CA2353804A1 - Control of myogenesis by modulation of wnt activity - Google Patents

Abstract

Systems, devices, and methods are disclosed in which Wnt activity modulators promote myogenesis for treatment or diagnosis of medical conditions, tissue repair, or creation of experimental models.

Description

CONTROL OF MYOGENESIS BY MODULATION OF WNT ACTIVITY
Background 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) (Perry and Rudnick 2000). In mammals, the MRF group includes MyoD, myogenin, MyfS, and MRF4/herculin/Myf6. All of the MRFs are expressed solely in skeletal muscle, becoming sequentially activated 1o throughout myogenesis. Ectopic expression of any one MRF in a wide variety of non-muscle cell types results in the conversion of these cells to the myogenic lineage (Weintraub et al. 1989).
During embryogenesis, cells become committed to the muscle lineage by expression of MRFs in the somites. Somites arise from the presegmental mesoderm adjacent to the neural tube. Extensive tissue interactions and signaling result in patterning of the somite to form the sclerotome, dermomyotome, and myotome (Cossu and Borello 1999; Currie and Ingham 1998; Tajbakhsh and Cossu 1997;
Gossler and Hrabe de Angelis 1998). Signals from the surface ectoderm, axial structures, including the dorsal neural tube and the notochord, and the lateral 2o mesoderm are involved in patterning the somite and regulating the differentiation of the myotome (Cossu et al. 1996; Munsterberg and Lassar 1995; Buffinger and Stockdale 1994; Buffinger and Stockdale 1995). Explant studies from avian embryos have shown that the inductive properties of the axial structures can be replaced by a combination of Sonic Hedgehog (SHH) and members of the Wnt family of signaling molecules (Munsterberg et al. 1995; Stern et al. 1995). However, SHH is not required in older explant cultures (Stern et al. 1995).
Wnts are encoded by a large gene family whose members have been found in round worms, insects, cartilaginous fish and vertebrates (Sidow, 1994). Wnts are thought to function in a variety of developmental and physiological processes since 3o many diverse species have multiple conserved Wnt genes (McMahon, Trends Genet., 8: 236-242 [1992]; Nusse and Varmus, Cell, 69: 1073-1087 [1992]). Wnt genes encode secreted glycoproteins that are thought to function as paracrine or autocrine signals active in several primitive cell types (McMahon, supra [1 992]; Nusse and Varmus, supra [1992]).
The Wnt growth factor family includes more than 10 genes identified in the mouse (Wnt-1, 2, 3a, 3b, 4, 5a, Sb, 6, 7a 7b, 8a, 8b, 10b, 11, 12) (see, e.g., Gavin et s al., Genes Dev., 4: 2319-2332 [1990]; Lee et al., Proc. Natl. Acad. Sci.
USA, 92:
2268-2272; Christiansen et al., Mech. Dev. 51: 341-350 [1995]) and at least 7 genes identified in the human (Wnt-1, 2, 3,4, Sa, 7a and 7b) by cDNA cloning (see, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 [1984]). The Wnt-1 proto-oncogene (int-1) was originally identified from mammary tumors induced by mouse mammary to tumor virus (MMTV) due to an insertion of viral DNA sequence (Nusse and Varmus, Cell, 31: 99-109 [1982]). In adult mice, the expression level of Wnt-1 mRNA is detected only in the testis during later stages of sperm development. Wnt-1 protein is about 42 KDa and contains an amino terminal hydrophobic region, which may function as a signal sequence for secretion (Nusse and Varmus, supra). The 1 s expression of Wnt-2/irp is detected in mouse fetal and adult tissues and its distribution does not overlap with the expression pattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis. The expression of Wnt-3 in mouse embryos detected in the neural tubes and in the limb buds. Wnt-Sa transcripts are detected in the developing fore- and hind limbs at 9.5 through 14.5 days and highest levels are 2o concentrated in apical ectoderm at the distal tip of limbs (Nusse and Varmus, supra [1992]. Recently, a Wnt growth factor, termed Wnt-x, was described (PCT/US94/14708; W095/17416) along with the detection of Wnt-x expression in bone tissues and in bone-derived cells. Also described was the role of Wnt-x in the maintenance of mature osteoblasts and the use of the Wnt-x growth factor as a 2s therapeutic agent or in the development of other therapeutic agents to treat bone-related diseases.
Wnts may play a role in local cell signaling. Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium (Papkoff and 3o Schryver, Mol. Cell. Biol., 10: 2723-2730 [1990]; Bradley and Brown, EMBO
J., 9:
1569-1575 [1990]).
Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt gene (Rijsenijk et al., Cell. 50: 649-657 [1987]) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth (Morata and Lawrence, Dev. Biol., 56: 227-240 [1977]; Baker, Dev. Biol., 125: 96-108 [1988];
Klingensmith and Nusse, Dev. Biol., 166: 396-414[1994]). In Caenorhabditis elegans, lin-44 encodes a Wnt which is required for asymmetric cell divisions (Herman and Horvitz, Development, 120: 1035-1047 [1994]). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, Cell, 62:
1073-1085 [1990]; Thomas and Cappechi, Nature, 346: 847-850 [1990]), and the outgrowth of embryonic primordia for kidney (Stark et al., Nature, 372: 679-[1994]), tail bud (Takada et al., Genes Dev., 8: 174-189 [1994]), and limb bud (Parr and McMahon, Nature, 374: 350-353 [1995]). Overexpression of Wnts in the mammary gland can result in mammary hyperplasia (McMahon, supra (1992]; Nusse and Varmus, supra [1992]), and precocious alveolar development (Bradbury et al., Dev. Biol., 170: 553-563 [1995]). A role for Wnts in mammalian hematopoiesis has not previously been suggested or considered.
Wnt-Sa and Wnt-Sb are expressed in the posterior and lateral mesoderm and the extraembryonic mesoderm of the day 7-8 murine embryo (Gavin et al., supra [1990]). These embryonic domains contribute to the AGM region and yolk sac tissues 2o from which multipotent hematopoietic precursors and HSCs are derived (Dzierzak and Medvinsky, supra [1995]; Zon, supra [1995], Kanatsu and Nishikawa, Development, 122: 823-830 [1996]). Wnt-Sa, Wnt-lOb, and other Wnts have been detected in limb buds, indicating possible roles in the development and patterning of the early bone microenvironment as shown for Wnt-7b (Gavin et al., supra [1990];
Christiansen et al., Mech. Devel., 51: 341-350 [1995]; Parr and McMahon, supra [1995]).
The regulation of myf5 expression by the dorsal neural tube during myotome formation can be replaced by cells expressing Wntl (Tajbakhsh et a1.1998).
Moreover, the double knock-out of Wntl and Wnt3a in mice ablates the dorsal medial 3o region of the dermomyotome and results in the loss of normal early expression of myf 5 but not MyoD (Ikeya and Takada 1998). This indicates an essential role for these signaling molecules in the regulation of myogenic gene expression within the dorsal medial dermomyotome. MyoD expression in the more lateral region of the myotome is thought to be controlled preferentially by signals from the dorsal ectoderm (Cossu and Borello 1999). Cells expressing Wnt7a are capable of replacing the dorsal ectoderm and regulating the expression of MyoD in the more lateral regions of the dermomyotome ((Tajbakhsh et a1.1998).
Systems and methods including the control of cell differentiation by modulation of Wnt activity 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 1o purposes.
Summary of the Invention In an embodiment, a system for implanting muscle tissue has a plurality of myogenic precursor cells and an effective amount of Wnt (or decrease in Wnt inhibitor) to stimulate myogenesis in the plurality of cells.
In another embodiment, a method of stimulating myogenesis includes delivering an effective amount of Wnt (or decrease in Wnt inhibitor) to cells capable of differentiating into muscle cells.
In yet another embodiment, a method for treating or preventing a disease or 2o 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 Wnt (or decrease in Wnt 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 Wnt (or decrease in Wnt inhibitor) to the plurality of cells to stimulate myogenesis.
Also within the scope of the invention are in vitro methods for stimulating the 3o proliferation of myoblasts and for stimulating the differentiation of myoblasts into myocytes. For example, myocytes can be obtained from a subject, incubated in vitro with a pharmaceutically efficient amount of an inhibitor of a Wnt protein, e.g., an inhibitor of Wnt3a, such that myoblast differentiation is inhibited and proliferation of the myoblasts is stimulated. These populations of myoblasts can then be induced to differentiate into myocytes in vitro by (i) removing a significant amount of the Wnt inhibitor and/or by (ii) adding to the culture an amount of Wnt agonist sufficient to s stimulate the differentiation of the myoblasts into myocytes. Populations of myoblasts or myocytes may be administered into a subject, which can be the same or a different subject from whom the myoblasts had first been obtained. The myoblasts and myocytes can also be modified prior to administration, e.g., by introducing into a significant amount of the cells nucleic acids, e.g., vectors, expressing proteins of interest.
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 fibrodysplasia ossificans progressiva.
Brief Description of Figures Fig. 1. P19[Mgn] and P19[MyoD+Mgn] cells require cellular aggregation to differentiate into skeletal muscle. P19 control cells (A, B), P19 [Mgn] cells (C, D), 2o and P19[MyoD+Mgn] cells (E, F) were aggregated for 4 days with (A, C, E) or without (B, D, F) Me2S0 and fixed on Day 6. Immunofluorescent images are shown after reaction with an anti-MyHC antibody, MF20 (magnification, x 16).
Fig. 2. Myogenin activity appears to be regulated post-translationally in a cell type-specific manner. P19 control cells (A-D) and P19[Mgn] cells (E-L) were fixed as monolayers on Day 0 (A, B, and E-H) and following aggregation without Me2S0 on Day 6 (C, D, I L). Coverslips were reacted with an anti-Mgn antibody (B, D, F, H, and .>) or an anti-MyHC antibody (L). The corresponding Hoechst staining is shown in A, C, E, G, I, and K (magnification, x 16 for A-F and I L; x 40 for G and H).
Fig. 3. P19[MyoD] and P19[Mgn] cell lines required 4 days of aggregation 3o for efficient myogenesis. Cells were aggregated for 1-4 days with Me2S0, and total RNA was harvested 1 day after transfer to tissue culture dishes (A ). Northern blots containing 6 ~g of total RNA were probed with cardiac a-actin. Expression levels were quantitated by densitometry and shown in B.
Fig. 4. The temporal expression pattern of factors involved in somite patterning during MRF induced myogenesis in P19 cells. P19[MyoD] and P19 control cell lines were aggregated for 4 days in the absence of Me2S0 and plated.
Total RNA
was isolated from a time course of differentiation from days 0 through 6 during the differentiation. Identical Northern blots containing 6 ~g of total RNA were probed with the cDNAs indicated on the right. The loading was standardized by hybridization to an 18 S probe (~.
Fig. 5. The temporal pattern of expression of somite-patterning factors during ~o Me2S0-induced skeletal myogenesis in P19 cells. P19 parental cells were aggregated in the presence of 0.8% MeZSO for 4 days and plated on tissue culture dishes.
Total RNA was isolated from a time course of differentiation from days 0 through 9 during the differentiation. Identical Northern blots containing 6 ~g of total RNA
were probed with the cDNAs indicated on the right. The loading was standardized by hybridization to an 18 S probe (~.
Fig. 6. Monolayers of P19[Mgn] cells show enhanced differentiation in the presence of Wnt3a but not in the presence of BMP-4. P19[Mgn] cells were mixed with P19 control cells (A-D) or P19[Wnt3a] cells (E-H) in the absence of BMP-4 (A, B, E, F), or in the presence of 5 ng/ml BMP-4 (C, D, E, H). Cells were fixed after 6 days of growth in monolayer. A, C, E, and G show Hoechst staining of the nuclei in the cultures. B, D, F, and H show bipolar myocytes reacted with the MyHC
antibody MF20. Magnification, x 16.
Fig. 7. Wnt3a significantly increases the number of MyHC-positive cells present in MyoD and Myogenin cultures. P19[MyoD] and P19[Mgn] cell lines were mixed with P19[Wnt3a] or P19 control cell lines in the presence and absence of 5 ng/ml BMP-4. The number of MyHC-positive cells were counted in a 25-mm2 representative area of each coverslip as indicated for each bar. The number of MyHC-positive cells in either P19[MyoD] or P19[Mgn] cultures were used to normalize the data. Error bars represent the standard error of the mean.
Detailed Description of the Invention _7_ 1. General The present invention is based at least in part on the finding that inhibition of Wnt activity suppresses myogenesis of myogenic precursors, while differentiation of myocytes requires Wnt. Therefore, proliferation and developmental progression of s myogenic precursors may be independently controlled by modulating the activity of Wnts.
Using a clonal mesodermal P19 cell line, which efficiently differentiates into skeletal muscle, it has been shown that overexpression of Wnt antagonists, such as Frzbs, blocked both differentiation into myocytes and expression of muscle-specific 1o genes in the presence of dimethyl sulfoxide (DMSO). Subsequent addition of excess Wnt, or decrease in the Wnt inhibitor, restored the ability of P 19 cells to differentiate into myocytes, demonstrating that Wnts, are indispensable for myocyte differentiation.
Because cells in culture may facilitate more experimental and analytical 15 approaches to studying muscle development, a number of studies have attempted to isolate clonal mesodermal cell lineages; however, none of these cell lines were experimentally as tractable and versatile as a tissue culture system.
Embryonal carcinoma (EC) cells, on the other hand, have become an attractive alternative to the embryo in the identification and analysis of components and events that occur during 2o myogenesis. P19 cells are EC cells capable of differentiating into a variety of cell types, representative of all three germ layers, in suspension culture with several chemical inducers (for review see Skerjanc 1999). P19 can be induced to differentiate into myogenic precursors. It is believed that the cell-cell interactions occurring during aggregation influences the extent of differentiation, since P19 cells do not 2s differentiate efficiently when cultured in monolayer. The myocytes derived from P19 cells display the biochemical and physiological properties that are expressed during early embryonic development. Aggregation of P19 cells induces the expression of the mesoderm marker, Brachyury T (Vidricaire et al. 1994), but few of these cells continue to differentiate. Aggregates treated with dimethyl sulfoxide differentiate into 3o cardiac and skeletal muscle along with other mesodermal and endodermal cell types (Edwards et al. 1983). Cardiocytes first appear on day 5 following DMSO
treatment, whereas skeletal muscle does not appear until day 9 following treatment. The _g_ appearance of cardiac and skeletal muscle is dependent both on the presence of DMSO and on unknown factors in the fetal calf serum (Wilton and Skerjanc 1999).
In co-culture experiments, skeletal muscle development in P19 cells was regulated by factors secreted from the neural tube (Angello et al. 1997). Thus, P19 cells provide an easily manipulatable system to examine early developmental events in tissue culture.
Previous studies examined how the ectopic expression of MyoD affects the developmental potential of P19 cells (Skerjanc et al. 1994). P19 cells expressing MyoD (termed P19[MyoD] cells), retained stem cell characteristics and did not 1o differentiate into skeletal muscle until the cells were aggregated, either with or without DMSO. These results suggested that mesoderm induction, via cellular aggregation, was essential for MyoD activity. Similar results were obtained by others using embryonic stem cells (Sham et al. 1992). Studies of P19[MyoD] cells have shown that MyoD protein is present and capable of binding DNA both before and after aggregation (Armour et a1.1999). This finding suggests that cellular aggregation is responsible for initiating signaling cascades that regulate MyoD directly.
Alternatively, aggregation may indirectly effect MyoD activity, possibly by inducing the expression of an essential cofactor or by altering chromatin structure at muscle-specific promoters. Previous studies have shown that myogenin, like MyoD, 2o requires cellular aggregation to initiate myogenesis (Ridgeway et al.
2000).
A tissue culture system capable of emulating early embryonic events that occur during somitogenesis is valuable for further analysis of the mechanisms involved. The P 19 cell culture system is such a system, because the differentiation of these stem cells simulates the biochemical and morphological processes that occur during early embryonic development (McBurney et al. 1982; Skerjanc 1999;
Rudnicki and McBurney 1987). P19 cells originate from the inner cell mass of day 6 murine embryos (Rudnicki and McBurney 1987). As such, these cells have been isolated before the occurrence of gastrulation and muscle specification and are therefore pluripotent in nature. Clearly, the differentiation of pluripotent stem cells 3o into skeletal myocytes must involve the creation of a specific cellular environment in which only a subset of cells is permissive to MRF-induced myogenesis. This is reminiscent of the complexity of regulatory mechanisms that control multiple lineage determinations during embryogenesis.
Aggregation of P19 cells induces the expression of the mesoderm marker, Brachyury T (Vidricaire et a1.1994), but few of these cells continue to differentiate.
Aggregates treated with dimethyl sulfoxide (DMSO) differentiate into cardiac and skeletal muscle along with other mesodermal and endodermal cell types (Edwards et al. 1983). Previous studies have shown that the expression of the MRFs MyoD or myogenin could bypass the requirement for DMSO but not for cellular aggregation.
We have shown that Wnts control the function of MyoD and myogenin, bypassing the requirement for cellular aggregation. Thus, P 19 cells provide an easily manipulatable to system to examine early developmental events in tissue culture. In particular, P19 cells can be manipulated by varying levels of Wnts to produce muscle cells.
Experiments reported herein (see Examples) utilized the P 19 cell culture system to examine the potential mechanisms involved in regulating MRF activity during cellular aggregation. These experiments show that myogenin activity is regulated in a cell lineage-specific and post-translational manner. During aggregation, somite-patterning factors such as Wnt3a, BMP-2/4, and Pax3 are expressed. In monolayer cultures, Wnt3a, but not Pax3 or BMP-4, can activate MRF-induced myogenesis in P19 cells, bypassing the requirement for aggregation.
Wnts control the function of myoD and myogenin.
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. This definition includes but is not limited to adult stem cells, embryonic stem (ES) cells, mesenchymal stem cells, embryonal carcinoma (EC) cells, and P19 cells.
As used herein, the term "Wnts" refers to members of the Wnt family, 3o including but not limited to any other members of the Wnt family of proteins whether they function as Wnts per se or not. The Wnt growth factor family includes more than 10 genes identified in the mouse (Wnt-1, 2, 3a, 3b, 4, Sa, Sb, 6, 7a 7b, 8a, 8b, - to 10b, 1 l, 12) (see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 [1990]; Lee et al., Proc.
Natl. Acad. Sci. USA, 92: 2268-2272; Christiansen et al., Mech. Dev. 51: 341-[1995]) and at least 7 genes identified in the human (Wnt-1, 2, 3,4, Sa, 7a and 7b) by cDNA cloning (see, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 [1984]).
The terms "Wnts" or "Wnt gene product" or "Wnt polypeptide" when used herein encompass native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides. Optionally, the Wnt polypeptide is not associated with native glycosylation. "Native glycosylation" refers to the carbohydrate moieties which are covalently attached to Wnt polypeptidewhen it to is produced in the mammalian cell from which it is derived in nature.
Accordindy, a human Wnt polypeptide produced in a non-human cell is an example of a Wnts which is "not associated with native glycosylation". Sometimes, the Wnt polypeptide is unglycosylated (e.g., as a result of being produced recombinantly in a prokaryote).
A "native sequence" polypeptide is one which has the same amino acid is sequence as a polypeptide (e.g., Wnt polypeptide) derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptidecan have the amino acid sequence of naturallyoccurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.
2o The term "native sequence Wnt polypeptide" includes those Wnt polypeptides from any animal species (e.g., human, murine, rabbit, cat, cow, sheep, chicken, procine, equine, etc.) as occurring in nature. The definition specifically includes human Wnt polypeptides, Wnt-1, 2, 3, 4, Sa, 7a and 7b and murine Wnt polypeptides, Wnt-1, 2, 3a, 3b, 4, Sa, Sb, 6, 7a, 7b, 8a, 8b, 10b, 11 and 12. The term "native 25 sequence Wnt protein" includes the native proteins with or without the initiating N-terminal methionine (Met), and with or without the native signal sequence. The native sequence human and murine Wnt polypeptides known in the art are from about 348 to about 389 amino acids long in their unprocessed form reflecting variability (particularly at the poorly conserved amino-terminus and several internal sites), 3o contain 21 conserved cysteines, and have the features of a secreted protein (see, e.g., Wnt polypeptides as in Gavin et al., supra; Lee et al., supra; Christiansen et al., supra;
PCT/LJS94/14708 [WO 95/17416]). The molecular weight of a Wnt polypeptide is about 38-42 kD in a monomeric form.
A "variant" polypeptide means a biologicallyactive polypeptideas defined below having less than 100% sequence identity with a native sequence polypeptide.
Such variants include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active Wnt variant will have to an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence Wnt polypeptide, preferably at least about 95%, more preferably at least about 99%.
Methods for production of numerous members of the Wnt family useful in the present invention are known and/or described in the literature. The structure and I5 methods for production of many Wnts can be found in publications and patents, e.g., 6,043,053 (describing Wnt3) and 6,159,462.
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 2o 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) Wnt. A Wnt agonist can be a wild-type Wnt protein or derivative thereof having at least one biological activity of Wnt.
25 "Antagonist" as used herein is meant to refer to an agent that downregulates (e.g. suppresses or inhibits) a Wnt bioactivity. An antagonist can also be a compound that downregulates expression of a Wnt gene or which reduces the amount of Wnt protein present. A Wnt antagonist can be a dominant negative form of a Wnt polypeptide. The Wnt antagonist can also be a nucleic acid encoding a dominant 3o negative form of a Wnt polypeptide, a Wnt antisense nucleic acid, a Wnt RNAi or a ribozyme capable of interacting specifically with a Wnt RNA. Yet other Wnt antagonists are molecules which bind to a Wnt polypeptide and inhibit its action. Yet other Wnt antagonists include antibodies interacting specifically with an epitope of a Wnt molecule, and inhibit or decrease its biological activity. In yet another preferred embodiment, a Wnt antagonist is a small molecule, such as a molecule capable of inhibiting the interaction between a Wnt polypeptide and a polypeptide with which it interacts.
The term "Wnt therapeutic" refers to a compound which increases or decreases a biological activity of Wnt.
"Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular 1o 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.
By "enhancing differentiation of a cell" is meant the act of increasing the extent of the acquisition or possession of one or more characteristics or functions which differ from that of the original cell (i.e., cell specialization). This can be 2o detected by screening for a change in the phenotype of the cell (e.g., identifying morphological changes in the cell and/or surface markers on the cell).
The phrase "enhancing proliferation of a cell" encompasses the step of increasing the extent of growth and/or reproduction of the cell relative to an untreated cell either in vitro or in vivo. An increase in cell proliferation in cell culture can be detected by counting the number of cells before and after exposure to a molecule of interest. The extent of proliferation can be quantified via microscopicexamination of the degree of confluency. Cell proliferation can also be quantified using a thymidine incorporation assay.
"Percent amino acid sequence identity" is defined herein as the percentage of 3o amino acid residues in the candidate sequence that are identical with the residues in the native sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the candidate sequence shall be construed as affecting sequence identity or homology.
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 to 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 2o 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).
"Myoblasts" are cells committed to a skeletal muscle fate but that have not undergone myogenesis. They may express lineage markers characteristic of cells 3o fated for skeletal muscle, such as MRFs, but do not display phenotypic characteristics of terminally differentiated skeletal muscle cells, such as sarcomere formation, or muscle fiber formation. As used herein, "myoblasts" and "myocytes" have the same definition as in the art. Such cells can be identified by specific markers, e.g., cell surface markers, such as those described in the Examples.
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 Wnt polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
to 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 Wnt" means that the activation state of Wnt in cells treated with the agent will be at least statistically significantly different from that in untreated cells.
An "inhibitor" of a Wnt is any molecule which decreases the activity of the Wnt or decreases the protein level of the Wnt. Thus, a Wnt inhibitor can be a small molecule which decreases activity of the Wnt, e.g., by interfering with interaction of the Wnt with another molecule, e.g., its substrate. It can also be a small molecule 2o which decreases expression of the gene encoding the Wnt. An inhibitor can also be an antisense nucleic acid, a ribozyme, an antibody, or a dominant negative mutant of the Wnt.
"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 Wnt.
3o 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 Wnt polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the Wnt polypeptide is disrupted.
As used herein, the term "transgene" means a nucleic acid sequence (encoding, e.g., a Wnt polypeptide, 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 to 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 is 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 2o 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 25 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 3o 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 Wnt activity may be accomplished at the DNA, RNA, and protein levels. Inhibitors of Wnt protein include Secreted Frizzled-related proteins (SFRPs), which are soluble forms of the Wnt receptors, and apparently act by competitive binding of Wnt. The SFRPs are also known as Secreted-apoptosis-related proteins, or SARPs. There are 5 members in the family: SFRP-1 /FrzA
/SARP-2; SFRP-2 /SDF-5 /SARP-1; SFRP-3 /Fritz /Frezzled /FrzB /Frzb-1; SFRP-4;
and SFRP-5 /SARP-3. Crescent and Sizzled (SZl) genes also encode members of this family. Other inhibitors of Wnt are the Cerberus and Cerberus-like proteins, as well 1o as Dickkopf (Dkk) and Dickkopf related proteins. Dickkopf proteins were formerly referred to as the Cysteine-rich secreted proteins (CRSPs or CRISPYs). At least some Dkk-related proteins are also known as Soggy proteins.
The structures of several proteins encoded by a gene family designated as frizzled, have previously been elucidated. Frizzled protein family members have been shown to bind to the Wingless (Wg) protein, the Drosophila prototype of the Wnt family. Bhanot et al., Nature, 382:225-230 (1996). In mammals and other species, the Frizzled family of proteins are membrane bound receptor molecules which bind proteins produced by the family of wnt genes. Wang et al., J. Biol. Chem., 271:4468-4476 (1996). Wnt genes play multiple roles in cell proliferation and differentiation 2o and are expressed in a variety of adult and embryonic tissues and organs, for example, brain, lung, pancreas, liver, spleen, kidney, intestines and/or other tissues and organs.
Several Frizzled-related genes (frzbs) have been identified which encode proteins resembling Frizzleds, i.e., receptors lacking most of the transmembrane domain(s). It has been postulated that Frzbs function as soluble antagonists of Wnt 2s signals. Hoang, et al., JBC 271:26131 (1996), identified a frizzled homologue (which they named frzb) expressed primarily in the cartilaginous cores of developing long bones which was thought to be involved in morphogenesis of the mammalian skeleton. Unlike the genes encoding the Frizzled family of proteins, the frzb gene does not contain seven transmembrane domains. The bovine and human Frzb are 94%
3o identical. Ratner, et al., PNAS 94:2859 (1997) identified in mouse eye cDNA
libraries three DNA sequences encoding secreted frizzled-related proteins (sFRP-1, SFRP-2, and sFRP-3). Wang, et al., Cell 88:757 (1997), isolated a Xenopus homologue of Frzb containing an amino-terminal Frizzled motif and which was soluble and secretable. It was also found to bind and inhibit Wnt-8 (a ventral inducer) and to act as a functional inhibitor of Wnt signalling through direct extracellular binding. Leyns, et al., Cell 88:747 (1997), have identified a secreted protein, Frzb-1, also having sequence similarity to the extracellular domain of Frizzled.
Wnt inhibition may also be achieved by using antibodies against Wnt proteins, or antibodies to Wnt receptors, such as the above-described proteins, or use of antibodies to remove/inhibit Wnt inhibitors. Alternatively, expression of Wnt receptor can also be blocked, as described, e.g., in U.S. patent 5,994,098 and WO
l0 98/54325.
Wnts may also be antagonized by administration of homologous antisense oligonucleotides to disrupt Wnt RNA processing and translation. In addition, dominant-negative variants of Wnt DNA may be introduced into target cells to overwhelm endogenous Wnt activity. Such an introduction may be accomplished with a vector, possibly a retroviral 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 2o cells, by any route of administration known to practitioners of ordinary skill in the art.
Another composition or therapeutic of the invention comprises an essentially pure population of myoblasts. In a preferred embodiment, the composition comprises a population of cells, in which at least about 70% of the cells are myoblasts;
more preferably, at least about 80%, 90%, 95%, or 99% of the cells are myoblasts.
Myoblasts are cells committed to a skeletal muscle fate but that have not undergone myogenesis. They may express lineage markers characteristic of cells fated for skeletal muscle, such as MRFs, but do not display phenotypic characteristics of terminally differentiated skeletal muscle cells, such as sarcomere formation, or muscle fiber formation.
3.1. Antisense, riboz~ triplex techniques, and RNAi Set forth below is a description of how to design and prepare antisense and ribozymes, for modulating in particular the Wnt pathway. The nucleotide and amino acid sequences of the subject Wnt polypeptides are known in the art. Although in a preferred embodiment, the target of the antisense is Wnt, the discussion below applies to the design of antisense and triplex molecules targeting any other polypeptide.
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 Wnt family, or protein regulating such, so as to inhibit expression of the member of the Wnt family or protein regulating such, 1o 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 2o 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 3o therapy have been reviewed, for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. With respect to antisense DNA, 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 DNA or RNA) that are complementary to a target mRNA. The antisense oligonucleotides will bind to the target mRNA 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 DNA 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, 1o the longer the hybridizing nucleic acid, the more base mismatches with an RNA 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 mRNA, 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-2o 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 3o are first performed 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 ~ o 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., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, 2o 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, 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, S-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 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.
s 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 1o 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 is 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 a-anomeric 20 oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-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 (moue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (moue et al., 1987, FEBS Lett.
2s 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.
30 (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 1o 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 2o 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 1o Publication W090/11364, 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 2o 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. W088/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech-type 3o 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 Wnts.

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 to "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, non-functional 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 2o modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive Wnt (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.
3o 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 1o 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' t 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 2o 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 25 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.
3o 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.
Also within the scope of the invention are double stranded small interfering RNAs (siRNAs), which mediate sequence specific mRNA degradation. RNA
interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. In vivo, long dsRNA is cleaved by ribonuclease III to generate 21- and 22-nucleotide siRNAs. It has been shown that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et al. Nature 2001 ;411(6836):494-8).
Accordingly, Wnt expression can be inhibited by administering to a cell small double stranded RNAs, such as to obtain post-transcriptional silencing of Wnt genes.
3.2 Antibodies for use in the invention Antibodies binding specifically to Wnts can be used to inhibit activation of Wnts according to the methods of the invention. Antibodies can also be used for detecting Wnts and for use in assays for isolating compounds which inhibit the 2o activity of Wnts.
Antibodies, including anti-Wnt antibodies, can be prepared according to methods known in the art. For example, by using immunogens derived from a Wnt 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 _2~_ 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 Wnt 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 1o 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 Wnt family member, e.g.
a 2o Wnt 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 a Wnt 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 Wnt or upstream or downstream kinase in their pathways. Production and use of such 3o antibodies is known in the art, as well as gene therapy methods for administering to a subject constructs) encoding such.

3.3. Methods for identifying modulators of the Wn~athways Modulators of Wnt pathways and in particular, of a Wnt 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 Wnt family member and a protein interacting with it (referred to as "binding partner"), and which activates or inactivates the Wnt.
Exemplary binding partners include Wnt receptors, e.g., SFRP proteins described herein. Alternatively, an in vitro Wnt assay comprising a Wnt and a binding partner of Wnt or upstream effector, can be performed and test compounds added to the to 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 Wnt or binding partner, to thereby modify the activity of the Wnt or binding partner. Such a compound can, e.g., modify the structure of a Wnt or binding partner and thereby affect its activity.
Accordingly, one exemplary screening assay of the present invention includes the steps of contacting a Wnt or functional fragment thereof or a Wnt 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 2o marker. Interaction of a test compound with a Wnt or fragment thereof or Wnt 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 3o cell. A solution containing the Wnt, 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 Wnt or portion thereof, (ii) a Wnt binding partner (e.g., substrate or directly upstream effector), and (iii) a test compound; and (b) detecting interaction of the Wnt and the Wnt binding protein. The Wnt and Wnt 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 Wnt and Wnt binding protein in the presence of the test compound, relative to the interaction in the absence l0 of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of Wnt bioactivity for the test compound. The compounds of this assay can be contacted simultaneously. Alternatively, a Wnt can first be contacted with a test compound for an appropriate amount of time, following which the Wnt 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 Wnt or binding partner is added to a composition containing the Wnt binding partner or Wnt, and the formation of a complex is quantitated in the absence 2o of the test compound.
Complex formation between a Wnt 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 Wnts or binding partners, by immunoassay, or by chromatographic detection.
Typically, it will be desirable to immobilize either the Wnt 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 Wnt to a binding partner, can be accomplished in any vessel suitable for containing the 3o 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/Wnt (GST/Wnt) 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 35S-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex s 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 to matrix, separated by SDS-PAGE, and the level of Wnt 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 Wnt or its cognate binding partner can be is immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated Wnt 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 well plates (Pierce Chemical). Alternatively, antibodies reactive with the Wnt can be 2o derivatized to the wells of the plate, and the Wnt trapped in the wells by antibody conjugation. As above, preparations of a binding protein and a test compound are incubated in the Wnt 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, 2s include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the Wnt 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 3o 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 terahydrochloride or 4-chloro-1 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) J Biol 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-Wnt 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 Wnt to 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, N~.
Other assays for identifying Wnt modulators include cell based assays. In an exemplary embodiment, a cell expressing a Wnt of interest, is incubated with a test compound, and the activity of the Wnt is measured, e.g., by measuring growth or 2o differentiation of a Wnt-responsive cell population. Detection can be done on isolated protein or on the cell.
In certain embodiments, the Wnt inhibitors are derivatives of Wnt, such as dominant negative mutants. A preferred dominant negative mutant is a dominant negative mutant of Wnt. Mutants can be obtained by screening libraries of Wnt analogs, such as Wnts having amino acid substitutions. In one embodiment, the variegated library of Wnt 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 Wnt sequences are expressible as 3o individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of Wnt sequences therein.
There are many ways by which such libraries of potential Wnt 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 Wnt 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 3rd 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 ~o 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 Wnt clone in order to generate a variegated population of Wnt fragments for screening and subsequent selection of inhibitors such as dominant negative forms of the Wnt.
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 2o be generated by (i) treating a double stranded PCR fragment of a Wnt 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 3o 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 s 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 Wnt 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 1026 molecules. Combinatorial libraries of this 1o size may be technically challenging to screen even with high throughput screening assays. To overcome this problem, a new technique has been developed recently, recursive 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 15 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 from Nature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al., 20 1993, Protein Engineering 6(3):327-331).
The invention also provides for reduction of the Wnt proteins to generate mimetics, e.g., peptide or non-pepide agents, such as small molecules, which are able to disrupt binding of a Wnt of the present invention with a molecule, e.g. a Wnt receptor. Thus, such mutagenic techniques as described above are also useful to map 2s the determinants of the Wnts which participate in protein-protein interactions involved in, for example, binding of the subject Wnts to a receptor. To illustrate, the critical residues of a subject Wnts which are involved in molecular recognition of its binding partner, e.g., receptor, can be determined and used to generate Wnt derived peptidomimetics or small molecules which competitively inhibit binding of the 3o authentic Wnt with that moiety. By employing, for example, scanning mutagenesis to map the amino acid residues of the subject Wnt proteins which are involved in binding other proteins, peptidomimetic compounds can be generated which mimic those residues of the Wnt which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of a Wnt. 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 1o et al. in Peptides: Structure and Function (Proceedings of the 9t" 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 Wnt 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 2o 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 and proliferation of myogenic progenitors such as myoblasts or 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 a stimulator of a Wnt polypeptide. Exemplary stimulators are described further herein.
3o The methods of the invention can be applied to mammalian subjects, preferably humans, mice, ovines, bovines, porcines, felines, and canines.
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 an inhibitor of Wnt. Exemplary inhibitors of Wnt include Wnt antagonists.
Modulation of Wnt 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 the halting or 1o 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, 2o 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 myogenic precursors are provided for introduction into the defect and are caused to differentiate and/or undergo maturation into functioning muscle by modulation of Wnt activity.
Alternatively, myogenic precursors may be treated with a Wnt modulator 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. Myogenic precursors treated with Wnt modulator may also facilitate repair of damage to a fetus caused by 3o 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 Wnt modulation 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 Wnt modulator-treated myogenic precursors. A medical device or delivery system may be deployed for introducing the myogenic precursors alone or in combination with other agents active upon the myoblasts or in combination with supporting structures for arranging the myoblasts. Such treatment may be carried out ex vivo, before implantation, or in vivo, after implantation, to promote growth and differentiation of muscle tissue exceeding to the muscle development possible in the absence of a Wnt modulator. 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 inhibited from differentiation by a Wnt inhibitor and caused to proliferate.
Once a is population of myogenic precursors is obtained that is adequate for restoration of the lost muscle function, the Wnt inhibition may then be removed, and the myogenic precursors allowed to differentiate. Cells during differentiation may exit the cell cycle, enlarge, polarize, align, aggregate, fuse, form myotubes, muscle fibers, and ultimately, fully functional muscle. 'rhe scaffold is introduced into the body at some 2o stage of myocyte development, either before or after treatment with a Wnt inhibitor.
In an embodiment, the scaffold may be an implantable prosthetic, such as a vascular graft, within which muscle is stimulated to proliferate by administration of a Wnt inhibitor, or to differentiate by removal of the inhibition, so as to recapitulate the muscle tone of an artery, urethra, bladder, diaphragm, or any other muscular structure, 25 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 Wnt modulators. 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 3o penumbra of injured tissue that may go on to die or to recover. Treatment of injured tissue with a Wnt modulator may help the tissue recover by stimulating myogenesis.
Alternatively, a Wnt 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.
Wnt modulators 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 stimulation of Wnt may enable halting or reversing the growth of muscle tumors such as rhabdomyomas, 1o 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 Wnt stimulator could be delivered to tumor cells, through any route of administration familiar to practitioners of ordinary skill in the art. The Wnt stimulator 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.
2o 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 3o stimulator, is a cumbersome, incapacitating, and often painful process.
Modulation of Wnt may provide a new therapy for preventing or reversing atrophy. In some disease states, such as Bell's Palsy, following which muscle function is only partially or imprecisely restored, Wnt modulation 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.
Other diseases, characterized by muscle atrophy or weakness, may also be amenable to treatment by Wnt modulators. Manipulation of Wnt 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 1o 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 Wnt modulators. An inhibitor may antagonize development of typically affected cell lineages. Examples of such diseases include I5 scleroderma, ataxia-telangiectasia, and neoplasms of cell types other than muscle.
Wnt modulators 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 a modulator and measuring the amount, duration, and/or functional qualities of any resulting 2o myogenesis. Such a system may also be employed to test the myogenic capacity of a 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.
Wnt modulators as disclosed herein may also provide standards to which new candidate agonists or inhibitors may be compared for efficacy.
25 Wnt modulators 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 3o 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 modulation of Wnt.
Alternatively, existing cultures could be stimulated to continue proliferation by administration of an inhibitor. 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 myogenic 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 to 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 2o 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 3o 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, LGMD1A, was mapped to Sq31-q33 (Speer, M. C. et al., Am. J. Hum. Genet. 50:121 l, 1992; Yamaoka, L. Y.
et al., Neuromusc. Disord.4:471, 1994), while six genes involved in the autosomal recessive forms were mapped to 15q15.1 (LGMD2A)(Beckmann, J. S. et al., C. R.
to Acad. Sci. Paris 312:141, 1991), 2p16-p13 (LGMD2B)(Bashir, R. et al., Hum.
Mol.
Genet. 3:455, 1994), 13q12 (LGMD2C)(Ben Othmane, K. et al., Nature Genet.
2:315, 1992; Azibi, K. et al., Hum. Mol. Genet. 2:1423, 1993), 17q12-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), 4q12 (LG1MD2E)(Lim, L. E., et. al., Nat.
Genet. 11:257, 1994; Bonnemann, C. G. et al. Nat. Genet. 11:266, 1995), and most recently to Sq33-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 2o 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 2p13.
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 3o 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 Wnt.
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 I o 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 1 s 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 2o 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 2s 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, 3o 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 1o 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 I5 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 2o 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 25 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, e.g., cardiac myopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy and 3o restrictive cardiomyopathy. At least certain 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).
1o 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 Wnt protein. In one embodiment, the protein is a dominant negative mutant of an enzyme, e.g., a dominant negative mutant of a Wnt.
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 myogenic cells. 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 to 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 2o 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 3o 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 (Felgner, 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 1o 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 i5 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 2o 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.
25 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 3o 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. 5,858,31. 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 to 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 practising 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 t5 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 Wnt is administered at a 20 location where one desires to stimulate myogenesis, e.g., a tissue comprising precursors 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 Wnt can be administered by injection into the site, or by topical administration.
2s 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, 3o 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 1o 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.
2o 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 3o 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 to 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 2o 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 Wnt 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 3o 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 Wnt 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).
to 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 1s 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 Wnt or inhibitor polypeptide or mRNA or its biological activity.
7. Other utilities for the invention 3o Modulators of Wnt 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, adult stem cells, embryonic stem cells, embryonal carcinoma (EC) cells, or myogenic progenitors.
Wnt inhibitors may also facilitate generation of model systems for identifying therapeutics for muscle disease or for performing experiments on myoblasts. A
Wnt inhibitor may be applied to progenitor cells at the myoblast stage to halt development at the blast stage. This method provides a source of myoblasts for experimental, therapeutic, or diagnostic use. The myoblasts can be of mammalian origin, preferably 1o human, mouse, ovine, bovine, canine, or feline. Treatment of such cells with a Wnt inhibitor may also help resolve difficulties of maintaining myocyte or myocyte progenitor cultures beyond a few days.
An inhibitor such as those disclosed herein may enable the creation of large numbers of muscle or myogenic progenitor cell cultures with greater longevity.
t5 Primary muscle cell explants, such as those described herein, or muscle stem cells may be obtained, inhibited from differentiation by administration of a Wnt inhibitor, grown in vitro to the desired density, and then allowed to develop by abrogating the inhibitor. Alternatively, existing cultures could be stimulated to continue myogenesis by addition of a Wnt agonist.
2o For example, adult stem cells could be differentiated into myoblasts by any means, and then treated with Wnt antagonist. A population would result that may comprise an essentially pure population of myoblasts or may be a mixture of myoblasts and other cells. The myoblasts could then be purified by fluorescence activated cell sorting (FACS) or any other method.
25 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 immortal myoblast and myocyte cell lines in which myogenesis is readily inducible. Such applications may enable further studies in the generation of in vitro muscle preparation for implantation, tissue 3o engineering of muscle and other such bioorganic prostheses, and facilitate further elucidation of muscle structural biology and molecular mechanics.
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 1o 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 Wnt, for example, can contain a reagent such as antibody binding to Wnt, and, if desired, a labeled second antibody, a suitable solution such as a buffer for performing, for example, an 2o immunohistochemical reaction and a known control sample for comparison to the test sample.
A kit for detecting altered expression of mRNA encoding Wnt 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 Wnt, 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.
3o 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 Wnt, for example in a pharmaceutical composition, and optionally a method of administration of the modulator or Wnt.
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.
1o A kit for producing an essentially pure population of myoblasts may include precursors of myoblasts, e.g., as a frozen composition, and a Wnt inhibitor.
The kit may further comprise markers specific for myogenic progenitors at a specific stage of differentiation, e.g., the myoblast stage.
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 2o 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°d 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, 3o 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 -s3-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).
s Examples 1. Wnt signaling regulates the function of the MRFs MyoD and myogenin We have examined potential mechanisms involved in regulating MRF activity 1o during cellular aggregation. Previous studies examined how the ectopic expression of MyoD affects the developmental potential of P19 cells (Skerjanc et al. 1994).

cells expressing MyoD (termed P19[MyoD] cells), retained stem cell characteristics and did not differentiate into skeletal muscle until the cells were aggregated, either with or without DMSO. Similar results were obtained by others in embryonic stem t5 cells (Sham et al. 1992). Previous studies also showed that myogenin, like MyoD, required cellular aggregation to initiate myogenesis (Ridgeway et al. 2000).
Here (in these Examples and in Exhibit A, incorporated herein by reference), we show that during aggregation, somite-patterning factors such as Wnt3a, BMP-2/4, and Pax3 are expressed. Furthermore, the temporal pattern of expression of somite-20 patterning factors, Wnt3a, WntSb, BMP-2/4, and Pax3, is correlated with the activation of MRF function. In monolayer cultures, Wnt3a, but not Pax3 or BMP-4, can activate MRF- induced myogenesis in P 19 cells, bypassing the requirement for aggregation.
Methods:
2s The abbreviations used are: MRF, myogenic regulatory factor; SHH, Sonic Hedgehog signaling molecule; Fzl, Frizzled I receptor; MEF2C, myocyte enhancer factor 2C; LEFI, lymphoid enhancer factor I; TCF, T cell factor; BMP, bone morphogenic protein;
kb, kilobase(s); PBS phosphate-buffered saline, MyHC, myosin heavy chain.
3o Plasmid Constructs:
All cDNAs in expression vectors are driven by the phosphoglycerate kinase (pgk-1) promoter (Adra et a1.1987). The DNA construct PGK-MyoD contains a 1.7-kb EcoRI fragment containing the complete open reading frame of MyoD cDNA
(Davis et al. 1987), as described (Pari et al. 1991 ). The construct PGK-myogenin contains a 1.4-kb EcoRI fragment containing the complete open reading frame of rat myogenin cDNA (Wright et al. 1989). The construct PGK-Pax3 contains a 2.3-kb EcoRI fragment containing the complete open reading frame of Pax3 cDNA
(Goulding et al. 1991). The construct PGK-Wnt3a contains a 1.4-kb EcoRI
fragment containing the complete open reading frame of Wnt3a (Roelink and Nusse 1991).
The construct PGK-Puro contains the gene encoding puromycin resistance, as described (Skerjanc et al. 1994). The construct PGK-LacZ contains the gene 1o encoding - galactosidase. PGK-vector DNA is a plasmid containing the pgk-1 promoter alone.
Cell Culture and DNA Transfections:
P19 embryonal carcinoma cells were cultured as described (Rudnicki and McBurney 1987; Wilton and Skerjanc 1999) in 5% Cosmic calf serum (Hyclone, Logan, UT) and 5% fetal bovine serum (CanSera,Rexdale, Ontario). Cells were transfected by the calcium phosphate method (Chen and Okayama 1987) unless otherwise stated. Stable cell lines expressing myogenin, MyoD, or Wnt3a were generated as described previously (Ridgeway et al. 2000; Ridgeway et al.
1999).
Duplicate transfections were performed with 8 ~g of PGK-myogenin or 8 ~g of 2o PGK-MyoD, 1 ~g of PGK-Puro, 1 ~g of PGK-LacZ, and 2.5 ~g of B 17 (McBurney et al. 1994). To isolate P 19 control cell lines, duplicate transfections were performed with 8 ~g of PGK-vector, 1 ~g of PGK-Puro, 1 ~.g of PGK-LacZ, and 2.5 ~g of B17.
To generate cells expressing both MyoD and myogenin, duplicate transfections were performed with 4.5 ~g of PGK-MyoD, 4.5 gg of PGK-myogenin, 1 ~g of PGK-Puro, 1 ~g of PGK-LacZ, and 2.5 ~g of B 17. After 24 h, (3-galactosidase assays were performed on one set to ensure high transfection efficiency, and 2 x 106 cells were plated in a 150-mm dish and selected for puromycin resistance (2 ~g/ml). After days, colonies were isolated for further studies. Cells expressing MyoD, myogenin, and both MyoD and myogenin are termed P19[MyoD] (Skerjanc et al. 1994), 3o P19[Mgn] (Ridgeway et al. 2000), and P19[MyoD+Mgn], respectively.
Differentiation was induced by plating 5 x 105 P19 control, P19[MyoD], P19[Mgn], or P19[MyoD + Mgn] cells into 60-mm bacterial dishes containing either 0.8%

DMSO or no DMSO. The presence or absence of DMSO had no effect on the ability of the MRFs to induce skeletal myogenesis. However, only in the presence of DMSO
will control cells differentiate into cardiac muscle on day 5 and skeletal muscle on day 9. Cells were cultured as aggregates for 4 days and then plated in tissue culture dishes and harvested for RNA, protein, or fixed for immunofluorescence, at the time indicated. In the aggregation time course experiment, cells were aggregated for 1-4 days and harvested 1 day after transfer into tissue culture dishes.
To determine the effect of Pax3 expression on the activity of MyoD or myogenin, PGK-Pax3 was transiently transfected into 4 P19[Mgn] and 4 P19[MyoD]
1 o cell lines. 7 ~g of PGK-Pax3 and 1 ~g of PGK-LacZ were transfected into P
19[Mgn]
and P19[MyoDJ cells using the FuGene 6 transfection system (Roche Molecular Biochemicals) according to the manufacturer's protocol. After 24 h, cells were plated onto coverslips and allowed to grow in monolayer and fixed on day 6. To produce cells that stably expressed MyoD and Pax3, P19[MyoD] cells were transfected with t5 10 ~g of PGK-Pax3, 1 ~g of PGK-Puro, 1 pg of PGK-LacZ, and 2.5 pg of B17, using the CaP04 transfection method (Chen and Okayama 1987). Transfection efficiencies were confirmed to be high (as above), and clones were selected in puromycin (2 ~g/ml) for 10 days. Clones were isolated, and those expressing both MyoD and Pax3 were differentiated as described above.
2o To determine the effects of BMP-4 on the ability of MyoD and myogenin to induce myogenesis, P19[MyoDJ and P19[Mgn] cells were grown in monolayer and differentiated (described above) in the presence and absence of 1, 5, 25, 100, and 200 ng/ml BMP-4 (Genetics Institute, Cambridge, MA) and fixed after 2, 4, or 6 days in monolayer culture or after 6 days of differentiation.
25 To determine the effects of Wnt3a on MRF activity, P19[MRF] cells were mixed with P19[Wnt3a] cells in ratios of 1:1 or 1:2 depending on the experiment. A
total of 150,000 cells was plated onto gelatin-coated coverslips in 35-mm dishes.
Cultures were grown for 6 days before fixing for immunofluorescence. These mixes were also grown in the presence of 5 ng/ml BMP-4 where indicated.
3o Immunofluorescence:
Cells were fixed in either methanol at 20°C for 5 min or Lana's fixative (4%
paraformaldehyde, 14% v/v saturated picric acid, 125 mM sodium phosphate) for min, rehydrated in PBS for 30 min at room temperature, and then incubated with the appropriate antibody. For total muscle myosin staining, 50 ~1 of a mouse anti-MyHC
monoclonal antibody supernatant, MF20 (Bader et al. 1982), was incubated for 1 h at room temperature. For myogenin staining, 100 ~1 of the anti-myogenin monoclonal s antibody supernatant, FSD (Wright et al. 1996), containing 0.03% Triton X-100 and 5% fetal calf serum, was incubated at 4 °C for 24 h. After three 5-min washes in PBS, cells were incubated for 1 h in 50 ~1 of PBS with 1 ~1 of goat anti-mouse IgG(H+L) Cy3-linked antibody (Jackson Immunoresearch Laboratories, PA).
Coverslips were mounted in a solution of 50% glycerol, 40% PBS, 9.9%
to p-phenylenediamine, and 0.1% Hoechst stain. Immunofluorescence was visualized with a Zeiss Axioskop microscope, and images were captured with a Sony 3CCD
color video camera, processed using Northern Exposure, Adobe Photoshop, and Corel Draw software, and printed with a dye sublimation phaser 450 Tektronic printer.
Immunofluorescence experiments were repeated at least twice with two P19 control 15 cell lines and four P19[Mgn] cell lines.
Northern Blot Anal, Total RNA was isolated from differentiated P19 control, P19[MyoD],and P19[Mgn] cell cultures by the lithium chloride/urea extraction method before and after differentiation (Auffray and Rougeon 1980). Northern blot analysis was 2o performed as described previously (Skerjanc et al. 1994). Total RNA (6 fig) was separated on a 1 % agarose, formaldehyde gel. Transfer to Hybond-N (Amersham Pharmacia Biotech, Oakville, Canada) occurred by capillary blotting, and RNA
was cross-linked by UV irradiation. The membrane was hybridized to DNA probes labeled to over 109 cpm/~g with [a-32P]dCTP using a multiprime labeling kit 25 (Amersham Pharmacia Biotech). The probes were purified on a spin column of Sephadex G-50 (Amersham Pharmacia Biotech) and hybridized for 16 h at 42°C.
Washing was performed for 5 x 5 min at room temperature in 2x SSC, 0.2% SDS
and for 15 min at 65 °C in 0.2x SSC, 0.2% SDS (0.1x SSC, 0.2% SDS for WntSb blots).
Hybridization was visualized by autoradiography and with a PhosphorImager SI
from 3o Molecular Dynamics. Densitometry was carried out using ImageQuaNT vl.ll software from Molecular Dynamics.
The probes used were: a 600-by PstI fragment from the human cardiac a-actin last exon (Rudnicki et al. 1990), a 1.8-kb EcoRI fragment from the mouse MyoD
cDNA (Davis et al. 1987), a 695-by EcoRI/PstI fragment from the rat myogenin cDNA (Wright et al. 1989), a 1.6-kb EcoRIBamHI fragment of the mouse Brachyury T cDNA (Herrmann et al. 1990), a 2.3-kb EcoRI fragment of Pax3 cDNA (Goulding et al. 1991), a 1.2-kb EcoRI fragment of mouse BMP-2 Cdna, a 1-kb HindIII/BamHI
fragment of mouse BMP-4 cDNA, a 1.4-kb EcoRI fragment of the Wnt3a cDNA
(Roelink and Nusse 1991 ), a 440-by PstI/SmaI fragment of the WntSb EST (IMAGE
clone ID 439000, obtained from ATCC catalog no. 896114), a 2.2-kb full-length Wnt 1 cDNA (Tajbakhsh et al. 1998), and a 1.6-kb full-length Wnt7a cDNA (Tajbakhsh et 1o al. 1998). The skeletal muscle-specific probe was a 600-by EcoRI fragment from the rat MLC 1 /3 cDNA (Garfinkel et al. 1982). All blots were standardized using a 750-by EcoRI fragment of rabbit 18 S cDNA.
Results:
Expression of Both MyoD and Myogenih Does Not Bypass the Requirement for Cellular Aggregation Previous studies have shown that stable P19 cell lines expressing either MyoD
or myogenin required aggregation to initiate myogenesis (Skerjanc et al. 1994;
Ridgeway et al. 2000). To examine whether the expression of both MyoD and myogenin could bypass the requirement for cellular aggregation, stable cell lines were 2o isolated that expressed both MRFs. Similar to P19[MyoD] cells (Skerjanc et al.
1994) and P19[Mgn] cells (Ridgeway et al. 2000), P19[MyoD+Mgn] cell lines did not express significant levels of MyHC when grown as a monolayer, as indicated by immunoreaction with the anti-MyHC antibody MF20 (data not shown). However, after 4 days of aggregation with (Fig. 1, A, C, and E) or without (Fig. l, B, D, and F) DMSO, P19[Mgn] (Fig. 1, C and D) and P19[MyoD+Mgn] (Fig. 1, E and F) cells appeared bipolar and expressed MyHC on day 6. P 19 control cells did not differentiate into skeletal muscle either with (Fig. 1A) or without DMSO (Fig.
1B) on day 6. P19 control cells differentiated into cardiac muscle in the presence of DMSO
(Fig. 1A), as described previously (Rudnicki and McBurney 1987). Consequently, 3o the activity of myogenin protein, alone or in combination with MyoD, was regulated by cellular aggregation.

Myogenin Is Post-translationally Regulated in a Cell Type-specifrc Manner Because myogenin mRNA was present in P19 stem cells on day 0 before aggregation (Ridgeway et al. 2000), the inability of myogenin to initiate differentiation in monolayer suggests that the myogenin protein was either not present or not functional. It is possible that post-transcriptional regulation prevented myogenin protein from being expressed. To examine this question, immunofluorescence with an anti-myogenin antibody was performed on P19[Mgn]
and P19 cells before and after aggregation (Fig. 2). Myogenin protein (Fig. 2, F, H, and J) was present in the Hoechst stained nuclei (Fig. 2, E, G, and I) of P19[Mgn]
1o cells before (Fig. 2, E-H) and after (Fig. 2, I and J) aggregation. In all cell lines examined, myogenin was found to be present in >90% of the nuclei (quantitated by counting cells on two coverslips from each of four cell lines). A higher magnification shows that some myogenin protein (Fig. 2H) was also present in the cytoplasm when compared with the Hoechst staining of the nuclei (Fig. 2G), which is probably due to a saturation of the nuclear transport machinery by the high levels of exogenous myogenin expression. P19 control cells (Fig. 2, A-D) did not express myogenin protein (Fig. 2, B and D) before (Fig. 2, A and B) or after (Fig. 2, C and D) aggregation. Because myogenin protein is present in the nucleus of cells before aggregation, a form of post-translational regulation may modify the activity of the 2o protein.
Not all of the myogenin-positive cells differentiated into skeletal muscle, because only 30-45% of the Hoechst-stained nuclei (Fig. 2K) expressed MyHC
(Fig.
2L), quantitated by counting cells on two coverslips from each of four cell lines. This suggests that the potential post-translational regulation of myogenin during the differentiation of P19 cells could be cell lineage-specific. Consequently, only a subset of the P19[Mgn] cells have the proper cellular environment permissive for full myogenin activity. It seems likely, therefore, that there is a subset of cells that express factors involved in positively regulating MRF activity.
Expression of Factors Involved in Somite Patterning Correlates with MRFActivation 3o To determine the optimal length of time required for skeletal myogenesis, a time course of aggregation was performed for P19[MyoD] and P19[Mgn] cells.
Cultures were aggregated for 1-4 days in the presence of DMSO, and harvested for RNA 1 day after being transferred into tissue culture dishes. Northern blots were examined for cardiac -actin expression to determine the extent of myogenesis under each condition (Fig. 3A), and the results were quantitated by densitometry (Fig. 3B).
Optimal skeletal myogenesis occurred after 4 days of aggregation, with an enhancement of myogenesis in the range of 3- to 8-fold. These results indicate that factors required to activate the MRFs may be expressed optimally between 3 and days of aggregation.
To identify candidate molecules that may be involved in regulating MyoD and myogenin activity, a time course of skeletal muscle development was analyzed in o P19, P19[MyoD], and P19[Mgn] cells aggregated for 4 days without DMSO. The expression of factors involved in somite patterning, such as Wntl, -3a, -Sb, and -7a, BMP-2 and -4, and Pax3, were examined by Northern blot analysis. The results obtained for P19[MyoD] (Fig. 4) and P19[Mgn] (data not shown) cells were found to be similar compared with P19 control cells (Fig. 4). MyoD was expressed throughout the time course from day 0 to day 6 in P19[MyoD] cells and not in the control cell line (Fig. 4A). The skeletal muscle-specific marker MLC 1/3 was expressed following aggregation on day 5 and increased on day 6 (Fig. 4B) in cells expressing MyoD. The mesoderm marker Brachyury T was expressed on days 2 through 4 in P 19 and P19[MyoD] cells (Fig. 4C), indicating the induction of mesoderm, as previously 2o reported (48). Expression of Brachyury T in P 19 cells treated without DMSO
did not lead to any further differentiation of these cells. WntSb was expressed from days 2 through 5, peaking on days 2 and 3, and then decreasing (Fig. 4D). WntSb was also expressed at lower levels in the control cells (Fig. 4D) indicating that aggregation alone up-regulates WntSb expression. Wnt3a was expressed from days 2 through 6, peaking on day 4 (Fig. 4E). Wntl and -7a expression was undetectable or at very low levels during the time course (data not shown). The expression of BMP-2 and appeared on day 3 (Fig. 4, F and G), and Pax3 expression first appeared on day 4 (Fig.
4H). Therefore, the expression of factors involved in somite patterning was activated by aggregation of P 19, P 19[MyoD], and P 19[Mgn] cells at the appropriate time to 3o make these factors candidates for regulating MRF activity.
Factors Involved in Somite Patterning Are Expressed during DMSO-induced Skeletal Myogenesis A time course of DMSO-induced skeletal myogenesis was analyzed for the expression of factors shown to be present during MRF-induced myogenesis. P19 parental cells were aggregated in the presence of 0.8% DMSO for 4 days under serum conditions, which enhanced the population of skeletal myocytes and decreased the number of cardiomyocytes formed (Wilton and Skerjanc 1999). Northern blots were performed on RNA harvested from each day during the differentiation. P19 cells aggregated in DMSO expressed Brachyury T at high levels on days 1 through 3 (Fig.
5A). WntSb was expressed from days 1 through 4, peaking on day 3 (Fig. 5B).
Wnt3a was the next factor expressed from days 2 through 4 (Fig. SC), and Wnt7a was 1o not expressed at significant levels during the DMSO-induced differentiation program (data not shown). BMP-4 was expressed from days 3 through 9 (Fig. SD), and Pax3 from days 4 through 9 (Fig. SE). The timing of the expression of each of these factors is similar to their expression in the MRF-induced time course shown in Fig.4.
This indicates that the expression patterns of these early factors during myogenesis is ordered in a specific manner.
During the endogenous differentiation pathway, MEF2 and MRF family members were also expressed. MEF2C, previously shown to synergize with the MRF
family of factors (Molkentin et al. 1995), was expressed from days 5 through 9 (Fig.
SF). The MRFs, MyoD and myogenin, were expressed from days 7 through 9 (Fig.
5, 2o G and H).
Wnt3a but Not BMP or Pax3 Can Activate MyoD and Myogenin Due to the findings that myogenin was regulated in a post-translational and cell type-specific manner in P19[Mgn] cells and that there was an ordered expression pattern for factors expressed during aggregation in both MRF-induced and DMSO-induced skeletal myogenesis (Figs. 4 and 5), we hypothesized that a factors) expressed during aggregation may be involved in regulating the activity of the MRFs.
Expression of this factors) should consequently bypass the requirement for cellular aggregation. To test this hypothesis, mixing experiments were carried out without aggregation. Monolayers of P19[Mgn] cells were mixed with various combinations of P19[Wnt3a] cells and P19 control cells in the presence and absence of BMP-4 for 6 days. P19[Mgn] cells mixed with P19 control cell lines differentiated into a very low percentage of MyHC-positive cells (Fig. 6B). When the same mixture was grown in the presence of BMP-4 (5 ng/ml), no increase in the number of MyHC-positive cells was observed (Fig. 6D). P19[Mgn] cultures mixed with P19[Wnt3a] cells, showed an increase in the number of MyHC-positive bipolar myocytes present (Fig. 6F).
The transcription factor Pax3 was also expressed during aggregation, before myogenesis (Figs. 4 and 5). The possibility that Pax3 could directly or indirectly regulate MRF activity was tested by transiently expressing Pax3 in P19[MyoD]
and P19[Mgn] cell lines. After transfection these cells were plated onto coverslips and grown in monolayer for 6 days. No increase in the number of MyHC-positive cells occurred after transient Pax3 expression (data not shown). The involvement of Pax3 1o in MRF activation was further tested by stably expressing Pax3 in P19[MyoD]
cells.
Again, no increase in myogenesis occurred in these cell lines either grown in monolayer or aggregated to induce myogenesis (data not shown).
To quantitate results observed in Fig. 6, the number of MyHC-positive cells present on a coverslip were counted, and the results of these counts are shown in a bar graph (Fig. 7). The presence of Wnt3a-expressing cells increased the number of MyHC-positive cells in P19[MyoD] cultures 5-fold 01, n = 9) and P19[Mgn]
cultures 8-fold 02, n = 10) (Fig. 7). The number of MyHC-positive cells decreased slightly in P19[MyoD] cells by 0.6-fold (t0.3, n = 4) and in P19[Mgn] cells by 0.3-fold 00.01, n = 2) in the presence of 5 ng/ml BMP-4 (Fig. 7). Furthermore, the presence of 2o BMP-4 in co-cultures of P19[MRF] cells and P19[Wnt3a] cells inhibited Wnt3a activation of MyoD and myogenin function (Fig. 7). In addition, P19[MyoD] and P19[Mgn] cell lines aggregated in the presence of various concentrations of (1, 5, 25, 100, and 200 ng/ml) did not show increases in the number of skeletal myocytes formed (data not shown). These findings indicate that Wnt3a expression but not Pax3 or BMP can lead to an activation of MRF function in P 19 cells.
Furthermore, BMP expression can antagonize the ability of Wnt to induce MRF
function.
2. The ability of anti-Wnts to proliferate a skeletal myoblast population 3o P 19 cells can differentiate into skeletal muscle after cellular aggregation in the presence of DMSO. Previous studies have shown that the expression of either MyoD
or myogenin could bypass the requirement for DMSO but not for cellular aggregation.

Furthermore, as described herein, Wnt3a could activate MyoD and myogenin in the absence of aggregation. Therefore, Wnts control the function of MyoD and myogenin.
Inhibition of Wnt signaling with anti-Wnts, such as Frzbs, caused the proliferation of skeletal myoblasts. P 19 cells were differentiated with DMSO
to form skeletal myoblasts, indicated by the expression of MyoD and myogenin. At this stage, cells expressing anti-Wnts or control cells were mixed with the skeletal myoblasts and the cultures were allowed to continue differentiating. Cultures containing control cells formed skeletal myocytes whereas cultures containing anti-1o Wnts remained as skeletal myoblasts. Addition of excess Wnt3a allowed the skeletal myoblasts to differentiate into skeletal myocytes.
Methods:
Cell culture:
Differentiation was induced as described previously (Skerjanc et al. 1998) by aggregating 0.5_106 cells in 60 mm bacterial dishes on day 0 with 0.8% DMSO.
The presence of DMSO induces the differentiation of endogenous skeletal muscle after 9 days and the expression of MyoD and myogenin by day 7 (Ridgeway et al. 2000;
Ridgeway, Wilton, and Skerjanc, 2000; Ridgeway and Skerjanc 2001). In order to determine if anti-Wnts can proliferate a skeletal myoblast population, cells were 2o aggregated for 4 days and then plated in 150 mm culture dishes. On day 7, equal numbers of P19 control cells or P19 cells expressing anti-Wnts were added to the cultures. RNA was harvested on day 9 and cells were fixed for immunofluorescence.
To determine if the proliferation of a skeletal myoblast population was reversible, cells were treated as described above. On day 9, excess cells expressing Wnt3a protein was added to the cultures. Cells were cultured for an additional 3 days, after which RNA was harvested and cells were fixed for immunofluorescence.
Northern Blots:
Northern blots were performed as described previously (Skerjanc et al. 1998).
Total RNA was isolated from day 0 and day 6 P19 cell lines and control cells by the lithium chloride/urea extraction method. Separation of total RNA (6 pg) was performed on a 1% agarose, formaldehyde gel by electrophoresis at 24V
overnight.
RNA was transferred to Hybond-N (Amersham Canada, Ltd., Oakville, Canada) by capillary blotting overnight and crosslinked by UV irradiation. Hybridization was performed with DNA probes labeled to over 109 cpm/~g with [a-32P]-dCTP using a multiprime labeling kit (Amersham Canada, Ltd., Oakville, Canada). The probes were purified on a G-50 Sephadex spin column (Pharmacia Biotech Inc., Baie d-Urfe, Quebec, Canada) and denatured by boiling then cooled on ice before use. The blots were prehybridized for 3-4h and hybridized by adding probe for 16h at 42 °C in either NorthernMax (Ambion) or a standard hybridization buffer [5x SSPE, Sx Denhardt's solution, 0.1% SDS, 100 ~g/ml denatured salmon sperm DNA (ssDNA; GIBCO), and 50% deionized formamide]. At the conclusion of the hybridization period, the blots 1o were washed for 5x5 min at 42 °C in 2x SSC, 0.1% SDS, and for 15 min at 65 C in 0.2x SSC, 0.1 % SDS. Hybridization was visualized by autoradiography and with a Phosphorimager SI from Molecular Dynamics.
The probes used were: a 600 by PstI fragment from the human cardiac a-actin last exon, a 1.8 Kb EcoRI fragment from the mouse MyoD cDNA, a 695 by EcoRl/Pstl fragment from the rat myogenin cDNA. All blots were standardized using a 750 by EcoRI fragment of rabbit 18S cDNA.
Immunofluorescence:
Cells were fixed in methanol at -20 °C for 5 min, rehydrated in PBS
for 15 min at room temperature, and then incubated with antibody, as described previously (Skerjanc et al. 1998). The fixed cells were incubated in the presence of 50 ~l of a mouse anti-MyHC monoclonal antibody supernatant, MF20, for 1h at room temperature in a humidified chamber. After three 5 min PBS washes, cells were similarly incubated for 1h at room temperature in the dark in 50 ~1 of PBS
with 1 ~1 of goat anti-mouse IgG(H+L) Cy3-linked antibody (Jackson Immunoresearch Laboratories, PA). The cells were washed with PBS and the coverslips were mounted in a solution of 50% glycerol, 40% PBS, 9.9% paraphenylene diamine and 0.1%
Hoechst stain onto glass slides. Immunofluorescence was visualized with a Zeiss Axioscope microscope, images were captured with a Sony 3CCD color video camera, and processed using Northern Eclipse, Adobe Photoshop 5.5 and Canvas 7 (Deneba) 3o software. Immunofluorescence experiments were carried out in duplicate.
Results:
Cells treated with anti-Wnts on day 7 expressed MyoD and myogenin but did not express cardiac a-actin or myosin heavy chain. After addition of excess Wnt3a, cells continued to express MyoD and myogenin but also expressed cardiac a-actin and myosin heavy chain.
Eauivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the 1o following claims.
Experimental methods employed in Examples Cell Culture P 19 cells were obtained from the American Type Culture Collection (ATCC
is CRL-1825). Cells were cultured in -minimum essential medium ( -MEM, GIBCO) supplemented with 5% (v/v) Cosmic Calf Serum (HyClone, Logon, Utah), 5% (v/v) fetal bovine serum (CanSera, Rexdale, Ontario) and 100 ~g/ml gentamycin (GIBCO) to prevent bacterial contamination. Cells were seeded on tissue culture grade dishes at a density of 1x105 cells per milliliter of supplemented media and subcultured every 2 2o days.

References cited Adra, C. N., Boer, P. H., and McBurney, M. W. ( 1987) Gene 60, 65-74 Angello, J. C., Stern, H. M., and Hauschka, S. D. (1997) Dev. Biol. 192, 93-98 s Armour, C., Garson, K., and McBurney, M. W. (1999) Exp. Cell Res. 251, 79-91 Auffray, C., and Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314 Bader, D., Masaki, T., and Fischman, D. A. (1982) J. Cell Biol. 95, 763-770 Buffinger, N., and Stockdale, F. E. (1994) Development 120, 1443-1452 Buffinger, N., and Stockdale, F. E. (1995) Dev. Biol. 169, 96-108 1o Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752 Cossu, G., and Borello, U. (1999) EMBO J. 18, 6867-6872 Cossu, G., Kelly, R., Tajbakhsh, S., Di Donna, S., Vivarelli, E., and Buckingham, M.
(1996) Development 122, 429-437 Currie, P. D., and Ingham, P. W. ( 1998) Mech. Dev. 73, 3-21 15 Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Cell 51, 987-1000 Edwards, M. K., Harris, J. F., and McBurney, M. W. (1983) Mol. Cell. Biol. 3, Garfinkel, L. L, Periasamy, M., and Nadal-Ginard, B. (1982) J. Biol. Chem.
257, 2o Gossler, A., and Hrabe de Angelis, M. (1998) in Current Topics in Developmental Biology (Pederson, R. A. , and Schatten, G. P., eds), Vol. 38 , pp. 225-287, Academic Press, Toronto Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R., and Gruss, P.
(1991) EMBO J.10, 1135-1147 2s Herrmann, B. G., Labeit, S., Poustka, A., King, T. R., and Lehrach, H.
(1990) Nature 343, 617-622 Ikeya, M., and Takada, S. ( 1998) Development 125, 4969-4976 McBurney, M. W., Jones-Villeneuve, E. M., Edwards, M. K., and Anderson, P. J.
(1982) Nature 299, 165-167 3o McBurney, M. W., Fournier, S., Schmidt-Kastner, P. K., Jardine, K., and Craig, J.
(1994) Somat. Cell. Mol. Genet. 20, 529-540 Molkentin, J. D., Black, B. L., Martin, J. F., and Olson, E. N. (1995) Cell 83, Munsterberg, A. E., Kitajewski, J., Bumcrot, D. A., McMahon, A. P., and Lassar, A.
B. (1995) Genes Dev. 9, 2911-2922 Munsterberg, A. E., and Lassar, A. B. (1995) Development 121, 651-660 Pari, G., Jardine, K., and McBurney, M. W. (1991) Mol. Cell. Biol. 1 l, Perry, R.L. and M.A. Rudnick. (2000). Front Biosci 5, D750-67 Ridgeway, A. G., Petropoulos, H., Siu, A., Ball, J. K., and Skerjanc, I. S.
(1999) FEBS Lett. 456, 399-402 1o Ridgeway, A. G., Petropoulos, H., Wilton, S., and Skerjanc, I. S. (2000) JBiol Chem 275(42), 32398-405 Ridgeway, A. G., Wilton, S., and Skerjanc, I. S. (2000) JBiol Chem 275(1), 41-Ridgeway, A. G., and Skerjanc, I. S. (2001) JBiol Chem 21, 21 Roelink, H., and Nusse, R. ( 1991 ) Genes Dev. 5, 3 81-3 88 Rudnicki, M. A., and McBurney, M. W. (1987) in Teratocarcinomas and Embryonic stem Cells. A Practical Approach (Robertson, E. J., ed) , pp. 19-49, IRL
Press, Oxford Rudnicki, M. A., Jackowski, G., Saggin, L., and McBurney, M. W. (1990) Dev.
Biol.
138, 348-358 2o Shani, M., Faerman, A., Emerson, C. P., Pearson-White, S., Dekel, L, and Magal, Y.
(1992) Symp. Soc. Exp. Biol. 46, 19-36 Skerjanc, I. S., Slack, R. S., and McBurney, M. W. (1994) Mol. Cell. Biol.
1464, Skerjanc, I. S., Petropoulos, H., Ridgeway, A. G., and Wilton, S. (1998) JBiol Chem 273(52), 34904-34910 Skerjanc, I. S. (1999) Trends Cardiovasc. Med. 9(5), 139-143 Stern, H. M., Brown, A. M., and Hauschka, S. D. (1995) Development 121, Tajbakhsh, S., and Cossu, G. (1997) Curr. Opin. Genet. Dev. 7, 634-641 3o Tajbakhsh, S., Borello, U., Vivarelli, E., Kelly, R., Papkoff, J., Duprez, D., Buckingham, M., and Cossu, G. (1998) Development 125, 4155-4162 Tajbakhsh, S., Borello, U., Vivarelli, E., Kelly, R., Papkoff, J., Duprez, D., Buckingham, M., and Cossu, G. (1998) Development 125, 4155-4162 Vidricaire, G., Jardine, K., and McBurney, M. W. (1994) Development 120, Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adam, M. A., Lassar, A.
B., and Miller, A. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5434-5438 Wilton, S., and Skerjanc, I. S. (1999) In Vitro Cell. Dev. Biol. Anim. 35, 175-Wright, W. E., Sassoon, D. A., and Lin, V. K. (1989) Cell 56, 607-617 Wright, W. E., Dackorytko, I. A., and Farmer, K. (1996) Dev. Genet. 19, 131-The above described embodiments are merely illustrative of the systems and 1o methods of the invention. Accordingly, it will be understood that the invention is not to be limited to the embodiments disclosed herein.

Claims (9)

1.A method for producing a myoblast, comprising:
contacting a cell capable of committing to a myogenic fate with an effective amount of a Wnt inhibitor; wherein said Wnt inhibitor suppresses myogenesis in said cell.

2. A method of inhibiting myogenesis, comprising:
contacting a cell capable of differentiating into a muscle cell with an effective amount of a Wnt inhibitor.

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 Wnt inhibitor to said plurality of cells to suppress myogenesis;
restoring the activity of Wnt upon 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 Wnt inhibitor to said plurality of cells to suppress myogenesis and to permit proliferation of said plurality of cells; and restoring the activity of Wnt upon 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 cardiac muscular defect, cardiac muscle death, cardiac muscle cell tumor, cardiac muscle cell cancer, or cardiac muscle hypertrophy.

6. A myoblast produced by the method of claim 1.

7. A population of cells, in which at least 80% of the cells are myoblasts.

8. The population of cells of claim 7, in which at least 90% of the cells are myoblasts.

9. A composition comprising myoblasts and a Wnt inhibitor.