WO2000012763A1 - Prostate specific regulatory nucleic acid sequences - Google Patents

Abstract

Nucleic acid encoding a regulatory sequence of human prostate specific antigen (PSA) and human glandular kallikrein (hK2), distal enhancer element 2 (DEE2) is provided. A nucleic acid construct including a functional distal enhancer element 2 (DEE2), operably linked to a nucleic acid sequence encoding a heterologous protein is provided. Expression vectors, host cells, and transgenic mice including this construct are also provided. A method for producing a transgenic nonhuman animal having a phenotype characterized by expression of a transgene in the prostate, by introducing a transgene including a distal enhacer element 2 (DEE2) into an embryo of an animal is provided. A method for providing transcription of a nucleic acid sequence in the prostate cell of an animal by introducing to a cell a nucleic acid construct including a distal enhancer element 2 (DEE2) operably associated with a nucleic acid sequence which encodes a product is also provided. The invention also provides a method of treating a subject having or at risk of having a prostate disorder by administering to the subject a therapeutically effective amount of a nucleic acid construct including a distal enhancer element 2 (DEE2).

Description

PROSTATE SPECIFIC REGULATORY NUCLEIC ACID SEQUENCES

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Grant Nos.

CA70218 and CA28332 and HL48170 and T32AI07285 awarded by the Public Health Service, and Grant No. T32CA09363 awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION This application relates generally to expression of genes in the prostate and more specifically to nucleic acid sequences encoding the distal enhancer element 2 (DEE2), and transgenic nonhuman animals that contain nucleic acid constructs including the distal enhancer element 2.

BACKGROUND OF THE INVENTION Prostate carcinoma is the most frequently diagnosed cancer in American men, and is the second leading cause of cancer deaths in that population (Parker, S.L., et al. , CA Cancer J. Clin. 47:5-27, 1997). Although this'disease is rarely seen in men under the age of 50, the incidence of prostate cancer increases rapidly in subsequent decades of life. Localized prostate tumors are treated relatively effectively with surgery and radiotherapy, whereas the treatment of metastatic disease is usually ultimately ineffective (Catalona, W.J., Cancer 75:1903-1908, 1995). Immunotherapy mediated through cytotoxic T lymphocytes (CTL) offers a promising treatment avenue, since T cells, in principle, can migrate throughout the body and specifically recognize and destroy metastatic tumor cells in an antigen specific manner. Prostate cancer cells express a well characterized antigen, prostate-specific antigen (PSA), whose expression is widely used clinically as a marker for prostate cancer . PSA, a kallikrein with serine protease activity, has a highly restricted tissue distribution and is expressed in the normal epithelial cells of the prostate gland, the same cell type from which most prostate tumors arise. Neither the regulation of PSA expression nor the role of PSA in normal or neoplastic prostate cells is well understood.

The genetic organization of the PSA gene, as well as its tight hormonal responsiveness and tissue specificity (Lee, C, et al, Endocrinology 136:796-803. 1995; Wolf, D.A., et al, Mol Endocrinol 6:753-762, 1992; Young, C.Y., et al,

Cancer Res. 51:3748-3752, 1991), suggests complex regulation. PSA is a member of a small multigene family of human kallikrens consisting of 3 known members (Clements, J.A., Mol. Cell. Endocrinol. 99:Cl-6, 1994; Riegman, P.H., et al, Genomics 14:6-11, 1992). The gene which encodes PSA is on chromosome 19, just upstream from the closely related (80% amino acid identity) kallikrein gene called hK2 (hGK-1). The expression of hK2 is also prostate-specific, although it is produced at approximately 10-50% the level of PSA (Young, CN., et al, Biochemistry 31:818- 824, 1992; McCormack, R.T., et al, Urology 45:729-744, 1995). Recently, the protease activity of hK2 has been shown to be capable of removing the 7 amino acid propeptide from the amino-terminus of PSA, resulting in the mature, active form (Lovgren, J., et al, Biochem. Biophys. Res. Commun. 238:549-555. 1997; Takayama, T.K., et al, J. Biol. Chem. 272:21582-21588, 1997).

In vitro analysis using portions of the PSA promoter in transient transfection assays has delineated several genomic regions which are involved in regulating PSA. These include a promoter region within 600 bp of the transcriptional start site which contains two androgen-responsive elements (ARE) as well as a distal enhancer element (DEE1) which contains one ARE and probably some other as yet unidentified elements (Schuur, E.R., et al, J. Biol Chem. 271:7043-7051, 1996; Cleutjens, K.B., et al, Mol. Endocrinol. 11:148-161, 1997; Zhang, S., et al, Biochem. Biophys. Res. Commun. 231:784-788, 1997; Cleutjens, K.B., et α/., J Biol. Chem. 271:6379-6388, 1996; Riegman, P.H., et al, Mol Endocrinol, 5:1921-1930, 1991). Transgenic mouse lines which contained the entire PSA gene (PSA-1 transgene) have been generated (Wei, C.W., et al, Proc. Natl. Acad. Sci. USA 94:6369-6374, 1997). These mice showed highly specific prostate expression of human PSA.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of distal enhancer element 2 (DEE2), a novel regulatory element of the human PSA gene. In vivo, DEE2 is located from about -5.2 kb to -3.6 kb relative to the transcriptional start site of the hK2 gene.

In a first embodiment, an isolated nucleic acid sequence containing a distal enhancer element 2 (DEE2) thereof is provided.

In another embodiment, the invention provides a nucleic acid construct including a functional distal enhancer element 2 (DEE2), operably linked to a nucleic acid sequence encoding a heterologous protein. The nucleic acid construct may optionally include a nucleic acid sequence encoding distal enhancer 1 (DEE1). Expression vectors, host cells, and transgenic mice including this construct are also provided.

In yet another embodiment, the invention provides a method for producing a transgenic nonhuman animal having a phenotype characterized by expression of a transgene in the prostate otherwise not naturally occurring, by introducing a transgene including a distal enhancer element 2 (DEE2) into an embryo of an animal, and then transplanting the embryo into a pseudopregnant animal. The embryo is allowed to develop to term, and transgenic offspring carrying the DEE2 transgene is identified. In a further embodiment, a method for providing transcription of a nucleic acid sequence in the a prostate cell of an animal by introducing into a cell of the animal a nucleic acid construct comprising a distal enhancer element 2 (DEE2), operably associated with a nucleic acid sequence which encodes a product, is provided. The product can be a biologically active polypeptide, a therapeutic agent, a cytotoxic agent, an antisense RNA, or a ribozyme, for example.

The invention also provides a method of treating a subject having or at risk of having a prostate disorder by administering to the subject a therapeutically effective amount of a nucleic acid construct comprising a distal enhancer element 2 (DEE2).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and B show a schematic diagram of the PSA-1 and PSA-2 transgene constructs. The three lambda clones (11-1P1, 60-2.1P1 and 33-1.4P1) and the assembled PSA-1 and PSA-2 transgene constructs are depicted in relation to the genomic map. Solid boxes indicate exons, and the open boxes indicate the 3' untranslated regions of the PSA and hK2 genes. The 5' untranslated regions are not depicted because of their small size. The 0.8ES probe used to detect the incorporation of PSA-2 transgene and the DEE probe are depicted as open boxes under the genomic map. Restriction sites are abbreviates as follows: B, BamHI; E, EcoRI; H, Hindlll; S, Sail; Sm, Smal. Note that the Smal site is not unique; it is included to define the 3' boundary of the PSA-2 construct.

FIG. 2A and B show the location of cross-hybridizing region based on the restriction map of PSA-2. Hatched box indicates the location of cross-hybridizing region termed DEE2. Dotted boxes indicate plasmid vector sequence.

FIG. 3 is the sequence of the DEE2 element. Underlining indicates the position of a putative ARE. FIG. 4A and B show sequence comparisons of the ARE elements from DEE1, DEE2 and a mutant DEE1^* isolated from a prostate cancer patient with an extremely high level of serum PSA (Pang et al, Cancer Res. 57:495-499, 1997).

FIG. 5 illustrates a proposed model for coregulation of PSA and hK2. This model illustrates a possible mechanism by which the DEE1 and DEE2 enhancers (ovals) up-regulate transcription of PSA and hK2 by interacting with the promoters (open squares). The cross-hatched rectangles indicate the structural genes of PSA and hK2.

FIG. 6 shows the induction of PSA in a low copy number transgenic mouse. Serum PSA levels at day 0 (castration), 2, 4, 7 (testosterone implant), 9,11 and 14 are illustrated. "Tg" denotes a trangenic animal (square, circle) and "NTG" (triangle) is a non-trangenic control animal.

FIG. 7 shows the enhancer activity of an 880 bp Pstl fragment of DEE2 when linked to a heterologous minimal S V40 promoter and downstream luciferase reporter gene, as assessed in transiently transfected LNCaP cells. "II" denotes the luciferase reporter containing the 880 bp Pst I fragment of DEE2 with the SV40 promoter and "hK2p" denotes the luciferase reporter containing the minimal hK2 promoter; "II- hK2p" reporters contain both the 880 bp Pst I fragment of DEE2 and the hK2 promoter, "down" indicates that the 880 bp Pst I fragment of DEE2 is positioned downstream (i.e. 3') of the luciferase gene; "rev" indicates that the 880 bp Pst I fragment of DEE2 is located upstream (i.e. 5') of the luciferase gene, but in reverse orientation relative to genomic DEE2; and +/- indicates the presence or absence of androgen. Luciferase reporter activity is shown as "light units." DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel enhancer element, distal enhancer element 2 (DEE2), which is a positive regulatory element of the human prostate- specific antigen (PSA) gene. The presence of DEE2 abrogates the dependence of high PSA gene copy-number for high levels of PSA expression in transgenic mice which have incorporated a PSA gene. Thus, the identification of DEE2 allows the production of transgenic mice expressing high levels of polypeptides in the prostate. Identification of DEE2 also allows construction of specific gene therapy vectors for targeting the cells of the prostate. For example, DEE2 can be used alone, or in combination with other PSA regulatory sequences, in conjunction with therapeutic genes or "suicide" genes to treat diseases of the prostate.

The gene that encodes PSA is on chromosome 19, just upstream from the closely related (80% identity at the amino acid level) kallikrein gene called hK2 (hGK-1). The term "DEE2" or "distal enhancer element 2" refers to the nucleotide sequence of SEQ ID NO: 1 (Fig.3), as well as complementary sequences and sequences which exhibit at least about 80% sequence identity, preferably at least 85% sequence identity, more preferably at least about 95% sequence identity with SEQ ID NO:l by BLAST analysis. In vivo, DEE2 is located from -5.2 kb to -3.6 kb relative to the transcriptional start site of the hK2 gene. BLAST sequence homology searches of this region (Altschul, S.F., et al., J. Mol. Biol. 215:403-410, 1990, herein incorporated by reference) show this region to be novel. A dot blot analysis of DEE 1 and DEE2 reveal extensive homology. It should be noted that DEE2 includes multiple active domains. For example, a stretch of sequence with high identity is located cluster in a region of about 400 base-pairs in length, which has been identified as the "core enhancer" of DEE1 (Schuur, E.R., et al., J Biol. Chem. 271:7043-7051, 1996;

Cleutjens, K.B., et al, Mol Endocrinol 11:148-161, 1997; Zhang, J.Y., et al, Nucleic Acid Res. 25:3143-3150, 1997, all herein incorporated by reference), a putative androgen responsive element (ARE) is located in the core enhancer region. As used herein, the term "regulatory sequence" or "regulatory element" or "expression control sequence" refers to a nucleic acid sequence capable of controlling the transcription of an operably associated gene. The term "operably associated" refers to functional linkage between the regulatory sequence and a gene, such as a structural gene, regulated by the regulatory sequence. The components so described are thus in a relationship permitting them to function in their intended manner. A regulatory sequence may include a promoter, an enhancer, a transcriptional or translational start site, a transcriptional or translational termination region, and/or a silencer, for example. Therefore, placing a gene under the regulatory control of a promoter, enhancer, or other regulatory element means positioning the gene such that the expression of the gene is controlled by the regulatory sequence(s). The term "promoter" as used herein refers to the minimal nucleotide sequence sufficient to direct transcription. Included are those promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue specific, or inducible by external signals or agents. Regulatory elements may be located in the 5' or 3' regions of the native gene, or in the introns.

A functional DEE2 or functional fragment thereof serves as an enhancer element. An "enhancer" is a regulatory element which is able increase the expression of an operably linked gene. An enhancer can be positioned to either side of the gene or within the coding region, and can occur on the template or coding strand. An enhancer is activated upon binding by regulatory proteins. The preferred positioning of a regulatory element, such as an enhancer, with respect to a heterologous gene placed under its control generally reflects its natural position relative to the structural gene it naturally regulates. Specific enhancers are effective in only particular types of cells. DEE2 is effective in the cells of the prostate gland.

Regulatory sequence functions during expression of a gene under its regulatory control and can be tested at the transcriptional stage using DNA/RNA and RNA/RNA hybridization assays (e.g., in situ hybridization, nucleic acid hybridization in solution or solid support) and at the translational stage using specific functional assays for the protein synthesized (e.g., by enzymatic activity, by immunoassay of the protein, by in vitro translation of mRNA or expression in microinjected xenopus oocytes).

In one embodiment, a distal enhancer element 1 (DEE1) enhancer can be operably linked to nucleic acid sequences encoding DEE2, which are operably linked to a nucleic acid sequence encoding a heterologous protein. The term "DEE1 " refers to a previously isolated distal enhancer element located at -5.3 to -3.7 kb to the PSA gene (Schuur, E.R., et α/., J Biol. Chem. 221:7043-7051, 1996; Cleutjens, K.B., et al, Mol. Endocrinol. 11:148-161, 1997; Wei, C.W., et al, Proc. Natl. Acad. Sci. USA 94:6369-6374, 1997; Zhang, J.Y., et al, Nucleic Acid Res. 25:3143-3150, 1997, all herein incorporated by reference).

The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 10 bases in length. By "isolated polynucleotide" is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g. , a cDNA) independent of other sequences. The nucleotides of the invention can be of various lengths, including, for example, 15, 20, 30 or 50 nucleotides or more and which preferably have enhancer function analagous with DEE2 set forth herein. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA. Nucleic acids expressing the products of interest can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit. Polynucleotide or nucleic acid sequences of use with the DEE2 enhancer of the invention include DNA, RNA and cDNA sequences.

Nucleic acid sequences utilized in the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features and 3) synthesis by the polymerase chain reaction (PCR). Sequences for specific genes can also be found in GenBank, National Institutes of Health computer database.

The phrase "nucleic acid sequence expressing a product of interest" refers to a structural gene which expresses a product such as biologically active protein of interest or an antisense RNA for example. The term "structural gene" excludes the non-coding regulatory sequence which drives transcription. The structural gene may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. A structural gene may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides. The structural gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions. The structural gene may also encode a fusion protein. It is contemplated that introduction into animal tissue of nucleic acid constructs of the invention will include constructions wherein the structural gene and its regulatory sequence, e.g., prostate specific antigen (PSA) regulatory sequence, are each derived from different animal species. The term "heterologous nucleic acid sequence" as used herein refers to at least one structural gene which is operably associated with the regulatory sequence of the invention. The nucleic acid sequence originates in a foreign species, or, in the same species if substantially modified from its original form. For example, the term "heterologous nucleic acid sequence" or includes a nucleic acid originating in the same species, where such sequence is operably linked to a regulatory sequence that differs from the natural or wild-type regulatory sequence (e.g., DEE2 regulatory sequence). The heterologous sequence can encode a protein, such as a therapeutic agent. A "therapeutic agent" is any polypeptide which, when expressed, can be used to treat the cause, or ameliorate the symptoms of, a disorder. The term "ameliorate" denotes a lessening of the detrimental effect of a disorder in the subject receiving therapy. "Gene expression" means the process by which a nucleotide sequence undergoes successful transcription and translation such that detectable levels of the delivered nucleotide sequence are expressed in an amount and over a time period so that a functional biological effect is achieved. "Expressible genetic construct" as used herein means a construct which has the DEE2 positioned with a nucleic acid encoding a desired product, such that the nucleic acid is expressed.

Examples of genes encoding therapeutic agents which can be used in the invention construct include biologically active polypeptides. One specific, nonlimiting example of a biologically active polypeptide is an antibody which specifically binds an antigen found in a prostate tumor. The term "antibody" as used herein includes intact molecules as well as fragments thereof, which are capable of binding the epitopic determinant. Another specific, nonlimiting example for uses such as immunotherapy or cancer therapy, are genes encoding cytotoxic agents. The term "cytotoxic agent" refers to a protein or other molecule having the ability to inhibit, kill, or lyse a particular cell. Cytotoxic agents include proteins such as ricin, abrin, diphtheria toxin, or the like. Expression of such proteins intracellularly results in inhibition of protein synthesis or death of the cell. The nucleic acid sequence encoding a protein of interest in the invention construct includes biologically active peptides such as immunomodulators and other biological response modifiers. The term "biological response modifiers" encompasses substances which are involved in modifying the immune response in such manner as to enhance the destruction of tumor, for example. Examples of immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, the interleukins, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon, amongst others. Included in this category are immunopotentiating agents including nucleic acids encoding a number of the cytokines classified as "-interleukins". These include, for example, interleukins 1 through 12. Also included in this category, although not necessarily working according to the same mechanisms, are interferons, and in particular gamma interferon (γ-IFN), tumor necrosis factor (TNF) and granulocyte- macrophage-colony stimulating factor (GM-CSF). Nucleic acids encoding growth factors, toxic peptides, ligands, receptors, suicide factors (e.g., TK) or other physiologically important proteins can also be expressed in cells of the prostate.

Sense or antisense nucleic acids can be used in the invention construct. For example, a sense polynucleotide sequence (the DNA coding strand) encoding a polypeptide can be introduced into the cell to increase expression of a "normal" gene. Other cell disorders can also be treated with nucleic acid sequences that interfere with expression at the translational level. This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme. Alternatively, the method includes administration of a reagent that mimics the action or effect of a gene product or blocks the action of the gene. Therefore, when a prostate tumor is etiologically linked with over expression of a polynucleotide, it would be desirable to administer an inhibiting reagent such as an antisense polynucleotide. The term "inhibit" or "inhibiting" refers to a measurable reduction in activity, preferably a reduction of at least 10% versus control, more preferably a reduction of 50% or more, still more preferably a reduction of 80% or more.

The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (see, e.g., Marcus-Sakura, Anal. Biochem. , 172:289, 1988). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40. 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell.

Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al, Antisense Res. and Dev., 1(3):227, 1991; Helene, C, Anticancer Drug Design, 6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type

(Hasselhoff, Nature, 334:585. 1988) and "hammerhead"-type. Tetrahymena-type, ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-typQ ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.

Nucleic acid sequences comprising DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein can be expressed in vitro by DNA transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. In a preferred embodiment, the eukaryotic cell is a human cell. The term "host cell" also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

In the present invention, the nucleic acid sequences encoding DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein may be inserted into an expression vector. The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the DEE2 nucleic acid sequences. Polynucleotide sequence which encode DEE2 can be operatively linked to other expression control sequences, such as a promoter, a transcriptional initiation region, a translational initiation region, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, a transcriptional termination region, and a translational termination region, amongst others. The term "control sequences" is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Both constitutive and inducible promoters, are included in the invention (see, e.g., Bitter et al, Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retro virus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.

In the present invention, a nucleic acid construct encoding DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7 -based expression vector for expression in bacteria (Rosenberg et al, Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J Biol. Chem.. 263:3521. 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).

Polynucleotide sequences encoding DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein can be introduced into either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.

By "transformation" is meant a genetic change induce in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, the genetic change is generally achieved by introduction of the DNA into the genome of the cell (i.e., stable). By "transformed cell" is meant a cell into which (or into an ancestor of which has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding DEE2 operably linked to a nucleic acid sequence encoding a heterologous protein of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.

The present invention also provides gene therapy for the treatment of a tumor or disease which is in the prostate. Such therapy would achieve its therapeutic effect by introduction of the appropriate polynucleotide which contains a therapeutic gene for example, into cells of subjects having the disorder. Delivery of invention constructs can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.

Gene therapy methods as described herein can be performed in vivo or ex vivo. In addition, it may be preferable to remove the majority of a tumor prior to gene therapy, for example surgically or by radiation.

Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retro virus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) is utilized. A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a sequence (including promoter region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome, for example, to allow target specific delivery of the retroviral vector containing the polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retro virus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to Ψ2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.

Another targeted delivery system for the invention construct is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al, Trends Biochem. Sci., 6:77, 1981). In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to nontarget cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotech- niques, 6:682, 1988).

TRANSGENIC ANIMALS AND METHODS OF MAKING THE SAME

In one embodiment, the present invention provides nonhuman transgenic animals having a transgene including DEE2. The term "animal" as used herein denotes all species except human. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Farm animals (e.g., bovine, porcine, ovine and avian animals (cow, pig, sheep and birds), and other animals such as horses, rabbits and the like), rodents (such as mice), and domestic pets (e.g., cats and dogs) are included within the scope of the present invention. In a preferred embodiment the animal is a mouse or a rat.

A "transgenic" animal is any animal containing cells that bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with recombinant virus. These genetic manipulation results in the inclusion of exogenous genetic material in cells of the animal. By "exogenous" is meant genetic material that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. "Transgenic" in the present context does not encompass classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule. Although it is highly preferred that this molecule be integrated within the animal's chromosomes, the present invention also contemplates the use of extrachromosomally replicating DNA sequences, such as might be engineered into yeast artificial chromosomes. The term "transgenic animal" also includes a "germ cell line" transgenic animal. A germ cell line transgenic animal is a transgenic animal in which the genetic information has been taken up and incorporated into a germ line cell, therefore conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals. A "transgenic" animal can be produced by cross-breeding two chimeric animals which include exogenous genetic material within cells used in reproduction. Twenty-five percent of the resulting offspring will be transgenic i.e., animals which include the exogenous genetic material within all of their cells in both alleles. 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.

Various methods to make the transgenic animals of the subject invention can be employed. Generally speaking, three such methods may be employed. In one such method, an embryo at the pronuclear stage (a "one cell embryo") is harvested from a female and the transgene is microinjected into the embryo, in which case the transgene will be chromosomally integrated into both the germ cells and somatic cells of the resulting mature animal. In another such method, embryonic stem cells are isolated and the transgene incorporated therein by electroporation, plasmid transfection or microinjection, followed by reintroduction of the stem cells into the embryo where they colonize and contribute to the germ line. Methods for microinjection of mammalian species is described in United States Patent No. 4,873,191. In yet another such method, embryonic cells are infected with a retrovirus containing the transgene whereby the germ cells of the embryo have the transgene chromosomally integrated therein.

When the animals to be made transgenic are avian, because avian fertilized ova generally go through cell division for the first twenty hours in the oviduct, microinjection into the pronucleus of the fertilized egg is problematic due to the inaccessibility of the pronucleus. Therefore, of the methods to make transgenic animals described generally above, retrovirus infection is preferred for avian species, for example as described in U.S. 5,162,215. If microinjection is to be used with avian species, however, a recently published procedure by Love et al , (Biotechnology, 12(l):60-63. 1994) can be utilized whereby the embryo is obtained from a sacrificed hen approximately two and one-half hours after the laying of the previous laid egg, the transgene is microinjected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until maturity.

When the animals to be made transgenic are bovine or porcine, microinjection can be hampered by the opacity of the ova thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy. To overcome this problem, the ova can first be centrifuged to segregate the pronuclei for better visualization.

Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al, Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.

In the microinjection method useful in the practice of the subject invention, the transgene is digested and purified free from any vector DNA, e.g., by gel electrophoresis. It is preferred that the transgene include an operatively associated promoter which interacts with cellular proteins involved in transcription, ultimately resulting in constitutive expression. Promoters useful in this regard include those from cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus, as well as those from the genes encoding metallothionin, skeletal actin, Penolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine kinase. Promoters for viral long terminal repeats (LTRs) such as Rous Sarcoma Virus can also be employed. The PSA promoter can also be employed. When the animals to be made transgenic are avian, preferred promoters include those for the chicken β-globin gene, chicken lysozyme gene, and avian leukosis virus. Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements well known in the art such as enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosome binding sites to permit translation.

Retroviral infection can also be used to introduce transgene into a non-human animal, as described above. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264. 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al. in Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al, Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, et al, Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, 1985, supra; Stewart, et al, EMBO J. 6:383-388, 1987). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner, D., et al, Nature 2r 98:623-628, 1982). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic nonhuman animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (D. Jahner et al, supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans, M.J., et al, Nature 222:154-156, 1981; Bradley, M.O., et al, Nature 309:255-258, 1984; Gossler, et al, Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; and Robertson et al, Nature 322:445-448, 1986). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review, see Jaenisch, R., Science 240:1468-1474, 1988).

A "transgene" is any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Included within this definition is a transgene created by the providing of an RNA sequence which is transcribed into DNA and then incorporated into the genome. The transgenes of the invention include DNA sequences including DEE2, which may be expressed in a transgenic non-human animal.

EXAMPLE 1 MATERIALS AND METHODS Generation of PSA Transgene Constructs and Transgenic Mice. Isolation of the two lambda clones encompassing the PSA gene (clones 11-1P1 and 60-2.1P1) was described previously (Wei, et al, 1997, supra). Using the PSA cDNA as a probe to screen a human fetal liver genomic library (ATCC 37333), a third lambda clone (clone 33-1.4P1) was isolated. By restriction mapping and partial DNA sequencing analyses, this clone was mapped to the intergenic region between the PSA and hK2 genes with its 3' end extending into the third exon of the hK2 gene. The PSA-1 transgene construct cloned in the pBluescript vector (pBS/PSA-1) was built from lambda clones 11-1P1 and 60-2.1P1 as described previously (Wei, 1997, supra). To generate the PSA-2 transgene construct, the 4.3-kb EcoRI fragment from clone 60-2.1P1 was assembled with fragments from 33-1.4P1 into the pBluescript vector. The resulting plasmid clone was designated pBS/PSA-2, which harbored a 14.4-kb PSA-2 transgene construct starting from the EcoRI site immediately downstream from the PSA gene and ending at the Smal site in the second intron on the hK2 gene.

After removing the vector sequence, equal numbers of the PSA-1 and PSA-2 transgene constructs were microinjected into the pronuclei of fertilized embryos from the intercross of (C57BL/6J X DBA/2J) F, hybrid mice as described (Wei, 1997, supra). The incorporation of transgenes in the potential founders was identified by Southern blotting on tail DNA using PSA cDNA and probe 0.8ES (standing for the 0.8 kb EcoRI/Sall fragment within the PSA-2 construct) as probes, which were specific for PSA-1 and PSA-2, respectively. Both probes were labeled by the random hexamer method.

RNA Analysis. Total RNA isolated from the prostate and various other tissues was analyzed by Northern blotting for the PSA transgene expression. RNA isolated from the human prostatic cell line LNCaP (ATCC, Rockville, MD) was used as a positive control.

lmmunocytochemistry . Prostates of nontransgenic or transgenic mice were removed, fixed in formalin, embedded in paraffin, and 5 μm sections were placed on poly-L-lysine-coated slides. After quenching of endogenous peroxidases, sections were blocked with normal goat serum and stained with polyclonal rabbit-anti-human PSA (DAKO, Carpinteria, CA) or normal rabbit immunoglobulin. This was followed by horseradish peroxidase (HRP)-conjugated goat-anti-rabbit Ig (DAKO, Carpinteria, CA) and staining was visualized by addition of metal-enhanced diaminobenzidine (Pierce, Rockford, IL).

PSA and Total Protein Analyses. Prostate tissue was homogenized in

200μl of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, ImM MgCl₂ and 1% NP-40). After the particulate debris was removed by centrifugation at 12,000 x g for five minutes, the resulting lysate was then analyzed for PSA protein expression by a PSA specific ELISA (Wei, C, et al, Cancer Immunol. Immunother., 42:362-368. 1996) and for total protein by BCA assay (Pierce, Rockford, IL). Serum PSA levels were determined using the ELISA.

In Vitro Transient Transfection Assays. Heterologous luciferase reporter plasmids containing an 880 bp fragment of DEE2 linked to the SV40 minimal promoter was constructed as follows: The 880bp Pstl fragment from DEE2 was subcloned into pBluescript and, subsequently subcloned into various orientations of the SV40 promoter plasmid pGL2 (Promega) containing the luciferase reporter. For the native 5' to 3' orientation, this insert was excised using the Xho I and Sac I restriction sites of pBluescript flanking the Pst I fragment and cloned into the compatible sites of the pGL2 plasmid. The other constructs were made similarly. The sites used in pGL2 were as follows: for reverse orientation, the Bgl II and Kpn I sites were used; for downstream orientation, the BamHI and Sal I sites in pGL2 were used.

The plasmids containing a minimal promoter of hK2 were made by using PCR to generate a 490 bp fragment of the hK2 proximal promoter into the Hind III and Bgl II site of PGL2 in place of the SV40 minimal promoter (referred to as hK2p in FIG. 7). To construct the II-hK2p plasmid, the 880 Pst I fragment of DEE2 was excised from pBluescript using the restriction enzymes Xho I and Sac I and cloned into the compatible sites of hK2p plasmid containing the minimal hK2 promoter (referred to as II-hK2p in FIG. 7)

Reporter plasmids having this 880 bp fragment of DEE2 were transiently transfected into human prostate cancer LNCaP cells using DOTAP (Boehringer Manheim). Luciferase activity was determined using the Promega Luciferase assay kit as described by the manufacturer. Briefly, about 4 X 10⁵ cells were plated into 60mm dishes and allowed to attach. After 48 hours, 30μl of DOTAP and 5μg of DNA were prepared and added to the cells. Following transfection, cells were washed and new media with and without androgen (lOnm R1881) was added. After 24 hours, cells were harvested, lysed using lysis buffer provided by the manufacturer, and the clarified lysate assayed for luciferase activity. The light units were determined by analysis in a luminometer normalizing to cell number.

DNA Sequence Analysis. The nucleotide sequence for DEE2 was entered into the BLAST program (Altschul, S.F., et al, J. Mol. Biol. 215:403-410, 1990) and searched against all nonredundant sequences in GenBank, EMBL, DDB J and PDB databases. The dot blot analysis of DEE 1 and DEE2 was conducted using the Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI).

EXAMPLE 2 GENERATION AND CHARACTERIZATION OF PSA4 TRANSGENIC MICE. Generation ofPSA4 transgenic mice

Six independent lines of PSA1 transgenics, which had incorporated various copies of the 14-kb PSA-1 transgene construct (Wei, et al, 1997, supra), were previously generated. The PSA-1 transgene construct contains approximately 6 kb of 5' flanking sequence and 2 kb of 3' flanking sequence, in addition to all the exons and introns of the PSA gene (FIG. 1). PSA transgene expression is restricted to the epithelial cells of the prostate tissues in these mice, closely following the human expression pattern (McCormack et al, 1995, supra). In humans, another kallikrein gene, the hK2 gene, also encodes a prostate-specific product (Young et al, 1992, supra).

The hK2 gene is located directly downstream from the PSA gene (Riegman et al, 1992, supra) and appears to be coregulated. In order to determine if the intergenic region between these two genes contains regulatory elements responsible for either tissue specificity or expression levels the PSA-2 transgene construct was generated, which covers 14.4-kb of genomic sequence that spans the entire intergenic region and extends into the second intron of the hK2 gene (FIG. 1). Transgenic mice were generated by co-injecting equal numbers of both the PSA-1 and PSA-2 DNA molecules. Of the 118 offspring generated and screened, 14 founders were identified which had incorporated both the PSA-1 and PSA-2 transgenes. These founders were collectively called PSA4 transgenics in order to distinguish them from PSA1 transgenics, which only had the PSA-1 transgene (Wei et al, 1997, supra). Of these founders, all but P4-8 were able to transmit both transgenes through their germline and thus 13 PSA4 transgenic lines were established. These transgenic lines varied in the number of copies of the two transgenes they had incorporated, and could be divided into three groups based on these numbers. The first group had high copy- number (greater than 10) of each transgene per diploid genome, including lines P4-4, P4-5, P4-6, P4-7, P4-9, P4-10, P4-11 and P4-13. Low copy-number lines, such as P4-1, P4-2, P4-3, and P4-12, had less than 2 copies of each transgene per diploid genome, constituting the second group. The third group, represented by line P4-14, had high copy-number of PSA-1 but low copy-number of PSA-2.

Characterization of the PSA4 Transgenic Mice.

To analyze the expression of the human PSA transgene in the prostate of the PS A4 transgenic mice, total RNA from the prostate of P4- 1 , P4- 14 and P4-4 mice, each representing the three different groups of PSA4 transgenics, was isolated and analyzed by Northern blotting. The PSA cDNA probe did not cross-react with any mouse gene product, as neither prostate nor other tissues from the nontransgenic control mouse hybridized to the probe. However, the three PSA4 transgenic lines tested all expressed the 1.5 kb major transcript of human PSA mRNA in the prostate at levels comparable to those of PI -9 transgenic mouse, which possesses only the PSA-1 construct, and the human prostatic cell line LNCaP.

To examine the tissue distribution of PSA mRNA, Northern blot analysis was performed on a panel of tissues isolated from males of these three independent transgenic lines (P4-1, P4-14 and P4-4). Like PI -9 transgenics, all three PSA4 transgenic lines showed a highly restricted tissue distribution of the PSA transgene, with PSA mRNA being detected only in the prostate and coagulating gland even after the blots were intentionally over-exposed. No expression could be documented in Northern blots of 5 μg of total RNA isolated from the testis, seminal vesicle, spleen, kidney, liver, thymus, heart, lung, salivary glands, and brain of P4-1, P4-14, and P4-4 transgenic mice. Expression of the transgene in the coagulating gland of the PSA transgenics is expected since it is the mouse equivalent of the middle lobe of human prostate (Price, D., Natl. Cancer Inst. Monogr., 12:1-27, 1963). In these experiments, 5 μg of LNCaP total RNA was used in each blot to serve as an internal control. It is worth nothing, however, that salivary glands and kidney did not express the transgene even though they are known to express high levels of mouse kallikreins (Clements, J.A., Endocr. Rev., 10:393-419, 1989; van Leeuwen, B.H., et al., J. Biol. Chem. 261:5529-5535. 1986). Furthermore, no transgene expression was observed by Northern blot analysis in the same panel of tissues isolated from the female counterparts of these PSA4 transgenic lines, including ovary and uterus (data not shown). Therefore, these data show that the human PSA transgene is specifically expressed in the prostate and coagulating gland but is not expressed detectably in other tissues of the PSA4 transgenics.

To identify the cell type in the prostate of the transgenics that expresses PSA, immunocytochemistry was performed. PSA-specific staining was seen in the ductal epithelium of transgenic mice of the PI -9 line as well as the P4-4 line in a remarkably similar fashion to that of human prostate tissue. Sections of prostates of nontransgenic were used as controls. Some background staining was observed in the nontransgenic prostate tested, however, this staining was localized to the nuclei of epithelial cells, rather than the ductal lining as seen in the transgenic prostate tissue. As a further control, normal rabbit Ig was used instead of rabbit-anti-human PSA. When normal rabbit Ig was used instead of rabbit-anti-human PSA, no staining was observed in any of the prostate sections. Taken together, the highly restricted tissue distribution of the PSA transgene in both the PSA1 and PSA4 transgenics is remarkably similar to the PSA expression pattern reported for humans. Since the addition of the 14.4-kb PSA-2 transgene construct did not alter the tissue distribution of PSA, it suggests that the PSA-1 transgene construct contains all the regulatory elements required for prostate-specific expression.

EXAMPLE 3 ANALYSIS OF THE LEVEL OF PSA EXPRESSION IN TRANSGENIC MICE BY ELISA

The high copy-number PSA1 transgenic lines (Pl-6, Pl-8 and Pl-9) and the PSA4 lines with various copy-numbers of both transgene constructs (P4-1, P4-14 and P4-4) exhibited comparable levels of PSA mRNA in the prostate by Northern blot analysis (see Example 2, above). In order to determine the levels of PSA protein in the prostates of the transgenic mice, tissues were removed, homogenized and PSA expression was analyzed by a sandwich ELISA assay (Wei et al. 1996, supra). Prostate homogenates from 11 independent lines of transgenic mice with different copy numbers of PSA-1 and PSA-2 constructs were analyzed for PSA and total protein levels by ELISA and BCA assay, respectively. The group with low copy- number of PSA-1 construct included two PI -2 and one PI -7 mice. The group with high copy-number of PSA-1 construct included one each of the PI -4, Pl-6 and Pl-8 and four Pl-9 mice. The group with low copy-numbers of PSA-1 and PSA-2 constructs included two P4-1 and one each of P4-2 and P4-12 mice. The group with high copy-number of PSA-1 and various copy-number of PSA-2 constructs included two each of the P4-4 and P4-14 mice. The total protein content of the same sample was also determined and used to normalize the PSA expression.

There were a number of interesting points regarding the PSA expression pattern. First, the expression levels of PSA in the PSAl transgenic lines correlated with the copy-numbers of the PSA-1 transgene construct. The low copy-number lines (PI -2 and PI -7) had much lower levels of PSA protein in the prostate than the high copy-number lines (PI -4, Pl-6, Pl-8 and Pl-9) (FIG. 5), an observation also reflected in the mRNA levels (Wei et al, 1997, supra). Secondly, the levels of PSA expression in all the PSA4 transgenic lines tested (P4-1, P4-2, P4-4, P4-12 and P4-14) were very similar to one another, and were comparable to those of the high copy-number PSAl lines. These results strongly suggested the presence of additional regulatory elements contained in the PSA-2 construct, which, even when present at low copy-number in lines such as P4-1, P4-2 and P4-12, alleviated the requirement for many copies of the PSA-1 construct for high levels of PSA expression in vivo.

Two transgenic mice from the P4-1 pedigree were further analyzed for induction of PSA expression by androgens. Briefly, the mice were castrated (FIG. 6, day 0) resulting in a decrease of serum PSA levels. At day 7, a testosterone pellet was implanted into the mice; during implantation surgery one of the two mice died. For the second mouse, serum PSA levels increased significantly after implantation (FIG. 6, days 8-9). These results confirm that the PSA construct in transgenic animals is hormonally responsive.

EXAMPLE 4

ISOLATION OF a DISTAL ENHANCER ELEMENT IN THE INTERGENIC

REGION BETWEEN THE PSA AND HK2 GENES Previous work using reporter constructs and in vitro transfection assays have characterized several DNA elements involved in PSA expression (Schuur et al,

1996, supra; Cleutjens et al, 1997, supra; Zhang et al, 1997, supra; Cleutjens et al, 1996, supra; Riegman et al, 1991, supra). One critical element, important for both androgen responsiveness as well as tissue specificity, is located between -5.3 to -3.7 kb of the PSA gene (Schuur et al, 1996, supra; Cleutjens et al, 1997, supra; Zhang et al, 1997, supra). The importance of this element, DEE1 (Willis, R.A., et al, Int. J. Mol. Med., 1:379-386, 1998), was revealed by transgenic approaches. Transgenic mice made using 652 bp of the PSA promoter coupled to the ras oncogene did not result in prostate tumors, nor expression of ras in the prostate (Schaffner, D.L., et al, Lab. Invest., 22:283-290, 1995). Instead, these mice developed carcinomas of the salivary gland and gastrointestinal tract, presumably due to the expression of the transgene. In contrast, two independent reports where a larger 5' flanking regions was used, including the DEE1, showed highly specific expression of either PSA itself (Wei et al, 1997, supra) or LacZ (Cleutjens, K., et al, Mol. Endocrinol, 11 :1256- 1265, 1997) in transgenic mice.

It is known that PSA and hK2 are tightly regulated, and have a very similar and highly restricted tissue distribution. Previous in vitro and in vivo reports, indicated the presence of a distal enhancer element (DEE1) located at -5.3 kb to -3.7 kb in the PSA gene (Schuur et al, 1996, supra; Cleutjens et al, 1997, supra; Wei et al, 1991, supra; Zhang, JN., et al, Nucleic Acids Res., 25:3143-3150, 1997). Based on our in vivo results (see Examples 1-3, above), it was hypothesized that a related element might be present in the PSA-2 transgene construct. In order to isolate the positive element, a 0.9 kb Pstl fragment was subcloned from within the DEE element of the PSA gene (now called DEE1), and used as a DEE probe to identify elements capable of cross-hybridization in the PSA-2 genomic clone. For Southern blot analysis of the PSA-2 genomic clone pBS/PSA-2 was digested with either (a) EcoRI and Hindlll, (b) EcoRI and Sail, © Sail and BamHI, (d) EcoRI, (e) Hindlll, and (f) Sail. The resulting digests were resolved on an agarose gel, transferred to a nylon membrane, probed with the 0.9 kb Pstl fragment of PSA-1, then washed under low stringency, and hybridizing bands were visualized by autoradiography. These blots revealed sequences which were able to cross-hybridize to the DEE probe. Of these cross-hybridizing restriction fragments, the 1.6 kb Sall/EcoRI fragment (-5.2 kb to -3.6 kb relative to the transcriptional start of the hK2 gene) was isolated and termed DEE2 (FIG. 2). The DNA sequence of this DEE2 fragment is presented in FIG. 3. A BLAST sequence homology search (Altschul et al, 1990, supra) of the databases using this sequence revealed that it was novel. Since it was isolated by cross- hybridization, as expected, it showed significant homology to the DEEl fragment of the PSA gene.

Dot blot analysis of DEEl from the PSA gene versus DEE2 in the intergenic region between PSA2 and hK2 was performed. A dot blot analysis of DEEl and DEE2 revealed extensive identity throughout the whole region. Notably, a stretch of sequence with a high degree of identity is clustered at a region of about 400 bp in length, which has been previously identified as the core enhancer of DEEl (Schuur et al, 1996, supra; Cleutjens et al, 1997, supra; Zhang et al, 1997, supra). Included in the core enhancer region of the DEEl is an ARE, which is essential for the androgen-induced enhancer activity. A putative ARE is also identified in the corresponding region of DEE2, which differs from that of DEEl by one nucleotide. This single nucleotide substitution at the last position brings it closer to the consensus ARE sequence (10 out of 12 bases are identical, FIG. 4). Even more striking is that it is identical to the sequence of a mutant ARE found in a prostate cancer patient with an extremely high level of PSA (Pang, S., et al, Cancer Res., 52:495-499, 1997), suggesting that this base conversion is of functional significance.

DNA sequencing showed that the DEE2 element is highly homologous (76% identical) to DEEl. Sequence analysis has identified several potential AREs in the DEE2. Among these potential AREs, one exhibits a closer match to the consensus ARE (Roche, P.J., Mol. Endocrinol. , 6:2229-2235, 1992) than the corresponding sequence in DEEl in the 5' region of the PSA gene. Taken together, the data indicate that a novel additional enhancer which is capable of increasing the expression of PSA in vivo has been identified. The location of the DEE2, 3.6 kb upstream from the transcriptional start of the hK2 gene, also suggests that this element is an enhancer able to regulate the hK2 gene.

The in vivo expression data as well as the DNA sequence data strongly support the idea that the DEE2 is an enhancer element. Intriguingly, the presence of two copies of DEE2 in the P4-1, P4-2 and P4-12 lines results in same levels of PSA expression in the prostate as the PSAl lines with high copy-number of DEEl, suggesting that the two enhancers may work synergistically with each other. These transgenic experiments do not directly address the relative strength of the DEEl and DEE2. However, it is striking that the ARE of DEE2 is closer to the consensus ARE sequence than the ARE of DEEl by one base pair. This one base pair change may be very significant. The ARE of DEE2 is identical to the ARE found in a mutant DEEl from the PSA gene of a prostate cancer patient with an extremely high serum PSA level (Pang et al, 1997, supra). This finding suggests that improved androgen receptor binding may contribute to higher enhancer activity and androgen inducibility. This is also consistent with a previous report which showed a greater degree of androgen inducibility exhibited by the hK2 gene compared to the PSA gene (Henttu, P., et al, Endocrinology 130:766-772, 1992).

The DEE2 sequence is unique, although it has significant homology to DEEl . In addition, DNA sequences with weaker homology to DEE2 in the databases have been identified using the BLAST search. The DEE2 sequence shows significant homology (54% identical) to the 5' flanking region of the canine arginine esterase gene (Chapdelaine, P., et al, DNA Cell Biol, 10:49-59, 1991). This canine gene is androgen-regulated and is largely prostate-specific (Chapdelaine et al, 1991, supra; Gauthier, E.R., et al, Mol. Cell. Endocrinol, 94:155-163, 1993), suggesting that this regulatory region is conserved throughout evolution. Other more limited homologies (72% identical over approximately 100 base pairs) to sequences determined through the Human Genome Project from chromosome 21 (accession number AF013725) and chromosome 17 (accession number AC002091) were also revealed by the BLAST search. The functional significance of these homologies remains to be determined.

EXAMPLE 5 ENHANCER FUNCTION ANALYSTS OF DEE2 To confirm that DEE2 functions as an enhancer element, a series of transfection assays using a heterologous reporter were performed. SV40 luciferase reporters having an 880 bp Pstl fragment of the DEE2 enhancer positioned either downstream of the luciferase gene or upstream of the luciferase gene, but in the reverse orientation, were transfected into LNCaP cells. Androgen treated (+) and untreated (-) transfected cells were assayed for luciferase activity (FIG. 7). Luciferase activity was induced by androgen in cells having the 880 bp Pstl fragment of the

DEE2 enhancer positioned upstream of the luciferase in its native orientation (FIG. 7; II SV40+). In addition, the 880 bp Pstl fragment of DEE2 (II) conferred androgen inducibility when located either downstream of the luciferase gene (FIG. 7; II (down)) or upstream in reverse (rev) orientation (FIG. 7; II (rev)). These results confirm that the 880 bp Pstl fragment of the DEE2 enhancer element has enhancer function, as evidenced by its ability to function independently of its position relative to the gene and its 5 '-3' orientation.

Without being bound by theory, it is likely that the conservation between PSA and hK2 genes not only includes the coding and immediate upstream regions (Riegman et al, 1991, supra; Riegman, P.H., et al, Biochem. Biophys. Res.

Commun.. 159:95-102. 1989), but also the distal upstream enhancer region. It is possible that a gene duplication event arose in which a primordial kallikrein gene and its prostate specific regulatory elements were duplicated as a unit. After duplication, the genes diverged into a chymotryptic protease (PSA) and a trypsin-like protease (hK2) which is involved in regulating PSA. The duplication of the distal regulatory elements may have ensured that tissue specificity is maintained. This may also provide an additional means of ensuring optimal relative expression levels of the PSA and hK2 proteins. Both PSA and hK2 are synthesized in a zymogen form, and the propeptide fragment needs to be proteolytically removed to generate the active form. The recent observation that pro-PSA can be activated by the trypsin-like protease activity of hK2 supports the theory that hK2 is a component of a cascade that leads to the activation of PSA (Lovgren et al, 1997, supra; Takayama et al, 1997, supra). A coordinate regulation of these two genes provides a layer of control over the PSA expression and activity.

Without being bound by theory, given the proximity and coregulated nature of PSA and hK2, and the observation that DEE2 can enhance the expression of PSA (see Example 3), it is possible that DEE2 works in concert with DEEl to regulate the levels of PSA expression in vivo normally. A model for this is outlined in FIG. 5. In this model, the DEEl element regulates the expression of the PSA gene. In contrast, the DEE2 element regulates the expression of both the PSA and hK2 genes. This could result from the proximity of DEE2 to the PSA and hK2 promoters (approximately 14 kb and 4 kb respectively). In contrast, while the DEEl is in close proximity to the start of the PSA gene (approximately 4 kb), it is more than 22 kb from the transcriptional start of the hK2 gene. Thus, the distance from the promoter could determine the effectiveness of the enhancers. Alternatively, a stochastic model can be envisioned in which the enhancer interacts, perhaps via specific transcription proteins, in a bidirectional manner with the first promoter elements it encounters. In effect, this would mean that the PSA gene would have 1.5 functional enhancers, whereas hK2 gene would effectively have only one-half an enhancer, since it shares it with the PSA gene. This would be the first example of this type of regulation, although there are some examples of common or "shared" enhancers between genes. For example, differential use of an enhancer located between two genes has been suggested in the regulation of the hox b3 and hox b4 genes though in this situation there has only been one enhancer identified for both genes (Gould, a., et al, Genes Dev., 11:900-913,1997). The evolutionarily related GM-CSF and IL-3 genes have a similar genetic organization to PSA and hK2. The enhancer located between the GM-CSF and IL-3 genes has been suggested to act on both the IL-3 and GM-CSF promoters (Cockerill, P.N., et al, Proc. Natl. Acad. Sci. USA, 90:2466-2470, 1993; Nishida, J., et al, Int. Immunol, 3:245-254, 1991), although other studies have proposed that they may function independently (Osborne, C.S., et al, J. Immunol, 155:226-235. 1995). The model in FIG. 5 also explains why there appears to be more PSA than hK2 expressed, even though both genes appear to have strong enhancers located in similar positions relative to their respective promoters. This model explains not only the overlapping tissue specificity but also the disparity in the expression levels between PSA and hK2 in vivo. Likewise, it also helps to explain the PSA levels observed in the transgenics. For instance, PSA is up-regulated by both DEEl and DEE2 in the P4-1, P4-2 and P4-12 mice, resulting in high levels of PSA which can only be achieved with high copy-numbers of DEEl alone.

The in vivo approach of using flanking regions of the PSA gene to generate transgenic mice has identified critical regulatory regions of the PSA and hK2 gene complex. By varying the flanking sequences used to generate sets of transgenic mice, a potential interplay between two enhancer elements has been demonstrated. Understanding the regulation of this gene family can also lead to a better understanding of how these proteases function in normal as well as cancerous prostate cells. Furthermore, the identification of elements which confer prostate specific expression can have significant implications for cancer gene therapy.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. An isolated nucleic acid sequence, comprising a distal enhancer element 2 (DEE2).

2. An isolated polynucleotide selected from the group consisting of: a) SEQ ID NO: 1; b) SEQ ID NO: 1 , wherein T is U; c) nucleic acid sequences complementary to a) or b) ; and d) fragments of a), b), or c) having at least 15 nucleotides.

3. An isolated polynucleotide having at least 80% sequence identity with the isolated polynucleotide of claim 2.

4. An isolated polynucleotide having at least 85% sequence identity with the isolated polynucleotide of claim 2.

5. An isolated polynucleotide having at least 95% sequence identity with the isolated polynucleotide of claim 2.

6. The isolated polynucleotide of claim 2, wherein said fragment comprises a Pst I fragment of DEE2.

7. The isolated polynucleotide of claim 2, wherein said fragment has enhancer function.

8. A nucleic acid construct, comprising: a distal enhancer element 2 (DEE2) or functional fragment thereof having at least 15 nucleotides operably linked to a nucleic acid sequence encoding a heterologous protein.

9. The nucleic acid construct of claim 8, further comprising a distal enhancer element 1 (DEEl).

10. The nucleic acid construct of claim 8, further comprising a transcriptional and translational initiation region.

11. The nucleic acid construct of claim 8, further comprising a transcriptional and translational termination region.

12. The nucleic acid construct of claim 8, wherein the nucleic acid sequence encodes a therapeutic reagent.

13. The nucleic acid construct of claim 8, wherein the nucleic acid sequence encodes a biologically active polypeptide.

14. The nucleic acid construct of claim 8, wherein the nucleic acid sequence encodes an antibody.

15. The nucleic acid construct of claim 8, wherein the nucleic acid sequence encodes an member of the group selected from an antisense RNA and a ribozyme, for disruption expression of an endogenous coding sequence.

16. The nucleic acid construct of claim 8, wherein the nucleic acid sequence encodes a cytotoxic agent.

17. An expression vector comprising the nucleic acid construct of claim 8.

18. The vector of claim 17, wherein the vector is a plasmid.

19. The vector of claim 17, wherein the vector is a viral vector.

20. A host cell containing the vector of claim 17.

21. The host cell of claim 20, wherein the cell is prokaryotic.

22. The host cell of claim 20, wherein the cell is eukaryotic.

23. The host cell of claim 22, wherein the cell is a human cell.

24. A transgenic nonhuman animal comprising the nucleic acid construct of claim 8 chromosomally integrated into the germ cells of the animal.

25. The transgenic animal of claim 24, wherein the animal is selected from the group of species consisting of murine, avian, bovine, ovine, piscine, and porcine.

26. The transgenic animal of claim 24, wherein the species is murine.

27. A method for producing a transgenic nonhuman animal having a phenotype characterized by expression of a transgene in the prostate otherwise not naturally occurring, comprising a) introducing a transgene into an embryo of an animal, said transgene comprising a distal enhancer element 2 (DEE2) or functional fragment thereof operably linked to a nucleic acid sequence encoding a heterologous protein; b) transplanting the embryo of a) into a pseudopregnant animal; c) allowing the embryo to develop to term; and d) identifying at least one transgenic offspring from c).

28. The method of claim 27, wherein said transgene further comprises a distal enhancer element 1 (DEEl).

29. The method of claim 27, wherein the animal is selected from the group of species consisting of murine, avian, bovine, ovine, piscine, and porcine.

30. The method of claim 27, wherein the animal is a mouse.

31. The method of claim 27, wherein said introducing of said transgene into said embryo results in expression of said heterologous protein in a cell of the prostate of said animal.

32. The method of claim 27, wherein the introduction of the transgene into the embryo is by introducing an embryonic stem cell containing the transgene into the embryo.

33. The method of claim 27, wherein the introduction of the transgene into the embryo is by infecting the embryo with a virus containing the transgene.

34. The method of claim 33, wherein the virus is a retrovirus.

35. A method for providing transcription of a nucleic acid sequence in a prostate cell of an animal, comprising: introducing to a cell of the animal a nucleic acid construct comprising a distal enhancer element 2 (DEE2) or functional fragment thereof operably associated with a nucleic acid sequence which encodes a product.

36. The method of claim 35, wherein said product is selected from the group consisting of a biologically active polypeptide, a therapeutic agent, a cytotoxic agent, an antisense RNA, and a ribozyme.

37. The method of claim 35, wherein said nucleic acid sequence further comprises a distal enhancer element 1 (DEEl).

38. A method of treating a subject having or at risk of having a prostate disorder, comprising administering to the subject a therapeutically effective amount of a nucleic acid construct comprising a distal enhancer element 2 (DEE2) or functional fragment thereof.