WO1999033986A1 - MAMMALIAN ENHANCER OF POLYCOMB (epc) ACTS AS A TUMOR SUPPRESSOR - Google Patents

Abstract

A mammalian tumor suppressor gene termed enhancer of polycomb and its expression products can be used as therapeutic, prognostic, and diagnostic tools for proliferative and developmental disorders. Nucleotide sequences of human enhancer of polycomb genes can also be used to identify a p10-11 region of human chromosome 10 and a q13.3 region of human chromosome 22.

Description

MAMMALIAN ENHANCER OF POLYCOMB (epc) ACTS AS A TUMOR SUPPRESSOR

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of tumor suppression. More particularly, the invention relates to tumor suppressor genes and proteins.

BACKGROUND OF THE INVENTION Mutations in tumor suppressor genes play an important role in the development of neoplasias. Manipulation of tumor suppressor gene expression can be used to prevent or treat neoplasias. Detection of mutations in tumor suppressor genes can also be used to detect neoplastic cells and genetic predispositions to neoplasias. Thus, there is a need in the art for the identification of mammalian tumor suppressor genes which can be used in methods of diagnosing, prognosing, and treating neoplastic cells in humans and other mammals.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods for diagnosing and treating neoplasia. These and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is an isolated and purified enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

Another embodiment of the invention is an isolated and purified polypeptide comprising at least 33 contiguous amino acids as shown in SEQ ID NO:2.

Still another embodiment of the invention is an enhancer of polycomb fusion protein comprising a first protein segment and a second protein segment fused together by means of a peptide bond. The first protein segment consists of at least 33 contiguous amino acids of an enhancer of polycomb protein as shown in SEQ ID NO:2.

Even another embodiment of the invention is a preparation of antibodies which specifically bind to a mammalian enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

Yet another embodiment of the invention is a cDNA molecule which encodes an enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

A further embodiment of the invention is a cDNA molecule which encodes at least 33 contiguous amino acids of SEQ ID NO:2. Another embodiment of the invention is a cDNA molecule comprising at least

447 contiguous nucleotides of SEQ ID NO:l.

Even another embodiment of the invention is a cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO: 1. Percent identity is determined using a Smith- Waterman homology search algorithm as implemented in a MPSRCH program using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

Still another embodiment of the invention is an isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO:l after washing with 0.2X SSC at 65 °C. The nucleotide sequence encodes a enhancer of polycomb protein having the amino acid sequence of SEQ ID NO:2.

A further embodiment of the invention is a construct comprising a promoter and a polynucleotide segment encoding at least 33 contiguous amino acids of an enhancer of polycomb protein as shown in SEQ ID NO:2. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter. Yet another embodiment of the invention is a host cell comprising a construct comprising a promoter and a polynucleotide segment encoding at least 33 contiguous amino acids of an enhancer of polycomb protein as shown in SEQ ID NO:2.

Even another embodiment of the invention is a recombinant host cell comprising a new transcription initiation unit. The new transcription initiation unit comprises in 5' to 3* order (a) an exogenous regulatory sequence, (b) an exogenous exon, and (c) a splice donor site. The new transcription initiation unit is located upstream of a coding sequence of a mammalian epc gene. The coding sequence of the mammalian epc gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, and 4. The exogenous regulatory sequence controls transcription of the coding sequence of the epc gene.

Another embodiment of the invention is a polynucleotide probe comprising at least 12 contiguous nucleotides selected from the group consisting of SEQ ID NOS:l, 3, and 4 and a detectable label. Still another embodiment of the invention is a method of identifying a neoplastic tissue of a human. Expression of a first epc gene in a first tissue of a human suspected of being neoplastic is compared with expression of a second epc gene in a second tissue of the human which is normal. The second epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. Decreased expression of the first epc gene relative to the second epc gene identifies the first tissue as being neoplastic.

Even another embodiment of the invention is a method to aid in the diagnosis or prognosis of neoplasia in a human. A first epc gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic is compared with a second epc gene, mRNA, or protein in a second tissue of a human which is normal. The second epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. A difference between the first and second epc genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue.

Yet another embodiment of the invention is a method to aid in detecting a genetic predisposition to neoplasia in a human. An epc gene, mRNA, or protein in the fetal tissue of a human is compared with a wild-type epc gene, mRNA, or protein. The wild- type epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. A difference between the epc gene, mRNA, or protein in the fetal tissue of the human and the wild-type human epc gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.

A further embodiment of the invention is a method of identifying a human chromosome 10. A preparation of metaphase human chromosomes is contacted with a polynucleotide probe comprising at least 12 contiguous nucleotides selected from a nucleotide sequence as shown in SEQ ID NO:3. A chromosome which specifically hybridizes to the polynucleotide probe is detected. A chromosome which specifically hybridizes to the polynucleotide probe is identified as a human chromosome 10.

A further embodiment of the invention is a method of identifying a human chromosome 22. A preparation of metaphase human chromosomes is contacted with a polynucleotide probe comprising at least 12 contiguous nucleotides selected from a nucleotide sequence as shown in SEQ ID NO:4. A chromosome which specifically hybridizes to the polynucleotide probe is detected. A chromosome which specifically hybridizes to the polynucleotide probe is identified as a human chromosome 22.

The present invention thus provides the art with mammalian tumor suppressor genes called enhancer of polycomb (epc). epc genes and proteins can be used, ter alia, as therapeutic, prognostic, and diagnostic tools for proliferative and developmental disorders and to identify a pi 0-11 region of a human chromosome 10 and to identify a ql3.3 region of a human chromosome 22.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is a discovery of the present invention that mammalian enhancer of polycomb

(Epc) proteins function as tumor suppressors and thus are implicated in neoplasias and other proliferative disorders, such as dysplasias and hyperplasias. Epc genes, proteins, and mRNAs can be used as diagnostic, prognostic, and therapeutic tools for these disorders. Coding sequences of human epc genes can also be used to identify a human chromosome 10 and a human chromosome 22. The coding region of one mouse epc gene, Epcl, has the nucleotide sequence shown in SEQ ID NO:l. Mouse Epcl mRNA comprises a 3.9 kb transcript containing 5' and 3' untranslated sequences, including a poly(A) tail, and a 2.3 kb open reading frame which is termed Epcl-L (Stankunas et al, Devel. 125, 4055-66, 1998). Epcl-L encodes a 764 amino acid protein as shown in SEQ ID NO:2. A second mouse epc gene, Epc2, encodes an amino acid sequence shown in SEQ ID NO:5.

One human epc gene, EPC1, comprises the nucleotide sequence shown in SEQ ID NO:3. A second human epc gene, EPC2, comprises the nucleotide sequence shown in SEQ ID NO:4. Any naturally occurring variants of the products of epc genes which may occur in mammals and which have, for example, tumor suppressive or antiproliferative activity, are within the scope of this invention.

Non-naturally occurring mammalian Epc variants which contain conservative amino acid substitutions relative to SEQ ID NOS:2 or 5 or to the amino acid sequences of Epc proteins encoded by polypeptides comprising SEQ ID NO: 3 or SEQ ID NO:4 but which retain substantially the same tumor suppressive activity as naturally occurring Epc variants are also biologically active mammalian Epc variants of the invention.

Naturally or non-naturally occurring mammalian Epc protein variants preferably are at least 85%, 90%, or 95% identical to SEQ ID NOS:2 or 5 or to Epc proteins encoded by polypeptides comprising SEQ ID NO:3 or SEQ ID NO:4 and have similar tumor suppressive functions. More preferably, the molecules are 98% or 99% identical. Percent sequence identity is determined using the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1. The Smith- Waterman homology search algorithm is taught in Smith and

Waterman, Adv. Appl. Math. (1981) - 482-489. Biologically active mammalian Epc variants include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. Covalent conjugates are prepared by linkage of functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residues of Epc proteins by means well known in the art. Epc variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect the tumor suppressive function of mammalian Epc proteins are also encompassed.

A subset of mutants, called muteins, is a group of proteins in which neutral amino acids such as serine are substituted for cysteine residues which do not participate in disulfide bonds. These mutants may be stable over a broader temperature range than naturally occurring forms of mammalian Epc. See Mark et ah, U.S. Pat. No. 4,959,314.

Biologically active human Epc polypeptides or polypeptide variants can comprise at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 33, 35, 40, 41, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 or more contiguous amino acids of the amino acid sequence shown in SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 25, 33, 35,

40, 41, 50, 75, 100, 125, or 150 contiguous amino acids of SEQ ID NO:5, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 32, 35, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or 350 or more contiguous amino acids of the amino acid sequences encoded by a polynucleotide comprising SEQ ID NOS:3 or 4. Polypeptide molecules having substantially the same amino acid sequence as human Epc protein but possessing minor amino acid substitutions which do not substantially affect the ability of the Epc polypeptides to interact with Epc-specific molecules, such as antibodies, are within the definition of biologically active Epc polypeptide variants.

Preferably, biologically active human Epc polypeptides or polypeptide variants are at least 65%, 75%, 85%, 90%, 95%, 98%, or 99% identical to Epc polypeptide fragments of SEQ ID NOS:2 or 5 or the amino acid sequences encoded by polynucleotides comprising SEQ ID NOS:3 or 4. Percent identity is determined as described above.

Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software. Preferably the amino acid changes in Epc protein or polypeptide variants are conservative amino acid changes, i.e., changes of similarly charged or uncharged amino acids. Conservative replacements are those which take place within a family of amino acids which are related in their side chains. Genetically encoded amino acids are generally divided into four families: acidic (aspartate, glutamate); basic (lysine, arginine, histidine); non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. It is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding properties of the resulting Epc molecule, especially if the replacement does not involve an amino acid at a binding site involved in an interaction of the Epc protein and another protein.

Tumor suppressive effects of mammalian Epc protein or polypeptide variants can be assessed or measured, for example, by transforming neoplastic cells in vitro with DNA constructs encoding a putative Epc variant and observing loss of the neoplastic phenotype by morphological or biochemical means. The neoplastic phenotype is familiar to those of skill in the art and includes aberrant cell cycle control, alterations in size and morphology, and biochemical changes, such as the appearance of tumor-specific markers and the over- or under-expression of tumor-associated gene products.

Mammalian Epc proteins and polypeptides can be isolated and purified from mammalian cells such as heart, brain, pancreas, skeletal muscle, placenta, spleen, thymus, prostate, testis, peripheral blood lymphocytes, lung, liver, kidney, bone marrow, appendix, or small intestine cells, and cells of the mucosal lining of the colon, using biochemical methods which are standard in the art. These methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, crystallization, electrofocusing, and preparative gel elecfrophoresis. The skilled artisan can readily select methods which will result in a preparation of Epc protein or polypeptide which is substantially free from other proteins and from carbohydrates, lipids, or subcellular organelles. A preparation of isolated and purified Epc protein is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Mammalian Epc proteins, polypeptides, or variants can be produced by recombinant DNA methods or by synthetic chemical methods. For production of recombinant Epc proteins or polypeptides, Epc coding sequences selected from the nucleotide sequences shown in SEQ ID NOS:l, 3, or 4 can be expressed in known prokaryotic or eukaryotic expression systems. Bacterial, yeast, insect, or mammalian expression systems can be used, as is known in the art. Alternatively, synthetic chemical methods, such as solid phase peptide synthesis, can be used to synthesize mammalian Epc protein, polypeptides, or variants. General means for the production of peptides, analogs or derivatives are outlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS - A SURVEY OF RECENT DEVELOPMENTS, B. Weinstein, ed. (1983). Substitution of D-amino acids for the normal L-stereoisomer of Epc can be carried out to increase the half-life of the molecule.

Fusion proteins comprising at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 33, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 or more contiguous amino acids of the amino acid sequence shown in SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 25, 33, 35, 40, 41, 50, 75, 100, 125, or 150 contiguous amino acids of SEQ ID NO:5, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 32, 35, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or 350 or more contiguous amino acids of the amino acid sequences encoded by a polynucleotide comprising SEQ ID NOS:3 or 4 can also be constructed. Epc fusion proteins are useful for generating antibodies against Epc amino acid sequences and for use in various assay systems. For example, Epc fusion proteins can be used to identify proteins which interact with Epc and which influence its tumor suppressive activity. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions such as the yeast two-hybrid or phage display systems, can also be used for this purpose. Such methods are well known in the art and can also be used as drug screens.

A Epc fusion protein comprises two protein segments fused together by means of a peptide bond. The first protein segment consists of at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 33, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 or more contiguous amino acids of the amino acid sequence shown in SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 25, 33, 35, 40, 41, 50, 75, 100, 125, or 150 contiguous amino acids of SEQ ID NO:5, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 32, 35, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or 350 or more contiguous amino acids of the amino acid sequences encoded by a polynucleotide comprising SEQ ID NOS:3 or 4. The first protein segment can also be a full-length mammalian Epc protein. The first protein segment can be N-terminal or C-terminal, as is convenient.

The second protein segment can be a full-length protein or a protein fragment or polypeptide. Proteins commonly used in fusion protein construction include β- galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Epitope tags can be used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.

Mammalian Epc fusion proteins can be made by covalently linking the first and second protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare mammalian Epc fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NOS:l, 3, or 4 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies which supply research labs with tools for experiments, including, for example, Promega Corporation (Madison, WI), Sfratagene (La Jolla, CA), Clontech (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Isolated mammalian Epc proteins, polypeptides, biologically active variants, or fusion proteins can be used as immunogens, to obtain a preparation of antibodies which specifically bind to epitopes of Epc proteins. The antibodies can be used, inter alia, to detect Epc protein in mammalian, particularly human, tissue or in fractions thereof. The antibodies can also be used to detect the presence of mutations in epc genes which result in under- or over-expression of Epc proteins or in expression of a mammalian Epc protein with altered size or electrophoretic mobility. By binding to an Epc protein, antibodies can also alter the tumor-suppressive function of mammalian Epc.

Antibodies which specifically bind to epitopes of mammalian Epc proteins, polypeptides, fusion proteins, or biologically active variants can be used in immunochemical assays, including but not limited to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Typically, antibodies of the invention provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in such immunochemical assays. Preferably, antibodies which specifically bind to mammalian Epc epitopes do not detect other proteins in immunochemical assays and can immunoprecipitate mammalian Epc proteins or polypeptides from solution.

Epc-specific antibodies specifically bind to epitopes present in a mammalian Epc protein having an amino acid sequence shown in SEQ ID NOS:2 or 5, to human Epc proteins having an amino acid sequence encoded by a polynucleotide comprising SEQ ID NOS:3 or 4, or to biologically active variants of those sequences. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids. Preferably, Epc epitopes are not present in other mammalian proteins.

Epitopes of mammalian Epc which are particularly antigenic can be selected, for example, by routine screening of Epc polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein to the amino acid sequences shown in SEQ ID NOS:2 or 5 or encoded by polynucleotides comprising SEQ ID NOS:3 or 4. Such methods are taught, for example, in Hopp and Wood, Proc. Natl. Acad. Sci. U.S.A. 78, 3824-28 (1981), Hopp and Wood, Mol. Immunol. 20, 483-89 (1983), and Sutcliffe et al., Science 219, 660-66 (1983). By reference to the amino acid and nucleotide sequences disclosed herein, regions of a mammalian Epc protein which could generate antibodies which might cross-react with other mammalian Epc proteins can be avoided.

Any type of antibody known in the art can be generated to bind specifically to mammalian Epc epitopes. For example, preparations of polyclonal and monoclonal antibodies can be made using standard methods which are well known in the art. Similarly, single-chain antibodies can also be prepared. Single-chain antibodies which specifically bind to mammalian Epc epitopes can be isolated, for example, from single-chain immunoglobulin display libraries, as is known in the art. The library is "panned" against mammalian Epc amino acid sequences, and a number of single chain antibodies which bind with high-affinity to different epitopes of a mammalian Epc protein can be isolated. Hayashi et al, 1995, Gene 160:129-30. Single-chain antibodies can also be constructed using a DNA amplification method, such as the polymerase chain reaction (PCR), using hybridoma cDNA as a template. Thirion et al., 1996, Ewr. J. Cancer Prev. 5:507-11.

Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma and Morrison, 1997, Nat. Biotechnol. 75:159-63. Construction of bivalent, bispecific single-chain antibodies is taught inter alia in Mallender and Noss, 1994, J. Biol. Chem. 269: 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DΝA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology. Nerhaar et al., 1995, Int. J. Cancer 57:497-501; Νicholls et α/., 1993, J Immunol. Meth. 755:81-91.

Monoclonal and other antibodies can also be "humanized" in order to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between, for example, rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences, for example, by site directed mutagenesis of individual residues, or by grafting of entire complementarity determining regions. Alternatively, one can produce humanized antibodies using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to Epc epitopes can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Other types of antibodies can be constructed and used in methods of the invention. For example, chimeric antibodies can be constructed as disclosed, for example, in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, can also be prepared.

Antibodies of the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passing the antibodies over a column to which a Epc protein, polypeptide, biologically active variant, or fusion protein is bound. The bound antibodies can then be eluted from the column, using a buffer with a high salt concentration.

Mammalian Epc-specific binding polypeptides other than antibodies can also be generated. Epc-specific binding polypeptides are polypeptides which bind with mammalian Epc or its variants and which have a measurably higher binding affinity for Epc and variants of Epc than for other polypeptides tested for binding. Higher affinity by a factor of 10 is preferred, more preferably a factor of 100. Such polypeptides can be found, for example, using the yeast two-hybrid system.

Mammalian epc polynucleotides according to the invention are subgenomic and contain less than a whole chromosome. Preferably, the subgenomic polynucleotides are intron-free. epc subgenomic polynucleotides can be isolated and purified free from other nucleotide sequences using standard nucleic acid purification techniques. For example, restriction enzymes and probes can be used to isolate subgenomic polynucleotide fragments which comprise the epc coding sequences. Isolated and purified subgenomic polynucleotides are in preparations which are free or at least 90% free of other molecules.

Isolated and purified epc subgenomic polynucleotides of the invention can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 447, 448, 450, 500, 1000, 1500, 2000, 2500, 3000, 3500, or 3750 or more contiguous nucleotides selected from SEQ ID NO:l, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000 or more contiguous nucleotides selected from either SEQ ID NO:3 or SEQ ID NO:4, or can comprise SEQ ID NOS:l, 3, or 4. Such polynucleotides can be used, for example, as primers or probes or for expression of epc proteins or polypeptides.

The complement of the nucleotide sequences shown in SEQ ID NOS:l, 3, and 4 are contiguous nucleotide sequences which form Watson-Crick base pairs with a contiguous nucleotide sequence as shown in SEQ ID NOS:l, 3, or 4. The complements of SEQ ID NOS: 1, 3, or 4 can be used, for example, to provide epc antisense oligonucleotides, primers, and probes.

Mammalian epc antisense oligonucleotides, primers, and probes can consist of at least 11, 12, 15, 20, 25, 30, 50, or 100 contiguous nucleotides which are complementary to the coding sequences shown in SEQ ID NOS:l, 3, or 4. A complement of the entire coding sequence or a mammalian epc gene can also be used. Double-stranded subgenomic polynucleotides which comprise all or a portion of the nucleotide sequences shown in SEQ ID NO:l, 3, or 4, as well as polynucleotides which encode Epc-specific antibodies or ribozymes, are also epc subgenomic polynucleotides.

Degenerate nucleotide sequences which encode amino acid sequences of mammalian Epc proteins or biologically active Epc variants, as well as homologous nucleotide sequences which are at least 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences shown in SEQ ID NOS:l, 3, or 4, are also mammalian epc subgenomic polynucleotides. Percent sequence identity is determined using computer programs which employ the Smith- Waterman algorithm, for example as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with the following parameters: a gap open penalty of 12 and a gap extension penalty of 1. Nucleotide sequences which hybridize to the coding sequences shown in SEQ ID NOS:l, 3, or 4 or their complements with at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35% basepair mismatches are also epc subgenomic polynucleotides. For example, using the following wash conditions~2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous epc sequences can be identified which contain at most about 25-30% basepair mismatches with SEQ ID NOS: 1 , 3, or 4 or their complements. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of mammalian epc subgenomic polynucleotides, as well as full-length genes which encode mammalian, particularly human, Epc proteins can also be identified by making suitable probes or primers and screening cDNA expression libraries from human or other mammalian species. It is well known that the T_m of a double- stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Homologous epc human polynucleotides or epc polynucleotides of other species can therefore be identified, for example, by hybridizing a putative homologous epc polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l, 3, or 4, comparing the melting temperature of the test hybrid with the melting temperature of a hybrid comprising a polynucleotide having a nucleotide sequence of SEQ ID NO:l, 3, or 4 and a polynucleotide which is perfectly complementary to SEQ ID NO:l, 3, or 4 and calculating the number of basepair mismatches within the test hybrid.

Nucleotide sequences which hybridize to the coding sequences shown in SEQ ID NOS:l, 3, or 4 or their complements following stringent hybridization and/or wash conditions are also epc subgenomic polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51. Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. It is well known that the T_m of a double- stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). The T_m of a hybrid between the epc sequences shown in SEQ ID NOS:l, 3, or 4 and a polynucleotide sequence which is 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T_m = 81.5 °C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600//), where / = the length of the hybrid in basepairs. Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C. Complementary DNA (cDNA) molecules which encode mammalian Epc proteins are also epc subgenomic polynucleotides of the invention, epc cDNA molecules can be made with standard molecular biology techniques, using mammalian epc mRNA as a template. Mammalian epc cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al., 1989. An amplification technique, such as the polymerase chain reaction (PCR), can be used to obtain additional copies of epc subgenomic polynucleotides, using, for example, human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize epc subgenomic polynucleotide molecules. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a mammalian Epc protein having the amino acid sequences shown in SEQ ID NOS:2 or 5, an amino acid sequence encoded by a polynucleotide comprising SEQ ID NOS:3 or 4, or a biologically active variant of those proteins.

The invention also provides polynucleotide probes which can be used to detect epc sequences, for example, in hybridization protocols such as Northern or Southern blotting or in situ hybridizations. Polynucleotide probes of the invention comprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 or more contiguous nucleotides selected from SEQ ID NOS:l, 3, or 4. Polynucleotide probes of the invention can comprise a detectable label, such as a radioisotopic, fluorescent, enzymatic, or chemiluminescent label.

If desired, expression of a mammalian epc gene can be altered using an antisense oligonucleotide. The sequence of the antisense oligonucleotide is complementary to at least a portion of the coding sequences shown in SEQ ID NOS:l, 3, or 4. Preferably, the antisense oligonucleotide is at least six nucleotides in length, but can be at least 8, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Longer sequences, such as the complement of a nucleotide sequence shown in SEQ ID NOS:l, 3, or 4, can also be used. Antisense oligonucleotides can be provided in an epc construct of the invention and introduced into tumor cells, using transfection techniques known in the art. Mammalian epc antisense oligonucleotides can be composed of deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux, 1994, Meth. Mol. Biol. 26:1-12; Uhlmann et al, 1990, Chem. Rev. 90:543-583.

Precise complementarity is not required for successful duplex formation between an antisense molecule and the complementary coding sequence of a mammalian epc gene. Antisense molecules which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a mammalian epc coding sequence, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent epc coding sequences, can provide targeting specificity for epc mRNA. Preferably, each stretch of contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular epc coding sequence. Mammalian epc antisense oligonucleotides can be modified without affecting their ability to hybridize to an epc coding sequence. These modifications can be internal or at one or both ends of the antisense oligonucleotide. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, can also be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. Agrawal et al., Trends Biotechnol. 70:152-158, 1992; Uhlmann et al, Chem. Rev. 90:543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 275:3539-3542, 1987.

Expression of a mammalian epc gene can also be decreased using a ribozyme, an RNA molecule with catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends Genet. 12: 510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art {e.g., Haseloff et al, U.S. 5,641,673).

The coding sequences shown in SEQ ID NOS:l, 3, and 4 can be used to generate a ribozyme which will specifically bind to mammalian epc mRNA. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al, Nature 53^:585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201). Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target. Mammalian epc ribozymes can be introduced into cells as part of a DNA construct, as is known in the art. The DNA construct can also include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of the ribozyme in the cells. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce the epc ribozyme-containing DNA construct into cells in order to decrease epc expression. Alternatively, if it is desired that the cells stably retain the DNA construct, it can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.

A mammalian epc subgenomic polynucleotide can be used in a construct, such as a DNA or RNA construct, epc constructs can be used, for example, to express all or a portion of a mammalian Epc protein in a host cell. The host cell comprising the expression construct can be prokaryotic or eukaryotic. A variety of host cells for use in bacterial, yeast, insect, and human expression systems are available and can be used to express the expression construct. Expression systems in bacteria include those described in Chang et al, Nature (1978) 275: 615, Goeddel et al, Nature (1979) 257: 544, Goeddel et al, Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S. 4,551,433, deBoer et al, Proc. Nat Acad. Sci. USA (1983) 80: 21-25, and Siebenlist et al, Cell (1980) 20: 269.

Expression systems for epc in yeast include those described in Hinnen et al, Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al, J. Bacteriol. (1983) 755: 163; Kurtz et al, Mol. Cell. Biol. (1986) 6: 142; Kunze et al, J. Basic Microbiol (1985) 25: 141; Gleeson et al, J. Gen. Microbiol. (1986) 732: 3459, Roggenkamp et al, Mol. Gen. Genet. (1986) 202 :302) Das et al, J. Bacteriol. (1984) 75S: 1165; De Louvencourt et al, J. Bacteriol. (1983) 154: 737, Nan den Berg et al, Bio/Technology (1990) 8: 135; Kunze et al, J. Basic Microbiol. (1985) 25: 141; Cregg et al, Mol. Cell. Biol. (1985) 5: 3376, U.S. 4,837,148, US 4,929,555; Beach and Nurse, Nature (1981) 300: 706; Davidow et al, Curr. Genet. (1985) 70: 380, Gaillardin et al, Curr. Genet. (1985) 70: 49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 772: 284-289; Tilburn et al, Gene (1983) 26: 205-221, Yelton et al., Proc. Natl. Acad. Sci. USA (1984) 81: 1470- 1474, Kelly and Hynes, EMBOJ. (1985) 4: 475479; EP 244,234, and WO 91/00357.

Expression of epc coding sequences in insects can be accomplished as described in U.S. 4,745,051, Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, and Vlak et al, J. Gen. Virol. (1988) 69: 765-776, Miller et al, Ann. Rev. Microbiol. (1988) 42: 111, Carbonell et al, Gene (1988) 73: 409, Maeda et al, Nature (1985) 375: 592-594, Lebacq-Verheyden et al., Mol Cell. Biol. (1988) 8: 3129; Smith et al, Proc. Natl. Acad. Sci. USA (1985) 82: 8404, Miyajima et al, Gene (1987) 58: 273; and Martin et al, DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al, Bio/Technology (1988) 6: 47-55, Miller et al, in GENERIC ENGINEERING (J.K. Setlow et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al, Nature, (1985) 375: 592-594. Expression of mammalian epc constructs in mammalian cells can be accomplished as described in Dijkema et al, EMBO J. (1985) 4: 761, Gorman et al, Proc. Natl. Acad. Sci. USA (1982b) 79: 6777, Boshart etal, Cell (1985) 41: 521 and U.S. 4,399,216. Other features of mammalian expression can be facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58: 44, Barnes and Sato, Anal. Biochem. (1980) 702: 255, U.S. 4,767,704, US 4,657,866, US 4,927,762, US 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

Constructs of the invention can be introduced into the host cells using any technique known in the art. These techniques include transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and calcium phosphate-mediated transfection.

A mammalian epc expression construct comprises a promoter which is functional in the particular host cell selected. The skilled artisan can readily select an appropriate promoter from the large number of cell type-specific promoters known and used in the art. A mammalian epc expression construct can also contain a transcription terminator which is functional in the host cell. The expression construct comprises a polynucleotide segment which encodes all or a portion of a human Epc protein. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter. The expression construct can be linear or circular and can contain sequences, if desired, for autonomous replication.

Mammalian epc subgenomic polynucleotides can also be used in gene delivery vehicles, for the purpose of delivering a mammalian epc mRNA or oligonucleotide (either with the sequence of a native mammalian epc mRNA or its complement), full- length Epc protein, Epc fusion protein, Epc polypeptide, or Epc-specific ribozyme or single-chain antibody, into a cell preferably a eukaryotic cell. According to the present invention, a gene delivery vehicle can be, for example, naked plasmid DNA, a viral expression vector comprising an epc polynucleotide, or a mammalian epc subgenomic polynucleotide in conjunction with a liposome or a condensing agent. In one embodiment of the invention, the gene delivery vehicle comprises a promoter and a mammalian epc subgenomic polynucleotide. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which can be activated by infection with a virus, such as the α- and β-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.

A mammalian epc gene delivery vehicle can comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxo virus, parvo virus, picornavirus, poxvirus, retro virus, togavirus or adeno virus. In a preferred embodiment, the mammalian epc gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al, Cell 33: 153, 1983, Cane and Mulligan, Proc. Natl. Acad. Sci. USA 81 :6349, 1984, Miller et al. , Human Gene Therapy 1:5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Nile and Hart, Cancer Res. 53:3860-3864, 1993; Nile and Hart, Cancer Res. 53:962-967, 1993; Ram et al, Cancer Res. 53:83-88, 1993; Takamiya et α/., J. Neurosci. Res. 33:493-503, 1992; Baba et al, J. Neurosurg. 79:729-735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805). Particularly preferred retroviruses are derived from retroviruses which include avian leukosis virus (ATCC Nos. NR-535 and NR-247), bovine leukemia virus (NR- 1315), murine leukemia virus (MLN), mink-cell focus-inducing virus (Koch et al, J. Vir. 49:828, 1984; and Oliff et al, J. Vir. 48:542, 1983), murine sarcoma virus (ATCC Νos. NR-844, 45010 and 45016), reticuloendotheliosis virus (ATCC Νos NR-994, VR-770 and 45011), Rous sarcoma virus, Mason-Pfizer monkey virus, baboon endogenous virus, endogenous feline retrovirus (e.g., RD114), and mouse or rat gL30 sequences used as a retroviral vector. Particularly preferred strains of MLV from which recombinant retroviruses can be generated include 4070A and 1504A (Hartley and Rowe, J Vir. 79:19, 1976), Abelson (ATCC No. NR-999), Friend (ATCC No. VR-245), Graffi (Ru et al. , J. Vir. 67:4122, 1993; and Yantchev Neoplasma 26:391, 1979), Gross (ATCC No. VR-590), Kirsten (Albino et al, J. Exp. Med. 164:1110, 1986), Harvey sarcoma virus (Manly et al, J. Vir. 52:3540, 1988; and Albino et al, J. Exp. Med. 164:1110, 1986) and Rauscher (ATCC No. VR-998), and Moloney MLV (ATCC No. VR-190). A particularly preferred non-mouse retrovirus is Rous sarcoma virus. Preferred Rous sarcoma viruses include Bratislava (Manly et al, J. Vir. 52:3540, 1988; and Albino et al, J. Exp. Med. 754:1710, 1986), Bryan high titer (e.g., ATCC Nos. VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard (ATCC No. VR-140), Carr-Zilber (Adgighitov et al, Neoplasma 27:159, 1980), Engelbreth-Holm (Laurent et al, Biochem BiophysActa 908:241, 1987), Harris, Prague (e.g., ATCC Nos. VR-772, and 45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354) viruses.

Any of the above retroviruses can be readily utilized in order to assemble or construct retroviral gene delivery vehicles for mammalian epc subgenomic polynucleotides given the disclosure provided herein and standard recombinant techniques (e.g. , Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., Cold Spring Harbor Laboratory Press, 1989, and Kunkle, Proc. Natl. Acad. Sci.

U.S.A. #2:488, 1985) known in the art. Portions of retroviral expression vectors can be derived from different retroviruses. For example, retrovector LTRs can be derived from a murine sarcoma virus, a tRNA binding site from a Rous sarcoma virus, a packaging signal from a murine leukemia virus, and an origin of second strand synthesis from an avian leukosis virus. These recombinant retroviral vectors can be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see Serial No. 07/800,921, filed November 29, 1991). Recombinant retroviruses can be produced which direct the site-specific integration of the recombinant retroviral genome into specific regions of the host cell DNA. Such site-specific integration can be mediated by a chimeric integrase incorporated into the retroviral particle (see Serial No. 08/445,466 filed May 22, 1995). It is preferable that the recombinant viral gene delivery vehicle is a replication-defective recombinant virus. Packaging cell lines suitable for use with the above-described retroviral gene delivery vehicles can be readily prepared (see Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266) and used to create producer cell lines (also termed vector cell lines or "NCLs") for production of recombinant viral particles. In particularly preferred embodiments of the present invention, packaging cell lines are made from human (e.g., HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviral gene delivery vehicles which are capable of surviving inactivation in human serum. The construction of recombinant retroviral gene delivery vehicles is described in detail in WO 91/02805. These recombinant retroviral gene delivery vehicles can be used to generate transduction competent retroviral particles by introducing them into appropriate packaging cell lines (see Serial No. 07/800,921). Similarly, adenovirus gene delivery vehicles can also be readily prepared and utilized given the disclosure provided herein (see also Berkner, Biotechniques 5:616-627, 1988, and Rosenfeld et al, Science 252:431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).

A mammalian epc gene delivery vehicle can also be a recombinant adenoviral gene delivery vehicle. Such vehicles can be readily prepared and utilized given the disclosure provided herein (see Berkner, Biotechniques 5:616, 1988, and Rosenfeld et al, Science 252:431, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).

Adeno-associated viral gene delivery vehicles can also be constructed and used to deliver Epc amino acids or nucleotides. The use of adeno-associated viral gene delivery vehicles in vitro is described in Chatterjee et al, Science 258: 1485-1488 (1992), Walsh et al, Proc. Nat'l. Acad. Sci. 89: 7257-7261 (1992), Walsh et al, J. Clin. Invest. 94: 1440-1448 (1994), Flotte et al, J. Biol. Chem. 268: 3781-3790 (1993), Ponnazhagan et al, J. Exp. Med. 179: 733-738 (1994), Miller et al, Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994), Einerhand et al, Gene Ther. 2: 336-343 (1995), Luo et al, Exp. Hematol 23: 1261-1267 (1995), and Zhou et al, Gene Therapy 3: 223-229 (1996). In vivo use of these vehicles is described in Flotte et al, Proc. Nat'l Acad. Sci. 90: 10613-10617 (1993), and Kaplitt et al, Nature Genet. 5:148-153 (1994).

In another embodiment of the invention, a mammalian epc gene delivery vehicle is derived from a togavirus. Preferred togaviruses include alphaviruses, in particular those described in U.S. Serial No. 08/405,627, filed March 15, 1995, WO 95/07994. Alpha viruses, including Sindbis and ELVS viruses can be gene delivery vehicles for mammalian epc subgenomic polynucleotides. Alpha viruses are described in WO

94/21792, WO 92/10578 and WO 95/07994. Several different alphavirus gene delivery vehicle systems can be constructed and used to deliver mammalian epc subgenomic polynucleotides to a cell according to the present invention. Representative examples of such systems include those described in U.S. Patents 5,091,309 and 5,217,879. Particularly preferred alphavirus gene delivery vehicles for use in the present invention include those which are described in WO 95/07994, and U.S. Serial No. 08/405,627.

Preferably, the recombinant viral vehicle is a recombinant alphavirus viral vehicle based on a Sindbis virus. Sindbis constructs, as well as numerous similar constructs, can be readily prepared essentially as described in U.S. Serial No. 08/198,450. Sindbis viral gene delivery vehicles typically comprise a 5' sequence capable of initiating Sindbis virus transcription, a nucleotide sequence encoding Sindbis non-structural proteins, a viral junction region inactivated so as to prevent fragment transcription, and a Sindbis RNA polymerase recognition sequence. Optionally, the viral junction region can be modified so that polynucleotide transcription is reduced, increased, or maintained. As will be appreciated by those in the art, corresponding regions from other alphaviruses can be used in place of those described above.

The viral junction region of an alphavirus-derived gene delivery vehicle can comprise a first viral junction region which has been inactivated in order to prevent transcription of the polynucleotide and a second viral junction region which has been modified such that polynucleotide transcription is reduced. An alphavirus-derived vehicle can also include a 5' promoter capable of initiating synthesis of viral RNA from cDNA and a 3' sequence which controls transcription termination.

Other recombinant togaviral gene delivery vehicles which can be utilized in the present invention include those derived from Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Patents 5,091,309 and 5,217,879 and in WO 92/10578. The Sindbis vehicles described above, as well as numerous similar constructs, can be readily prepared essentially as described in U.S. Serial No. 08/198,450.

Other viral gene delivery vehicles suitable for use in the present invention include, for example, those derived from poliovirus (Evans et al, Nature 339:385, 1989, and Sabin et al, J. Biol. Standardization 7:115, 1973) (ATCC VR-58); rhinovirus (Arnold et al, J. Cell. Biochem. L401, 1990) (ATCC VR-1110); pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al, Proc. Natl. Acad. Sci. U.S.A. 86:311, 1989; Flexner et α/., Ann. NY. Acad. Sci. 559:86, 1989; Flexner et al, Vaccine 8:11, 1990; U.S. 4,603,112 and U.S. 4,769,330; WO 89/01973) (ATCC VR-111; ATCC VR-2010); SV40 (Mulligan et al, Nature 277:108, 1979) (ATCC VR-305), (Madzak et al, J. Gen. Vir. 73:1533, 1992); influenza virus (Luytjes et al, Cell 59:1107, 1989; McMicheal et al, The New England Journal of Medicine 309:13, 1983; and Yap et al, Nature 273:238, 1978) (ATCC VR-797); parvovirus such as adeno-associated virus (Samulski et α/., J Vir. 53:3822, 1989, and Mendelson et α/. , Virology 755:154, 1988) (ATCC VR-645); herpes simplex virus (Kit et al, Adv. Exp. Med. Biol. 275:219, 1989) (ATCC VR-977; ATCC VR-260); Nature 277: 108, 1979); human immunodeficiency virus (EPO 386,882, Buchschacher et al, J. Vir. 55:2731, 1992); measles virus (EPO 440,219) (ATCC VR-24); A (ATCC VR-67; ATCC VR-1247), Aura (ATCC VR-368), Bebaru virus (ATCC VR-600; ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC VR-926), Y-62-33 (ATCC VR-375), ONyong virus, Eastern encephalitis virus (ATCC VR-65; ATCC VR- 1242), Western encephalitis virus (ATCC VR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus (Hamre et al, Proc. Soc. Exp. Biol. Med. 727:190, 1966) (ATCC VR-740).

A mammalian epc subgenomic polynucleotide can also be combined with a condensing agent to form a gene delivery vehicle. In a preferred embodiment, the condensing agent is a polycation, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Many suitable methods for making such linkages are known in the art (see, for example, Serial No. 08/366,787, filed December 30, 1994).

In an alternative embodiment, a mammalian epc subgenomic polynucleotide is associated with a liposome to form a gene delivery vehicle. Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter. Under appropriate conditions, a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm. Prior to interaction with the surface of a cell, however, the liposome membrane acts as a relatively impermeable barrier which sequesters and protects its contents, for example, from degradative enzymes. Additionally, because a liposome is a synthetic structure, specially designed liposomes can be produced which incorporate desirable features. See Stryer, Biochemistry, pp. 236- 240, 1975 (W.H. Freeman, San Francisco, CA); Szoka et al, Biochim. Biophys. Ada 500:1, 1980; Bayer et al, Biochim. Biophys. Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol 149:119, 1987; Wang et al, PROC. NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et al.,Anal Biochem. 775:420, 1989, and U.S. Patent 4,762,915. Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and expression constructs comprising epc polynucleotides such those disclosed in the present invention.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al, Proc. Natl. Acad. Sci. USA 84:1413-1416, 1987), mRNA (Malone et al, Proc. Natl. Acad. Sci. USA #5:6077-6081, 1989), and purified transcription factors (Debs et al, J. Biol. Chem. 255:10189-10192, 1990), in functional form. Cationic liposomes are readily available. For example, N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. See also Feigner et al, Proc. Natl. Acad. Sci. USA 91: 5148-5152.87, 1994. Other commercially available liposomes include Transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al, Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al, METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al, Proc. Natl. Acad. Sci. USA #7:3410-3414, 1990; Papahadjopoulos et al, Biochim. Biophys. Acta 394:483, 1975; Wilson et al, Cell 17:11, 1979; Deamer and Bangham, Biochim. Biophys. Acta 443:629, 1976; Ostro et al, Biochem. Biophys. Res. Commun. 75:836 , 1977; Fraley et al, Proc. Natl. Acad. Sci. USA 75:3348, 1979; Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA 75:145, 1979; Fraley et al, J. Biol Chem. 255:10431, 1980; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75:145, 1979; and Schaefer- Ridder et α/., Science 275:166, 1982.

In addition, lipoproteins can be included with an epc polynucleotide for delivery to a cell. Examples of such lipoproteins include chylomicrons, HDL, IDL, LDL, and

VLDL. Mutants, fragments, or fusions of these proteins can also be used. Modifications of naturally occurring lipoproteins can also be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are included with a polynucleotide, no other targeting ligand is included in the composition.

In another embodiment, naked mammalian epc subgenomic polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859. Such gene delivery vehicles can be either DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al, Hum. Gene. Ther. 3 : 147- 154, 1992. Other suitable vehicles include DNA-ligand (Wu et al , J. Biol. Chem. 254:16985-16987, 1989), lipid-DNA combinations (Feigner et al, Proc. Natl Acad. Sci. USA #4:7413 7417, 1989), liposomes (Wang et al, Proc. Natl. Acad. Sci. #4:7851-7855, 1987) and microprojectiles (Williams et al, Proc. Natl. Acad. Sci. 88:2126-2130, 1991). One can increase the efficiency of naked mammalian epc subgenomic polynucleotide uptake into cells by coating the polynucleotides onto biodegradable latex beads. This approach takes advantage of the observation that latex beads, when incubated with cells in culture, are efficiently transported and concentrated in the perinuclear region of the cells. The beads will then be transported into cells when injected into muscle. Mammalian epc subgenomic polynucleotide-coated latex beads will be efficiently transported into cells after endocytosis is initiated by the latex beads and thus increase gene transfer and expression efficiency. This method can be improved further by treating the beads to increase their hydrophobicity, thereby facilitating the disruption of the endosome and release of mammalian epc subgenomic polynucleotides into the cytoplasm. In one embodiment of the invention, expression of an endogenous mammalian epc gene in a cell can be altered by introducing in frame with the endogenous mammalian epc gene a DNA construct comprising a transcription unit by homologous recombination to form a homologously recombinant cell comprising the transcription unit. The transcription unit comprises a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. This method of affecting endogenous gene expression is taught in U.S. Patent No. 5,641,670.

The targeting sequence is a segment of at least 10, 12, 15, 20, or 50 contiguous nucleotides selected from one of the nucleotide sequences shown in SEQ ID NOS:l, 3, and 4. The transcription unit is located upstream to a coding sequence of the endogenous mammalian epc gene. The exogenous regulatory sequence directs transcription of the coding sequence of the mammalian epc gene. Preferably, the homologously recombinant cell is a differentiated cell, for example, a cell of the brain, thymus, testis, heart, prostate, small intestine, placenta, spleen, skeletal muscle, kidney, liver, lung, pancreas, or the mucosal lining of the colon. It is also preferred that the exogenous regulatory sequence directs increased transcription of the coding sequence of the mammalian epc gene. The human EPC1 gene maps to human chromosome region lOpl 1-12. The human EPC2 gene maps to human chromosome region 22ql3.3. Polynucleotide probes selected from SEQ ID NOS:3 or 4 can therefore be used to identify these chromosome regions in metaphase spreads of human chromosomes. Preparations of human metaphase chromosomes can be prepared using standard cytogenetic techniques from human primary tissues or cell lines. Polynucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 3 are used to identify human chromosome 10. Polynucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO:4 are used to identify human chromosome 22. The polynucleotide probes can be labeled, for example, with a radioactive, fluorescent, biotinylated, or chemiluminescent label, and detected by well known methods appropriate for the particular label selected. Protocols for hybridizing polynucleotide probes to preparations of metaphase chromosomes are also well known in the art. A polynucleotide probe will hybridize specifically to nucleotide sequences in the chromosome preparations which are complementary to the nucleotide sequence of the probe.

A polynucleotide probe which hybridizes specifically to human chromosome region lOpl 1-12 or 22ql3.3 hybridizes to nucleotide sequences present in the human EPC1 or EPC2 genes, respectively, and not to nucleotide sequences present in other human genes. A polynucleotide probe which hybridizes specifically to an EPC1 or EPC2 gene provides a detection signal at least 5-, 10-, or 20-fold higher than the background hybridization provided with non-EPCl or -EPC2 coding sequences.

A human chromosome which specifically hybridizes to a polynucleotide probe selected from SEQ ID NO: 3 is identified as a human chromosome 10. Preferably, the polynucleotide probe identifies the short arm of human chromosome 10. More preferably, the polynucleotide probe identifies a pi 1-12 region of human chromosome 10. A human chromosome which specifically hybridizes to a polynucleotide probe selected from SEQ ID NO:4 is identified as a human chromosome 22. Preferably, the polynucleotide probe identifies the long arm of human chromosome 22. More preferably, the polynucleotide probe identifies a ql3.3 region of human chromosome 22. The present invention also provides a method to identify neoplastic tissue in a human. The expression of an EPCl or EPC2 gene can be compared between a first tissue which is suspected of being neoplastic and a second tissue of the human which is normal. The normal tissue can be any tissue of the human, especially those which express the EPCl or EPC2 gene, including but not limited to, brain, thymus, testis, heart, prostate, placenta, spleen, small intestine, skeletal muscle, pancreas, and the mucosal lining of the colon. The tissue suspected of being neoplastic can be derived from a different tissue type of the human, but preferably it is derived from the same tissue type, for example an intestinal polyp or other abnormal growth. Without being limited to any particular theory, the inventor believes that a difference between the epc gene, mRNA, or protein in the two tissues which are compared, for example in molecular weight, amino acid or nucleotide sequence, indicates a somatic mutation in the EPCl or EPC2 gene. Alternatively, a difference in relative abundance of an EPCl or EPC2 mRNA or Epc protein may indicate a somatic mutation in a gene which regulates the expression, half- life, or degradation of EPCl or EPC2 mRNA or protein.

The EPCl or EPC2 genes in the two tissues can be compared by any means known in the art. For example, the two genes can be sequenced, and the sequence of the EPCl or EPC2 gene in the tissue suspected of being neoplastic can be compared with the wild-type EPCl or EPC2 sequence in the normal tissue. The EPCl or EPC2 genes or portions of the genes in the two tissues can be amplified, for example using nucleotide primers selected from the nucleotide sequences shown in SEQ ID NOS:3 or 4 in the polymerase chain reaction (PCR) or other amplification technique. The amplified genes or portions of genes can be hybridized to polynucleotide probes selected from the nucleotide sequences shown in SEQ ID NO:3 and 4. The polynucleotide probes can be labeled by a variety of methods, such as radiolabeling, biotinylation, or labeling with fluorescent or chemiluminescent tags, and detected by standard methods known in the art. A difference in the nucleotide sequence of the EPCl or EPC2 gene in the tissue suspected of being neoplastic compared with the wild-type EPCl or EPC2 nucleotide sequence is indicative of neoplasia. Alternatively, EPCl or EPC2 mRNA in the two tissues can be compared. PolyA⁺ RNA can be isolated from the two tissues as is known in the art. For example, one of skill in the art can readily determine differences in the size or amount of EPCl or EPC2 mRNA transcripts between the two tissues that are compared, using Northern blots and polynucleotide probes selected from the nucleotide sequences shown in SEQ ID NO:3 and 4. Decreased expression of EPCl or EPC2 mRNA in a tissue sample suspected of being neoplastic compared with the expression of EPCl or EPC2 mRNA in a normal tissue is indicative of neoplasia.

Any method for analyzing proteins can be used to compare two Epc proteins from matched samples. Sizes of the Epc proteins in the two tissues can be compared, for example, using antibodies of the present invention to detect Epc proteins in Western blots of protein extracts from the two tissues. Other changes, such as expression levels and subcellular localization, can also be detected immunologically using antibodies of the invention. A lower Epc protein expression level in a tissue suspected of being neoplastic compared with the Epc protein expression level in a normal tissue is indicative of neoplasia.

Similarly, comparison of EPCl or EPC2 gene sequences or of EPCl or EPC2 gene expression products, e.g., mRNA and protein, between a tissue of a human which is suspected of being neoplastic and a normal tissue of a human can be used to diagnose or prognose neoplasia in the human. Such comparisons of EPCl or EPC2 genes, mRNA, or protein can be made as described above. Decreased expression of the EPCl or EPC2 gene in the tissue suspected of being neoplastic indicates the presence of neoplastic cells in the tissue. The degree of decreased expression of the EPCl or EPC2 gene in the neoplastic tissue relative to wild-type expression of the gene in normal tissue or differences in the amount of decreased expression of the EPCl or EPC2 gene in the neoplastic tissue over time can be used to prognose the progression of the neoplasia in that tissue or to monitor the response of the neoplastic tissue to various therapeutic regimens over time.

In addition, a genetic predisposition to neoplasia in a human can be detected by comparing an EPCl or EPC2 gene, mRNA, or protein in a fetal tissue with a wild-type EPCl or EPC2 gene, mRNA, or protein. Fetal tissues which can be used for this purpose include, but are not limited to, amniotic fluid, chorionic villi, blood, and the blastomere of an in v/^'tro-fertilized embryo. The wild-type EPCl or EPC2 gene can be obtained from any tissue. The mRNA or protein can be obtained from a normal tissue of a human in which the EPCl or EPC2 gene is expressed. Such tissues are disclosed above.

Differences such as alterations in the nucleotide sequence or size of the fetal EPCl or EPC2 gene or mRNA, or alterations in the molecular weight, amino acid sequence, or relative abundance of fetal Epc protein, indicate a germline mutation in the EPCl or EPC2 gene of the fetus which indicates a genetic predisposition to neoplasia. Kits for use in the diagnostic or chromosome identification methods described above are also provided, epc diagnostic kits comprise reagents which specifically bind to a human epc gene or expression product and which can be used in methods of the invention, such as epc subgenomic polynucleotide probes or antibodies. Means for labeling the probes or antibodies, reagents for use in the methods, such as buffers, and instructions for using the kits can also be included.

Mammalian epc subgenomic polynucleotides or proteins can also be used to change the pattern of differentiation of a cell, e.g., in order to study the process of differentiation and to test compounds which affect this process. Changes in patterns of differentiation are also desirable, for example, in the treatment of anaplastic tumors, which are composed of cells which have lost some of their differentiated characteristics. Cells in which the patterns of differentiation can be changed using an epc composition comprising epc subgenomic polynucleotides, proteins, polypeptides, or fusion proteins include, but are not limited to, erythropoietic stem cells, neuroblasts, chrondroblasts, melanoblasts, myoblasts, and neural crest cells. Patterns of differentiation of differentiated cells can also be changed, for example, by contacting a differentiated cell with a composition comprising a molecule which specifically binds to Epc amino acid sequences, such as an antibody.

Neoplastic cells transformed with wild-type epc subgenomic polynucleotides can be used as model systems to study cancer remission and drug treatments which promote such remission. A variety of neoplastic cell lines, such as HeLa, HL60, SW480, G361, A549, U937, THP-1, and UOCM1, are available and can be used for this purpose. Suppression of the neoplastic phenotype is a process which involves alterations in gene expression in the transformed cells. These alterations in gene expression will be reflected in morphological and biochemical changes in the transformed cells. Morphological changes can be studied, for example, by observing the transformed cells microscopically and comparing the appearance of the transformed cells with cells which have not received a wild-type epc subgenomic polynucleotide. Biochemical alterations can be studied, inter alia, by comparing the proteins which are expressed by the cells before and at various times after transformation with the wild-type epc subgenomic polynucleotide. Methods of comparing proteins between two cells, such as one- or two-dimensional polyacrylamide gel electrophoresis, are well known in the art.

Neoplastic cells transformed with wild-type epc subgenomic polynucleotides and in the process of suppressing neoplastic growth can also be exposed to various drug treatments to determine which treatments promote the morphological or biochemical changes which accompany suppression of the neoplastic phenotype. Similarly, progenitor cells transformed with wild-type epc subgenomic polynucleotides can also be used to study changes which accompany cellular differentiation in progenitor cells and responses of these cells to test compounds which affect differentiation. Progenitor cells such as erythropoietic stem cells, neural crest cells, neuroblasts, chondroblasts, and myoblasts, can be used for this purpose.

Therapeutic compositions comprising mammalian, preferably human, epc subgenomic polynucleotides can be used to treat neoplastic cells. The invention provides a therapeutic composition for providing an epc tumor suppressor function in a cell. The therapeutic composition suppresses neoplasia, dysplasia, or hyperplastic cell growth. The cell to be treated can be any cell of a human which has decreased expression of an epc tumor suppressor gene, for example, a neoplastic cell. The therapeutic composition comprises a subgenomic polynucleotide encoding all or a portion of human EPCl or EPC2 gene in a pharmaceutically acceptable carrier. The subgenomic polynucleotide can be, e.g. , mRNA or DNA. Pharmaceutically acceptable carriers are well known to those in the art. Such carriers include, but are not limited to, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Pharmaceutically acceptable salts can also be used in the composition, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes, such as those described in U.S. 5,422,120, WO 95/13796, WO 91/14445, or EP 524,968 Bl, can also be used as a carrier for the therapeutic epc composition.

Typically, the therapeutic epc composition is prepared as an injectable, either as a liquid solution or suspension, however solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition can also be formulated into an enteric coated tablet or gel capsule according to known methods in the art, such as those described in U.S. 4,853,230, EP 225,189, AU 9,224,296, and AU 9,230,801.

Proliferative disorders, such as neoplasias, dysplasias, and hyperplasias, can be treated by administration of the therapeutic epc composition. Neoplasias which can be treated with the therapeutic composition include, but are not limited to, cervical cancers, melanomas, colorectal adenocarcinomas, Wilms' tumor, retinoblastoma, sarcomas, myosarcomas, lung carcinomas, breast carcinomas, ovarian carcinomas, leukemias, such as chronic myelogenous leukemia, promyelocytic leukemia, monocytic leukemia, and myeloid leukemia, and lymphomas, such as histiocytic lymphoma. Proliferative disorders which can be treated with the therapeutic composition include disorders such as anhydric hereditary ectodermal dysplasia, congenital alveolar dysplasia, epithelial dysplasia of the cervix, fibrous dysplasia of bone, and mammary dysplasia. Hyperplasias, for example, endometrial, adrenal, breast, prostate, or thyroid hyperplasias or pseudoepitheliomatous hyperplasia of the skin can be treated with epc therapeutic compositions. Even in disorders in which epc mutations are not implicated, increased expression of an epc can have therapeutic application. In these disorders, increasing expression of an epc gene can help to suppress tumors. Similarly, in tumors where epc expression is not aberrant, increasing epc expression can suppress metastases.

Both the dose of the therapeutic epc composition and the means of administration can be determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. Administration of the therapeutic agents of the invention can include local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, the therapeutic composition contains an expression construct comprising a promoter and a polynucleotide segment of at least 12, 22, 25, 30, or 35 contiguous nucleotides of the coding strand of an epc gene. Within the expression construct, the polynucleotide segment is located downstream from the promoter, and transcription of the polynucleotide segment initiates at the promoter. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion can be located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a tumor can be identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor which has a necrotic center can be aspirated and the composition injected directly into the now empty center of the tumor. The therapeutic epc composition can be directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging can be used to assist in certain of the above delivery methods. Receptor-mediated targeted delivery of therapeutic compositions containing epc polynucleotides or proteins to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al. (1993), Trends in Biotechnol 11, 202-05; Chiou et al. (1994), GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.); Wu & Wu (1988), J. Biol. Chem. 263, 621-24; Wu et ed. (1994), J. Biol. Chem. 269, 542-46; Zenke et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59; Wu et al. (1991), J Biol. Chem. 266, 338-42. Preferably, receptor-mediated targeted delivery of therapeutic compositions containing all or a portion of an epc protein is used to deliver the protein to specific tissues. Many tumors, including breast, lung, and ovarian carcinomas, over-express antigens specific to malignant cells, such as glycoprotein pi 85"^. Antibodies which specifically bind to these antigens can be bound to liposomes which contain an antibody of the invention. When injected into the bloodstream of a patient, the anti- plδS¹¹^ antibody directs the liposomes to the target cancer cells, where the liposomes are endocytosed and thus deliver their contents to the neoplastic cell (see Kirpotin et al, Biochem. 36: 66, 1997). In a preferred embodiment, a p 185™^ antibody targeted delivery system is used to deliver all or a portion of an epc protein in a cancer cell. Liposomes can be loaded with the protein, as is known in the art (see Papahadjopoulos et al, Proc. Natl. Acad. Sci. U.S.A. 88: 11640, 1991; Gabizon, Cancer Res. 52: 891, 1992; Lasic and Martin, Stealth Liposomes, 1995; Lasic and Papahadjopoulos, Science 267: 1275, 1995; and Park et al, Proc. Natl Acad. Sci. U.S.A. 92: 1327, 1995).

Alternatively, a therapeutic composition containing epc subgenomic polynucleotides can be introduced into human cells ex vivo, and the cells can then be replaced into the human. Cells can be removed from a variety of locations including, for example, from a selected tumor or from an affected organ. In addition, the therapeutic composition can be inserted into non-tumorigenic cells, for example, dermal fibroblasts or peripheral blood leukocytes. If desired, particular fractions of cells, such as a T cell subset or stem cells, can also be specifically removed from the blood (see, for example, PCT WO 91/16116). An epc-containing therapeutic composition can then be contacted with the removed cells utilizing any of the above-described techniques, followed by the return of the cells to the human, preferably to or within the vicinity of a tumor. The methods described above can additionally comprise the steps of depleting fibroblasts or other non-contaminating tumor cells subsequent to removing tumor cells from a human, and/or the step of inactivating the cells, for example, by irradiation.

Therapeutic compositions containing epc subgenomic polynucleotides can be administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the epc subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of epc polynucleotides or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Serial No. 08/869,309, which is expressly incorporated herein.

The invention provides a knock-out mammal in which an endogenous epc gene is not expressed. Methods ofmaking knock-out mammals are well known in the art.

The mammal can be any experimental mammal, such as a mouse, rat, or rabbit; however, a mouse is preferred. The endogenous wild-type epc gene of the mammal can be deleted entirely, resulting in an absence of epc protein in the mammal. Alternatively, mutations such as deletions, insertions, missense substitutions, or inversions, can be introduced into the epc gene. Such mutations result in expression of truncated or otherwise aberrant forms of epc protein in the knock-out mammal. Preferably, the knock-out mammal does not express the endogenous wild-type epc gene in one or more of the following tissues: heart, brain, pancreas, skeletal muscle, placenta, spleen, thymus, prostate, testis, small intestine, and the mucosal lining of the colon. Knock-out mammals of the invention are useful as model systems for studying the effects of drugs in the absence of wild-type epc protein or in the presence of altered forms of the epc protein in the mammal. Knock-out mammals of the invention can also be used to develop therapeutic treatments for diseases associated with alterations in epc gene expression, such as neoplasia. An epc polynucleotide can also be delivered to subjects for the purpose of screening test compounds for those which are useful for enhancing transfer of epc polynucleotides to the cell or for enhancing subsequent biological effects of epc polynucleotides within the cell. Such biological effects include hybridization to complementary epc mRNA and inhibition of its translation, expression of an epc polynucleotide to form epc mRNA and/or Epc protein, and replication and integration of an epc polynucleotide. The subject can be a cell culture or an animal, preferably a mammal, more preferably a human.

Test compounds which can be screened include any substances, whether natural products or synthetic, which can be administered to the subject. Libraries or mixtures of compounds can be tested. The compounds or substances can be those for which a pharmaceutical effect is previously known or unknown. The compounds or substances can be delivered before, after, or concomitantly with an epc polynucleotide. They can be administered separately or in admixture with an epc polynucleotide. Integration of a delivered epc polynucleotide can be monitored by any means known in the art. For example, Southern blotting of the delivered epc polynucleotide can be performed. A change in the size of the fragments of a delivered polynucleotide indicates integration. Replication of a delivered polynucleotide can be monitored wter alia by detecting incorporation of labeled nucleotides combined with hybridization to an epc polynucleotide probe. Expression of an epc polynucleotide can be monitored by detecting production of epc mRNA which hybridizes to the delivered polynucleotide or by detecting Epc protein. Epc protein can be detected immunologically. Delivery of epc polynucleotides according to the present invention therefore provides an excellent system for screening test compounds for their ability to enhance transfer of epc polynucleotides to a cell, by enhancing delivery, integration, hybridization, expression, replication or integration in a cell in vitro or in an animal, preferably a mammal, more preferably a human.

The complete contents of all references cited in this disclosure are expressly incorporated herein by reference.

Claims

1. An isolated and purified enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2, wherein percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

2. The isolated and purified enhancer of polycomb protein of claim 1 which comprises amino acid sequence shown in SEQ ID NO:2.

3. An isolated and purified polypeptide comprising at least 33 contiguous amino acids as shown in SEQ ID NO:2.

4. An enhancer of polycomb fusion protein comprising a first protein segment and a second protein segment fused together by means of a peptide bond, wherein the first protein segment consists of at least 33 contiguous amino acids of an enhancer of polycomb protein as shown in SEQ ID NO:2.

5. A preparation of antibodies which specifically bind to a mammalian enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2, wherein percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

6. A cDNA molecule which encodes an enhancer of polycomb protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2, wherein percent identity is determined using a Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

7. A cDNA molecule which encodes at least 33 contiguous amino acids of SEQ ID NO:2.

8. The cDNA molecule of claim 7 which encodes SEQ ID NO:2.

9. The cDNA molecule of claim 8 which comprises SEQ ID NO: 1.

10. A cDNA molecule comprising at least 447 contiguous nucleotides of SEQ ID NO:l.

11. A cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:l, wherein percent identity is determined using a Smith- Waterman homology search algorithm as implemented in a MPSRCH program using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.

12. An isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO:l after washing with 0.2X SSC at 65 ┬░C, wherein the nucleotide sequence encodes an enhancer of polycomb protein having the amino acid sequence of SEQ ID NO:2.

13. A construct comprising : a promoter; and a polynucleotide segment encoding at least 33 contiguous amino acids of an enhancer of polycomb protein as shown in SEQ ID NO:2, wherein the polynucleotide segment is located downstream from the promoter, wherein transcription of the polynucleotide segment initiates at the promoter.

14. A host cell comprising a construct which comprises: a promoter and: a polynucleotide segment encoding at least 33 contiguous amino acids of an enhancer of polycomb protein having an amino acid sequence as shown in SEQ ID NO:2.

15. A recombinant host cell comprising a new transcription initiation unit, wherein the new transcription initiation unit comprises in 5' to 3' order:

(a) an exogenous regulatory sequence;

(b) an exogenous exon; and

(c) a splice donor site, wherein the new transcription initiation unit is located upstream of a coding sequence of a mammalian epc gene, wherein the coding sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, and 4, wherein the exogenous regulatory sequence controls transcription of the coding sequence of the epc gene.

16. A polynucleotide probe comprising at least 12 contiguous nucleotides selected from the group consisting of SEQ ID NOS:l, 3, and 4 and a detectable label.

17. A method of identifying a neoplastic tissue of a human, comprising the step of: comparing expression of a first epc gene in a first tissue of a human suspected of being neoplastic with expression of a second epc gene in a second tissue of the human which is normal, wherein the second epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, wherein decreased expression of the first epc gene relative to the second epc gene identifies the first tissue as being neoplastic.

18. A method to aid in the diagnosis or prognosis of neoplasia in a human, comprising the step of: comparing a first epc gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic with a second epc gene, mRNA, or protein in a second tissue of a human which is normal, wherein the second epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, wherein a difference between the first and second epc genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue.

19. A method to aid in detecting a genetic predisposition to neoplasia in a human, comprising the step of: comparing an epc gene, mRNA, or protein in the fetal tissue of a human with a wild-type epc gene, mRNA, or protein, wherein the wild-type epc gene comprises a coding sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:4, wherein a difference between the epc gene, mRNA, or protein in the fetal tissue of the human and the wild-type human epc gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.

20. A method of identifying a human chromosome 10, comprising the steps of: contacting a preparation of metaphase human chromosomes with a polynucleotide probe comprising at least 12 contiguous nucleotides selected from a nucleotide sequence as shown in SEQ ID NO:3; and detecting a chromosome which specifically hybridizes to the polynucleotide probe, wherein a chromosome which specifically hybridizes to the polynucleotide probe is identified as a human chromosome 10.

21. The method of claim 20 wherein the region of the chromosome which specifically hybridizes to the nucleotide probe is identified as the short arm of a human chromosome 10.

22. The method of claim 21 wherein the region of the chromosome which specifically hybridizes to the nucleotide probe is identified as a pi 0-11 region of a human chromosome 10.

23. A method of identifying a human chromosome 22, comprising the steps of: contacting a preparation of metaphase human chromosomes with a polynucleotide probe comprising at least 12 contiguous nucleotides selected from a nucleotide sequence as shown in SEQ ID NO:4; and detecting a chromosome which specifically hybridizes to the polynucleotide probe, wherein a chromosome which specifically hybridizes to the polynucleotide probe is identified as a human chromosome 22.

24. The method of claim 23 wherein the region of the chromosome which specifically hybridizes to the nucleotide probe is identified as the long arm of a human chromosome 22.

25. The method of claim 24 wherein the region of the chromosome which specifically hybridizes to the nucleotide probe is identified as a ql3.3 region of a human chromosome 22.