WO1999029850A1 - HUMAN KISMET PROTEIN (hkis) ACTS AS AN ONCOGENE - Google Patents

Abstract

A human oncogene termed hkis and its protein product can be used, inter alia, as diagnostic and prognostic tools for neoplastic disorders. The human hkis gene can also be used to identify a q13 region of human chromosome 16.

Description

HUMAN KISMET PROTEIN (hkis) ACTS AS AN ONCOGENE

TECHNICAL AREA OF THE INVENTION The invention relates to the area of oncogenesis. More particularly, the invention relates to oncogenes and proteins.

BACKGROUND OF THE INVENTION

Mutations which activate expression of oncogenes play an important role in the development of neoplasias. Detection of mutations in oncogenes can be used to detect neoplastic cells and genetic predispositions to neoplasias. Thus, there is a need in the art for the identification of mammalian oncogenes 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 hum∑in kismet protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity between the first and second human kismet proteins is determined using a Smith- Waterman homology search algorithm using an affme gap search with a gap open penalty of 12 .and a gap extension penalty of 1.

Another embodiment of the invention is an isolated polypeptide comprising at least 8 contiguous .amino acids as shown in SEQ ID NO:2.

Even .another embodiment of the invention is a kismet 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 8 contiguous amino acids of a human kismet protein having an amino acid sequence as shown in SEQ ID NO:2. Yet another embodiment of the invention is a preparation of antibodies which specifically bind to a hum.an kismet protein having .an amino acid sequence as shown in SEQ ID NO:2.

Still .another embodiment of the invention is a cDNA molecule which encodes a hum.an kismet 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.

Even another embodiment of the invention is a cDNA molecule which encodes at least 8 contiguous amino acids of SEQ ID NO:2.

Another embodiment of the invention is a cDNA molecule which comprises at least 12 contiguous nucleotides of SEQ ID NO:l.

Yet .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 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 kismet protein having the amino acid sequence of SEQ ID NO:2.

Even .another embodiment of the invention is a construct comprising a promoter and a polynucleotide segment encoding at least 8 contiguous amino acids of a hum.an kismet 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.

Another embodiment of the invention is a host cell comprising a construct. The construct comprises a promoter and a polynucleotide segment which encodes at least 8 contiguous amino acids of a human kismet protein having an amino acid sequence as shown in SEQ ID NO:2. Yet another embodiment of the invention is a recombinant host cell comprising a new transcription initiation unit. The new tr.anscription initiation unit comprises in 5' to 3' order: an exogenous regulatory sequence, an exogenous exon, and a splice donor site. The new transcription initiation unit is located upstream of a coding sequence of an hkis gene having a coding sequence as shown in SEQ ID NO: 1. The exogenous regulatory sequence controls transcription of the coding sequence of the hkis gene.

Still another embodiment of the invention is a polynucleotide probe comprising at least 12 contiguous nucleotides of SEQ ID NO:l.

A further embodiment of the invention is a method of identifying a neoplastic tissue of a hum.an. Expression of a first hkis gene in a first tissue of a human suspected of being neoplastic is compared with expression of a second hkis gene in a second tissue of the human which is normal. Increased expression of the first hkis gene relative to the second hkis gene identifies the first tissue as being neoplastic.

Another embodiment of the invention is a method to aid in the diagnosis or prognosis of neoplasia in a human. A first hkis gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic is compared with a second hkis gene, mRNA, or protein in a second tissue of a human which is normal. A difference between the first and second hkis genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue. Still another embodiment of the invention is a method to aid in detecting a genetic predisposition to neoplasia in a human. An hkis gene, mRNA, or protein in the fetal tissue of a human is compared with a wild-type hkis gene, mRNA, or protein. A difference between the hkis gene, mRNA, or protein in the fet tissue of the human and the wild-type human hkis gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.

Yet .another embodiment of the invention is a method of identifying a human chromosome 16. 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:l. A chromosome which specifically hybridizes to the nucleotide probe is detected. A chromosome which specifically hybridizes to the nucleotide probe is identified as a human chromosome 16.

The present invention thus provides the art with a novel hum.an oncogene called "kismet" (hkis). The hkis gene and protein can be used, ter alia, as therapeutic and diagnostic tools for neoplasia and to identify a ql 3 region of a hum.an chromosome 16.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a discovery of the present invention that hkis functions as an oncogene and thus is implicated in neoplastic disorders. The hkis gene, protein, and mRNA can be used as therapeutic and diagnostic tools for these disorders. Coding sequences of hkis can also be used to identify hum.an chromosome 16.

Hum.an kismet protein has the amino acid sequence shown in SEQ ID NO:2. Human kismet polypeptides, biologically active polypeptides or protein variants, and fusion proteins, as described below, are all human kismet proteins of the invention. For example, any natur ly occurring variants of SEQ ID NO:2 which may occur in human tissues and which have oncogenic activity are biologically active kismet variants and are within the scope of this invention. Non-naturally occurring kismet variants which contain conservative amino acid substitutions relative to SEQ ID NO:2 but which retain substantially the same oncogenic activity as naturally occurring kismet variants are also biologically active human kismet variants of the invention.

Naturally or non-naturally occurring human kismet proteins preferably are at least 85%, 90%, or 95% identical to SEQ ID NO:2 and have similar oncogenic functions. More preferably, the molecules are 98% or 99% identical. Percent sequence identity between a putative hum,an kismet variant and the amino acid sequence of SEQ ID NO:2 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) 2:482-489. Biologically active human kismet variants include glycosylated forms of kismet, aggregative conjugates of kismet with other molecules, and covalent conjugates of kismet with unrelated chemical moieties. Kismet variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect the oncogenic function of kismet are also encompassed. 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 kismet by means well known in the art.

A subset of mutants, called muteins, is a group of polypeptides with the non- disulfide bond participating cysteines substituted with a neutral amino acid, generally, with serines. These mutants may be stable over a broader temperature r.ange th.an kismet. See M-ark et al, U.S. Pat. No. 4,959,314.

Biologically active hum-an kismet polypeptides or polypeptide variants can comprise at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 1100 contiguous amino acids of the amino acid sequence shown in SEQ ID NO:2.

Polypeptide molecules having substantially the same amino acid sequence as human kismet protein but possessing minor amino acid substitutions which do not substantially affect the ability of the kismet polypeptides to interact with kismet-specific molecules, such as antibodies, are within the definition of biologically active kismet polypeptides or variants.

Preferably, biologically active human kismet polypeptides or polypeptide variants are at least 65%, 75%, 85%, 90%, 95%, 98%, or 99% identical to kismet polypeptide fragments of SEQ ID NO:2. Percent identity of kismet polypeptides or polypeptide variants or derivatives with fragments of SEQ ID NO:2 is determined as described above. Preferably the amino acid changes in kismet 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, tryptoph,an); 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 kismet molecule, especially if the replacement does not involve an amino acid at a binding site involved in an interaction of kismet and another protein. Oncogenic effects of human kismet protein or polypeptide variants can be assessed or measured, for example, by transforming non-neoplastic cells in vitro with DNA constructs encoding a putative kismet variant and observing the acquisition of a 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.

Hum.an kismet proteins -and polypeptides can be isolated and purified from human cells such as heart, brain, pancreas, skeletal muscle, placenta, spleen, thymus, prostate, testis, or small intestine cells, and cells of the mucos-al 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 electrophoresis. The skilled artisan can readily select methods which will result in a preparation of kismet protein or polypeptide which is substantially free from other proteins and from carbohydrates, lipids, or subcellular organeiles. A preparation of isolated and purified kismet 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. Human kismet proteins, polypeptides, or variants can be produced by recombinant DNA methods or by synthetic chemical methods. For production of recombin.ant kismet proteins or polypeptides, kismet coding sequences selected from the nucleotide sequence shown in SEQ ID NO:l 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 human kismet 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, Weinstein, B. ed., M.arcell Dekker, Inc., publ., New York (1983). Substitution of D-amino acids for the normal L-stereoisomer of kismet 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, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 1100 contiguous kismet amino acids can also be constructed. Kismet fusion proteins are useful for generating antibodies against kismet amino acid sequences and for use in various assay systems. For example, kismet fusion proteins can be used to identify proteins which interact with kismet and which influence, for example, its oncogenic activity. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions such as the ye-ast 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 kismet fusion protein comprises two protein segments fused together by me-ans of a peptide bond. The first protein segment consists of at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 1100 contiguous amino acids of a kismet protein, or variant. The amino acids can be selected from the amino acid sequence shown in SEQ ID NO:2 or from a biologically active variant of that sequence, such as those described above. The first protein segment can also be a full-length kismet 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 acetyltr-ansferase (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 (DID) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.

Kismet fusion proteins can be made by covalently linking the first and second two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare kismet fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l 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), Stratagene (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 kismet 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 kismet protein. The antibodies can be used, inter alia, to detect kismet protein in hum-an tissue or in fractions thereof. The antibodies can also be used to detect the presence of mutations in the kismet gene which result in under- or over-expression of kismet protein or in expression of a kismet protein with .altered size or electrophoretic mobility. By binding to kismet, .antibodies can also alter the oncogenic function of kismet.

Antibodies which specifically bind to epitopes of kismet 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 kismet epitopes do not detect other proteins in immunochemical assays and can immunoprecipitate kismet protein or polypeptides from solution.

Kismet-specific antibodies specifically bind to epitopes present in a kismet protein having the .amino acid sequence shown in SEQ ID NO:2 or to biologically active variants of that sequence. 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, kismet epitopes are not present in other human proteins.

Epitopes of kismet which are particularly antigenic can be selected, for example, by routine screening of kismet polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein to the amino acid sequence shown in SEQ ID NO:2. 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).

Any type of .antibody known in the art can be generated to bind specifically to kismet 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 kismet epitopes can be isolated, for example, from single-chain immunoglobulin display libraries, as is known in the art. The library is "panned" against kismet amino acid sequences, .and a number of single chain antibodies which bind with high-affinity to different epitopes of kismet 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 z^'nter alia in Mallender and Voss, 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. Verhaar et al, 1995, Int. J. Cancer (57:497-501; Νicholls et al., 1993, J. Immunol Meth. 765:81-91.

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 kismet 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 therapeutically 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 kismet 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.

Kismet-specific binding polypeptides other than antibodies can also be generated. Kismet-specific binding polypeptides are polypeptides which bind with kismet or its variants and which have a measurably higher binding affinity for the kismet and polypeptide derivatives of kismet th-an 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. The coding region of the hkis gene h.as the nucleotide sequence shown in SEQ ID

NO:l. Isolated and purified hkis polynucleotides according to the invention are subgenomic contain less than the whole chromosome 16. Preferably, the polynucleotides are intron-free.

Isolated and purified hkis 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, 450, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 contiguous nucleotides selected from SEQ ID NO: 1 or can comprise SEQ ID NO: 1. Such polynucleotides can be used, for example, as primers or probes or for expression of kismet proteins or polypeptides. The complement of the nucleotide sequence shown in SEQ ID NO:l is a contiguous nucleotide sequence which forms Watson-Crick base pairs with a contiguous nucleotide sequence shown in SEQ ID NO: 1. The complement of SEQ ID NO: 1 is a subgenomic polynucleotide of the invention and can be used, for example, to provide hkis antisense oligonucleotides, primers, and probes. Antisense oligonucleotides and probes of the invention can consist of at least 11, 12, 15, 20, 25, 30, 50, or 100 contiguous nucleotides which are complementary to the coding sequence shown in SEQ ID NO: 1. A complement of the entire coding sequence can also be used. Double-stranded polynucleotides which comprise all or a portion of the nucleotide sequence shown in SEQ ID NO: 1, as well as polynucleotides which encode kismet-specific .antibodies or ribozymes, are also subgenomic polynucleotides of the invention.

Degenerate nucleotide sequences encoding amino acid sequences of kismet protein or biologically active kismet vari.ants, as well as homologous nucleotide sequences which are at least 65%, 75%, 85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO:l, .are also hkis subgenomic polynucleotides. Percent sequence identity between the nucleotide sequence of SEQ ID NO:l .and a putative homologous or degenerate hkis nucleotide sequence 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 sequence shown in SEQ ID NO:l or its complement with at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35% basepair mismatches are also hkis subgenomic polynucleotides of the invention. For ex.ample, 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 hkis sequences can be identified which contain at most about 25-30% basepair mismatches with SEQ ID NO:l or its complement. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Nucleotide sequences which hybridize to the coding sequence shown in SEQ ID NO:l or its complement following stringent hybridization and/or wash conditions are also hkis subgenomic polynucleotides of the invention. 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 hkis sequence shown in SEQ ID NO:l and a polynucleotide sequence which is 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:l 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) - 600/0, 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. hkis 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 hkis coding sequences. Isolated and purified hkis subgenomic polynucleotides .are in preparations which are free or at least 90% free of other molecules.

Human hkis mRNA comprises a major transcript which measures 15 kb on Northern blots of human polyA⁺ RNA probed with an hkis polynucleotide probe. The 15 kb transcript is expressed, for example, in heart, brain, pancreas, skeletal muscle, placenta, spleen, thymus, prostate, testis, small intestine, and the mucosal lining of the colon. The 15 kb hkis mRNA transcript is expressed at very high levels in the K562 (chronic myelogenous leukemia) cell line. The hkis tr.anscript is also expressed at high levels in a number of other cell lines, including HL60 (promyelocytic leukemia), human cancer cell lines, such as SW480 (colon carcinoma), G361 (melanoma), A549 (lung carcinoma), U937 (histiocytic lymphoma), THP-1 (monocytic leukemia), EBV, and UOCM1 (myeloid leukemia). HeLa cells (cervical carcinoma) do not express the 15 kb transcript, but express a 6 kb hkis tr.anscript.

Complementary DNA (cDNA) molecules which encode kismet proteins are also hkis subgenomic polynucleotides of the invention, hkis cD A molecules can be made with stand.ard molecular biology techniques, using hkis mRNA as a template, hkis 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 subgenomic polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize the hkis polynucleotide molecules of the invention. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a kismet protein having the .amino acid sequence shown in SEQ ID NO:2 or a biologically active variant of that sequence. All such nucleotide sequences are within the scope of the present invention.

The invention also provides polynucleotide probes which can be used to detect hkis 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 NO:l. Polynucleotide probes of the invention can comprise a detectable label, such as a radioisotopic, fluorescent, enzymatic, or chemiluminescent label.

An hkis polynucleotide of the present invention can be used in a construct, such as a DNA or RNA construct, hkis constructs can be used, for example, to express all or a portion of a kismet 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 for hkis 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. Natl. Acad. Sci. U.S.A. (1983) 80: 21-25, and Siebenlist et al, Cell (1980) 20: 269. Expression systems for hkis 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) 755: 1165; De Louvencourt et al, J. Bacteriol. (1983) 154: 737, Van den Berg et al, Bio/Technology (1990) 5: 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) 10: 380, Gaillardin et al, Curr. Genet. (1985) 10: 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. U.S.A. (1984) 57: 1470-1474, Kelly and Hynes, EMBOJ. (1985) 4: 475479; EP 244,234, and WO 91/00357.

Expression of hkis 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: 177, Carbonell et al, Gene (1988) 73: 409, Maeda et al, Nature (1985) 575: 592-594, Lebacq-Verheyden et al, Mol. Cell Biol. (1988) 5: 3129; Smith et al, Proc. Natl Acad. Sci. U.S.A. (1985) 52: 8404, Miyajima et al, Gene (1987) 55: 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. Mammalian expression of hkis can be accomplished as described in Dijkema et al, EMBOJ. (1985) 4: 761, Gorman et al, Proc. Natl. Acad. Sci. U.S.A. (1982b) 79: 6777, Boshart et al, 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) 55: 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 tr.ansfeπin-polycation-mediated DNA tr.ansfer, 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.

An hkis 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. An hkis expression construct can also contain a tr.anscription terminator which is functional in the host cell. The expression construct comprises a polynucleotide segment which encodes all or a portion of a hunran kismet 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.

Polynucleotides of the invention can also be used in gene delivery vehicles, for the purpose of delivering an hkis mRNA or oligonucleotide (either with the sequence of native hkis mRNA or its complement), full-length kismet protein, kismet fusion protein, kismet polypeptide, or kismet-specific ribozyme or single-ch n .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 hkis polynucleotide, or an hkis polynucleotide in conjunction with a liposome or a condensing agent.

In one embodiment of the invention, the gene delivery vehicle comprises a promoter and an hkis 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 .are activatable 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.

An hkis 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, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the hkis 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 55. 153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 57:6349, 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; Vile and Hart, Cancer Res. 55:3860-3864, 1993; Vile and Hart, Cancer Res. 55:962-967, 1993; Ram et al, Cancer Res. 55:83-88, 1993 ; Takamiya et al. , 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. VR-535 and VR-247), bovine leukemia virus (VR- 1315), murine leukemia virus (MLV), 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 Nos. VR-844, 45010 and 45016), reticuloendotheliosis virus (ATCC Nos VR-994, VR-770 .and 45011), Rous sarcoma virus, Mason-Pfizer monkey virus, baboon endogenous virus, endogenous feline retrovirus (e.g., RDl 14), 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. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al, J. Vir. 67:4722, 1993; and Yantchev Neoplasma 26:397, 1979), Gross (ATCC No. VR-590), Kirsten (Albino et al. , J. Exp. Med. 164:\7\0, \9S6), Harvey sarcoma virus

(Manly et al, J. Vir. 62:3540, 1988; and Albino et al, J. Exp. Med. 164:1710, 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. 62:3540, 1988; and Albino et al, J. Exp. Med. 164:1710, 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 Biophys Acta 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 retrovir hkis gene delivery vehicles given the disclosure provided herein and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring H.arbor Laboratory Press, 1989, and Kunkle, PROC. NATL. ACAD. SCI. U.S.A. 52:488, 1985) known in the art. Portions of retroviral hkis expression vectors can be derived from different retroviruses. For ex-ample, 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 str.and synthesis from an avian leukosis virus. These recombin.ant 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 recombin.ant 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 "VCLs") 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 6:6X6-627, 1988, and Rosenfeld et al, Science 252:431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).

An hkis 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 6:6X6, 1988, and Rosenfeld et al, Science 252:431, 1991 , WO 93/07283, WO 93/06223, and WO 93/07282). Adeno-associated viral hkis gene delivery vehicles can also be constructed and used to deliver Kismet 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, an hkis 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 hkis 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 hkis 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 simile 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 559:385, 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. 86:3X7, 1989; Flexner et al. , Ann. N. Y. Acad. Sci. 569:86, 1989; Flexner et al. , Vaccine 5:17, 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: X 08, 1979) (ATCC VR-305), (Madzak et al, J. Gen. Vir. 75:1533, 1992); influenza virus (Luytjes et al, Cell 59: 1107, 1989; McMicheal et al, The New England Journal of Medicine 509:13, 1983; and Yap et al, Nature 275:238, 1978) (ATCC VR-797); parvovirus such as adeno-associated virus (Samulski et α/., J. Vir. 65:3822, 1989, and Mendelson et α/., Virology 766:154, 1988) (ATCC VR-645); herpes simplex virus (Kit et al. , Adv. Exp. Med. Biol. 215:2 X 9, 1989) (ATCC VR-977; ATCC VR-260); Nature 277: 108, 1979); human immunodeficiency virus (EPO 386,882, Buchschacher et al, J. Vir. 66:2731, 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), CNyong 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). An hkis polynucleotide of the invention 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, an hkis 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. Acta 600:1, 1980; Bayer et al, Biochim. Biophys. Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol. 749:119, 1987; Wang et al, PROC. NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et al, Anal Biochem. 776:420, 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 hkis 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 84:74X3-74X6, 1987), mRNA (Malone et al, Proc. Natl. Acad Sci. USA 56:6077-6081, 1989), and purified transcription factors (Debs et al, J. Biol Chem. 265:10189-10192, 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-(trimethyl.ammonio)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 57:3410-3414, 1990; Papahadjopoulos et al, Biochim. Biophys. Acta 594:483, 1975; Wilson et al, Cell 17:77, 1979; Deamer and Bangham, Biochim. Biophys. Acta 443:629, 1976; Ostro et al, Biochem. Biophys. Res. Commun. 76:836 , 1977; Fraley et al, Proc. Natl Acad. Sci. USA 76:3348, 1979; Enoch .and Strittmatter, Proc. Natl Acad. Sci. USA 76:145, 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 al, Science 275:166, 1982.

In addition, lipoproteins can be included with an hkis 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 hkis 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 hkis DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al. , Hum. Gene. Ther. 5:147-154, 1992. Other suitable vehicles include DNA-ligand (Wu et al, J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413 7417, 1989), liposomes (Wang et al, Proc. Natl Acad. Sci. 54:7851-7855,

1987) and microprojectiles (Williams et al, Proc. Natl. Acad. Sci. 55:2726-2730, 1991). One can increase the efficiency of naked hkis 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, hkis 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 hkis polynucleotides into the cytopl.asm.

In one embodiment of the invention, expression of an endogenous hkis gene in a cell can be altered by introducing in frame with the endogenous hkis gene a DNA construct comprising a transcription unit by homologous recombination to form a homologously recombinant cell comprising the transcription unit. The tr.anscription 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, which is incorporated herein by reference. The targeting sequence is a segment of at least 10, 12, 15, 20, or 50 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO:l. The transcription unit is located upstream to a coding sequence of the endogenous hkis gene. The exogenous regulatory sequence directs tr.anscription of the coding sequence of the hkis gene. Preferably, the homologously recombinant cell is a differentiated cell, for example, a cell of the brain, thymus, testis, he.art, prostate, small intestine, placenta, spleen, skeletal muscle, 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 hkis gene.

The hkis gene maps to human chromosome region 16ql3. Polynucleotides of the invention can therefore be used to identify this chromosome region in metaphase spreads of hum.an chromosomes. Preparations of human metaphase chromosomes can be prepared using standard cytogenetic techniques from hum.an prim.ary tissues or cell lines. Polynucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO:l -are used to identify the human chromosome. 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 p-articular 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 probe which hybridizes specifically to human chromosome region 16ql3 hybridizes to nucleotide sequences present in the hkis gene .and not to nucleotide sequences present in other human genes. A probe which hybridizes specifically to an hkis gene provides a detection signal at least 5-, 10-, or 20-fold higher th.an the background hybridization provided with non-hkis coding sequences.

A hum-an chromosome which specifically hybridizes to an hkis polynucleotide probe is identified as a hum.an chromosome 16. Preferably, the polynucleotide probe identifies the long arm of human chromosome 16. More preferably, the polynucleotide probe identifies a ql3 region of human chromosome 16.

The present invention also provides a method to identify neoplastic tissue in a human. The expression of an hkis gene can be compared between a first tissue which is suspected of being neoplastic and a second tissue of the hum-an which is normal. The normal tissue can be any tissue of the human, especially those which express the hkis 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 hkis gene, mRNA, or kismet protein in the two tissues which are compared, for example in molecular weight, amino acid or nucleotide sequence, indicates a somatic mutation in the hkis gene. Alternatively, a difference in relative abundance of hkis mRNA or kismet protein may indicate a somatic mutation in a gene which regulates the expression, half-life, or degradation of hkis mRNA or kismet protein in the tissue of the human which was suspected of being neoplastic.

The hkis 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 hkis gene in the tissue suspected of being neoplastic can be compared with the wild-type hkis sequence in the normal tissue. The hkis genes or portions of the hkis genes in the two tissues can be amplified, for example using nucleotide primers selected from the nucleotide sequence shown in SEQ ID NO:l 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 sequence shown in SEQ ID NO:l. 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 hkis gene in the tissue suspected of being neoplastic compared with the wild-type hkis nucleotide sequence is indicative of neoplasia.

Alternatively, hkis 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 hkis mRNA transcripts between the two tissues that are compared, using Northern blots and polynucleotide probes selected from the nucleotide sequence shown in SEQ ID NO: 1. Increased expression of hkis mRNA in a tissue sample suspected of being neoplastic compared with the expression of hkis mRNA in a normal tissue is indicative of neoplasia.

Any method for analyzing proteins can be used to compare two hkis proteins from matched samples. Sizes of the kismet proteins in the two tissues can be compared, for example, using .antibodies of the present invention to detect hkis 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 higher hkis protein expression level in a tissue suspected of being neoplastic compared with the hkis protein expression level in a normal tissue is indicative of neoplasia.

Similarly, comparison of hkis gene sequences or of hkis gene expression products, e.g., mRNA .and protein, between a tissue of a hum.an which is suspected of being neoplastic and a normal tissue of a human can be used to diagnose or prognose neoplasia in the hum.an. Such comparisons of hkis genes, mRNA, or protein can be made as described above. Increased expression of the hkis gene in the tissue suspected of being neoplastic indicates the presence of neoplastic cells in the tissue. The degree of increased expression of the hkis gene in the neoplastic tissue relative to wild-type expression of the gene in normal tissue or differences in the amount of increased expression of the hkis 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 hkis gene, mRNA, or protein in a fetal tissue with a wild-type hkis 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 vitro- fertilized embryo. The wild-type hkis gene can be obtained from any tissue. The mRNA or protein can be obtained from a normal tissue of a human in which the hkis gene is expressed. Such tissues are disclosed above. Differences such as alterations in the nucleotide sequence or size of the fetal hkis gene or mRNA, or alterations in the molecule weight, amino acid sequence, or relative abundance of fetal kismet protein, indicate a germline mutation in the hkis gene of the fetus which indicates a genetic predisposition to neoplasia. Neoplastic cells transformed with wild-type hkis polynucleotides can be used as model systems to study oncogenesis and drug treatments which are effective in inducing tr.ansformation to a neoplastic phenotype. Oncogenesis 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 ex.ample, by observing the transformed cells microscopically and comparing the appearance of the transformed cells with non-neoplastic cells which have not received a wild-type hkis polynucleotide. Biochemical alterations can be studied, inter alia, by comp.aring the proteins which are expressed by the cells before and at various times after tr.ansformation with the wild-type hkis gene. Methods of comparing proteins between two cells, such as using SDS polyacrylamide electrophoresis, are well known in the art. Non-neoplastic cells transformed with a wild-type hkis polynucleotide .and in the process of acquiring a neoplastic phenotype can also be exposed to various drug treatments to determine which treatments promote or suppress morphological or biochemical changes which accompany acquisition of the neoplastic phenotype.

Growth of primary tumors and metastatic lesions can be suppressed by contacting the tumor with a reagent which specifically binds to an expression product of hkis. In one embodiment of the invention, expression of hkis is decreased using a ribozyme, an RNA molecule with catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, X990, 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 sequence shown in SEQ ID NO: 1 an be used to generate a ribozyme which will specifically bind to hkis mRNA. Methods of designing and constructing ribozymes which can cleave other RNA molecules in tr-ans in a highly sequence specific manner have been developed and described in the art (see Haseloff et αl., Nature 554:585-591, 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. hkis 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 tr.anscription of the ribozyme in the cells.

Mechanical methods, such as microinjection, liposome-mediated tr.ansfection, electroporation, or calcium phosphate precipitation, can be used to introduce the hkis ribozyme-containing DNA construct into cells in order to decrease hkis 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.

Expression of hkis can also be altered using an antisense oligonucleotide. The sequence of the antisense oligonucleotide is complementary to at least a portion of the coding sequence shown in SEQ ID NO:l. 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 the nucleotide sequence shown in SEQ ID NO:l, can also be used. Antisense oligonucleotides can be provided in an hkis construct of the invention and introduced into tumor cells, using transfection techniques known in the art. hkis 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 intemucleotide 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 26:1-72; Uhlmann et αl., 1990, Chem. Rev. 90:543-583.

Although precise complementarity is not required for successful duplex formation between an hkis .antisense oligonucleotide and the complementary coding sequence of hkis, antisense oligonucleotides with no more than one mismatch are preferred. One skilled in the art can easily use the calculated melting point of an hkis antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular coding sequence of hkis. hkis antisense oligonucleotides can be modified without affecting their ability to hybridize to an hkis 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.

Antibodies of the invention which specifically bind to kismet protein cm also be used to alter expression of hkis. Specific .antibodies bind to kismet and prevent the protein from functioning in the cell. Polynucleotides encoding single-chain antibodies of the invention can be introduced into cells, using standard transfection techniques. Alternatively, therapeutic antibodies of the invention can be targeted to a particular cell type, for example, by binding an antibody to a coupling molecule which is specific for both the antibody and the target, as disclosed in WO 95/08577. The coupling molecule can comprise immunoglobulin binding domains.

Antibodies which specifically bind to kismet protein, hkis .antisense oligonucleotides, or hkis polynucleotides which encode single-chain antibodies or ribozymes can be provided to a tumor in a therapeutic composition. Therapeutic compositions of the invention also comprise a pharmaceutically acceptable carrier.

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 ex.ample, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or be zoates. 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 hkis composition.

Typically, the therapeutic hkis 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 hkis composition. Neoplasias which can be treated with the therapeutic composition include, but are not limited to, cervical cancers, melanomas, colorectal adenocarcinom.as, 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 hkis therapeutic compositions. Even in disorders in which hkis mutations are not implicated, increased expression of hkis can have therapeutic application. In these disorders, increasing hkis expression can help to suppress tumors. Similarly, in tumors where hkis expression is not aberrant, increasing hkis expression can suppress metastases. Both the dose of a kismet composition and the means of its 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. Preferably, a therapeutic composition of the invention decreases the level of kismet protein in the tumor by at least 50%, 60%, 70%, or 80%. Most preferably, the level of kismet protein in the tumor is decreased by at least 90%, 95%, 99%, or 100%. The effectiveness of the therapeutic composition can be assessed using methods well known in the art, such as hybridization of polynucleotide probes to hkis mRNA, qu.antitative RT-PCR, or detection of kismet protein using specific .antibodies of the invention. If the composition contains kismet .antibodies, effective dosages of the composition are in the range of about 5 μg to about 50 μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg. Therapeutic compositions containing hkis polynucleotides can be administered in a range of about 100 ng to about 200 mg of DNA for local administration. 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.

Factors such as method of action and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the hkis polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of hkis polynucleotides or the same .amounts readministered 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.

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. A therapeutic hkis composition can be directly administered to the surface of the tumor, for ex-ample, 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, for example, hkis antisense oligonucleotides, ribozymes, or antibodies 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 al. (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 hkis protein is used to deliver the protein to specific tissues. Many tumors, including breast, lung, and ovarian carcinomas, overexpress 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- pi 85™^ 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 plδS™¹¹² antibody targeted delivery system is used to deliver all or a portion of an hkis 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, the therapeutic composition containing therapeutic reagents of the invention 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). The AHs-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.

An hkis therapeutic composition can also be used to change the pattern of differentiation of a cell, e.g., in order to study the process of de-differentiation and test compounds which .affect this process. Any differentiated cell, such as a heart, brain, pancreas, skeletal muscle, placenta, spleen, thymus, prostate, testis, small intestine, or peripheral blood cell, can be used for this purpose.

The invention provides a knock-out mammal whose endogenous hkis gene is not expressed. Methods of making 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 hkis gene of the mammal can be deleted entirely, resulting in an absence of hkis protein in the mammal. Alternatively, mutations such as deletions, insertions, missense substitutions, or inversions, can be introduced into the hkis gene. Such mutations result in expression of truncated or otherwise aberrant forms of hkis protein in the knock-out mammal. Preferably, the knock-out mammal does not express the endogenous wild-type hkis 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 hkis protein or in the presence of altered forms of the hkis protein in the mammal. Knock-out m.ammals can also be used to develop therapeutic treatments for diseases associated with alterations in hkis gene expression, such as neoplasia.

An hkis polynucleotide can also be delivered to subjects for the purpose of screening test compounds for those which are useful for enhancing transfer of hkis polynucleotides to the cell or for enhancing subsequent biological effects of hkis polynucleotides within the cell. Such biological effects include hybridization to complementary hkis mRNA and inhibition of its translation, expression of an hkis polynucleotide to form hkis mRNA and/or kismet protein, and replication and integration of an hkis 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 hkis polynucleotide. They can be administered separately or in admixture with an hkis polynucleotide. Integration of a delivered hkis polynucleotide can be monitored by any means known in the art. For example, Southern blotting of the delivered hkis 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 inter alia by detecting incorporation of labeled nucleotides combined with hybridization to an hkis probe. Expression of an hkis polynucleotide can be monitored by detecting production of hkis mRNA which hybridizes to the delivered polynucleotide or by detecting kismet protein. Kismet protein can be detected immunologically. Delivery of hkis polynucleotides according to the present invention therefore provides .an excellent system for screening test compounds for their ability to enhance tr.ansfer of hkis 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 human kismet protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2, wherein percent 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.

2. The isolated human kismet protein of claim 1 which has the .amino acid sequence shown in SEQ ID NO:2.

3. An isolated polypeptide comprising at least 8 contiguous amino acids as shown in SEQ ID NO:2.

4. A kismet 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 8 contiguous amino acids of a human kismet protein as shown in SEQ ID NO:2.

5. A preparation of antibodies which specifically bind to a human kismet protein having an amino acid sequence as shown in SEQ ID NO:2.

6. A cDNA molecule which encodes a human kismet 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 8 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 12 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 a kismet protein having the amino acid sequence of SEQ ID NO:2.

13. A construct comprising : a promoter; and a polynucleotide segment encoding at least 8 contiguous amino acids of a human kismet 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 8 contiguous amino acids of a hum.an kismet 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 tr.anscription initiation unit is located upstream of a coding sequence of an hkis gene as shown in SEQ ID NO:l, wherein the exogenous regulatory sequence controls transcription of the coding sequence of the hkis gene.

16. A polynucleotide probe comprising at least 12 contiguous nucleotides of SEQ ID NO: 1.

17. The polynucleotide probe of claim 16 which comprises a detectable label.

18. A method of identifying a neoplastic tissue of a human, comprising the step of: comparing expression of a first hkis gene in a first tissue of a human suspected of being neopl.astic with expression of a second hkis gene in a second tissue of the human which is normal, wherein increased expression of the first hkis gene relative to the second hkis gene identifies the first tissue as being neoplastic.

19. A method to aid in the diagnosis or prognosis of neoplasia in a human, comprising the step of: comparing a first hkis gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic with a second hkis gene, mRNA, or protein in a second tissue of a human which is normal, wherein a difference between the first and second hkis genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue.

20. A method to aid in detecting a genetic predisposition to neoplasia in a human, comprising the step of: comparing an hkis gene, mRNA, or protein in the fetal tissue of a human with a wild-type hkis gene, mRNA, or protein, wherein a difference between the hkis gene, mRNA, or protein in the fetal tissue of the human and the wild-type human hkis gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the huπran.

21. A method of identifying a human chromosome 16, 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: 1 ; 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 16.