untreated cultures while continuing to increase in response to SB202190. Mef2c is also more abundant in SB202190-treated cultures by day 3 compared to the untreated control.

SB202190 inhibited activity of the pGL3-4X481uc in a dose-dependent manner (FIG. 2A), while enhancing activity of all of the muscle reporters (FIG. 2B). The induction of pGL2-E4Luc was much more dramatic than the induction of pGL3- myogeninLuc, or pGL3-c-actinLuc, which were only modestly affected by SB202190. It is possible that these differences result from there being more components that can bind and cause transactivation of the relatively non-specific E-box. Alternatively, these differences may represent a specific mechanism whereby the induced myogenesis is primarily due to an increase in other MRFs such as MyoD, Myf5, or MRF, with activation of myogenin and cardiac actin representing a minor effect of the inhibitor, or a consequence of other MRF activation.

In addition to examining the reporters mentioned, the activities of MEF2A and

MEF2C were studied by co-transfecting constructs containing the DNA binding domain of Gal4 fused to either MefZa or Mef2c, with a luciferase reporter containing Gal4 response elements (pG5-Luc). SB202190 induced luciferase activity in cells co- transfected with either Gal4-Mef2a, or Gal4-Mef2c, indicating activation of these two MEFs is involved in the myogenic response to p38 inhibition (FIG. 2C). Northern blot analysis to assess the endogenous expression of cartilage- and muscle-specific genes indicated similar results observed from luciferase assays (FIG. 2D). The normal increase in col II expression that takes place over time in primary limb cultures was completely blocked in SB202190-treated cultures, whereas expression of myogenin decreased in untreated cultures while increasing in those cultures treated with the inhibitor. Administration of SB202190 also enhanced Mef c expression by day 3 in the treated cultures, correlating with the increase in MEF2c activity revealed by luciferase assays.

3. Inhibition of p38 enhances myogenesis of myogenic progenitors

To identify the population of cells that contribute to the increased muscle seen in response to SB202190, cells of the proximal portion of the developing limb from El 1.5 embryos were cultured separately from those of the distal portion (FIG. 3). The proximal region of the limb bud at this stage contains considerably more somitic- derived myogenic cells compared to the distal portion which consists predominantly of prechondrogenic cells. Somitic mesoderm-derived cells are typical examples of myogenic progenitors. Cultures were established from distal limb mesenchyme (top panels) or proximal limb mesenchyme (bottom panels). At the time of initiation, the proximal cultures contain more MF20-positive cells compared to distal cultures (see MF20 staining on day 0). In 6 days, there are few detectable myoctyes in untreated distal cultures compared to proximal cultures, but many more cartilage nodules form from distal mesenchyme than from proximal mesenchyme. Following 6 days of treatment with SB202190, in both distal and proximal cultures, there is a decrease in nodule formation, and an increase in the formation of foci of MF20-positive cells. The myocytes in treated proximal cultures, however, are much more prevalent, stain more intensely, and are more highly organized into parallel arrays. Scale bars in FIG. 3 are as follows: bright field, 1.5mm; fluorescence images 0.5mm. If p38 inhibition acts to re-direct prechondrogenic cells to the myogenic lineage, we would expect to see the dramatic myogenic effect of SB202190 to a similar extent in distal cultures. In contrast, however, the magnitude of the myogenic response to the inhibitor appears to be directly proportional to the number of MF20-positive cells that are present in the culture at the time of initiation. Specifically, there was substantially more muscle seen in the proximal cultures after 6 days of SB202190 treatment compared to the distal cultures. This strongly suggests that p38 inhibition promotes myogenesis of cells that migrate into the limb from the somites and not the chondrogenic cells derived from the distal tip of the limb bud. Further support for this comes from the almost complete absence of MF20-positive cells in response to treatment with SB202190 in limb mesenchymal cultures derived from E10 embryos (data not shown). At initiation, these cultures are almost completely devoid of MF20- positive cells, as these cells have not yet migrated into the developing limb from the somite.

4. Inhibition of p38 induces rapid changes in morphology and re-organization of myocytes.

To further examine the effects of SB202190 on myogenesis, cells were fixed and analyzed for MF20 expression at earlier time points following treatment (FIG. 4). Primary whole-limb cultures, both untreated and SB202190-treated (10 μM) were fixed and analyzed for My He expression at various times within 24 hrs. In control cultures, at all time points examined, MF20-positive cells are only slightly bipolar, with small cellular extensions, are randomly oriented, and distributed throughout the culture. Within only a few hours of SB202190 addition, MF20-positive cells acquire an enhanced bipolarity with the bipolar cellular extensions oriented in the same direction. These bipolar cells are also in very close proximity to each other. As early as 6 hrs following treatment, this polarization and aggregation is pronounced, as indicated by the presence of foci of myocytes that become more discernable by 24 hrs. Scale bars for FIG. 4 are as follows: top 8 panels, 0.125 mm; bottom panel, 0.50 mm.

The increased muscle formation appears to be a result of rapid advancement of pre-existing myocytes. Even after one hour of treatment with SB 202190, these

MF20-positive cells aggregate together and polarize, forming distinct foci of myocytes within 6 hours. The appearance of these discrete aggregations of bipolar cells is even more striking following 24 hours of treatment. Interestingly, these dramatic changes in myogenesis are not accompanied by changes in the number of MF20-positive cells, suggesting that the enhanced muscle formation observed is not due to an increase in proliferation of myogenic precursors, or terminally differentiated myocytes, but rather an advance in myogenesis of preexisting myocytes. Thus, inhibition of p38 may play a role in post-differentiation stages of muscle formation, by coordinating the tremendous organization that myocytes typically undergo, including polarization, fusion, and aggregation. 5. BMPs inhibit SB202190-induced polarization but not aggregation of myocytes.

Close examination of the behavior of myocytes in response to SB202190 strongly suggests that this inhibitor induces two major events: polarization and aggregation. Whether the enhanced myocyte fusion is a direct result of the inhibitor, or an indirect result of the cells coming within closer proximity to one another is not known. In an attempt to understand the role of p38 in the overall context of myogenesis we investigated the effects of p38 inhibition on the response to BMP-4, another factor known to modulate myogenesis (Perry and Rudnick 2000). In response to BMP-4, there is an increase in the number of MF20-positive cells, and in the number of cartilage nodules. When treated with BMP-4 and SB202190, however, there are even more myocytes, while the chondrogenic effect of BMP-4 is attenuated. Interestingly, these myocytes are much shorter than in cultures treated with SB202190 alone, with smaller processes, suggesting the cells have not yet polarized. These non- polarized cells aggregate together however, suggesting that BMP-4 prevents the polarization but not the aggregation effect of SB202190. Further support for this comes from the response of cells to Noggin and SB202190. In cultures treated with Noggin (lOOng/ml) alone, there are almost no MF20-positive cells, however, when treated in combination with SB202190, few myocytes are detectable, but those present have undergone dramatic polarization extending long processes bidirectionally.

With reference to FIG. 5, Top panel: Primary mesenchymal cultures were established and stained with alcian blue after 4 days in culture. Addition of Noggin (100 ng/ml) or SB202190 (10 μM) alone or together to these cultures inhibits the formation of alcian blue-stained cartilage nodules, whereas the addition of BMP-4 (lOng/ml) promotes cartilage nodule formation. Middle panels: The addition of Noggin also decreases the number of MF20-positive cells, which can be partly rescued by the addition of SB202190. These doubly-treated cultures also exhibit greater fusion and long myotubes. The addition of BMP-4 increases the number of MF20-positive cells and in the presence of SB202190 the cells exhibit a less-stellate appearance consistent with an early myocyte phenotype. Bottom panel: B-geo-expressing G8 cells exhibit a phenotype similar to that of the MF20-positive cells under all treatment conditions. Untreated cultures present with and abundance of G8 (purple) cells randomly distributed throughout the culture, whereas addition of Noggin results in fewer G8 cells. The addition of BMP-4 and/or SB202190 promotes the appearance of organized G8 cells and SB202190 can rescue the effects of Noggin on G8 cells. Scale bar: top panel, 1 mm; middle top panel, 0.25mm; middle bottom panel, 0.06mm; bottom panel, 0.5mm.

Interestingly, all of these effects of SB202190 in primary cultures are reproduced in G8 myoblasts that have been introduced into the cultures. When G8 myoblasts were cultured on their own, inhibition of p38 had no effect on their progression (data not shown). When mixed in with primary cultures to comprise 5% of the total cells in the cultures, these retro virally tagged cells responded to SB202190 in a manner very similar to myocytes of the developing limb. These results strongly suggest that muscle-promoting effects of the inhibitor rely heavily on factors provided in the limb mesenchymal environment.

The above described embodiments are merely illustrative of the systems and methods of the invention. Accordingly, it will be understood that the invention is not to be limited to the embodiments disclosed herein, but is to be understood from the following claims, which are to be interpreted as broadly as allowed under the law.

Experimental methods employed in Examples

Establishment, treatment and transient transfection of primary limb mesenchymal cultures

Limb mesenchymal cells were harvested from embryonic age (E) 11.25-E11.75 mouse embryos as previously described (Cash et al. 1997; Weston et al. 2000), with some modifications. To establish proximal or distal cultures the proximal half of each limb bud was separated from the distal half, and each pool of limb fragments was processed separately. For these cultures, the cells were resuspended at a density of 1.5 X 10⁷ cells/ml for seeding of 24-well culture plates (Corning). Cell media was replaced daily or every other day and the media was supplemented with inhibitors or vehicle (DMSO). The ρ38 inhibitors SB202190 and SB203580 were obtained from Calbiochem and dissolved in DMSO. Cell mixing experiments were performed by adding G8-beta geo cells to resuspended primary cells such that 5% of the entire cell suspension consisted of the G8-bgeo myoblasts. This mixture was used to seed 24-well culture plates in 10 μl volumes, similar to plating primary cells alone. For transfection purposes the cells were resuspended at 2.5 X 10⁷ cells/ml and mixed with a DNA/FuGene6 mixture in a 2:1 ratio. FuGene6-DNA mixtures were prepared according to the manufacturer's instructions (Roche Biomolecular). Briefly, 1 μg of reporter, 1 μg of expression vector and 0.05 mg of pRLSV40 (Promega) were mixed for a total of ~ 2 μg DNA in 100 μl of media and FuGene6. Fifty microliters of the DNA mixture was transferred into a sterile 1.5 ml microcentrifuge tube, followed by 100 μl of cells. Cells were gently triturated and 10 ml was used to seed a single well of a 24-well culture dish. After 1.5 hrs in a humidified CO₂ incubator, 1 ml of medium was added to each well. Twenty-four hours following transfection the media was replaced and the appropriate supplements were added.

Analysis of reporter gene activity was carried out using the Dual Luciferase Assay System according to the manufacturer's instructions (Promega). Briefly, ~ 48 hrs post-transfection cells were washed once with PBS and lysed in 100 μl of Passive

Lysis Buffer for 20 min. Firefly and renilla luciferase activities were determined by using 40 μl of the cell lysate in a 96-well format Molecular Devices luminometer.

Construction of expression and reporter plasmids

To increase reporter gene sensitivity the Sox9 responsive reporter gene from 4X48-p89 luciferase (Lefebvre et al. 1996) was subcloned into pGL3-basic (Promega). A fragment containing the reiterated (4 X 48) Sox9 binding sequence upstream of the mouse Col II minimal promoter (-89 to +13) was liberated from the 4X48-p89 plasmid by digestion with BamHl and Hindϊll. This fragment was subcloned into the BgRl and Hindlϊl sites of pGL3-basic (Promega) to generate pGL3-4X48. The cardiac actin promoter-luciferase vector was generated by subcloning a fragment of the cardiac actin promoter from -440 to +6 into pGL3 -basic. The myogenin promoter-luciferase construct was made by subcloning a 1.14 kB fragment of the myogenin promoter (containing the region from pGZ1092, (Yee and Rigby 1993)) from plasmid pGBB into pGL3 -basic.

Immunofluorescence, in situ beta-galactosidase and alcian blue staining of cultures Mouse anti-MyHC monoclonal antibody supernatant was used to observe those cells within primary limb mesenchymal cultures that are myogenic (Bader et al. 1982). Briefly, cells were washed with PBS, fixed for 5 min. with cold methanol, rehydrated with PBS for 30 min. then incubated with the MF20 supernatant for 1 hour at room temperature (Ridgeway et al. 2000). Following antibody incubation, cells were washed 3 times for 5 min. each prior to incubation in a goat anti-mouse IgG(H+L) Cy 3 -linked antibody (Jackson Immunoresearch Laboratories, PA) at a 1 :50 dilution in PBS for 30 min.. Immunofluorescent cells were observed using a Zeiss Axiovert microscope, and images were captured by a DVC1300c color digital camera using the Northern Exposure software program.

To follow localization of LacZ-expressing cells in primary cultures, cells were briefly fixed and stained with Magental Gal (BioShop Inc.) as previously described (Weston et al. 2000). Alcian blue staining was performed on fixed cultures also as described (Cash et al. 1997).

Generation of G8-βgeo cells

G8 embryonic myoblasts (American type-culture collection, ATTC) were maintained in Dulbecco's Modified Eagle's Media supplemented with 10% fetal bovine serum and 10% horse serum (Christian et al. 1977). Cultures were subcultured prior to reaching ~80% confluence to minimize the loss of myoblasts. For generation of G8 beta-geo cells, G8 cells were infected with pMSV-beta-geo. The pMSV-beta-geo is an MSV -based retrovirus containing the beta-geo gene under the control of an internal minimal TK promoter (Underhill unpublished). To infect G8 myoblasts, the retrovirus was added directly to the G8 cells for 3 hrs in the presence of lOμg/ml polybrene. Within 4 hours of infection, ~ 2 volumes of media was added to the cells. One day post-infection the media was exchanged for fresh media containing 600 μg/ml active G418. Cells were subcultured thrice during the next 10 days of selection in G418. At the end of the culture period the >95 % of the cells were found to be positive for β- galactosidase activity.

Northern blot analysis

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Claims

We Claim:

1. A system for implanting muscle tissue, comprising: a plurality of myogenic precursor cells; and an effective amount of a p38 mitogen activated protein kinase (MAPK) inhibitor; wherein said p38 MAPK inhibitor stimulates myogenesis in said plurality of cells.

2. A method of stimulating myogenesis, comprising: delivering an effective amount of a p38 MAPK inhibitor to cells capable of differentiating into muscle cells.

3. A method for treating or preventing a disease or condition that is caused or contributed to by diseased or defective muscle tissue, comprising: providing a plurality of myogenic precursor cells; delivering an effective amount of a p38 MAPK inhibitor to said plurality of cells to stimulate myogenesis; and introducing said plurality of cells into a subject.

4. A method for treating or preventing a disease or condition that is caused or contributed to by diseased or defective muscle tissue, comprising: providing a plurality of myogenic precursor cells; introducing said plurality of cells into a subject; and delivering an effective amount of a p38 MAPK inhibitor to said plurality of cells to stimulate myogenesis.

5. A method according to claims 3 or 4, wherein the disease or condition comprises at least one of a congenital heart defect, damage to a fetus caused by a teratogen, a fetal muscular defect, muscle tissue death, muscle tissue excision, muscle atrophy, muscle cell tumor, muscle cell cancer, muscle overgrowth, muscle denervation, or fibrodysplasia ossificans progressiva.

6. A use of a pharmaceutically effective amount of an inhibitor of a p38 polypeptide for the manufacture of a medicament to treat a disease or condition that would benefit from stimulation of production of muscle cells.

7. A use of a pharmaceutically effective amount of a stimulator of p38 for the manufacture of a medicament to treat a disease or condition that would benefit from a reduction or inhibition of myogenesis.